Tough adhesion type composite hydrogel material as well as preparation method and application thereof

Abstract

The invention discloses a tough adhesion type composite hydrogel material as well as a preparation method and application thereof, and belongs to the technical field of material preparation. The gelatin, the tannic acid and other raw materials are utilized, the composite hydrogel is prepared through physical crosslinking, the process is simple and convenient, and the prepared composite hydrogel is excellent in mechanical property, has good flexibility and can provide protection for wounds and resist the influence of external force; the coating has super-strong toughness, is not easy to crack, has strong adhesion with the surface of a titanium material, and is not easy to fall off. And the composite hydrogel can effectively remove excessive reactive oxygen species (ROS), eliminate inflammation, improve a bone repair microenvironment under pathological conditions of diabetes mellitus, and promote bone repair of diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the technical field of material preparation, and particularly relates to a tough and adhesive composite hydrogel material, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes mellitus (DM) is one of the chronic metabolic diseases affecting human health. The repair of bone defects in diabetic patients faces many challenges, mainly due to various pathophysiological changes caused by DM, such as abnormal immune function, chronic inflammation, accumulation of reactive oxygen species (ROS), vascular system damage, lack of insulin / insulin-like growth factor (IGF), and accumulation of advanced glycation end products (AGEs). The combined action of these factors leads to a significant delay in the fracture healing process of diabetic patients, and even nonunion may occur.

[0003] Currently, the drugs for clinical treatment of DM mainly focus on reducing inflammation (such as injecting insulin and taking hypoglycemic drugs including insulin secretagogues (such as sulfonylurea drugs, meglitinide drugs, etc.)) and using titanium alloy bone nails to promote bone repair. However, these drugs often cause drug resistance, long-term side effects, and a long treatment time, making it difficult to achieve an ideal treatment effect. In the current field of diabetic bone repair, when titanium implants are used in diabetic patients, the implants often do not bind firmly to human bones. At the same time, due to the changes in the immune microenvironment such as hyperglycemia, increased reactive oxygen species, and local inflammation caused by diabetes, simply using titanium alloy bone nails cannot effectively repair bone defects under diabetic conditions. Moreover, diabetes can also lead to endoplasmic reticulum (ER) stress. In a hyperglycemic environment, the glycosylation and abnormal folding of proteins increase, resulting in an increased protein load in the ER lumen, triggering ER stress, and then causing apoptosis of osteocytes, which is not conducive to bone repair.

[0004] Since bone defect healing involves immune regulation mainly by macrophages, which form a bone immune regulation cascade with downstream osteoblast-related cells, how to prepare an environmentally responsive hydrogel to achieve intelligent sensing of the diabetic microenvironment has become a major problem. Finding a safe and effective method to inhibit inflammation and promote bone repair is very important for the treatment of DM. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a tough and adhesive composite hydrogel material, a preparation method thereof, and an application thereof, so as to solve the technical problem of poor bone repair effect of existing hydrogel materials.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing a tough and adhesive composite hydrogel material is provided, including the following steps: Dissolve the first raw material in a solvent, then sequentially add a polyhydroxy compound, the second raw material, and a photoinitiator and mix them evenly. Finally, crosslink under ultraviolet light for 10 - 20 minutes to obtain a tough and adhesive composite hydrogel material (GPT); the first raw material is gelatin, hyaluronic acid, sodium alginate, or silk fibroin, and the second raw material is N-hydroxyethyl acrylamide (HEAA), β-hydroxyalkylamide (HAA), N,N-dimethylacrylamide (DMAA), or diacetone acrylamide (DAAM). The content of the second raw material is related to the amount of hydrogen bond content and will significantly affect the water content and mechanical properties of the hydrogel; the ratio of the first raw material, polyhydroxy compound, second raw material, and photoinitiator is 0.05 - 0.15 g : 5 - 15 μL : 0.5 - 0.7 g : 7 - 9 g.

[0007] Based on the above technical solution, the present invention can also be improved as follows:

[0008] Further, the ratio of the first raw material, polyhydroxy compound, second raw material, and photoinitiator is 0.1 g : 10 μL : 0.6 g : 8 g.

[0009] Further, the gelatin is type B gelatin. Type B gelatin is a biocompatible bioactive macromolecule with good properties, which can simulate the extracellular matrix and effectively promote the synthesis and mineralization of the extracellular matrix.

[0010] Further, the polyhydroxy compound is tannic acid, dopamine, gallic acid, catechin, or anthocyanin.

[0011] Further, the concentration of tannic acid is 1 - 2 wt%.

[0012] Further, the photoinitiator is I2959.

[0013] The present invention also discloses a tough and adhesive composite hydrogel material prepared by the above preparation method.

[0014] The present invention also discloses the application of the tough and adhesive composite hydrogel material in the preparation of bone repair materials.

[0015] Further, the tough and adhesive composite hydrogel material can also be used as a carrier to prepare bone repair drugs.

[0016] The beneficial effects of the present invention are as follows:

[0017] 1. The present invention prepares a composite hydrogel by physical crosslinking. Reversible covalent bond hydrogen bonds are formed through the interaction of amino groups in raw material one, phenolic hydroxyl groups in polyhydroxy compounds, and double bond oxygen structures in raw material two to stabilize gel formation. Therefore, the composite material has good elasticity and toughness, can provide protection for wounds, resist the influence of external forces, has super toughness, is not easy to break, has a strong adhesion force to the titanium material surface, and is not easy to fall off.

[0018] 2. The composite hydrogel prepared by the present invention can logically respond to intracellular and extracellular cascade signals, effectively scavenge excessive reactive oxygen species (ROS), eliminate inflammation, promote the polarization of M1 macrophages to M2 macrophages, improve the bone repair microenvironment under diabetic pathological conditions. Through the polyphenolic hydroxyl structure in the hydrogel, it can specifically scavenge reactive oxygen species, eliminate inflammation, etc., and can specifically intervene in intracellular stress responses, effectively reducing the protein folding load of the endoplasmic reticulum, thereby protecting cells from the damage of toxic endoplasmic reticulum stress, which is beneficial to diabetic bone repair.

[0019] 3. The composite hydrogel prepared by the present invention can also load therapeutic drugs and can better repair the affected area. Description of the Drawings

[0020] Figure 1 SEM image of hydrogel GPT-5;

[0021] Figure 2 Adhesion test results of the hydrogel;

[0022] Figure 3 Self-healing experiment results of the hydrogel;

[0023] Figure 4 Contact angle test diagram of the hydrogel;

[0024] Figure 5 Tensile stress-strain test diagram of the hydrogel;

[0025] Figure 6 Intracellular reactive oxygen species scavenging test results of the hydrogel in macrophages;

[0026] Figure 7 Detection results of the expression content of inflammatory factor IL-6;

[0027] Figure 8 Detection results of the expression content of inflammatory factor TNF-α;

[0028] Figure 9 Detection results of the expression content of inflammatory factor IL-10;

[0029] Figure 10 Rat femoral repair pictures after 4 weeks of repair. Detailed Embodiments

[0030] The specific embodiments of the present invention will be described below to facilitate those skilled in the art to understand the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained by purchasing in the market. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those ordinary skilled in the art in the technical field, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0031] Example 1

[0032] A preparation method of a tough and adhesive composite hydrogel material comprises the following steps: Dissolve 0.05 g of hyaluronic acid in 1 mL of distilled water, and fully dissolve it in a water bath at 55 °C for 70 min, and then cool it to room temperature; then successively add 15 μL of dopamine, 0.7 g of β-hydroxyalkylamide, and 5% (w / w) I2959 and mix them evenly. Coat the prepared hydrogel precursor solution evenly on the surface of a titanium sheet treated by ultrasonic treatment, and finally crosslink it under ultraviolet light for 10 min to form a tough and adhesive composite hydrogel material adhering to the surface of the titanium sheet.

[0033] Example 2

[0034] A preparation method of a tough and adhesive composite hydrogel material comprises the following steps: Dissolve 0.15 g of sodium alginate in 1 mL of distilled water, and fully dissolve it in a water bath at 65 °C for 50 min, and then cool it to room temperature; then successively add 10 μL of gallic acid, 0.5 g of N,N-dimethylacrylamide, and 5% (w / w) I2959 and mix them evenly. Coat the prepared hydrogel precursor solution evenly on the surface of a titanium sheet treated by ultrasonic treatment, and finally crosslink it under ultraviolet light for 20 min to form a tough and adhesive composite hydrogel material adhering to the surface of the titanium sheet.

[0035] Example 3

[0036] A preparation method of a tough and adhesive composite hydrogel material, comprising the following steps: Dissolve 0.1 g of type B gelatin in 1 mL of distilled water, and fully dissolve it in a water bath at 60 °C for 1 h, and then cool it to room temperature; then sequentially add 5 μL of tannic acid solution (concentration 1.5 wt%), 0.6 g of N - hydroxyethyl acrylamide and 5% (w / w) I2959 and mix evenly. Coat the prepared hydrogel precursor solution evenly on the surface of a titanium sheet treated by ultrasonic treatment, and finally crosslink it under ultraviolet light for 15 min to form a tough and adhesive composite hydrogel material (GPT - 5) adhering to the surface of the titanium sheet. The surface of the hydrogel is milky white and slightly transparent, and has strong viscoelasticity. Its SEM image ( Figure 1 ) shows a three - dimensional network porous structure with hydrogel characteristics, indicating that the hydrogel can be successfully prepared by the method of the present invention.

[0037] Example 4

[0038] The difference between this example and Example 3 is that: the addition amount of the tannic acid solution is adjusted to 10 μL, and the remaining implementation conditions are the same as those in Example 3, to obtain a tough and adhesive composite hydrogel material (GPT - 10).

[0039] Example 5

[0040] The difference between this example and Example 3 is that: the addition amount of the tannic acid solution is adjusted to 15 μL, and the remaining implementation conditions are the same as those in Example 3, to obtain a tough and adhesive composite hydrogel material (GPT - 15).

[0041] Taking the tough and adhesive composite hydrogel materials (GPT - 5, GPT - 10 and GPT - 15) prepared in Examples 3 - 5 as examples, the following experiments are carried out.

[0042] Experimental Example 1 Mechanical Properties

[0043] 1. Adhesion

[0044] Carry out a titanium sheet lap shear force experiment on GPT - 5, GPT - 10 and GPT - 15 materials. The test steps are as follows: Cut the titanium sheet into rectangular titanium sheets with a length of 2 cm × width of 1 cm, and then ultrasonically clean the cut titanium sheets in water and 75% alcohol for 15 min to remove surface dust. Apply 20 μL of the hydrogel precursor solutions prepared in Examples 3 - 5 evenly on the surface of a 1 cm 2 titanium sheet. After crosslinking by ultraviolet irradiation for 15 min, then cover the titanium sheet without coating the hydrogel solution after ultrasonic treatment on the titanium sheet with the hydrogel coating, and let it stand for 2 h. After the hydrogel on the titanium sheet is slightly dry, use a mechanical testing instrument to test the tensile force.

[0045] The results are as Figure 2As shown, it can be seen from the figure that the adhesion of the hydrogel to the titanium sheet is very strong, and a great deal of force is required to separate the adhered titanium sheets. This strong adhesion property mainly stems from the dynamic hydrogen bonds and coordination bonds formed between the hydrogel and the surface of the titanium sheet, indicating that the composite hydrogel materials prepared by the present invention all have good adhesion.

[0046] 2. Self-healing property

[0047] The self-healing properties of the GPT-5, GPT-10, and GPT-15 materials were tested. The test steps were as follows: The hydrogel sample was cut, and the hydrogel sample was cut into two segments or multiple parts to simulate damage; then the cut segments of the same sample were brought into contact and joined together. After standing for 2 h, it was found through tensile testing that the cut part of the hydrogel sample was adhesively joined together again ( Figure 3 ), which is because hydrogen bonds will reform between the hydroxyl groups on the hydrogel molecular chains to achieve self-healing, which further shows that the composite hydrogel materials prepared by the present invention are rich in hydrogen bonds and have the ability of self-healing.

[0048] 3. Contact angle measurement

[0049] The contact angle properties of the GPT-5, GPT-10, and GPT-15 materials were tested. The hydrogel sample was placed on the measurement platform to ensure that the surface of the sample was parallel to the platform, and the position of the sample was adjusted so that it was within the contact angle measurement area. Using a micro syringe or an automatic droplet spray head, a liquid was dropped onto the surface of the hydrogel. The liquid droplet should be kept as round as possible to avoid excessive disturbance. After the liquid droplet was stable, the automatic analysis function of the measuring instrument was started, and the contact situation between the liquid droplet and the sample surface was captured by a camera, and the contact angle value was recorded. The wettability of the hydrogel surface was judged according to the contact angle value: a contact angle less than 90° is hydrophilic, and greater than 90° is hydrophobic. Hydrophilic hydrogels have excellent biocompatibility, can reduce friction and mechanical damage to surrounding tissues, and hydrophilic hydrogel materials have good biodegradability and can gradually decompose and be absorbed in the body.

[0050] The results are as Figure 4 shown. The contact angles of all the hydrogel samples are less than 90°, so it can be inferred that the hydrogel material has good biocompatibility.

[0051] 4. Toughness

[0052] Tensile stress-strain experiments were carried out on the GPT-5, GPT-10, and GPT-15 materials. The test steps were as follows: The hydrogel precursor solution prepared in Examples 3-5 was injected into a dumbbell-shaped mold and formed by ultraviolet irradiation. The hydrogel sample was fixed on the fixture, and the hydrogel sample was stretched by a tensile testing machine, and the stress, strain and other parameter changes of the hydrogel sample were recorded in real time.

[0053] The results are as follows Figure 5 As shown, the strain value at material fracture can be obtained from the stress-strain diagram analysis, which also reflects the ductility of the material. The material can withstand large deformations before fracture, indicating good ductility. The energy absorption capacity of the material before fracture can be measured by the area under the stress-strain curve: a large area under the curve means good toughness and more energy absorption of the material; a small area under the curve means poor toughness and less energy absorption of the material. It can be seen from the figure that the composite hydrogel material has excellent toughness.

[0054] Experimental Example 2 Scavenging Effect of Intracellular Reactive Oxygen Species

[0055] The ability of GPT-5, GPT-10, and GPT-15 materials to scavenge reactive oxygen species was tested in macrophages. The test steps were as follows: Macrophages were inoculated into culture dishes or cell plates and cultured using DMEM medium containing 10% fetal bovine serum, and placed in a cell culture incubator at 37 °C and 5% CO 2 . When the cells grew to 70%-80% confluence, macrophages were treated with hydrogen peroxide (500 μM) or other stimulants to induce the cells to produce ROS. The hydrogel samples were added to the cell culture medium and co-incubated with macrophages for 24 h. Different treatment groups were set up, such as the Control group (control group), the H 2 O 2 treatment group, the H 2 O 2 + 0.1 g / mL each hydrogel treatment group, etc. The cells were stained with DCFH-DA (Solarbio kit). After DCFH-DA entered the cells, it was oxidized to the fluorescent substance DCF, and the fluorescence intensity was proportional to the intracellular ROS level. After 5 min of incubation, the cells were washed with PBS to remove the dye that did not enter the cells. The intracellular fluorescence intensity was detected using a fluorescence microscope or a flow cytometer to evaluate the ROS level.

[0056] The results are as follows Figure 6 As shown, the intracellular reactive oxygen fluorescence level of macrophages treated with the hydrogel material decreased, which was significantly lower than that of the experimental group treated only with hydrogen peroxide, and was comparable to that of the Control group without any treatment, indicating that the composite hydrogel material has the effect of scavenging reactive oxygen species and repairing the diabetic microenvironment.

[0057] Experimental Example 3 Inflammatory Clearance Ability

[0058] The inflammatory clearance ability of GPT-5, GPT-10, and GPT-15 materials was tested. It was tested using an ELISA kit (purchased from Xinbosheng Biotechnology Co., Ltd.). ELISA (enzyme-linked immunosorbent assay) is an immunological detection method based on antigen-antibody reactions and is commonly used to detect inflammatory factors in blood or tissues. The experiment was divided into Control, LPS, GPT-5, GPT-10, and GPT-15 groups. The Control group was macrophages cultured in a normal high-glucose medium without any treatment. The remaining four groups were all treated with 100 ng / mL lipopolysaccharide (LPS, sigma) to simulate the conditions of cellular inflammation. The GPT-5, GPT-10, and GPT-15 groups were respectively added with 1 mL of the hydrogel material extract (GB / T 16886.12—XXXX / ISO10993-12:202) at a concentration of 0.1 g / mL. After co-culturing with the cells for 24 h, the test samples were centrifuged to obtain the supernatant, and the inflammatory factors were extracted from it. The processed samples were added to the microplate, and standards were added simultaneously as controls, and then incubated. The microplate was incubated at 37 °C for 5 min to allow the antigen and antibody to bind. After incubation, the microplate was washed with the washing buffer to remove unbound substances. An enzyme-labeled antibody was added to the microplate and incubated again; the microplate was washed again. A substrate solution was added and incubated for 5 min, and finally, a stop solution was added to terminate the reaction. The absorbance value (450 nm) of the microplate was read using an enzyme-labeled immunosorbent assay reader. A standard curve was plotted based on the absorbance values of the standards, and the concentration of the inflammatory factors in the test samples was calculated.

[0059] Figures 7 - 9 The detection results of the expression levels of inflammatory factors (IL-6, TNF-α, and IL-10) in the tough adhesion type composite hydrogel material showed that the material had a downregulating effect on the concentrations of pro-inflammatory factors (IL-6, TNF-α) in macrophages of the existing inflammatory model and an upregulating effect on the anti-inflammatory factor (IL-10). This indicates that the tough adhesion type composite hydrogel material has the ability to regulate the immune microenvironment and has an anti-inflammatory function. Indirectly, it reflects that the material has the effect of polarizing the macrophage phenotype from M1 pro-inflammatory macrophages to M2 anti-inflammatory macrophages.

[0060] Experimental Example 4 Detection of Bone Repair Ability

[0061] Six-week-old Sprague-Dawley rats (purchased from Jiangsu Huachuang Xinnuo Medical Technology Co., Ltd.) were fed a high-fat diet for 2 weeks. They were fasted for 12 h before surgery and then intraperitoneally injected with streptozotocin (STZ, purchased from Guangzhou Meilun Biotechnology Co., Ltd.) dissolved in sodium citrate buffer at a dose of 60 mg / kg / day for one week. After injection, they were allowed to eat and drink for 2 h and then resumed their diet. The fasting blood glucose levels of the rats were measured daily, and rats with fasting blood glucose levels exceeding 16.7 mM during the experimental period (one week) were selected as experimental subjects for the diabetes model. The successfully modeled diabetic rats were divided into a diabetes group, a diabetes-hydrogel GPT-5 group, a diabetes-hydrogel GPT-10 group, and a diabetes-hydrogel GPT-15 group. Finally, a rat femoral defect model was established, and the settings for each group of rats were as follows:

[0062] (1) Diabetes group: Diabetic rats;

[0063] (2) Diabetes-hydrogel GPT-5: Diabetic rats + GPT-5;

[0064] (3) Diabetes-hydrogel GPT-10: Diabetic rats + GPT-10;

[0065] (4) Diabetes-hydrogel GPT-15: Diabetic rats + GPT-15;

[0066] 50 μL of the hydrogel precursor solution prepared in Examples 3-5 was evenly coated on the surface of the titanium rod that had been ultrasonically cleaned, and cross-linked by ultraviolet irradiation for 15 min to form a uniform hydrogel titanium sheet coating. The treated titanium rod was inserted into the femur of the rat to form a rat femoral defect model. In the diabetes group model, the ultrasonically cleaned titanium rod was directly inserted.

[0067] The results were as Figure 10 shown. Compared with other groups, the bone repair effect of the diabetes-hydrogel GPT-15 group was the most obvious, and the osteogenic effect was more obvious than that of the diabetic rats in the diabetes group, indicating that this hydrogel material can significantly improve the diabetic inflammatory microenvironment and effectively promote diabetic bone repair.

Claims

1. A method for preparing a strong and adhesive composite hydrogel material, characterized in that: The following steps are involved: The raw material one is dissolved in a solvent, and then a polyhydroxy compound, a raw material two and a photoinitiator are added in sequence and mixed evenly, and finally cross-linked under ultraviolet light for 10-20 minutes to obtain a strong and tough adhesive composite hydrogel material; the raw material one is gelatin, hyaluronic acid, sodium alginate or silk fibroin, and the raw material two is N-hydroxyethyl acrylamide, β-hydroxyalkylamide, N,N-dimethylacrylamide or diacetone acrylamide; the ratio of the raw material one, the polyhydroxy compound, the raw material two and the photoinitiator is 0.05-0.15g: 5-15μL: 0.5-0.7g: 7-9g.

2. The method for preparing a strong and adhesive composite hydrogel material according to claim 1, characterized in that: The ratio of the raw material 1, the polyhydroxy compound, the raw material 2 and the photoinitiator is 0.1 g: 10 μL: 0.6 g: 8 g.

3. The method for preparing a strong and adhesive composite hydrogel material according to claim 1, characterized in that: The gelatin is type B gelatin.

4. The method for preparing a strong and adhesive composite hydrogel material according to claim 1 or 2, characterized in that: The polyhydroxy compound is tannic acid, dopamine, gallic acid, catechin or anthocyanin.

5. The method for preparing a strong and adhesive composite hydrogel material according to claim 4, characterized in that: The concentration of the tannic acid is 1-2 wt %.

6. The method for preparing a strong and adhesive composite hydrogel material according to claim 1 or 2, characterized in that: The photoinitiator is I2959.

7. A strong and adhesive composite hydrogel material, characterized in that: The strong and tough adhesive composite hydrogel material is prepared by the preparation method of any one of claims 1 to 6.

8. Use of the strong and adhesive composite hydrogel material according to claim 7 in the preparation of bone repair materials.

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