Traditional Chinese medicine composite hydrogel for repairing diabetic bone defects and its preparation method and application

Abstract

The present invention provides a traditional Chinese medicine composite hydrogel for repairing diabetic bone defects, as well as its preparation method and application. The hydrogel is made from the following components: 10-20 wt% methacryloyl gelatin, 2-5 wt% tannic acid, 2-5 wt% cerium oxide nanozyme, and the balance is water. The hydrogel overcomes the shortcomings of existing materials, such as poor mechanical properties, insufficient antioxidant and anti-inflammatory effects, and poor osteogenesis, significantly improving the efficiency and effectiveness of diabetic bone defect repair. It has important clinical application value and broad market prospects. The preparation process of the hydrogel is simple, the raw materials are readily available, and it is easy to industrialize. In addition, the mechanical properties, degradation rate, and functionality can be flexibly controlled by adjusting the reaction conditions, providing convenient conditions for large-scale production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a traditional Chinese medicine composite hydrogel for repairing diabetic bone defects, and a preparation method and application thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by chronically elevated blood sugar levels, often accompanied by a range of chronic complications, including cardiovascular disease, kidney disease, bone defects, and neuropathy. Bone defects are a common complication in diabetic patients, manifesting as a significant decrease in bone healing capacity and bone loss. This is primarily attributed to the hyperglycemic state of diabetic patients, which leads to disrupted bone metabolism, enhanced inflammatory responses, and elevated oxidative stress. These pathological processes result in delayed or even failed bone regeneration, significantly impacting patients' quality of life.

[0003] In recent years, hydrogels have become an important material for repairing bone defects due to their excellent biocompatibility, high water content, and excellent tissue integration. Bioactive hydrogels have shown great potential in diabetic bone defect repair. By incorporating functional molecules with antioxidant and anti-inflammatory properties, hydrogels can play a significant role in improving the local microenvironment, promoting cell proliferation and differentiation, and accelerating bone regeneration.

[0004] Although a variety of hydrogel materials have been developed for bone defect repair, their application in the field of diabetic bone defects still faces many challenges. For example, it is difficult to strike a balance between strength and bioactivity in existing materials, resulting in insufficient mechanical properties to support the physical environment required for bone regeneration, or insufficient bioactivity to significantly improve the repair effect. Sreya et al. developed a 3D-printed titanium scaffold with good mechanical support, but the biological inertness of titanium prevents it from having a good immune regulatory effect. Li et al. designed a double-network hydrogel composed of a polyvinyl alcohol network and a gelatin colloidal network, which is loaded with interleukin-10 (IL-10) and releases IL-10 early to reduce the production of endogenous ROS. However, the growth factor is easily inactivated, and its long-term stability and effectiveness in the body are insufficient.

[0005] Therefore, developing a hydrogel material with multifunctional properties such as high strength, antioxidant, and anti-inflammatory properties for repairing diabetic bone defects is a technical problem that needs to be urgently solved in this field. Summary of the Invention

[0006] To overcome the defects of the prior art, the present invention aims to provide a traditional Chinese medicine composite hydrogel for repairing diabetic bone defects, as well as a preparation method and application thereof.

[0007] The present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a traditional Chinese medicine composite hydrogel for repairing diabetic bone defects, which is made of the following components: 10-20 wt% of methacryloyl gelatin, 2-5 wt% of tannic acid and 2-5 wt% of cerium oxide nanozyme, and the balance is water;

[0009] Furthermore, the composite hydrogel is made of the following components: 15 wt% of methacryloyl gelatin, 3 wt% of tannic acid and 3 wt% of cerium oxide nanozyme, and the balance is water;

[0010] The second aspect of the present invention provides a method for preparing any of the above-mentioned traditional Chinese medicine composite hydrogels for repairing diabetic bone defects, comprising the following steps:

[0011] Synthesis of S1 methacryloyl gelatin: Gelatin was added to a phosphate buffer solution and stirred at 40-60°C and 200-400 rpm to completely dissolve the gelatin. Methacrylic anhydride (MA) was then added dropwise to the gelatin solution using a microsyringe pump and stirred at 200-400 rpm. The reaction was allowed to proceed at 40-60°C for 2-4 h. PBS was then added to stop the reaction. After stirring for another 5-20 min, the reaction solution was poured into a dialysis tube and dialyzed in deionized water for 5-8 days, with the water changed 1-3 times per day to remove byproducts and unreacted methacrylic anhydride. The solution was then freeze-dried in a freeze dryer to obtain white sponge-like methacryloyl gelatin.

[0012] Furthermore, in S1, the mass ratio of gelatin to phosphate buffer solution is 1:8-12;

[0013] Furthermore, in S1, the gelatin:methacrylic anhydride (MA) mass volume ratio is 4-8 g:1 mL;

[0014] Furthermore, in S1, the dropping rate of the methacrylic anhydride (MA) is 0.1-0.3 mL / min;

[0015] S2 Synthesis of Cerium Oxide Nanozymes: Prepare a water-ethylene glycol solution of cerium nitrate to a concentration of 5-6 mmol / L, heat the solution, and quickly add 30-40 v / v% ammonia water when the temperature reaches 55-70°C. Stir the resulting mixture vigorously at 55-70°C until a yellow dispersion is obtained. Cool and centrifuge the yellow dispersion to obtain a yellow CeO2 precipitate, wash it with ethanol and water several times until the pH is neutral, and then freeze-dry it to obtain a light yellow nano-cerium oxide powder.

[0016] Preparation of S3 Traditional Chinese Medicine Composite Hydrogel: The methacrylated gelatin prepared in S1, tannic acid, and the cerium oxide nanozyme prepared in S2 were mixed according to the above weight ratio, fully dissolved in deionized water, and lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate LAP was added, and the volume was adjusted with deionized water, and the mixture was shaken to obtain the hydrogel.

[0017] Furthermore, in S2, the centrifugation parameter for precipitating the cerium oxide nanozyme is 10000-14000 rpm;

[0018] Furthermore, in S2, the volume ratio of water to ethylene glycol in the water-ethylene glycol solution is 1:1;

[0019] Furthermore, in S2, the amount of ammonia added is 0.1 to 0.2 of the solution volume;

[0020] Furthermore, in S2, the vigorous stirring time is 2 to 4 h;

[0021] Furthermore, in S2, the centrifugation parameter is 10000-14000 rpm;

[0022] Furthermore, in S2, the freeze-drying parameters are: the temperature of the freezing stage is -40°C to -80°C; the temperature of the sublimation stage is -20°C to -40°C; the temperature of the thawing stage is 20-30°C, and the time is 24-48 hours;

[0023] Furthermore, in S3, the amount of lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate added is 0.25-5% of the sum of the weight of methacrylated gelatin, tannic acid, and the cerium oxide nanozyme prepared in S2;

[0024] The third aspect of the present invention provides the use of any of the above-mentioned traditional Chinese medicine composite hydrogels for repairing diabetic bone defects in the preparation of bone repair drugs or medical devices for diabetic patients.

[0025] Beneficial effects of the present invention:

[0026] Gelatin Methacryloyl (GelMA) is a photocrosslinked hydrogel material widely used in biomedical engineering, tissue engineering, and 3D bioprinting. It is derived from gelatin modified by methacryloylation. Derived from natural gelatin (a hydrolyzate of collagen), methacryloyl gelatin supports cell adhesion, proliferation, and differentiation, and exhibits excellent biocompatibility. The methacryloyl groups on the molecular chain can polymerize under the influence of blue light (in combination with a photoinitiator such as LAP) to form a stable gel. By varying the concentration, degree of crosslinking, or illumination conditions, the hardness, porosity, and degradation rate of the gel can be adjusted, resulting in excellent mechanical properties.

[0027] Tannic acid is a natural polyphenolic compound found widely in plants (such as gallnut, tea, and oak bark). It possesses strong antioxidant, antibacterial, metal chelating, and protein-binding abilities. Tannic acid can bind to GelMA to enhance the performance of hydrogels: improving the mechanical strength of the hydrogel through hydrogen bonding / hydrophobic interactions between the phenolic hydroxyl groups and GelMA; imparting antioxidant and anti-inflammatory properties; and delaying hydrogel degradation by chelating with cerium ions.

[0028]

[0029] Cerium oxide nanozymes It can mimic the catalytic behavior of natural enzymes (such as catalase and superoxide dismutase), playing an important role in antioxidant and anti-inflammatory effects. Cerium ions chelate with tannic acid to form a metal polyphenol network, which not only enhances the mechanical properties of the traditional Chinese medicine composite hydrogel and prolongs its degradation time, but also enhances its antioxidant and anti-inflammatory effects, controls its release, and prolongs the drug's shelf life. The redox reaction is shown below:

[0030]

[0031] The functionalized Chinese medicine composite hydrogel prepared by combining the three overcomes the defects of existing materials such as poor mechanical properties, insufficient antioxidant and anti-inflammatory effects, and poor osteogenic effects, significantly improving the efficiency and effect of diabetic bone defect repair, and has important clinical application value and broad market prospects. The preparation process of the present invention is simple, the raw materials are easily available, and it is easy to industrialize. In addition, the mechanical properties, degradation rate and functionality can be flexibly controlled by adjusting the reaction conditions, providing convenient conditions for large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It shows that the traditional Chinese medicine composite hydrogel has good compressive mechanical properties;

[0033] Figure 2 The state of the Chinese medicine composite hydrogel before and after light irradiation;

[0034] Figure 3 Showing the antioxidant capacity of traditional Chinese medicine composite hydrogel;

[0035] Figure 4 It showed that the traditional Chinese medicine composite hydrogel can effectively remove reactive oxygen species in mouse embryonic osteoblast precursor cells and mouse macrophages;

[0036] Figure 5 It shows that Chinese herbal medicine composite hydrogel can promote the proliferation and differentiation of osteoblasts;

[0037] Figure 6It shows that Chinese medicine composite hydrogel can promote the repair of mandibular defects in diabetic rats. DETAILED DESCRIPTION

[0038] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0039] (1) Implementation

[0040] Example 1: Preparation of methacryloyl gelatin

[0041] Weigh 5 g of gelatin powder and add it to 50 mL of preheated phosphate buffered saline (PBS). Place the mixed solution in a 50°C water bath and stir continuously at 300 rpm using a magnetic stirrer until the gelatin is completely dissolved. Slowly add 4 mL of methacrylic anhydride (MA) dropwise to the dissolved gelatin solution at a rate of 0.2 mL / min using a microsyringe pump, maintaining stirring. Maintain the reaction temperature at 50°C and stirring at 300 rpm for 2 hours. Terminate the reaction by adding 50 mL of PBS and continue stirring for 10 minutes to ensure uniform mixing. The reaction solution is transferred to a dialysis bag and dialyzed against deionized water for 6 days, with the dialysis water replaced twice daily to remove byproducts and unreacted MA. After dialysis, add 100 mL of deionized water to the dialyzate and stir for 15 minutes to mix thoroughly. Aliquot the solution into 4 mL centrifuge tubes and freeze at -80°C. Freeze-dry for 48 hours to obtain a white, spongy GelMA. The product was stored in a desiccator at room temperature for future use.

[0042] Example 2: Preparation of Nano-Cerium Oxide

[0043] Dissolve 2.52 g (5.8 mmol) of cerium nitrate in 100 mL of water-ethylene glycol mixture (volume ratio 1:1). After the solution temperature rises to 60°C, quickly add 16 mL of 35% ammonia water. , and stirred vigorously at 60 ° C for 3 h until a yellow dispersion was formed. After the reaction was completed, the mixture was cooled and centrifuged at 12000 rpm to collect the yellow The solution was precipitated and then washed repeatedly with ethanol and water until neutral. The solution was freeze-dried at -30℃ for 24 h to obtain a light yellow powder of nano-cerium oxide.

[0044] Example 3: Preparation of composite hydrogel

[0045] Preparation of composite hydrogel: Weigh 1.5 g of methacryloyl gelatin (GelMA), 0.5 g of tannic acid (TA), and 0.5 g of cerium oxide nanozyme. , add 10 ml of deionized water, fully dissolve it at 37 °C, add 0.05 g of lithium phenyl-2,4, 6-trimethylbenzoylphosphinate (LAP), make up to volume with deionized water, and shake well to obtain a composite hydrogel.

[0046] (2) Experimental examples

[0047] Experimental methods:

[0048] 1. How to use Chinese medicine composite hydrogel

[0049] The traditional Chinese medicine composite hydrogel was injected into the bone defect site as needed and cured using 405 nm wavelength blue light irradiation. The power of the curing lamp was 1.5 W and the irradiation time was 30 s.

[0050] 2. Measuring the Mechanical Strength of Hydrogels

[0051] A Teflon mold with an inner diameter of 8 mm and a height of 15 mm was used to place methacryloyl gelatin, tannic acid-methacryloyl gelatin, and tannic acid-nanocerium oxide-methacryloyl gelatin into the mold, respectively. The hydrogels were irradiated with 405 nm blue light for 30 s. The cured hydrogels were taken out and placed in a mechanical testing machine to measure the pressure-deformation data.

[0052] 3. Observe the hydrogel curing effect

[0053] Place 1.5 ml of hydrogel in a small glass vial, tilt it, and take a photo. Then, place the vial upright and illuminate it with a curing light for 30 seconds. Observe the hydrogel, tilt the vial, and take a photo.

[0054] 4. Determination of DPPH absorption peak

[0055] Composite hydrogels with different concentrations of 0, 2.5, 5, 12.5, 25, and 50 ppm were mixed with DPPH solution of the same concentration. The color change was observed after 10 minutes, and the absorption peak at a wavelength of 517 nm was measured using a microplate reader.

[0056] 5. DCFH-DA Fluorescent Probe Staining

[0057] Mouse embryonic osteoblast precursor cells and mouse macrophages were cultured under in vitro simulated diabetic conditions (i.e., high glucose and high hydrogen peroxide). The cells were then incubated with methacryloyl gelatin, tannic acid-methacryloyl gelatin, and tannic acid-nanocerium oxide-methacryloyl gelatin for 12 h and then stained with the DCFH-DA fluorescent probe.

[0058] 6. Mouse Embryonic Osteogenic Precursor Cell Proliferation Experiment

[0059] Mouse embryonic osteoblast precursor cells were cultured under in vitro simulated diabetic conditions (i.e., high glucose and high hydrogen peroxide), and then incubated with methacryloyl gelatin, tannic acid-methacryloyl gelatin, and tannic acid-nanocerium oxide-methacryloyl gelatin. They were incubated with CCK-8 liquid for one hour on days 1, 3, 5, and 7, and the absorbance of the supernatant was measured at 450 nm.

[0060] 7. Treatment of bone defects

[0061] A diabetic bone defect model was established in rats, and streptozotocin (STZ) was injected intraperitoneally. Seven days later, blood glucose was greater than 16.7 mmol / L, which was considered a successful diabetes model. At this time, a 5 mm diameter bone defect was created in the rat mandible, and the composite hydrogel was injected and solidified. The wound was sutured, and samples were collected at 4 and 8 weeks for CBCT imaging.

[0062] Experimental results:

[0063] 1. Mechanical strength

[0064] like Figure 1 As shown, the tannic acid-nanocerium oxide-methacryloyl gelatin (traditional Chinese medicine composite hydrogel) of the present invention has greatly improved compression mechanical properties compared with simple gelatin, and the compression mechanical properties can reach 1.5 MPa.

[0065] 2. State of Chinese medicine composite hydrogel before and after illumination

[0066] like Figure 2 As shown, before irradiation, the tannic acid-nanocerium oxide-methacryloyl gelatin (TCM composite hydrogel) was in a liquid state, exhibiting the pale yellow color of the incorporated cerium oxide nanozyme. After irradiation, the TCM composite hydrogel solidified into a solid state.

[0067] 3. Antioxidant capacity of composite hydrogel

[0068] like Figure 3 As shown in the results, tannic acid-nanocerium oxide-methacryloyl gelatin (TCM composite hydrogel) can effectively remove 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH), showing strong antioxidant ability.

[0069] 4. Chinese medicine composite hydrogel can effectively remove active oxygen

[0070] like Figure 4 As shown in the results, tannic acid-nanocerium oxide-methacryloyl gelatin (TCM composite hydrogel) can effectively eliminate reactive oxygen species (ROS) in mouse embryonic osteoblast precursor cells and mouse macrophages.

[0071] 5. Traditional Chinese medicine composite hydrogel can promote the proliferation of osteoblasts

[0072] like Figure 5 As shown, tannic acid-nanocerium oxide-methacryloyl gelatin (traditional Chinese medicine composite hydrogel) can promote the proliferation of osteoblasts compared with the simple diabetes group.

[0073] 6. Composite hydrogel can promote the repair of diabetic bone defects

[0074] like Figure 6 As shown in the data, the newly repaired bone area and volume at the bone defect site of tannic acid-nanocerium oxide-methacryloyl gelatin (TCM composite hydrogel) were larger, indicating that TCM composite hydrogel can promote the repair of mandibular defects in diabetic rats.

[0075] The above is only a preferred embodiment of the present invention, but the present invention should not be limited to the contents disclosed in this embodiment. Therefore, any equivalent or modified implementations that do not depart from the spirit disclosed in the present invention fall within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composite hydrogel for repairing diabetic bone defects, characterized in that: The hydrogel is made of the following components: 10-20 wt% of methacryloyl gelatin, 2-5 wt% of tannic acid, 2-5 wt% of cerium oxide nanozyme, and the balance is water. The preparation method of the traditional Chinese medicine composite hydrogel includes the following steps: Synthesis of S1 methacryloyl gelatin: Gelatin was added to a phosphate buffer solution and stirred at 40-60°C and 200-400 rpm to completely dissolve the gelatin. Methacrylic anhydride (MA) was then added dropwise to the gelatin solution using a microsyringe pump and stirred at 200-400 rpm. The reaction was allowed to proceed at 40-60°C for 2-4 hours. PBS was then added to stop the reaction. After stirring for another 5-20 minutes, the reaction solution was poured into a dialysis tube and dialyzed in deionized water for 5-8 days, with the water changed 1-3 times per day to remove byproducts and unreacted methacrylic anhydride. The solution was then freeze-dried in a freeze dryer to obtain white sponge-like methacryloyl gelatin. S2 Synthesis of Cerium Oxide Nanozymes: Prepare a water-ethylene glycol solution of cerium nitrate to a concentration of 5-6 mmol / L. Heat the solution. When the temperature reaches 55-70°C, rapidly add 30-40 v / v% ammonia water. Stir the resulting mixture vigorously at 55-70°C until a yellow dispersion is obtained. Cool and centrifuge the yellow dispersion to obtain a yellow CeO2 precipitate. Wash the precipitate with ethanol and water several times until the pH is neutral, and then freeze-dry to obtain a light yellow nano-cerium oxide powder. Preparation of S3 traditional Chinese medicine composite hydrogel: The methacrylated gelatin prepared in S1, tannic acid, and cerium oxide nanozyme prepared in S2 were mixed according to the above component percentages, deionized water was added to fully dissolve them, and lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate LAP was added. The volume was adjusted with deionized water and the mixture was shaken to obtain the hydrogel.

2. The Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 1, characterized in that: The composite hydrogel is made of the following components: 15 wt% of methacryloyl gelatin, 3 wt% of tannic acid, 3 wt% of cerium oxide nanozyme, and the balance is water.

3. A method for preparing the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to any one of claims 1 to 2, characterized in that: The following steps are involved: Synthesis of S1 methacryloyl gelatin: Gelatin was added to a phosphate buffer solution and stirred at 40-60°C and 200-400 rpm to completely dissolve the gelatin. Methacrylic anhydride (MA) was then added dropwise to the gelatin solution using a microsyringe pump and stirred at 200-400 rpm. The reaction was allowed to proceed at 40-60°C for 2-4 hours. PBS was then added to stop the reaction. After stirring for another 5-20 minutes, the reaction solution was poured into a dialysis tube and dialyzed in deionized water for 5-8 days, with the water changed 1-3 times per day to remove byproducts and unreacted methacrylic anhydride. The solution was then freeze-dried in a freeze dryer to obtain white sponge-like methacryloyl gelatin. S2 Synthesis of Cerium Oxide Nanozymes: Prepare a water-ethylene glycol solution of cerium nitrate to a concentration of 5-6 mmol / L. Heat the solution. When the temperature reaches 55-70°C, rapidly add 30-40 v / v% ammonia water. Stir the resulting mixture vigorously at 55-70°C until a yellow dispersion is obtained. Cool and centrifuge the yellow dispersion to obtain a yellow CeO2 precipitate. Wash the precipitate with ethanol and water several times until the pH is neutral, and then freeze-dry to obtain a light yellow nano-cerium oxide powder. Preparation of S3 traditional Chinese medicine composite hydrogel: The methacrylated gelatin prepared in S1, tannic acid, and cerium oxide nanozyme prepared in S2 were mixed according to the above component percentages, deionized water was added to fully dissolve them, and lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate LAP was added. The volume was adjusted with deionized water and the mixture was shaken to obtain the hydrogel.

4. The method for preparing the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 3, characterized in that: In S1, the mass ratio of gelatin to phosphate buffer solution is 1:8-12.

5. The method for preparing the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 3, characterized in that: In S1, the mass volume ratio of gelatin to methacrylic anhydride (MA) is 4-8 g:1 mL.

6. The method for preparing the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 3, characterized in that: The dropping rate of the methacrylic anhydride MA is 0.1-0.3 mL / min.

7. The method for preparing the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 3, characterized in that: In S2, the amount of ammonia added is 0.1-0.2 of the solution volume.

8. The method for preparing the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 3, characterized in that: In S2, the freeze-drying parameters are: the temperature in the freezing stage is -40°C to -80°C; the temperature in the sublimation stage is -20°C to -40°C; the temperature in the thawing stage is 20~30°C, and the time is 24~48 hours.

9. The method for preparing the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 3, characterized in that: In S3, the amount of lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate added is 0.25-5% of the total weight of methacrylated gelatin, tannic acid, and cerium oxide nanozyme.

10. Use of the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to any one of claims 1 to 2 in the preparation of bone repair drugs or medical devices for diabetic patients.

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