Traditional Chinese medicine composite hydrogel for repairing diabetic bone defect as well as preparation method and application of traditional Chinese medicine composite hydrogel

Abstract

The invention provides traditional Chinese medicine composite hydrogel for repairing diabetic bone defects as well as a preparation method and application of the traditional Chinese medicine composite hydrogel. The traditional Chinese medicine composite hydrogel is prepared from the following components: 10-20wt% of methylacryloyl gelatin, 2-5wt% of tannic acid, 2-5wt% of cerium oxide nano-enzyme and the balance of water. The hydrogel provided by the invention overcomes the defects of poor mechanical properties, insufficient antioxidant and anti-inflammatory effects, poor osteogenesis promoting effect and the like of materials in the prior art, significantly improves the efficiency and effect of diabetic bone defect repair, and has important clinical application value and wide market prospects; the preparation process is simple and convenient, raw materials are easy to obtain, industrial production is easy, the mechanical property, degradation rate and functionality can be flexibly regulated and controlled by adjusting reaction conditions, and convenient conditions are provided for large-scale production and application.

Description

Technical Field

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

[0002] Diabetes is a metabolic disease characterized by long-term elevated blood glucose levels, often accompanied by a series of chronic complications, including cardiovascular diseases, kidney diseases, bone defects, and neuropathy, etc. Among them, bone defect is one of the common complications in diabetic patients, mainly manifested as a significant decline in bone healing ability and bone loss. This is mainly attributed to the bone metabolic disorder, enhanced inflammatory response, and elevated oxidative stress level caused by the hyperglycemic state of diabetic patients. These pathological processes lead to the delay or even failure of bone regeneration, greatly affecting the quality of life of patients.

[0003] In recent years, hydrogels have become an important material for repairing bone defects due to their good biocompatibility, high water content, and good integration ability with tissues. Especially in the field of repairing diabetic bone defects, bioactive hydrogels show great application potential. By introducing functional molecules with antioxidant and anti-inflammatory effects, hydrogels can play an important 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, there are still many challenges in their application in the field of diabetic bone defects. For example, it is difficult to balance the strength and bioactivity of 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 metal scaffold, which has good mechanical support, but the biological inertness of titanium metal makes it unable to have good immunomodulatory effects. Li et al. designed a double-network hydrogel composed of a polyvinyl alcohol network and a gelatin colloid network, and simultaneously loaded interleukin 10 (IL-10), releasing IL-10 early to reduce the production of endogenous ROS, but the growth factor is easily inactivated, and its long-term stability and effectiveness in vivo are insufficient.

[0005] Therefore, developing a hydrogel material with high strength, antioxidant, anti-inflammatory and other multifunctional properties for repairing diabetic bone defects is an urgent technical problem to be 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, its preparation method and application.

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

[0008] In the 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, 2-5 wt% of cerium oxide nanozyme, and the balance is water;

[0009] Further, 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;

[0010] In the second aspect of the present invention, there is provided a preparation method of any of the above traditional Chinese medicine composite hydrogels for repairing diabetic bone defects, comprising the following steps:

[0011] S1 Synthesis of methacryloyl gelatin: Gelatin is added to a phosphate buffer solution and stirred at 40-60 °C and 200-400 rpm until all the gelatin is dissolved. Then, methacrylic anhydride (MA) is added dropwise to the gelatin solution using a micro-injection pump and stirred at 200-400 rpm. The reaction is carried out at 40-60 °C for 2-4 h. Subsequently, PBS is added to stop the reaction, and after stirring for 5-20 min, the reaction solution is poured into a dialysis tube and dialyzed in deionized water for 5-8 days, changing the water 1-3 times a day to remove by-products and unreacted methacrylic anhydride in the reaction solution. Then, it is placed in a freeze dryer for freeze-drying to obtain white spongy methacryloylated gelatin;

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

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

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

[0015] S2 Synthesis of cerium oxide nanozyme: Prepare an aqueous-ethylene glycol solution of cerium nitrate to make its concentration reach 5-6 mmol / L. Heat the solution. When the temperature reaches 55-70 °C, quickly add ammonia water with a concentration of 30-40 v / v%. The resulting mixture is vigorously stirred at 55-70 °C until a yellow dispersion is obtained. The yellow dispersion is cooled and centrifuged to obtain a yellow CeO2 precipitate, which is washed with ethanol and water several times until the pH is neutral, and then freeze-dried to obtain light yellow nano cerium oxide powder;

[0016] Preparation of traditional Chinese medicine composite hydrogel: Mix the methacrylated gelatin prepared in S1, tannic acid, and the cerium oxide nanozyme prepared in S2 according to the above weight ratio, fully dissolve with deionized water, add lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and make up the volume with deionized water, then shake well to obtain it;

[0017] Further, in S2, the centrifugation parameters for precipitating the cerium oxide nanozyme are 10000 - 14000 rpm;

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

[0019] Further, in S2, the addition amount of ammonia water is 0.1 - 0.2 of the solution volume;

[0020] Further, in S2, the intense stirring time is 2 - 4 h;

[0021] Further, in S2, the centrifugation parameters are 10000 - 14000 rpm;

[0022] Further, 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 h;

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

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

[0025] Advantages of the present invention:

[0026] Gelatin Methacryloyl (GelMA) is a photocrosslinkable hydrogel material widely used in biomedical engineering, tissue engineering, 3D bioprinting and other fields. It is modified from gelatin by methacryloylation. Gelatin Methacryloyl is derived from natural gelatin (hydrolysis product of collagen), supports cell adhesion, proliferation and differentiation, and has good biocompatibility; the methacryloyl groups on the molecular chain can polymerize under the initiation of blue light (cooperating with a photoinitiator such as LAP) to form a stable gel. By changing the concentration, crosslinking degree or light irradiation conditions, the hardness, porosity and degradation rate of the gel can be adjusted to make it have good 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 is shown that the traditional Chinese medicine composite hydrogel can promote the repair of mandibular bone defects in diabetic rats. Detailed implementation manners

[0038] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal implementation manner, and do not constitute a limitation on the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0039] (I) Embodiments

[0040] Embodiment 1: Preparation of methacrylated gelatin (GelMA)

[0041] Weigh 5 g of gelatin powder and add it to 50 mL of preheated phosphate buffer solution (PBS). Place the mixed solution in a 50°C constant temperature water bath and continuously stir it with a magnetic stirrer at a speed of 300 rpm until the gelatin is completely dissolved. Slowly add 4 mL of methacrylic anhydride (MA) to the dissolved gelatin solution at a rate of 0.2 mL / min through a micro-injection pump while maintaining the stirring state. Keep the reaction temperature at 50°C and the stirring speed at 300 rpm, and let the mixed solution react for 2 hours. Add 50 mL of PBS to terminate the reaction, and continue to stir for 10 minutes to make the solution evenly mixed. Transfer the reaction solution to a dialysis bag and perform dialysis purification with deionized water (for 6 days), changing the dialysis water twice a day during this period to remove by-products and unreacted MA. After dialysis is completed, add 100 mL of deionized water to the dialysis solution and stir for 15 minutes to mix evenly. Aliquot the solution into 4 mL centrifuge tubes and freeze it at -80°C in an ultra-low temperature freezer. Freeze-dry for 48 hours to obtain white sponge-like GelMA. Store the product in a desiccator and keep it at room temperature for standby.

[0042] Embodiment 2: Preparation of nano-ceria

[0043] Dissolve 2.52 g (5.8 mmol) of cerium nitrate in 100 mL of a 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 vigorously stir and react at 60°C for 3 h until a yellow dispersion is formed. After the reaction is completed, cool the mixture, and centrifuge at 12000 rpm to collect the yellow precipitate, and then wash it repeatedly with ethanol and water until it is neutral. Freeze-dry at -30°C for 24 h to obtain pale yellow nano-ceria powder.

[0044] Embodiment 3: Preparation of the composite hydrogel

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

[0046] (II) Experimental examples

[0047] Experimental methods:

[0048] 1. Method of using the traditional Chinese medicine composite hydrogel

[0049] Inject the traditional Chinese medicine composite hydrogel into the bone defect site as needed, and irradiate it with blue light with a wavelength of 405 nm for curing. The power of the curing lamp is 1.5 w, and the irradiation time is 30 s.

[0050] 2. Measure the mechanical strength of the hydrogel

[0051] Use a Teflon mold with an inner diameter of 8 mm and a height of 15 mm. Place gelatin methacrylate, tannic acid-gelatin methacrylate, and tannic acid-nano cerium oxide-gelatin methacrylate into the mold respectively, irradiate with 405 nm blue light for 30 s, take out the cured hydrogel, and place it in a mechanical testing machine to measure the pressure-deformation data.

[0052] 3. Observe the curing effect of the hydrogel

[0053] Place 1.5 ml of the hydrogel in a small glass bottle, tilt it, and take a photo. Then place the small glass bottle upright, irradiate it with a light curing lamp for 30 s, then observe the state of the hydrogel, tilt the small glass bottle, and take a photo.

[0054] 4. Determine the DPPH absorption peak

[0055] Mix different concentrations of the composite hydrogel at 0, 2.5, 5, 12.5, 25, and 50 ppm with the DPPH solution at the same concentration respectively. Observe the color change after 10 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorption peak at a wavelength of 517 nm.

[0056] 5. DCFH-DA fluorescent probe staining

[0057] Under in vitro simulated diabetic conditions (i.e., high glucose and high hydrogen peroxide), culture mouse embryonic osteoblast precursor cells and mouse macrophages. Then incubate the cells with gelatin methacrylate, tannic acid-gelatin methacrylate, and tannic acid-nano cerium oxide-gelatin methacrylate for 12 h, and then use the DCFH-DA fluorescent probe for staining.

[0058] 6. Proliferation experiment of mouse embryonic osteoprogenitor cells

[0059] Under in vitro simulated diabetic conditions (i.e., high glucose and high hydrogen peroxide), mouse embryonic osteoprogenitor cells were cultured, and then the cells were co-incubated with methacrylated gelatin, tannic acid-methacrylated gelatin, and tannic acid-nano cerium oxide-methacrylated gelatin. They were incubated with CCK-8 solution for one hour at 1, 3, 5, and 7 days, and the absorbance of the supernatant at 450 nm was measured.

[0060] 7. Treatment of bone defects

[0061] A rat diabetic bone defect model was established by intraperitoneal injection of streptozotocin (STZ). Seven days later, if the blood glucose was greater than 16.7 mmol / L, the diabetic model was considered successful. At this time, a 5-mm diameter bone defect was created in the mandible of the rat. After injecting and solidifying the composite hydrogel, the wound was sutured. Samples were taken at the 4th and 8th weeks, and CBCT was taken.

[0062] Experimental results:

[0063] 1. Mechanical strength

[0064] As Figure 1 shown, the tannic acid-nano cerium oxide-methacrylated gelatin (traditional Chinese medicine composite hydrogel) of the present invention has greatly improved compressive mechanical properties compared with pure gelatin, and the compressive mechanical properties can reach 1.5 MPa.

[0065] 2. State of the traditional Chinese medicine composite hydrogel before and after light irradiation

[0066] As Figure 2 shown, before light irradiation, the tannic acid-nano cerium oxide-methacrylated gelatin (traditional Chinese medicine composite hydrogel) was in a liquid state, showing the light yellow color of the incorporated cerium oxide nanozyme. After light irradiation, the traditional Chinese medicine composite hydrogel solidified into a solid state.

[0067] 3. Antioxidant ability of the composite hydrogel

[0068] As Figure 3 shown, the tannic acid-nano cerium oxide-methacrylated gelatin (traditional Chinese medicine composite hydrogel) can effectively scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), showing strong antioxidant ability.

[0069] 4. Reactive oxygen species that the traditional Chinese medicine composite hydrogel can effectively scavenge

[0070] As Figure 4 shown, the tannic acid-nano cerium oxide-methacrylated gelatin (traditional Chinese medicine composite hydrogel) can effectively scavenge reactive oxygen species (ROS) in mouse embryonic osteoprogenitor cells and mouse macrophages.

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

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

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

[0074] As Figure 6 shown, the new repaired bone area and volume at the bone defect site of tannic acid-nano cerium oxide-methacrylated gelatin (traditional Chinese medicine composite hydrogel) are larger, indicating that the traditional Chinese medicine composite hydrogel can promote the repair of mandibular bone defects in diabetic rats.

[0075] The above are the preferred embodiments of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, all equivalent or modified implementations completed without departing from the spirit disclosed in the present invention fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composite hydrogel for repairing diabetic bone defects, characterized in that, It is made of the following components: 10 - 20 wt% methacryloyl gelatin, 2 - 5 wt% tannic acid, 2 - 5 wt% cerium oxide nanozyme, and the balance is water.

2. The traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 1, wherein, The composite hydrogel is made of the following components: 15 wt% methacryloyl gelatin, 3 wt% tannic acid, 3 wt% 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, It includes the following steps: S1 Synthesis of methacryloyl gelatin: Add gelatin to phosphate buffer solution, stir at 40 - 60 °C and 200 - 400 rpm until all gelatin is dissolved. Then, use a micro-injection pump to drop methacrylic anhydride (MA) into the gelatin solution and stir at 200 - 400 rpm. React at 40 - 60 °C for 2 - 4 h. Subsequently, add PBS to stop the reaction, stir for another 5 - 20 min, pour the reaction solution into a dialysis tube, and dialyze in deionized water for 5 - 8 days, changing water 1 - 3 times a day to remove by-products and unreacted methacrylic anhydride in the reaction solution. Then, place it in a freeze dryer for lyophilization to obtain white spongy methacryloylated gelatin. S2 Synthesis of cerium oxide nanozyme: Prepare an aqueous-ethylene glycol solution of cerium nitrate with a concentration of 5 - 6 mmol / L. Heat the solution. When the temperature reaches 55 - 70 °C, quickly add ammonia water with a concentration of 30 - 40 v / v%. Vigorously stir the obtained mixture 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 multiple times until the pH is neutral, and then freeze-dry to obtain light yellow nano cerium oxide powder. S3 Preparation of traditional Chinese medicine composite hydrogel: Mix the methacryloylated gelatin prepared in S1, tannic acid, and the cerium oxide nanozyme prepared in S2 according to the above component percentages, add deionized water to dissolve fully, add lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), make up the volume with deionized water, and shake well to obtain.

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

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

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

8. The preparation method of 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 h.

9. The preparation method of the traditional Chinese medicine composite hydrogel for repairing diabetic bone defects according to claim 3, characterized in that, In S3, the addition amount of the lithium phenyl-2,4,6-trimethylbenzoylphosphinate is 0.25 - 5% of the sum of the weights of the methacryloylated 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 - 2 in the preparation of drugs or medical devices for bone repair of diabetic patients.

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