A copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-enzyme activity and its preparation method and application

Abstract

A copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-enzyme activity, as well as its preparation method and application, is disclosed. In a neutral microenvironment, the material primarily exhibits catalase-like activity, scavenging H2O2 and alleviating oxidative stress. In a weakly acidic microenvironment, it primarily exhibits peroxidase-like activity, catalyzing H2O2 to produce the more toxic ·OH antibacterial compound. Simultaneously, copper's inherent osteogenic properties synergistically enhance bone regeneration. The present invention evaluates the SA / CuHP hydrogel's biocompatibility, antioxidant properties, osteogenic ability under oxidative stress conditions, and antibacterial efficacy under weakly acidic conditions through in vitro experiments. A periodontitis model in type 2 diabetic rats is established to verify its in vivo antibacterial and osteogenic effects and biosafety, providing a new strategy for the treatment of diabetic periodontitis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-type enzyme activity, and a preparation method and application thereof. Background Art

[0002] Alveolar bone is an important periodontal tissue. During the development of periodontitis, irreversible resorption and destruction of alveolar bone can lead to loose teeth and even tooth loss. 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 the body and peroxides that are easily formed into free radicals, including superoxide free radicals (O2 - ), hydroxyl radical (·OH), nitric oxide radical (·NO), singlet oxygen ( 1 O2) and hydrogen peroxide (H2O2), among others. When attacked by pathogenic bacteria, periodontal inflammatory tissues upregulate the production of ROS to eliminate microorganisms. However, excessive ROS cannot be promptly eliminated, resulting in an imbalance between the oxidative and antioxidant functions of periodontal tissues, a phenomenon known as oxidative stress. Hyperglycemia induces the production of ROS through pathways such as the polyol pathway, the hexosamine pathway, the protein kinase C pathway, and the advanced glycation end product pathway, exacerbating oxidative stress. In periodontal tissues, excessive ROS leads to increased apoptosis of osteoblast-related cells and decreased osteogenic differentiation through pathways such as lipid peroxidation, protein denaturation, and DNA damage. This interferes with bone matrix synthesis and mineralization, destroying the quality and structure of alveolar bone and accelerating alveolar bone resorption.

[0003] Diabetes complicates antimicrobial treatment of periodontitis. Under normal circumstances, the buffering effect of saliva maintains oral pH in the neutral range, which is crucial for maintaining oral health. However, under periodontitis conditions, the formation of bacterial biofilms and dental plaque results in a weakly acidic periodontal microenvironment (pH 4.5–6.5). Glucose levels in the gingival crevicular fluid of diabetic patients are significantly elevated, providing a rich source of nutrients for subgingival microorganisms, altering the composition and structure of the microbial community, shifting it toward a state conducive to pathogenic growth and promoting the development of periodontitis. This local microecological imbalance makes diabetic periodontitis more likely to progress to extensive lesions, involving the furcation zone, reducing the cleaning efficiency of traditional mechanical debridement instruments in this area. Therefore, controlling plaque microorganisms remains a challenge in the treatment of diabetic periodontitis.

[0004] Therefore, for diabetic periodontitis, it is urgent to develop treatment strategies that have antibacterial, antioxidant and bone repair functions to slow down the destruction of alveolar bone.

[0005] Currently, research on nanozymes for the treatment of diabetic periodontitis has focused on single enzyme activity and function, primarily on catalyzing the production of ROS or scavenging ROS. Therefore, designing novel multifunctional biomaterials based on the need for bidirectional regulation of ROS in the diabetic periodontitis microenvironment—that is, achieving antibacterial effects through ROS generation and alleviating oxidative stress damage by scavenging excess ROS—is beneficial for improving the therapeutic efficacy of diabetic periodontitis. Summary of the Invention

[0006] In order to solve the technical defects of single enzyme activity and function in the existing research on nanozymes for treating diabetic periodontitis, the present invention provides a copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-type enzyme activity, as well as its preparation method and application.

[0007] The technical solution adopted by the present invention is: a copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-type enzyme activity, wherein the copper hydrogen phosphate-loaded hydrogel is constructed by loading copper hydrogen phosphate (CuHP) on an injectable sodium alginate hydrogel (SA), wherein 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 0.5-1wt%.

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

[0009] A method for preparing a copper hydrogen phosphate-loaded hydrogel having pH-responsive multi-enzyme activity, characterized in that the method comprises the following steps:

[0010] (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. Dissolve copper sulfate (CuSO4) powder in double-distilled water, then add this solution to phosphate buffered saline (PBS) and shake the reaction to ensure uniform mixing and complete reaction. After the reaction is completed, separate the supernatant by centrifugation. Finally, wash and freeze-dry to obtain CuHP microflowers.

[0011] (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; finally, the mixed solution was added dropwise to the CaCl2 solution using a syringe to form SA / CuHP hydrogel.

[0012] Preferably, the concentration of SA in step (2) is 1.5 wt%.

[0013] Preferably, the sterilization condition of high temperature and high pressure sterilization in step (2) is sterilization at 121° C. for 15 minutes.

[0014] Preferably, the mass fraction of CuHP in step (2) is 0.5-1 wt%.

[0015] Application of a copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-enzyme activity in the preparation of bone repair materials for diabetic periodontitis.

[0016] 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 eliminate ROS.

[0017] The weak acid condition is pH=5, and the neutral condition is pH=7.4.

[0018] The POD-like enzyme reaction process of SA / CuHP hydrogel is shown in reaction formula (1) (2), and the CAT-like enzyme reaction process is shown in reaction formula (3) (4). The mechanism by which SA / CuHP exhibits POD-like and CAT-like enzyme activities under different pH conditions may be: (1) The variable valence state of copper ions (Cu + / Cu 2+ ): Cu in CuHP is the core of the catalytic active center. Under acidic conditions, Cu 2+ It tends to remain stable and catalyzes the heterolytic splitting of H2O2 to generate ·OH through a Fenton-like reaction, i.e. reaction (1); under neutral and weakly alkaline conditions, OH - As the concentration increases, Cu + It tends to remain stable, while H2O2 tends to dissociate into HO2·, which promotes the homolysis of H2O2 to generate O2. (2) Changes in the oxidizability of H2O2: Under acidic conditions, the oxidizability of H2O2 is stronger, and H2O2 is more easily oxidized by Cu + Reduction, generating OH; Under alkaline conditions, H2O2 has stronger reducing properties, and H2O2 is more easily absorbed by Cu 2+ Oxidation eventually produces O2.

[0019]

[0020] The beneficial effects of the present invention are: a copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-type enzyme activity, and its preparation method and application. The material mainly exhibits catalase-like activity in a neutral microenvironment, scavenging H2O2 and alleviating oxidative stress; in a weakly acidic microenvironment, it mainly exhibits peroxidase-like activity, catalyzing H2O2 to produce the more toxic ·OH antibacterial; at the same time, the inherent osteogenic properties of copper synergistically enhance bone regeneration. The present invention evaluates the biocompatibility, antioxidant properties, osteogenic ability under oxidative stress conditions, and antibacterial efficacy under weakly acidic 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, providing a new strategy for the treatment of diabetic periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the preparation process of SA / CuHP hydrogel and its application in the treatment of diabetic periodontitis.

[0022] Figure 2 Characterization of CuHP; (A) SEM image of CuHP (B) EDS image of Cu, P and O in CuHP (C) XRD characteristic peak diagram of CuHP.

[0023] Figure 3 Macroscopic (A) and SEM images (B) of SA / CuHP hydrogels with different concentrations.

[0024] Figure 4 The gelation (A), injectability (B) and in situ gelation (C) of SA / CuHP hydrogel.

[0025] Figure 5 POD enzyme activity of SA / CuHP hydrogel; (A) SA / CuHP hydrogels with different concentrations (B) different pH.

[0026] Figure 6 CAT-like enzyme activity of SA / CuHP hydrogel under different pH conditions; (A) O2 bubble generation (B) H2O2 clearance rate.

[0027] Figure 7 These are rat mandibular BMSCs.

[0028] 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.

[0029] Figure 9 BMSCs were stimulated with 300 μM or 500 μM H2O2 for 3 hours and then cultured for 24 hours. Cell viability was detected by CCK8 assay.

[0030] Figure 10 (A) DCFH-DA fluorescence images of BMSCs after stimulation with different treatments for 3 hours and continued culture for 24 hours. (B) Semi-quantitative analysis of intracellular ROS levels.

[0031] Figure 11 BMSCs were stimulated with different treatments for 3 hours and then cultured for 24 hours. Cell viability was detected by CCK8 assay.

[0032] Figure 12 (A) Live-dead cell staining images of BMSCs after stimulation with different treatments for 3 hours and continued culture for 24 hours. (B) Semi-quantitative analysis of live cell rate.

[0033] Figure 13 (A) BMSCs were stimulated with different treatments for 3 hours and then cultured for 24 hours. Scratch wound images at 0 and 24 hours were taken. (B) Semi-quantitative analysis of cell migration rate was performed.

[0034] Figure 14 (A) ALP staining images of BMSCs after 3 hours of stimulation with different treatments and 7 days of osteogenic induction culture. (B) Semi-quantitative analysis of ALP activity.

[0035] Figure 15 (A) Alizarin red staining images of BMSCs after 3 hours of stimulation with different treatments and 21 days of osteogenic induction culture. (B) Semi-quantitative analysis of calcium nodule formation.

[0036] Figure 16 (A) RUNX2 immunofluorescence staining images of BMSCs after 3 hours of stimulation with different treatments and 4 days of osteogenic induction culture. (B) Quantitative analysis of RUNX2 fluorescence intensity.

[0037] Figure 17 The expression levels of osteogenic-related mRNA in BMSCs after stimulation with different treatments for 3 hours and osteogenic induction culture for 4 days.

[0038] Figure 18 The expression levels of osteoblast-related proteins in BMSCs after being stimulated with different treatments for 3 hours and cultured for 4 days.

[0039] Figure 19 For (A) Aa Images of colony formation on the spot plates after 24 h of culture with different treatments (B) Colony counting.

[0040] Figure 20 for Aa Live and dead bacterial staining images after 24 hours of culture with different treatments.

[0041] Figure 21 for AaSEM images of cells cultured with different treatments for 24 h.

[0042] Figure 22 Body weight (A) and blood glucose levels (B) of SD rats on days 0, 3, and 7 after STZ injection.

[0043] Figure 23 The process of modeling periodontitis in the maxillary left second molar of SD rats; (A) before silk ligature (B) silk ligature (C) removal of the ligature.

[0044] Figure 24 (A) Image of gingival crevicular fluid smear colonies and (B) colony counts.

[0045] Figure 25 (A) Micro-CT 3D reconstruction, sagittal section, and X-ray image of the rat maxillary bone. (B) The distance between the alveolar bone crest and the cementoenamel junction (ABC-CEJ) of the rat maxillary second molar.

[0046] Figure 26 are the bone volume fraction (BV / TV) and trabecular separation (Tb.Sp) of the alveolar bone between the first and second molars of the rat maxilla.

[0047] Figure 27 HE staining of periodontal tissue of rat D.

[0048] Figure 28 Masson staining of rat periodontal tissue. DETAILED DESCRIPTION

[0049] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0050] Example 1 Preparation and characterization of CuHP-loaded hydrogel

[0051] Preparation of CuHP and CuHP-loaded hydrogels

[0052] CuHP was provided by the Shanghai Institute of Ceramics, China, and was prepared as follows. First, 0.768 g of copper sulfate powder was dissolved in 10 mL of double-distilled water, and this solution was then added to 500 mL of 0.02 M PBS. The reaction was then shaken for 6 hours to ensure uniform mixing and complete reaction. After the reaction, the supernatant was separated by centrifugation (10,000 rpm for 5 minutes). Finally, the solution was washed three times and lyophilized to obtain CuHP.

[0053] To prepare the SA / CuHP composite hydrogels, SA was first added to double-distilled water at a concentration of 1.5% (wt%), magnetically stirred until completely dissolved, sterilized under high temperature and high pressure (121°C for 15 minutes), and cooled to obtain the SA solution. 1g of calcium chloride and 100mL of deionized water were then weighed and stirred to prepare a 1% calcium chloride solution for later use. CuHP (0.25%, 0.5%, 1%, and 2% wt%) and the SA sol were then mixed using a two-way tube and syringe. Finally, this mixed solution was added dropwise to the CaCl2 solution using a 1mL syringe to form the SA / CuHP hydrogels, designated SA / CuHP (0.25%), SA / CuHP (0.5%), SA / CuHP (1%), and SA / CuHP (2%). The SA solution was then added dropwise directly to the CaCl2 solution using a 1mL syringe to form the SA hydrogels. Since the minimum filling accuracy of a 1 mL syringe is 10 µL and a volume of 10 µL can essentially fill the rat periodontal pocket when the hydrogel is applied in vivo, 10 µL hydrogel spheres were prepared for in vitro cell and bacterial experiments.

[0054] Characterization of CuHP-loaded hydrogels

[0055] The micromorphology of SA / CuHP hydrogels with different concentrations (0.25%, 0.5%, 1%, and 2% wt%) was observed using SEM. The injectability of the hydrogels was evaluated by gross observation.

[0056] POD 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. The resulting mixture was stirred for 20 minutes, and then 0.8 mL of 3% H₂O₂ solution was added. The reaction was allowed to proceed at room temperature for 10 minutes, and the absorbance change at 664 nm was measured using 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), reacted at room temperature for 10 minutes, and the absorbance change at 664 nm was measured using UV-vis-NIR spectrophotometry.

[0057] CAT-like enzyme activity: SA and SA / CuHP hydrogels were immersed in 10% H₂O₂ solutions at pH 5, 6, and 7.4, respectively. After incubation at 37°C for 5 minutes, the generation of O₂ bubbles was observed and recorded. Furthermore, a CAT assay kit was used to evaluate the CAT-like enzyme activity of SA / CuHP hydrogels at different pH conditions (5, 6, and 7.4). According to the assay principle, the CAT decomposition reaction of H₂O₂ can be rapidly terminated by the addition of ammonium molybdate. The remaining H₂O₂ reacts with the ammonium molybdate to form a pale yellow complex. The CAT activity can be calculated by measuring the absorbance change at 405 nm using a microplate reader.

[0058] Experimental results

[0059] Basic characteristics of CuHP

[0060] like Figure 2 As shown in A, the synthesized particles exhibit a micro-flower-like structure, consisting of numerous petal-like nanosheets. Figure 2 As shown in B, copper, phosphorus and oxygen elements are evenly distributed ( Figure 2 B). Figure 2 As shown in Figure C, the X-ray diffraction peaks of the sample precisely correspond to the diffraction peaks of Cu4H(PO4)3·3H2O (PDF No. 31-0458). These results demonstrate the successful synthesis of CuHP microflowers in this study.

[0061] Basic characteristics, injectability and enzyme-like activity of CuHP-loaded hydrogels

[0062] like Figure 3 As shown in A, pure SA hydrogel is colorless, and with the increase of CuHP incorporation, the blue color of SA / CuHP hydrogel gradually deepens. Figure 3 As shown in B, SA hydrogel has a porous structure, and CuHP with different concentrations is evenly distributed on the surface of SA hydrogel without destroying its porous framework structure.

[0063] like Figure 4 As shown in A, when SA / CuHP hydrogel comes into contact with CaCl2 solution, the hydrogel transforms from sol state to gel state. Figure 4 As shown in Figure 2, SA / CuHP hydrogel exhibits excellent injectability and can be formed into different shapes. Figure 4 As shown in C, SA / CuHP hydrogel can form gel in situ after injection into the gingival sulcus of the second molar of rats.

[0064] Next, the POD-like enzyme activity of SA / CuHP hydrogel was investigated. Figure 5As shown in A, as the concentration of SA / CuHP hydrogel increases, the absorbance at 664 nm gradually decreases and the blue solution gradually becomes lighter, indicating that the blue reduced state MB is oxidized to a colorless oxidized state. This shows that the POD-like enzyme activity of SA / CuHP hydrogel is positively correlated with its concentration, that is, the higher the concentration, the stronger the ability to catalyze the decomposition of H2O2 to generate ·OH, thereby more effectively oxidizing MB. Subsequently, SA / CuHP (1%) hydrogel was used to evaluate the POD-like enzyme activity under different pH conditions (pH = 5, 6, 7.4). Figure 5 As shown in Figure 2(B), the absorbance at 664 nm decreased with decreasing pH, indicating that the POD-like enzyme activity of SA / CuHP hydrogel was higher under acidic conditions.

[0065] The effects of different pH on the CAT-like enzyme activity of SA / CuHP hydrogel were further investigated. Figure 6 As shown in A, no obvious O2 bubbles were observed in the H2O2 solution with the addition of SA hydrogel; however, after incubation of SA / CuHP hydrogel for 5 minutes, H2O2 decomposed: at pH 5, only trace O2 bubbles were visible around the hydrogel, and at pH 6, the number of O2 bubbles in the solution increased significantly. When the pH rose to 7.4, dense O2 bubbles appeared in the solution. Figure 6 As shown in Figure B, the H2O2 scavenging rate increased with increasing pH (pH 7.4 > 6 > 5), with the scavenging efficiency being higher under neutral conditions than under acidic conditions. These results indicate that the SA / CuHP hydrogel possesses pH-sensitive CAT-like enzyme activity, and its catalytic efficiency in decomposing H2O2 into H2O and O2 is stronger in neutral environments than in acidic ones.

[0066] The present invention combines the characteristics of the weakly acidic periodontal microenvironment caused by bacterial infection in diabetic periodontitis to design a material with multiple enzyme activities under different pH conditions, SA / CuHP hydrogel. The micronized flower morphology of CuHP provides a large specific surface area, which is conducive to interaction with surrounding substances and enhances the catalytic reaction. SA / CuHP hydrogel exhibits pH-sensitive multiple enzyme activities. Under weak acid conditions, it mainly exhibits POD enzyme activity, catalyzing H2O2 to produce the more toxic ·OH. Under neutral conditions, it mainly exhibits CAT enzyme activity, converting H2O2 into non-toxic O2 and H2O. Compared with single-functional nanozymes, this multifunctional enzyme system is more in line with the treatment needs of the complex pathological process associated with diabetic periodontitis.

[0067] Example 2: Evaluation of in vitro biocompatibility and antioxidant properties of CuHP-loaded hydrogels

[0068] Morphology of rat jaw BMSCs

[0069] Rat mandibular BMSCs were isolated and cultured by tissue block adherence method. Under an optical microscope, BMSCs (P2) were observed to grow adherently and arranged in a vortex. The cell morphology was long spindle-shaped, with an elongated cell body and an oval nucleus in the center. The chromatin was evenly distributed ( Figure 7 This long spindle-shaped morphology facilitates the connection between cells in tissues and provides a structural basis for their proliferation, migration, osteogenic differentiation and other biological functions.

[0070] In vitro biocompatibility analysis

[0071] like Figure 8 As shown, low-concentration SA / CuHP hydrogels (0.25%, 0.5%, and 1% wt%) promoted cell proliferation, while high-concentration SA / CuHP hydrogels (2% wt%) exhibited cytotoxicity and inhibited cell proliferation. Among all groups, the SA / CuHP (1%) group exhibited the highest absorbance at 450 nm, indicating that BMSC proliferation was more effectively promoted in this group. Therefore, the SA / CuHP (1%) hydrogel was selected for subsequent experiments and is abbreviated as SA / CuHP hydrogel.

[0072] Construction of in vitro oxidative stress model

[0073] like Figure 9 As shown in the figure, compared with the control group, cell viability was enhanced after stimulation with 300 μM H2O2, while cell viability was significantly decreased after stimulation with 500 μM H2O2 for 3 hours (p<0.05). Therefore, in this study, 500 μM H2O2 was used to stimulate cells for 3 hours to establish a BMSCs oxidative stress model and simulate the pathological microenvironment of diabetic periodontitis.

[0074] In vitro antioxidant activity analysis

[0075] Analysis of intracellular ROS levels

[0076] The DCFH-DA fluorescent probe was used to detect the ROS level in each group of cells. Figure 10 As shown in A and B, compared with the control group, the H2O2 group and the H2O2+SA group showed significantly enhanced green fluorescence signals (p<0.05), indicating that the intracellular ROS level was significantly higher than that of the control group. Notably, the cell fluorescence intensity after SA / CuHP hydrogel intervention was significantly lower than that of the H2O2 group (p<0.05), indicating that SA / CuHP hydrogel can effectively remove excess ROS.

[0077] Cell viability analysis

[0078] The cell viability after different treatments was detected by CCK8 assay. Figure 11As shown in the results, H2O2 stimulation led to a decrease in cell viability (p<0.05); compared with the H2O2 group and the H2O2+SA group, the cell viability of the H2O2+SA / CuHP group was significantly enhanced (p<0.05). This result indicates that SA / CuHP hydrogel can alleviate the cytotoxicity caused by oxidative stress.

[0079] Cell viability analysis

[0080] Live-dead cell staining was used to further verify the effects of different treatments on cell viability. Figure 12 As shown, a large number of live cells with green fluorescence staining were detected in the control group; in the H2O2 group and H2O2+SA group, a decrease in green fluorescence was observed, while an increase in dead cells with red fluorescence signals was observed (p<0.05); after intervention with 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 live / dead cell staining results ( Figure 12 B). This result indicates that SA / CuHP hydrogel can reduce the damage of H2O2 to cell viability.

[0081] Cell migration ability analysis

[0082] In addition, cell scratch assay was used to evaluate cell migration ability. Figure 13 As shown in A and B, at 24 hours, the cell migration rate in the control group reached almost 100%, while the cell migration rate in the H2O2 group and the H2O2+SA group decreased (p<0.05). However, after SA / CuHP intervention, the cell migration rate was significantly improved (p<0.05). This result proves that SA / CuHP hydrogel can promote cell migration under oxidative stress conditions.

[0083] The SA / CuHP hydrogel prepared by the present invention has excellent antioxidant properties. Under oxidative stress conditions, the SA / CuHP hydrogel can reduce the generation of reactive oxygen species in cells and reduce the inhibitory effects of oxidative stress on cell proliferation, cell viability and cell migration.

[0084] Example 3: Evaluation of the in vitro osteogenic performance of CuHP hydrogel

[0085] ALP activity assay

[0086] ALP activity of cells after different treatments was analyzed by ALP staining. Figure 14As shown in A, the cells in the control group showed dense dark blue-purple ALP-positive areas in the cytoplasm, indicating that they had high early osteogenic differentiation activity. In the other three H2O2-induced groups, the staining area was reduced and the color intensity was weakened, suggesting that H2O2 can inhibit the ALP activity of BMSCs. Compared with the H2O2 group and the H2O2+SA group, the staining area of ​​the H2O2+SA / CuHP group was enlarged and the color depth was restored, proving that SA / CuHP can effectively antagonize the H2O2-induced ALP activity inhibition. ALP quantitative results are shown in Figure 2. Figure 14 As shown in Figure B, ALP activity was significantly reduced in the H2O2 group (P<0.05). Compared with the H2O2 group, ALP activity in the H2O2+SA group showed no significant change (P>0.05). SA / CuHP hydrogel intervention significantly enhanced ALP activity (P<0.05). These results, based on enzyme activity, confirm that SA / CuHP can ameliorate the inhibitory effect of H2O2 on the early osteogenic differentiation of BMSCs.

[0087] Calcium nodule formation analysis

[0088] ARS staining was used to detect the formation of calcium nodules after different treatments. Figure 15 As shown in A, the Control group formed dense orange-red calcium nodules, showing typical mineralized matrix deposition characteristics, indicating that the late osteogenic differentiation function of BMSCs was normal. In the other three H2O2-induced groups, the number of calcium nodules was significantly reduced and sparsely distributed, suggesting that the oxidative stress environment severely damaged the mineralization ability of BMSCs. Compared with the H2O2 group and the H2O2+SA group, the area of ​​calcium nodule deposition in the H2O2+SA / CuHP-treated group increased, indicating that SA / CuHP can effectively restore the late osteogenic differentiation function of BMSCs under oxidative stress conditions. ARS quantitative results are shown in Figure 2. Figure 15 As shown in Figure B, the relative calcium nodules in the H2O2 group were significantly reduced (P<0.05). There was no statistical difference between the H2O2+SA group and the H2O2 group (P>0.05). Compared with the H2O2 group, the relative calcium nodules in the H2O2+SA / CuHP group were significantly increased (P<0.05). These results indicate that SA / CuHP can significantly improve H2O2-induced late mineralization disorder.

[0089] RUNX2 expression analysis

[0090] The expression of RUNX2 in cells was analyzed by immunofluorescence staining. Figure 16As shown in A and B, the control group displayed a strong green fluorescence signal in the cell nucleus, indicating that RUNX2 protein was effectively activated during the early stages of osteogenic differentiation. H2O2 stimulation resulted in a significant decrease in nuclear RUNX2 fluorescence intensity compared with the control group. The H2O2+SA group showed a modest increase in RUNX2 fluorescence, but this did not significantly differ from the H2O2 group (P>0.05). However, the green fluorescence after H2O2+SA / CuHP hydrogel intervention was significantly stronger than that in the H2O2 group (P<0.05), approaching the level of the control group.

[0091] Expression analysis of osteogenesis-related genes (ALP, OCN, OSX)

[0092] The expression levels of osteogenesis-related genes ALP, OCN, and OSX were detected by qPT-PCR. Figure 17 As shown in the results, under H2O2-induced oxidative stress conditions, the expressions of osteogenesis-related genes ALP, OCN, and OSX were significantly decreased (P<0.05). Compared with the H2O2 group, the expressions of ALP, OCN, and OSX in the H2O2+SA group did not change significantly (P>0.05), while the expressions of ALP, OCN, and OSX in the H2O2+SA / CuHP group were significantly increased (P<0.05).

[0093] Analysis of osteogenesis-related protein expression (RUNX2, OSX)

[0094] The expression levels of osteoblast-related proteins (RUNX2, OSX) were detected by WB. Figure 18 As shown, compared with the Control group, the expressions of bone-related proteins RUNX2 and OSX were significantly downregulated in the H2O2 group (P<0.05). Compared with the H2O2 group, the expressions of RUNX2 and OSX in the H2O2+SA group did not change significantly (P>0.05), while the expressions of RUNX2 and OSX in the H2O2+SA / CuHP group were significantly upregulated (P<0.05).

[0095] in conclusion

[0096] A key clinical manifestation of diabetic periodontitis is alveolar bone resorption, the core mechanism of which involves impaired osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in an oxidative stress microenvironment. This study focused on oxidative stress, a key pathological component, and systematically evaluated the protective effects of SA / CuHP hydrogel on the osteogenic function of BMSCs. Within the osteogenic differentiation regulatory network, RUNX2 is a master transcription factor for osteogenic differentiation. By directly binding to the promoter regions of genes encoding bone-specific proteins such as ALP and OCN, it precisely regulates their transcriptional activity, thereby initiating and advancing the osteogenic differentiation program. ALP, a core marker of early osteogenic differentiation, catalyzes the release of inorganic phosphate, promotes the deposition of hydroxyapatite crystals, and initiates bone matrix mineralization. OSX, a key downstream effector of RUNX2, mediates the terminal differentiation of osteoblast precursors into functional osteoblasts and is a marker of mid-stage osteogenic differentiation. OCN, a specific marker of late osteogenic differentiation, is secreted primarily by mature osteoblasts. OCN, through its γ-carboxylated glutamic acid residues, specifically binds to Ca²⁺, mediating the oriented arrangement of hydroxyapatite crystals in the bone matrix. Therefore, the present invention investigated the effects of SA / CuHP on the expression 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 downregulation of the expression of proteins of BMSC osteogenesis-related genes, while SA / CuHP hydrogels promoted the expression of osteogenesis-related genes and proteins, reversing the inhibitory effect of oxidative stress on BMSC osteogenesis. In addition, ALP staining results showed that the ALP activity of cells increased after SA / CuHP hydrogel treatment, and ARS staining results showed that the formation of calcified nodules increased after SA / CuHP hydrogel treatment, once again demonstrating the osteogenic properties of SA / CuHP hydrogels under oxidative stress conditions.

[0097] The osteoprotective effect of SA / CuHP hydrogels may stem from the effective synergy between their CAT-like activity and the inherent osteogenic properties of copper. In an oxidative stress microenvironment, the material's CAT-like activity significantly improves the osteogenic microenvironment of BMSCs by targetedly scavenging H2O2, a key oxidative mediator. Studies have shown that excessive H2O2 accumulation directly inhibits the mineralization capacity of osteoblast precursor cells and induces apoptosis, while effective local H2O2 scavenging creates the necessary redox homeostasis for osteogenic differentiation. Furthermore, the inherent biological properties of copper offer unique advantages in bone repair. Copper has been shown to promote bone matrix protein secretion and calcium deposition by activating the expression profile of osteoblast-related genes, and its osteogenic effects have been extensively validated in the field of bone defect repair. In this study, SA / CuHP lowered the oxidative damage threshold through its CAT-like activity in a neutral microenvironment, while copper directly enhanced the osteogenic activity of BMSCs. This synergistic effect enhances the protective effect against osteogenesis under oxidative stress.

[0098] In an oxidative stress environment, SA / CuHP hydrogel can significantly alleviate the inhibitory effect of oxidative stress on osteogenesis, increase cell ALP activity, enhance bone mineralization, and promote the expression of osteogenesis-related genes and proteins.

[0099] Example 4: Evaluation of the in vitro antibacterial properties of CuHP-loaded hydrogels

[0100] Bacterial proliferation analysis

[0101] The bacterial proliferation after different treatments was investigated by bacterial spotting and colony counting. Figure 19 As 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 H2O2 or SA / CuHP added alone and the H2O2+SA group showed only weak antibacterial effects (p<0.05), suggesting that H2O2 and SA / CuHP alone are difficult to effectively inhibit Aa The colony count in the H2O2+SA / CuHP group was significantly lower than that in the H2O2 or SA / CuHP groups alone (p<0.05). These results indicate that H2O2+SA / CuHP exhibits a significant inhibitory effect on bacterial proliferation.

[0102] Bacterial viability analysis

[0103] Bacterial viability after different treatments was analyzed by bacterial live-dead staining. Figure 20 As shown, the control group was almost entirely green fluorescent (live bacteria); in the SA group, green fluorescence was dominant, with only sporadic red fluorescence (dead bacteria), indicating that SA itself did not exhibit significant antibacterial activity. In the H2O2 and H2O2+SA groups, the red fluorescence signal increased slightly compared to the control group, but green fluorescence was still dominant overall, suggesting that low-concentration H2O2 has a limited killing effect on bacteria. In the SA / CuHP group, the proportion of red fluorescence increased significantly; in the H2O2+SA / CuHP group, the red fluorescence signal intensity and coverage far exceeded those of the other groups. These results indicate that the bactericidal effect of SA / CuHP is enhanced in the presence of H2O2 in a weakly acidic environment.

[0104] Bacterial morphology observation

[0105] The effects of different treatments on cell morphology were observed by SEM. Figure 21 As shown, the bacterial surfaces in the control and SA groups were smooth and intact, appearing short rods. In the H2O2 and H2O2+SA groups, the bacterial surfaces showed slight wrinkling and deformation, indicating partial damage to the cell membrane integrity. SA / CuHP treatment resulted in irregular bacterial morphology and membrane damage, while bacteria in the H2O2+SA / CuHP group exhibited even more severe wrinkling. These results suggest that H2O2 and SA / CuHP synergistically aggravate bacterial cell membrane damage, enhancing antibacterial efficacy.

[0106] The experimental results showed that SA hydrogel did not show antibacterial activity; H2O2 and SA / CuHP hydrogel alone also showed weak antibacterial effects; while the H2O2+SA / CuHP group showed significant antibacterial effects, which were significantly stronger than the antibacterial effects of the H2O2 group or the SA / CuHP group. The antibacterial mechanism of SA / CuHP hydrogel may be mainly attributed to its POD enzyme activity. It was proved that the POD enzyme activity of SA / CuHP hydrogel dominated in a weak acid environment, catalyzing H2O2 to generate the more oxidative ·OH. Previous studies have shown that the antibacterial mechanism of ·OH mainly includes: (1) ·OH oxidizes cell membrane lipids, destroys membrane integrity, causes leakage of cell contents and osmotic pressure imbalance. (2) Destroys 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 synergistically exert antibacterial effects. Previous studies have shown that the antibacterial mechanisms of copper ions mainly include: (1) positively charged Cu²⁺ binds to the negatively charged bacterial cell membrane, destroying the membrane potential and increasing permeability, leading to ion imbalance and cytoplasmic leakage. (2) competitively binding to metal ions in the active center of enzymes, resulting in inactivation of key enzymes such as respiratory chain enzymes. (3) binding to electron transport chain components on mitochondria or cell membranes, hindering ATP synthesis and inhibiting energy metabolism. Therefore, SA / CuHP hydrogels, through the synergistic action of POD enzyme activity and Cu ions, jointly destroy membrane structures, key biomolecules (proteins, DNA) and metabolic pathways, thereby enhancing antibacterial effects.

[0107] Example 5: In vivo therapeutic effect and safety evaluation of CuHP-loaded hydrogels

[0108] Establishment of type 2 diabetes rat model

[0109] like Figure 22 As shown in Figures A and B, rats in the control group steadily gained weight, with random blood glucose levels below 16.7 mmol / L. However, after four weeks of high-fat diet and STZ injection combined to induce diabetes, rats in the experimental groups (PBS, SA, and SA / CuHP) experienced weight loss and significantly elevated random blood glucose levels, which remained above the diagnostic threshold of 16.7 mmol / L. The experimental groups exhibited typical diabetic symptoms: food and water intake were significantly higher than those in the healthy control group; polyuria resulted in increased bedding moisture and odor, requiring daily changes; and weight loss persisted. Physical and behavioral observations further revealed that the experimental groups exhibited dull, dry fur, decreased locomotor activity, and delayed reflexes. Changes in blood glucose, body weight, and physiological status preliminarily confirmed the successful establishment of a rat model of type 2 diabetes.

[0110] Establishment of a diabetic periodontitis model

[0111] Based on the type 2 diabetic rat model, a periodontitis model was established by silk ligation. Figure 23 As shown in the figure, the healthy periodontal tissue before silk ligation was characterized by a pink and tough gum, a gum margin close to the tooth surface, and a probe that did not bleed easily. Three weeks after silk ligation, the ligature was removed and it was found that a large amount of food debris accumulated in the interproximal space of the second molar, the gums were red, swollen and retracted, the gum margin was round and blunt and could not fit the tooth surface, and the probe was prone to bleeding, which preliminarily proved that the periodontitis model was successfully established.

[0112] Gingival crevicular fluid smear colony count results

[0113] The bacterial count in rat periodontal pockets was quantitatively analyzed using the plate count method. Figure 24 As shown, the number of bacterial colonies in the periodontal pockets of the control group was low, while the PBS group showed significant bacterial proliferation (P<0.05). The colonies on the plate were dense and widely distributed, indicating a 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 SA hydrogel alone group also showed some antibacterial effect (P<0.05).

[0114] Micro-CT analysis

[0115] like Figure 25 As shown in A and B, micro-CT three-dimensional reconstructions, sagittal sections, and X-ray images visually demonstrate the alveolar bone conditions in each group. In the control group, the ABC and CEJ anatomical positions were closely aligned, with intact bone contours. In contrast, the PBS group exhibited alveolar bone resorption and destruction, with a significant increase in the CEJ-ABC distance (P < 0.05), and bone loss and exposure in the furcation region, further confirming the successful establishment of a diabetic periodontitis model. While the SA intervention group showed a decreasing trend in the CEJ-ABC distance, the difference was not statistically significant compared with the PBS group (P > 0.05). The SA / CuHP group demonstrated significant improvement in alveolar bone destruction, with a significantly shorter CEJ-ABC distance compared with the PBS group (P < 0.05).

[0116] The alveolar bone between the first molar and the second molar was selected for bone microstructure parameter analysis, such as Figure 26As shown in A and B, the BV / TV ratio in the PBS group was significantly lower than that in the control group, and the trabecular bone separation (Tb.Sp) ratio was significantly increased (P < 0.05). Although the BV / TV and Tb.Sp parameters in the SA group were slightly improved compared with the PBS group, the differences did not reach statistical significance (P > 0.05). The SA / CuHP group demonstrated significant bone microarchitecture regulation: compared with the PBS group, the BV / TV ratio was significantly increased, and the Tb.Sp ratio was significantly decreased (P < 0.05). These results indicate that the SA / CuHP hydrogel effectively alleviates alveolar bone destruction caused by diabetic periodontitis.

[0117] HE staining analysis

[0118] like Figure 27 As shown in the data, the intermolar papillae of the control group were intact, the gingival epithelium was continuous without defects, the junctional epithelium was located at the cementoenamel junction and was tightly attached to the root surface, and the height of the alveolar ridge was consistent with the physiological bone contour. The silk ligature intervention areas all showed varying degrees of attachment loss, manifested as root-to-root displacement of the junctional epithelium. The junctional epithelium in the PBS and SA groups was loosely attached to the root surface, and the height of the alveolar bone ridge was significantly reduced. The periodontal pathological features of the SA / CuHP group were significantly improved: the connections between epithelial cells were tighter and stably attached to the cementum surface, and the alveolar bone height remained relatively intact.

[0119] Masson staining analysis

[0120] like Figure 28 As shown, the periodontal ligament fiber bundles in the control group were arranged regularly. The collagen fiber bundles in the PBS and SA groups were disordered and showed signs of fragmentation and dissolution. Although the periodontal ligament fibers in the SA / CuHP group were partially disordered, their overall continuity was better than that in the PBS and SA groups.

[0121] In vivo safety analysis

[0122] After four weeks of treatment, histopathological analysis of the rats' major organs revealed no significant inflammatory infiltration, necrosis, or structural abnormalities in the heart, liver, spleen, lungs, or kidneys in any of the groups, with no significant pathological differences observed compared to the control group. These results demonstrate that the SA / CuHP hydrogel exhibited good biocompatibility in vivo and suggest its potential safety for clinical application.

[0123] Summarize

[0124] (1) A high-fat diet-induced STZ intervention strategy was used to construct a type 2 diabetes rat model. The successful establishment of a type 2 diabetes rat model was confirmed by observing the changes in rat status, body weight, and blood sugar.

[0125] (2) Based on type 2 diabetic rats, regional periodontitis of the left maxillary second molar was induced by silk thread ligation, which effectively simulated the pathological process of clinical diabetic periodontitis. The model successfully reproduced the typical pathological characteristics of periodontal clinical attachment loss, probing bleeding and alveolar bone resorption.

[0126] (3) In the treatment of diabetic periodontitis rat model, SA / CuHP hydrogel showed excellent antibacterial properties, significantly reduced the destruction and absorption of alveolar bone, and was safe in vivo.

[0127] It should be noted that although the present invention has been described in the above specific embodiments, the inventive concept of the present invention is not limited thereto. Any improvements or variations based on the inventive concept are protected by this patent.

[0128] The above is only a preferred embodiment of the present invention, and the scope of protection 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 is included in the scope of protection of the present invention. It should be emphasized that for ordinary technicians in this technical field, without departing from the purpose and scope of the present invention, any modification or equivalent replacement should be regarded as part of the scope of protection of the present invention.

Claims

1. Application of a copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-enzyme activity in the preparation of diabetic periodontitis bone repair materials, characterized in that: The pH response conditions of the diabetic periodontitis bone repair material are: exhibiting POD enzyme activity under weak acid conditions, catalyzing the generation of ROS; exhibiting CAT enzyme activity under neutral conditions, scavenging ROS. The weak acid condition is pH=5, and the neutral condition is pH=7.

4. The copper hydrogen phosphate-loaded hydrogel with pH-responsive multiple enzyme activities is constructed by loading copper hydrogen phosphate (CuHP) on an injectable sodium alginate hydrogel (SA). 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 0.5-1wt%.

2. The use according to claim 1, characterized in that The mass fraction of copper hydrogen phosphate in the copper hydrogen phosphate-loaded hydrogel is 1 wt %.

3. The use according to claim 1, characterized in that The copper hydrogen phosphate-loaded hydrogel having pH-responsive multi-type enzyme activity is prepared by 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. Dissolve copper sulfate (CuSO4) powder in double-distilled water, then add this solution to phosphate buffered saline (PBS) and shake the reaction to ensure uniform mixing and complete reaction. After the reaction is completed, separate the supernatant by centrifugation. 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; finally, the mixed solution was added dropwise to the CaCl2 solution using a syringe to form SA / CuHP hydrogel.

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

5. The use 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 use according to claim 3, characterized in that The mass fraction of CuHP in step (2) is 0.5-1 wt%.

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