Injectable self-crosslinking hydrogel for repairing endometrial injury and preparation method of injectable self-crosslinking hydrogel

Abstract

The invention discloses a preparation method of injectable self-crosslinking hydrogel for repairing endometrial injury, which comprises the following steps: mixing a precursor solution containing oxidized dextran and a precursor solution containing lysozyme, and standing and curing at body temperature to obtain the injectable self-crosslinking hydrogel for repairing endometrial injury. The hydrogel disclosed by the invention is based on a self-crosslinking reaction of Schiff base, can form gel after standing under a body temperature condition, does not need to introduce an extrafrontal cross-linking agent and an initiation condition, can form a stable physical barrier in situ after being injected into a uterine horn, realizes prevention of tissue adhesion of an injured part and regulation and control of an inflammatory microenvironment, has remarkable anti-inflammatory activity, and is suitable for clinical application. The structure and function reconstruction of the endometrium can be obviously promoted through an antibacterial-anti-inflammatory synergistic effect mechanism.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to an injectable self-crosslinking hydrogel for endometrial injury repair and a preparation method thereof. Background Art

[0002] The endometrium is an important guarantee for maintaining female reproductive function. However, frequent intrauterine operations and intrauterine infections and other various reasons can damage the endometrium. Endometrial injury is the main cause of infertility in about 8%-12% of reproductive-age women worldwide. Traditional methods for repairing damaged endometrium include hysteroscopic adhesiolysis, hyaluronic acid gel, hormone therapy, etc. These treatment methods are prone to various complications, such as inflammatory reactions, endometriosis, endometrial cancer and other diseases, and cannot restore fertility. Designing a hydrogel that can anti-inflammatory and promote repair and can act as a physical barrier for endometrial injury repair is one of the main ways to solve the above problems.

[0003] A hydrogel is a crosslinked three-dimensional network that can absorb a large amount of water or biological fluids and has now become an ideal material for various biomedical applications. Among them, injectable hydrogels represent a class of hydrogels that can be injected into the body in a minimally invasive manner through a small-gauge needle and then solidify in situ at the target site. Due to their ability to conform to the shape of the treated tissue or organ, provide excellent tissue contact and integration and other characteristics, they have received extensive attention. Various injectable hydrogels and crosslinking strategies emerge in an endless stream. In the process of realizing the present invention, the inventors found that there are at least the following problems in the prior art:

[0004] 1. Poor biodegradability: Most injectable hydrogels are composed of polymers, which are difficult to biodegrade, and the degradation products may be cytotoxic and lack the biological activity of natural materials.

[0005] 2. Insufficient mechanical properties: The mechanical properties of existing hydrogels are poor, it is difficult to provide sufficient tissue support, and the adhesion to tissues is weak, which easily leads to repair failure.

[0006] 3. Unsafe crosslinking strategy: Currently commonly used crosslinking methods (such as UV irradiation) may cause potential harm to tissues, limiting their safety and effectiveness in the body. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an injectable self-crosslinking hydrogel for endometrial injury repair and a preparation method thereof in view of the above-mentioned deficiencies of the prior art. This hydrogel is prepared from polysaccharides and bio-derived lysozyme as raw materials. The material source is natural and safe, non-toxic and has no side effects. It can form a gel in situ after being injected into the uterine injury site, establish a physical barrier, and promote the repair of endometrial injury.

[0008] The present invention has the following advantages compared with the prior art:

[0009] 1. The injectable self-crosslinking hydrogel for endometrial injury repair of the present invention is based on the Schiff base self-crosslinking reaction and can form a gel by standing at body temperature without introducing additional crosslinking agents and initiation conditions.

[0010] 2. The injectable self-crosslinking hydrogel for endometrial injury repair of the present invention has excellent mechanical properties, with a shear modulus exceeding 5 kPa, high mechanical properties, and is biodegradable.

[0011] 3. The injectable self-crosslinking hydrogel for endometrial injury repair of the present invention is based on the imine bonds in the network, can undergo reversible hydrolysis in a physiological environment, realize the sustained release of lysozyme, and has a long-term antibacterial effect.

[0012] 4. The injectable self-crosslinking hydrogel for endometrial injury repair of the present invention can regulate the inflammatory microenvironment, has significant anti-inflammatory activity, and can promote endometrial repair based on the synergistic effect of antibacterial and anti-inflammatory.

[0013] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the nuclear magnetic resonance spectrum of oxidized dextran in step one of Example 3;

[0015] Figure 2 is a schematic diagram of the test results of the mechanical properties of each hydrogel;

[0016] Figure 3 is a schematic diagram of the dynamic mechanical analysis (DMA) results of the 75-5:5 hydrogel;

[0017] Figure 4 is a schematic diagram of the swelling test results of the hydrogel;

[0018] Figure 5 is a schematic diagram of the degradation performance test results of the hydrogel;

[0019] Figure 6 is a schematic diagram of the antibacterial performance test results of the hydrogel;

[0020] Figure 7 is a schematic diagram of the anti-inflammatory performance test results of the hydrogel;

[0021] Figure 8 is a schematic diagram of the test results of the animal experiments of the hydrogel. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] Next, in combination with the embodiments of the present application, the technical solutions will be described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0023] In the following description, the term "and / or" is used to describe the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: the case where A exists alone, the case where B exists alone, and the case where A and B exist simultaneously. Where A and B may be singular or plural.

[0024] In the following description, terms such as "include", "comprise", "have", and "contain" are all open-ended terms, that is, they are meant to include but not be limited to.

[0025] Those skilled in the art should understand that in the following description of the embodiments of the present application, the sequence numbers do not mean the order of execution, and some or all of the steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0026] Those skilled in the art should understand that the numerical range in the embodiments of the present application should be understood as specifically disclosing each intermediate value between the upper and lower limits of the range. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, the technical / scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application pertains. Although the present application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] The technical principle adopted by the present invention: Using dextran and lysozyme as the main raw materials, oxidized dextran containing aldehyde groups is obtained through oxidation modification, and covalently crosslinked with lysozyme to obtain a hydrogel.

[0029] Some embodiments provide a preparation method of an injectable self-crosslinking hydrogel for endometrial injury repair, including:

[0030] Oxidize dextran with sodium periodate solution to obtain oxidized dextran;

[0031] Mix the precursor solution containing oxidized dextran with the precursor solution containing lysozyme, and let it stand and solidify at body temperature to obtain an injectable self-crosslinking hydrogel for endometrial injury repair.

[0032] Lysozyme is an important non-specific immune factor in animals. It can directly kill bacteria in the matrix immune response and can also induce and regulate the synthesis and secretion of other immune factors. It is commonly present in human body fluids. Based on the biological characteristics of lysozyme, the present invention creatively crosslinks it with oxidized polysaccharides to obtain a novel injectable self-crosslinking hydrogel with the function of repairing endometrial injury.

[0033] In some embodiments, the molecular weight (MW) of the dextran is 100,000, and the MW of the structural unit is 396.42998.

[0034] In some embodiments, the mass of the sodium periodate solution is 0.1 to 0.5 times the mass of the dextran, and the mass percentage content of sodium periodate in the sodium periodate solution is 2% to 10%.

[0035] In some embodiments, the oxidation degree of the oxidized dextran is 25% to 100%, and / or the mass ratio of oxidized dextran to lysozyme is (9:1) to (1:9); preferably, the mass ratio of oxidized dextran to lysozyme is (9:1) to (2:8); more preferably, the oxidation degree is 75% to 100%, and the mass ratio is (4:6) to (2:8); further preferably, the oxidation degree is 75%, and the mass ratio is 2:8.

[0036] By oxidizing the amino groups in natural polysaccharides to aldehyde groups, the oxidized polysaccharides are promoted to react with lysozyme to form Schiff bases.

[0037] The degree of oxidation of oxidized dextran and the mass ratio of oxidized dextran to lysozyme directly affect the gelation effect. The inventors found through research that pure oxidized dextran or lysozyme alone cannot form a gel. The higher the degree of oxidation of oxidized dextran, the larger the range of the mass ratio of gellable oxidized dextran to lysozyme. When the degree of oxidation is 25%, the mass ratio of oxidized dextran to lysozyme is 8:2 to 6:4. When the degree of oxidation is 50%, the mass ratio is 9:1 to 4:6. When the degree of oxidation is 75%, the mass ratio range is 9:1 to 2:8. When the degree of oxidation is 100%, the mass ratio range of oxidized dextran to lysozyme is 9:1 to 1:9. It shows that the higher the degree of oxidation of oxidized dextran, the higher the content of gellable lysozyme. Generally, it is considered that lysozyme, as a substance with immunomodulatory effects, its content is positively correlated with the repair effect. During the research process, the inventors unexpectedly found that the performance of the hydrogel is not a linear relationship with the degree of oxidation and the mass ratio. When the degree of oxidation of oxidized dextran is 75% - 100% and the mass ratio of oxidized dextran to lysozyme is (4:6) - (2:8), the shear modulus of the hydrogel exceeds 5 kPa, having high mechanical properties. When the degree of oxidation of oxidized dextran is 75% and the mass ratio is 2:8, the hydrogel has significant antibacterial and bactericidal effects, inhibits macrophage polarization, and promotes the repair of endometrial injury.

[0038] In some embodiments, the lysozyme is egg white lysozyme.

[0039] Due to its property of specifically recognizing and binding to target substrates, lysozyme has a unique enzymatic hydrolysis mechanism and is usually used as a raw material for testing the safety and in vivo degradability of biological dressings such as hydrogels. The inventors found that lysozyme can crosslink in the hydrogel network to repair the damaged parts in the uterine cavity.

[0040] In some embodiments, the solvents in the precursor solution containing oxidized dextran and the precursor solution containing lysozyme are both buffer solutions.

[0041] In some embodiments, the curing time is 6 h.

[0042] On the other hand, provided is an injectable self-crosslinking hydrogel for endometrial injury repair prepared by the above method.

[0043] A series of experiments were conducted before the application of the present invention. Now, some test results are listed to further describe the invention in detail, and detailed descriptions will be given below in combination with embodiments.

[0044] In the following examples, the specific sources of lysozyme and dextran are not limited and can be, for example, commercially available or self-made. For example, in the following examples, the lysozyme is egg white lysozyme, ≥20000U / mg, L799345, purchased from Macklin, and the molecular weight (MW) of the dextran is 100,000, and the MW of the structural unit is 396.42998.

[0045] Example 1

[0046] This example provides a preparation method of an injectable self-crosslinking hydrogel for endometrial injury repair, including:

[0047] Step 1: Disperse 4 g of dextran in 20 mL of absolute ethanol, add 0.535 g of an aqueous sodium periodate solution with a mass percentage of 2.675%, magnetically stir in the dark at 25 °C for 6 h, dialyze the stirred system with ultrapure water in a dialysis bag with a molecular weight cut-off of 14,000 for 48 h, and freeze-dry to obtain oxidized dextran; the oxidation degree of the oxidized dextran is 25%;

[0048] The determination method of the oxidation degree is the hydroxylamine hydrochloride titration method, which specifically includes:

[0049] Prepare a hydroxylamine-methyl orange solution, including: dissolve 17.5 g of hydroxylamine hydrochloride in 150 mL of ultrapure water, add 6.0 mL of methyl orange reagent, dilute to 1 L, and adjust the pH value to 4.0 to obtain a hydroxylamine hydrochloride-methyl orange solution;

[0050] Dry the oxidized dextran to a constant weight, take 0.1 g of the dried sample and dissolve it in 25 mL of the hydroxylamine hydrochloride-methyl orange solution to obtain a mixed system, let the mixed system stand for 2 h, titrate with a standard sodium hydroxide solution, and determine the oxidation degree OD of the oxidized dextran according to the following formula;

[0051]

[0052] where OD is the oxidation degree of the oxidized dextran, in %;

[0053] V NaOH is the volume of the standard sodium hydroxide solution, in mL;

[0054] n NaOH is the concentration of the standard sodium hydroxide solution, in mol / L;

[0055] W [Dex-O] is the mass of the oxidized dextran, in g;

[0056] Step 2: Mix the precursor solution containing oxidized dextran and the precursor solution containing lysozyme according to the mass ratio of oxidized dextran to lysozyme of 1:9, and let it stand at 37°C for 6 h for solidification to obtain an injectable self-crosslinking hydrogel for endometrial injury repair, labeled as 25-1:9; the precursor solution containing oxidized dextran is prepared by dispersing oxidized dextran in PBS buffer solution with a pH of 7.5, and the concentration of oxidized dextran in the precursor solution containing oxidized dextran is 15 wt%; the precursor solution containing lysozyme is prepared by dispersing lysozyme in PBS buffer solution with a pH of 7.5; the mass percentage content of lysozyme in the precursor solution containing lysozyme is 15 wt%.

[0057] Example 2

[0058] This example provides a preparation method of an injectable self-crosslinking hydrogel for endometrial injury repair, including:

[0059] Step 1: Disperse 4 g of dextran in 20 mL of absolute ethanol, add 1.069 g of an aqueous sodium periodate solution with a mass percentage content of 5.345%, magnetically stir in the dark at 25°C for 6 h, dialyze the stirred system in a dialysis bag with a molecular weight cut-off of 14,000 using ultrapure water for 48 h, and freeze-dry to obtain oxidized dextran; the oxidation degree of the oxidized dextran is 50%;

[0060] Step 2: Mix the precursor solution containing oxidized dextran and the precursor solution containing lysozyme according to the mass ratio of oxidized dextran to lysozyme of 1:9, and let it stand at 37°C for 6 h for solidification to obtain an injectable self-crosslinking hydrogel for endometrial injury repair, labeled as 50-1:9; the precursor solution containing oxidized dextran is prepared by dispersing oxidized dextran in PBS buffer solution with a pH of 7.5, and the concentration of oxidized dextran in the precursor solution containing oxidized dextran is 15 wt%; the precursor solution containing lysozyme is prepared by dispersing lysozyme in PBS buffer solution with a pH of 7.5; the mass percentage content of lysozyme in the precursor solution containing lysozyme is 15 wt%.

[0061] Example 3

[0062] This example provides a preparation method of an injectable self-crosslinking hydrogel for endometrial injury repair, including:

[0063] Step 1: Dissolve 4 g of dextran in 20 mL of absolute ethanol, add 1.604 g of an aqueous sodium periodate solution with a mass percentage of 8.02%, stir magnetically in the dark at 25 °C for 6 h, dialyze the stirred system against ultrapure water in a dialysis bag with a molecular weight cut-off of 14,000 for 48 h, and freeze-dry to obtain oxidized dextran; the oxidation degree of the oxidized dextran is 75%.

[0064] Step 2: Mix the precursor solution containing oxidized dextran and the precursor solution containing lysozyme according to a mass ratio of oxidized dextran to lysozyme of 1:9, and let it stand at 37 °C for 6 h for curing to obtain an injectable self-crosslinking hydrogel for endometrial injury repair, labeled as 75-1:9; the precursor solution containing oxidized dextran is prepared by dispersing oxidized dextran in PBS buffer solution, the pH of the PBS buffer solution is 7.5, and the concentration of oxidized dextran in the precursor solution containing oxidized dextran is 15 wt%; the precursor solution containing lysozyme is prepared by dispersing lysozyme in PBS buffer solution, the pH of the PBS buffer solution is 7.5; the mass percentage of lysozyme in the precursor solution containing lysozyme is 15 wt%.

[0065] Example 4

[0066] This example provides a preparation method of an injectable self-crosslinking hydrogel for endometrial injury repair, including:

[0067] Step 1: Dissolve 4 g of dextran in 20 mL of absolute ethanol, add 2.139 g of an aqueous sodium periodate solution with a mass percentage of 10.695%, stir magnetically in the dark at 25 °C for 6 h, dialyze the stirred system against ultrapure water in a dialysis bag with a molecular weight cut-off of 14,000 for 48 h, and freeze-dry to obtain oxidized dextran; the oxidation degree of the oxidized dextran is 100%.

[0068] Step 2: Mix the precursor solution containing oxidized dextran and the precursor solution containing lysozyme according to a mass ratio of oxidized dextran to lysozyme of 1:9, and let it stand at 37 °C for 6 h for curing to obtain an injectable self-crosslinking hydrogel for endometrial injury repair, labeled as 100-1:9; the precursor solution containing oxidized dextran is prepared by dispersing oxidized dextran in PBS buffer solution, the pH of the PBS buffer solution is 7.5, and the concentration of oxidized dextran in the precursor solution containing oxidized dextran is 15 wt%; the precursor solution containing lysozyme is prepared by dispersing lysozyme in PBS buffer solution, the pH of the PBS buffer solution is 7.5; the mass percentage of lysozyme in the precursor solution containing lysozyme is 15 wt%.

[0069] Examples 5 - 20

[0070] Examples 5 to 20 respectively provide a preparation method of an injectable self-crosslinking hydrogel for endometrial injury repair, which is the same as that of Examples 1 to 4, and the differences are shown in Table 1.

[0071] Table 1 Parameters in the preparation methods of Examples 1 to 20

[0072]

[0073]

[0074] Comparative Example 1

[0075] This comparative example provides a method for preparing an injectable self-crosslinking hydrogel, including:

[0076] Using dextran and lysozyme as raw materials, and preparing according to the method of Step 2 of Example 1, wherein the mass ratios of dextran to lysozyme are 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 or 10:0 respectively.

[0077] Comparative Example 2

[0078] This comparative example examines the influence of the mass ratio of dextran to lysozyme on the properties of the hydrogel. It is the same as Example 1, except that the mass ratio is 1:9.

[0079] Comparative Example 3

[0080] This comparative example examines the influence of the mass ratio of dextran to lysozyme on the properties of the hydrogel. It is the same as Example 1, except that the mass ratio is 9:1.

[0081] The gelation situations of the methods of Examples 1 to 20 and Comparative Examples 1 to 3 are shown in Table 2.

[0082] Table 2 Gelation situations under different oxidation degrees of oxidized dextran and different mass ratios of oxidized dextran to lysozyme

[0083]

[0084] √: Gelation ×: No gelation

[0085] As can be seen from Table 2, in Comparative Example 1, dextran and lysozyme were used as raw materials, and the resulting product was in a liquid phase, and no hydrogel was obtained, indicating that dextran and lysozyme could not be gelated. When the raw material was only oxidized dextran or lysozyme, gelation could not occur. As the oxidation degree of oxidized dextran increased, the mass ratio range for gelation expanded. Specifically, when the oxidation degree was 25%, gelation could occur when the mass ratio of oxidized dextran to lysozyme was 8:2 to 6:4; when the oxidation degree was 50%, gelation could occur when the mass ratio was 9:1 to 4:6; when the oxidation degree was 75%, the mass ratio range for gelation was 9:1 to 2:8; when the oxidation degree was 100%, gelation could occur when the mass ratio of oxidized dextran to lysozyme was 9:1 to 1:9.

[0086] Performance evaluation

[0087] Figure 1 It is the nuclear magnetic resonance spectrum of oxidized dextran in Step 1 of Example 3. Compared with the nuclear magnetic resonance spectrum of dextran, characteristic peaks attributed to the protons at positions 2-6 of dextran appear at 4.0 ppm to 3.5 ppm, the peak at 5.0 ppm is attributed to the anomeric protons of C1, accounting for 2.5% of the total amount of dextran, and new peaks attributed to hemiacetals appear in the range of 5.8 ppm to 4.2 ppm. These peaks do not appear in dextran, and no peak attributed to aldehyde groups appears in the spectrum. The possible reason is that during the oxidation reaction of dextran hydroxyl groups, aldehyde groups are converted into hemiacetals.

[0088] Figure 2 It is the test results of the mechanical properties of each hydrogel. The test conditions are at 5% strain and 1 HZ. The results show that when the oxidation degree of oxidized dextran is 75% - 100% and the mass ratio is 4:6 - 2:8, the shear modulus of the hydrogel exceeds 5 kPa, showing high mechanical properties.

[0089] Figure 3 It is the DMA result of the 75-5:5 hydrogel. The results of the storage modulus and loss modulus when standing in a 37°C environment for 0 - 48 h show that as the curing time extends, the storage modulus of the system first increases and then decreases. When the curing time is 13 - 14 h, the hydrogel reaches complete mechanical strength, indicating that the hydrogel of the present invention can form a hydrogel with a stable three-dimensional network structure through self-crosslinking and withstand external pressure.

[0090] Figure 4 It is the swelling test result of the hydrogel. The test method includes: immersing 400 μL of the hydrogel in 3.6 mL of PBS buffer with a pH of 7.5, oscillating and incubating at 37°C until the preset time, updating the PBS buffer every day during the oscillating incubation process, and measuring the mass of the hydrogel after swelling by water absorption. Figure 4Among them, the swelling ratio is (mass after swelling - mass before swelling) / mass before swelling. The results show that the swelling ratio of the hydrogel first increases rapidly, then decreases and then levels off. In the initial stage of swelling, the mass of the hydrogel increases rapidly because the hydrogel of the present invention absorbs water and swells rapidly, and the swelling ratio reaches up to 55%. It has a high swelling ratio. The mass of the hydrogel decreases within 1-2 days. The reason may be that the hydrogel degrades, the Schiff base dynamic cross-linking in the hydrogel hydrolyzes, lysozyme is released, and the mass decreases. The weight change of the hydrogel in water is determined by the interaction between swelling and degradation. Swelling dominates in the initial stage, degradation dominates in the middle stage, and swelling and hydrolysis reach equilibrium in the later stage. The hydrogel of the present invention has a high swelling ratio, can rapidly absorb the exudate of the damaged part, minimize the wound infection rate, and has the characteristic of slow degradation, and can repair endometrial damage.

[0091] Figure 5 The results are for the degradation performance test of the hydrogel. The test method includes placing the hydrogel in ultrapure water, incubating at 37 °C, replacing the ultrapure water every 12 h, taking out the hydrogel and freeze-drying it at the preset time, measuring the dry weight, and determining the release amount of lysozyme. The results show that with the extension of the incubation time, the release amount of lysozyme in all hydrogels shows a trend of first increasing and then leveling off, indicating that the hydrogel of the present invention can stably release lysozyme within 34 days. Within 34 days, the highest degradation rate is only 19.7%, showing good potential for long-term treatment. Under the same degree of oxidation, with the increase of the content of oxidized dextran, the lysozyme release curve is flatter. Under the condition of the same mass ratio of oxidized dextran to lysozyme, with the increase of the degree of oxidation, the lysozyme release curve is more flat.

[0092] Figure 6 The results are for the antibacterial performance test of the hydrogel. The test method includes: placing single strains of Escherichia coli and Staphylococcus aureus in 12 mL of LB liquid medium, culturing in a shaker at 37 °C for 12 h, with the shaker vibration rate of 180 revolutions per minute, measuring the OD value at 630 nm with a microplate reader, diluting the bacterial solution to 10 6 CFU / mL, taking 500 μL of the diluted bacterial solution and co-culturing it with 200 mg of the incubated hydrogel sample for 2 h, diluting the co-cultured bacterial solution 10 3 times, taking 100 μL and spreading it on a solid medium plate, culturing in an incubator at 37 °C for 14 h, and recording the number of colony-forming units; the incubated hydrogel sample is obtained by incubating 200 mg of the hydrogel in 500 μL of physiological saline for 24 h. The results are as Figure 6 shown. According to Figure 6It can be seen that the hydrogel of the present invention can effectively inhibit the generation of bacteria, indicating that the hydrogel of the present invention can specifically prevent bacterial infections during endometrial repair, and has a more significant antibacterial effect on Staphylococcus aureus. The bacterial morphology after the above treatment was observed by scanning electron microscopy. The method for obtaining the observed sample was as follows: the cultured bacterial solution was centrifuged at 3000 rpm for 5 min, washed with PBS buffer, then fixed with 2.5% glutaraldehyde by mass percentage for 4 h, washed with PBS buffer again, and then dehydrated successively with ethanol with volume fractions of 30%, 50%, 70%, 85%, and 95% for 10 min each, dehydrated twice with 100% ethanol, 15 min each time. Finally, it was dried in a vacuum oven at 37 °C to obtain the bacteria to be observed. The observation results showed that the bacteria treated with the hydrogel of the present invention all showed varying degrees of wrinkling and damage, and the 75-2:8 hydrogel had more obvious shrinkage and damage of bacteria. The hydrogel of the present invention has good antibacterial effects.

[0093] The test results of the anti-inflammatory performance of the hydrogel are as Figure 7 shown. The test method includes: inducing and culturing 1 mL of RAW264.7 cells at 2×10 5 / mL with 1 mL of LPS solution at a concentration of 500 ng / mL for 24 h as the control group, and inducing and culturing the above cells with LPS solution containing the hydrogel for 24 h as the experimental group, and measuring the control group and the experimental group with a flow cytometer; in the LPS solution containing the hydrogel, the concentration of LPS is 500 ng / mL, the concentration of the hydrogel is 100 mg / mL, and the hydrogel is 75-2:8. The results showed that the hydrogel of the present invention can effectively weaken the polarization state of macrophages, and has the effects of reducing inflammation and promoting healing.

[0094] The animal experiment results of the hydrogel are as Figure 8As shown. The test method includes: Female SD rats at 8 weeks old with a normal estrous cycle and a weight of 200 - 250 g were selected. After the estrous cycle ended, absolute ethanol was injected into the uterus to establish a thin endometrium model in rodents. The process of establishing the uterine injury model is as follows: The rats were anesthetized by injecting a 2% sodium pentobarbital solution at an injection volume of 0.3 mL per 100 g. The abdominal incision was made to expose the uterine horns. The proximal and distal ends of the uterus were clamped with hemostatic forceps. 0.5 mL of a 95% ethanol solution by volume was injected into the uterine horns for 60 s. The vascular clamp was released, and extrusion was performed to remove the residual ethanol. The damaged area was washed with PBS buffer to further remove ethanol. The uterus was rinsed, and sutured with 6-0 absorbable sutures. The rats were randomly divided into three groups: 1. Sham operation group: No modeling treatment was performed, and only 50 μL of PBS buffer was injected; 2. Model group: 30 min after injecting 95% ethanol by volume, 50 μL of PBS buffer was injected through the uterine horns; 3. Experimental group: 30 min after injecting 95% ethanol by volume, 50 μL of 75-2:8 hydrogel was injected through the uterine horns to the damaged area. The results showed that the damaged uterine tissues in the model group showed obvious edema, inflammatory congestion, and structural deformation. Significant endometrial regeneration was observed in the rats injected with the hydrogel, and there was no obvious difference in the appearance of the uterus compared with the sham operation group. It is indicated that the hydrogel of the present invention can significantly promote the structural and functional reconstruction of the endometrium through the antibacterial and anti-inflammatory synergistic action mechanism. The hydrogel can form a stable physical barrier in situ after being injected into the uterine horns, realizing the prevention of tissue adhesion at the damaged site. At the same time, combined with the sustained release of lysozyme and the excellent anti-inflammatory activity of the hydrogel, it has long-term antibacterial effect, regulates the inflammatory microenvironment, prevents bacterial infection after endometrial injury, and promotes endometrial repair.

Claims

1. A method for preparing an injectable self-crosslinking hydrogel for repairing endometrial damage, characterized in that: The method comprises mixing a precursor solution containing oxidized dextran with a precursor solution containing lysozyme, and allowing the mixture to stand and solidify under body temperature conditions to obtain an injectable self-crosslinking hydrogel for repairing endometrial damage.

2. The method for preparing the injectable self-crosslinking hydrogel for repairing endometrial damage according to claim 1, characterized in that: The oxidized dextran is obtained by oxidizing dextran with a sodium periodate solution.

3. The method for preparing the injectable self-crosslinking hydrogel for repairing endometrial damage according to claim 2, characterized in that: The mass of the sodium periodate solution is 0.1 to 0.5 times the mass of the dextran.

4. The method for preparing the injectable self-crosslinking hydrogel for endometrial damage repair according to claim 2, characterized in that: The mass percentage of sodium periodate in the sodium periodate solution is 2% to 10%.

5. The method for preparing the injectable self-crosslinking hydrogel for repairing endometrial damage according to claim 1, characterized in that: The oxidation degree of the oxidized dextran is 25% to 100%, and / or the mass ratio of the oxidized dextran to lysozyme is (9:1) to (1:9).

6. The method for preparing the injectable self-crosslinking hydrogel for repairing endometrial damage according to claim 5, characterized in that: The mass ratio of the oxidized dextran to lysozyme is (9:1) to (2:8).

7. The method for preparing the injectable self-crosslinking hydrogel for repairing endometrial damage according to claim 6, characterized in that: The oxidation degree of the oxidized dextran is 75% to 100%, and / or the mass ratio of the oxidized dextran to lysozyme is (4:6) to (2:8).

8. The method for preparing the injectable self-crosslinking hydrogel for repairing endometrial damage according to claim 7, characterized in that: The oxidation degree of the oxidized dextran is 75%, and the mass ratio of the oxidized dextran to lysozyme is 2:

8.

9. The method for preparing the injectable self-crosslinking hydrogel for repairing endometrial damage according to claim 1, characterized in that: The lysozyme is egg white lysozyme.

10. A hydrogel prepared by the method for preparing an injectable self-crosslinking hydrogel for repairing endometrial damage as claimed in claims 1 to 9.

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