A viscosity-controllable chitosan-and-andrias skin secretion hydrogel as well as a preparation method and application thereof

Abstract

The present application provides a viscosity controllable chitosan-giant salamander skin secretion hydrogel and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The giant salamander skin secretion lyophilized powder is mixed and stirred with ascorbic acid solution respectively to obtain a giant salamander skin secretion solution and a chitosan solution; the giant salamander skin secretion solution and the chitosan solution are mixed in a mass ratio of 6-18:1 to obtain the viscosity controllable chitosan-giant salamander skin secretion hydrogel. The viscosity of the hydrogel can be effectively controlled, and the rheological properties of the hydrogel can be controlled by adjusting the mass ratio of the giant salamander skin secretion solution and the chitosan solution. The chitosan-giant salamander skin secretion hydrogel prepared by the present application has good physical properties, antibacterial hemostatic performance and biocompatibility, and can be widely used in the field of wound healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a viscosity-controllable chitosan-Andrias davidianus skin secretion hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Hemostasis and repair of deep wounds is an important research topic in the field of biomedicine, and suture is the most widely used method for hemostasis and repair of deep wounds, but it may cause additional tissue damage, increase the risk of infection and foreign body reaction, cause local inflammation, and is not conducive to wound recovery. Hydrogel provides a new solution for solving the hemostasis and repair of deep wounds due to its similar structure to human tissue extracellular matrix and high biocompatibility. In particular, natural biomass hydrogel with self-healing performance can not only effectively stop bleeding, but also solve the problem of wound cracking caused by movement during the healing process. Andrias davidianus can secrete a kind of mucus with high viscosity and wound healing performance when stimulated, which is a natural biomass material with wide application potential. However, the mucus has high viscosity, poor toughness and is difficult to dissolve, which is not conducive to processing and utilization, and greatly limits its application in deep wounds. Therefore, it is necessary to provide a method for preparing viscosity-controllable Andrias davidianus skin secretion hydrogel. SUMMARY

[0003] Therefore, the present application provides a viscosity-controllable chitosan-Andrias davidianus skin secretion hydrogel as well as a preparation method and application thereof. The hydrogel dressing prepared in the present application has multiple functions such as injectability, antibacterial property, self-healing and wound healing promotion, and solves the problems of high viscosity, difficulty in dissolving and processing and utilization of Andrias davidianus skin secretion.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a preparation method of a viscosity-controllable chitosan-Andrias davidianus skin secretion hydrogel, which comprises the following steps:

[0006] (1) Collecting Andrias davidianus skin secretion, freeze-drying to obtain Andrias davidianus skin secretion freeze-dried powder;

[0007] (2) Dissolving the Andrias davidianus skin secretion freeze-dried powder in an acetic acid solution, stirring to obtain a mixed solution, centrifuging the mixed solution, taking the supernatant, placing the supernatant in a dialysis bag for dialysis for 70-75 hours, freeze-drying the solution in the dialysis bag to obtain purified Andrias davidianus skin secretion freeze-dried powder;

[0008] (3) Mixing and stirring the purified Andrias davidianus skin secretion freeze-dried powder and chitosan with an ascorbic acid solution respectively to obtain an Andrias davidianus skin secretion solution and a chitosan solution;

[0009] (4) mixing the giant salamander skin secretion solution and the chitosan solution in a mass ratio of 6-18:1, and stirring to obtain the chitosan-giant salamander skin secretion hydrogel with controllable viscosity.

[0010] Preferably, the temperature for freeze-drying is -50 to -40℃, the vacuum degree is 10-20 Pa, and the freeze-drying time is 22-26 h.

[0011] Preferably, the molar concentration of the acetic acid solution is 0.4-0.6 M; and in the mixed solution, the mass concentration of the giant salamander skin secretion is 30-50 mg / mL.

[0012] Preferably, the centrifugal force for centrifugation is 4500-5500 g, and the centrifugation time is 8-12 min.

[0013] Preferably, the molecular cut-off of the dialysis bag is 280-320 Da.

[0014] Preferably, the molar concentration of the ascorbic acid solution is 0.6-0.9 M.

[0015] Preferably, in the giant salamander skin secretion solution, the mass concentration of the giant salamander skin secretion is 50-70 mg / mL; and in the chitosan solution, the mass concentration of the chitosan is 50-70 mg / mL.

[0016] Preferably, the degree of polymerization of the chitosan is 100-300.

[0017] The application further provides the chitosan-giant salamander skin secretion hydrogel with controllable viscosity prepared by the method.

[0018] The application further provides the use of the chitosan-giant salamander skin secretion hydrogel with controllable viscosity in preparing a dressing for promoting skin healing.

[0019] By adopting the above technical scheme, the application has the following beneficial effects: the giant salamander skin secretion lyophilized powder and chitosan are mixed with an ascorbic acid solution and stirred to obtain a giant salamander skin secretion solution and a chitosan solution; the giant salamander skin secretion solution and the chitosan solution are mixed in a mass ratio of 6-18:1 and stirred to obtain the chitosan-giant salamander skin secretion hydrogel with controllable viscosity. The application can effectively control the viscosity of the hydrogel and control the rheological properties of the hydrogel by adjusting the mass ratio of the giant salamander skin secretion solution and the chitosan solution. The chitosan-giant salamander skin secretion hydrogel prepared by the application has good physical properties, antibacterial hemostatic performance and biocompatibility, and can be widely used in the field of wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1The photo of the hydration reaction of SSAD in water.

[0021] Figure 2 The solubility of SSAD in different solvents.

[0022] Figure 3 The solubility of SSAD in acetic acid solution and VC solution.

[0023] Figure 4 The comparison chart of SSAD before and after purification.

[0024] Figure 5 The infrared spectrum of different hydrogels.

[0025] Figure 6 The SEM images of SSAD and CSAD-6, CSAD-12, CSAD-18 hydrogels.

[0026] Figure 7 The modulus and viscosity of CSAD hydrogel, where A is the modulus and B is the viscosity.

[0027] Figure 8 The swelling rate of SSAD and CSAD-6, CSAD-12, CSAD-18 hydrogels (different letters indicate significant differences, the same below).

[0028] Figure 9 The degradation rate of SSAD and CSAD-6, CSAD-12, CSAD-18 hydrogels.

[0029] Figure 10 The tissue adhesion performance, texture analyzer test tensile force and tissue adhesion strength results of CSAD-6, CSAD-12, CSAD-18 hydrogels; where A is the tissue adhesion performance, B is the texture analyzer test tensile force, and C is the tissue adhesion strength.

[0030] Figure 11 The self-healing performance and underwater self-healing performance of CSAD hydrogel; A and B are the self-healing performance; C is the underwater self-healing performance.

[0031] Figure 12 The antibacterial performance results of CS, SSAD, VC and CSAD-6, CSAD-12, CSAD-18 hydrogels.

[0032] Figure 13 The blood compatibility results of CSAD-6, CSAD-12, CSAD-18 hydrogels; where A is the hemolysis performance and B is the hemolysis index.

[0033] Figure 14Figures of coagulation test results of CSAD-6, CSAD-12, CSAD-18 hydrogels, wherein A is coagulation performance, and B is coagulation time.

[0034] Figure 15 Figure of coagulation index results of CSAD-6, CSAD-12, CSAD-18 hydrogels.

[0035] Figure 16 Figures of red blood cell adhesion and platelet adhesion results of CSAD-6, CSAD-12, CSAD-18 hydrogels; wherein A is red blood cell adhesion, and B is platelet adhesion.

[0036] Figure 17 Figures of bleeding conditions and bleeding amounts of mice in each group at different times after tail cutting and hydrogel injection; wherein A is bleeding condition, and B is bleeding amount.

[0037] Figure 18 Figures of bleeding conditions and bleeding amounts of liver bleeding mice in each group at different times after hydrogel injection; wherein A is bleeding condition, and B is bleeding amount.

[0038] Figure 19 Figures of cell survival rates of each group of hydrogels co-incubated with cells for 24 h and 48 h.

[0039] Figure 20 Figure of L929 live / dead cell fluorescence staining image (scale bar: 200 μm).

[0040] Figure 21 Figures of effects of CS, SSAD and CSAD-6, CSAD-12, CSAD-18 hydrogels on cell migration; wherein A is cell migration condition, and B is cell migration rate.

[0041] Figure 22 Figures of repair ability of CS, SSAD and CSAD-6, CSAD-12, CSAD-18 hydrogels on cell oxidative damage.

[0042] Figure 23 Figures of wound healing conditions of each group of hydrogels; wherein A is a picture of wound healing period, B is a wound healing rate, and C is a self-healing figure after hydrogel fracture. DETAILED DESCRIPTION

[0043] Anduan skin secretions (SSAD) will undergo hydration reaction when encountering water, rapidly absorbing water and forming a hydrogel. However, the cohesion of this gel is strong, resulting in poor ductility and elasticity of the gel when stretched Figure 1 ), which greatly limits the application of Anduan skin secretions in the medical field, especially in wound dressings requiring good stretchability and flexibility.

[0044] Based on this, the application provides a preparation method of the viscosity-controllable chitosan-and-giant salamander-skin-secretion hydrogel, comprising the following steps:

[0045] (1) collecting giant salamander skin secretions, freeze-drying to obtain giant salamander skin secretion freeze-dried powder;

[0046] (2) dissolving the giant salamander skin secretion freeze-dried powder in an acetic acid solution, stirring to obtain a mixed solution, centrifuging the mixed solution, taking supernatant, placing the supernatant in a dialysis bag for dialysis for 70-75 hours, freeze-drying the solution in the dialysis bag to obtain purified giant salamander skin secretion freeze-dried powder;

[0047] (3) mixing and stirring the purified giant salamander skin secretion freeze-dried powder and chitosan with ascorbic acid solutions respectively to obtain giant salamander skin secretion solution and chitosan solution;

[0048] (4) mixing the giant salamander skin secretion solution and chitosan solution at a mass ratio of 6-18:1, and stirring to obtain the viscosity-controllable chitosan-and-giant salamander-skin-secretion hydrogel.

[0049] The application first collects giant salamander skin secretions, freeze-dries to obtain giant salamander skin secretion freeze-dried powder; the freeze-drying temperature is-50 to-40℃, preferably-48 to-42℃, and more preferably-45℃; the vacuum degree is 10 to 20 Pa, preferably 12 to 18 Pa, and more preferably 15 Pa; and the freeze-drying time is 22 to 26 hours, preferably 23 to 25 hours, and more preferably 24 hours. Under the freeze-drying parameters, the water in the giant salamander skin secretions can be sufficiently removed, and the activity of the giant salamander skin secretions can be maintained.

[0050] The application obtains the freeze-dried powder of giant salamander skin secretion, and then purifies the freeze-dried powder. The freeze-dried powder of giant salamander skin secretion is dissolved in an acetic acid solution, and stirred until the freeze-dried powder is completely dissolved to obtain a mixed solution. The molar concentration of the acetic acid solution is 0.4-0.6 M, preferably 0.45-0.55 M, and more preferably 0.5 M. In the mixed solution, the concentration of the freeze-dried powder of giant salamander skin secretion is 30-50 mg / mL, preferably 35-45 mg / mL, and more preferably 40 mg / mL. Then, the mixed solution is centrifuged to remove insoluble particles or impurities possibly existing in the solution. The centrifugal force of the centrifugation is 4500-5500 g, preferably 4700-5200 g, and more preferably 5000 g. The centrifugation time is 8-12 min, preferably 9-11 min, and more preferably 10 min. After centrifugation, the supernatant is taken and poured into a dialysis bag, and the dialysis bag is placed in pure water for dialysis to remove small molecular impurities and acetic acid in the solution. The molecular cut-off of the dialysis bag is 280-320 Da, preferably 290-310 Da, and more preferably 300 Da. In order to keep the dialysate clean and the dialysis effect, the pure water needs to be replaced regularly, and the replacement frequency is 5-7 h, preferably 5.5-6.5 h, and more preferably 6 h. The dialysis time is 70-75 h, preferably 71-74 h, and more preferably 72 h, to obtain a purified giant salamander skin secretion solution. The purified giant salamander skin secretion solution is freeze-dried to obtain a purified freeze-dried powder of giant salamander skin secretion, and the freeze-drying conditions are the same as above.

[0051] The application dissolves the prepared purified freeze-dried powder of giant salamander skin secretion in an ascorbic acid solution to obtain a giant salamander skin secretion solution. The molar concentration of the ascorbic acid solution is 0.6-0.9 M, preferably 0.7-0.8 M, and more preferably 0.75 M. In the giant salamander skin secretion solution, the mass concentration of the giant salamander skin secretion is 50-70 mg / mL, preferably 55-65 mg / mL, and more preferably 60 mg / mL.

[0052] The application dissolves the prepared giant salamander skin secretion solution in an ascorbic acid solution to obtain a chitosan solution. The molar concentration of the ascorbic acid solution is 0.6-0.9 M, preferably 0.7-0.8 M, and more preferably 0.75 M. In the chitosan solution, the mass concentration of the chitosan is 50-70 mg / mL, preferably 55-65 mg / mL, and more preferably 60 mg / mL.

[0053] The ascorbic acid solution and the chitosan solution are mixed at room temperature, and stirred uniformly, so as to obtain the viscosity-controllable chitosan-giant salamander skin secretion hydrogel; the mass ratio of the giant salamander skin secretion solution and the chitosan solution is 6-18:1, preferably 9-15:1, and more preferably 12:1; the temperature of the room temperature is 24-26 DEG C, and more preferably 25 DEG C; and the degree of polymerization of the chitosan is 100-300, preferably 150-250, and more preferably 200.

[0054] The application further provides the viscosity-controllable chitosan-giant salamander skin secretion hydrogel prepared by the preparation method.

[0055] The application further provides application of the viscosity-controllable chitosan-giant salamander skin secretion hydrogel in preparation of a dressing for promoting skin healing.

[0056] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0057] The application selects healthy and well-grown 2-year-old artificially bred giant salamanders as experimental objects, and the scheme of the application is approved by the Experimental Animal Ethics Committee of Guiyang University (Experimental Animal Use License No. (GYU-BEE-2022-02).

[0058] The S. aureus and E. coli of the application are purchased from Shanghai Jianke Biological Technology Co., Ltd.

[0059] The L929 mouse fibroblasts of the application are purchased from Shanghai Enzyme-Linked Biological Technology Co., Ltd.

[0060] The mice used in the experiments of the application are SPF Kunming mice purchased from Sibeifeng (Beijing) Biotechnology Co., Ltd.

[0061] The experimental data of the application is statistically analyzed by using Origin and GraphPad Prism 9.0 software; independent sample T-test is used for comparison between two groups, and one-way ANOVA is used for comparison among three groups or more groups; and significant differences are indicated by different letters (P<0.05).

[0062] Example 1. Preparation of giant salamander skin secretion freeze-dried powder

[0063] (1) Andrias davidianus skin secretion collection: Select healthy and well-grown 2-year-old artificially bred Andrias davidianus as experimental objects, and use physical stimulation to make the Andrias davidianus secrete mucus. Immediately, use a sterilized medicine spoon to gently scrape the surface of the skin, and carefully collect the mucus. Transfer the collected Andrias davidianus skin secretion to a beaker, and then place it in a freeze dryer. The temperature of the freeze dryer is set at -45°C, the vacuum degree is 20 Pa, and the freeze drying time is 24 h. The freeze-dried Andrias davidianus skin secretion powder is obtained and stored at -80°C for later use.

[0064] (2) Andrias davidianus skin secretion purification:

[0065] ①Dissolve the freeze-dried Andrias davidianus skin secretion powder at a concentration of 40 mg / mL in acetic acid (0.5 M) and use a magnetic stirrer to stir at room temperature until the Andrias davidianus skin secretion powder is completely dissolved in the acetic acid solution. After complete dissolution, pour it into a centrifuge tube;

[0066] ②Place the centrifuge tube in a centrifuge and centrifuge at 5000 g for 10 min to remove insoluble particles or impurities that may exist in the solution;

[0067] ③After centrifugation, carefully remove the supernatant and pour it into a dialysis bag with a molecular weight cut-off of 300 Da;

[0068] ④Seal the dialysis bag and place it in a beaker containing sufficient pure water for dialysis. To ensure the complete removal of small molecular impurities and acetic acid from the Andrias davidianus skin secretion solution, replace the pure water every 6 h to maintain the cleanliness and dialysis effect of the dialysis solution. The dialysis process lasts for 3 d, and a pure Andrias davidianus skin secretion solution is obtained;

[0069] ⑤Freeze-dry the obtained Andrias davidianus skin secretion solution for 24 h to obtain an impurity-free Andrias davidianus skin secretion powder for subsequent experiments.

[0070] Example 2

[0071] Dissolve the Andrias davidianus skin secretion freeze-dried powder in a VC solution (0.75 M) at a concentration of 60 mg / mL to obtain an Andrias davidianus skin secretion solution. Meanwhile, dissolve chitosan with a degree of polymerization of 100-300 in a VC solution (0.75 M) at a concentration of 60 mg / mL to obtain a chitosan solution. Mix the Andrias davidianus skin secretion mucus and the chitosan solution at a mass ratio of 6:1 at room temperature (25°C) and stir uniformly to obtain a chitosan-Andrias davidianus skin secretion hydrogel, which is named CSAD-6.

[0072] Example 3

[0073] The Andrias skin secretion lyophilized powder was dissolved in VC solution (0.75 M) at a concentration of 60 mg / mL to obtain an Andrias skin secretion solution; at the same time, chitosan with a degree of polymerization of 100-300 was dissolved in VC solution (0.75 M) at a concentration of 60 mg / mL to obtain a chitosan solution. The Andrias skin secretion mucus and the chitosan solution were mixed at room temperature (25°C) at a mass ratio of 12:1, stirred uniformly, to obtain a chitosan-Andrias skin secretion hydrogel, designated as CSAD-12.

[0074] Example 4

[0075] The Andrias skin secretion lyophilized powder was dissolved in VC solution (0.75 M) at a concentration of 60 mg / mL to obtain an Andrias skin secretion solution; at the same time, chitosan with a degree of polymerization of 100-300 was dissolved in VC solution (0.75 M) at a concentration of 60 mg / mL to obtain a chitosan solution. The Andrias skin secretion mucus and the chitosan solution were mixed at room temperature (25°C) at a mass ratio of 12:1, stirred uniformly, to obtain a chitosan-Andrias skin secretion hydrogel, designated as CSAD-12.

[0076] Comparative Example 1

[0077] Unlike Example 2, water was used to replace the VC solution.

[0078] Comparative Example 2

[0079] Unlike Example 2, anhydrous ethanol was used to replace the VC solution.

[0080] Comparative Example 3

[0081] Unlike Example 2, 0.75 M sodium hydroxide solution was used to replace the VC solution.

[0082] Comparative Example 4

[0083] Unlike Example 2, 0.75 M sodium bicarbonate solution was used to replace the VC solution.

[0084] Comparative Example 5

[0085] Unlike Example 2, 0.75 M acetic acid solution was used to replace the VC solution.

[0086] Experimental Example 1

[0087] The Andrias skin secretion was dissolved in 0.75 M VC solution and the solutions of Comparative Examples 1-5 at a concentration of 60 mg / mL, respectively, to determine the dissolution rate of the Andrias skin secretion in different solvents, and the results are shown in Table 1. Figure 2 Figure 2 ​It is known that the dissolution rate of the giant salamander skin secretion in ethanol and sodium hydroxide solution is less than 20%. However, the dissolution rate of the giant salamander skin secretion in acetic acid and VC solution is higher than 95%.

[0088] The present application also tests the dissolution rate of the giant salamander skin secretion in acetic acid and VC, and the results are shown in Figure 3 Figure 3 It is shown that although the acetic acid solution can dissolve the giant salamander skin secretion, the dissolution rate is significantly lower than that of the VC solution.

[0089] Experimental Example 2

[0090] The CSAD-6, CSAD-12 and CSAD-18 prepared in Examples 2-4 are subjected to Fourier transform infrared spectroscopy analysis, scanning electron microscope (SEM) observation and rheological property test.

[0091] 1. Fourier transform infrared spectroscopy (FTIR) analysis

[0092] (1) After freeze-drying each group of mucus hydrogel for 24 h, grind into fine powder;

[0093] (2) Mix the mucus hydrogel powder with 1-2 mg of potassium bromide powder and press into a tablet;

[0094] (3) Adjust the FTIR spectrometer to record the projection mode, and record the spectral data as absorbance units. Parameters: wave number range 4000-400 cm -1 , resolution 4 cm 1 , scanning 16 times;

[0095] (4) Collect all the FTIR spectra by FTIR spectrometer under low air humidity (less than 40%) and room temperature conditions;

[0096] (5) Use Pesakfit4.12 software to analyze the secondary structure of the mucus hydrogel, and use Gaussian function to deconvolute, peak split and curve fit the amide I of the original spectrum (1600-1700 cm -1 ).

[0097] From the spectral data Figure 5 , it can be seen that the absorption band of 3420 cm -1 of the giant salamander skin secretion (SSAD) and 3436 cm -1 of chitosan (CS) respectively corresponds to the stretching vibration of -OH, and when the two are mixed to form a hydrogel, the absorption peak shifts to 3294 cm -1 , -OH moves to low wavelength, indicating that hydrogen bond plays a key role in the process of gel formation. In addition, 1648 cm -1 ​The absorption peaks at 2923 cm -1 The absorption peaks at 2923 cm -1 correspond to the stretching vibration of C-H, which is a unique structural feature of CS. The change in the characteristic peak also indicates that SSAD and CS form a network structure through intermolecular hydrogen bonding to form a hydrogel. These results show that the network structure of the CSAD hydrogel is formed by hydrogen bonding between SSAD and CS molecules. The original intermolecular hydrogen bonding in SSAD is replaced by CS, and the solvent dissolves to achieve viscosity control of the hydrogel system.

[0098] 2. Scanning Electron Microscope (SEM) Observation

[0099] (1) After freezing the prepared hydrogel in an ultra-low temperature refrigerator (-80°C) for 12 hours, use a freeze dryer to remove the water in the mucous hydrogel;

[0100] (2) Use a scalpel to cut the CSAD hydrogel after freeze-drying to obtain a hydrogel sheet and expose its internal structure;

[0101] (3) After gold spraying treatment, use a German Carl Zeiss AG Sigma300 scanning electron microscope to observe under the condition of 15kV.

[0102] The results of scanning electron microscope observation Figure 6 show that natural SSAD presents a blocky and lamellar structure and does not have a porous structure. CSAD-6 (SSAD / CS = 6:1) hydrogel has a small and dense pore structure, and CSAD-18 (SSAD / CS = 18:1) hydrogel has larger pores and local area collapse phenomenon, indicating that its internal structure stability is lower. The CSAD-12 ratio hydrogel has a moderate pore size and uniform distribution, showing a good network structure. It is shown that by changing the mixing ratio of CS and SSAD, the pore structure of the CSAD hydrogel can be controlled. Moderate pore structure not only helps to improve the mechanical strength of the hydrogel, but also can improve the biological performance, such as promoting cell penetration and nutrient delivery.

[0103] 3. Rheological property test

[0104] (1) Modulus test (oscillation mode), frequency test with fixed strain 1%, and frequency change range 0.1-100 rad / s;

[0105] (2) Kinetic viscosity test, test shear rate logarithmic change (0.1-1000 s -1 ).

[0106] Storage modulus (G′) reflects a material's ability to store energy during deformation, reflecting its elastic behavior. Loss modulus (G″) represents a material's ability to dissipate energy during deformation, reflecting its viscous behavior. In hydrogels with different stoichiometric ratios, the storage modulus (G′) is consistently higher than the loss modulus (G″). Figure 7 (A) indicates that these hydrogels all possess strong elastic properties and a relatively stable internal network structure. Kinetic viscosity tests show that ( Figure 7 In the B group, CSAD-6 exhibited the highest viscosity, while CSAD-18 had the lowest, but both were still higher than the viscosities of CS and SSAD. This indicates that adjusting the ratio of CS to SSAD solution can effectively control the viscosity of the hydrogel and thus its rheological properties. This method not only achieves injectable performance of the hydrogel but also ensures structural stability, making it applicable to various biomedical fields, including wound healing and tissue repair.

[0107] Example 3. Physical property characterization of chitosan-giant salamander skin secretion hydrogel

[0108] 1. Swelling properties

[0109] Hydrogels can effectively reduce dressing changes and nursing costs by absorbing wound exudate, and alleviate patient discomfort. Their excellent absorbency and water retention help maintain dressing moisture, and a moist environment helps remove necrotic tissue from the wound, thereby accelerating wound healing. Therefore, this invention tested the swelling properties of the hydrogel.

[0110] (1) The hydrogel samples and SSAD samples prepared in Examples 2-4 were freeze-dried for 24 hours to remove moisture;

[0111] (2) Weigh the freeze-dried sample and put it into PBS solution. After 30 minutes, take out the hydrogel, wipe off the moisture and weigh it again (n=3).

[0112] (3) Calculate the swelling ratio according to Equation 1:

[0113] Swelling rate (%) = Ws / Wo × 100% (Equation 1)

[0114] In the formula, Ws is the mass of the hydrogel after swelling at different times; Wo is the mass of the hydrogel before swelling.

[0115] The swelling ratios of the chitosan-giant salamander skin secretion hydrogels CSAD-6, CSAD-12, CSAD-18 and giant salamander skin secretion (SSAD) prepared in Examples 2-4 are as follows: Figure 8The results show that the proportion of CS in CSAD-6 is higher, and the network density is larger, resulting in smaller internal pores and lower swelling rate than CSAD-12, which indicates that the swelling performance of CSAD hydrogel can be regulated by adjusting the network density of the hydrogel.

[0116] 2. Degradation performance

[0117] In the wound healing process, the ideal hydrogel should have degradation performance, which can provide necessary protection for the wound in the early stage and gradually degrade during tissue repair to provide space for the growth of new tissues, so the degradation performance is measured:

[0118] (1) The hydrogel samples prepared in Examples 2-4 and SSAD samples are respectively placed in PBS solution for immersion;

[0119] (2) At 6, 12, 24, 48 and 60h, respectively, wipe off the moisture and weigh (n = 3);

[0120] (3) The degradation rate is calculated according to formula 2:

[0121] Degradation rate (%) = (Wt-Wb) / Wb x 100% (Formula 2)

[0122] In the formula, Wt represents the weight of the hydrogel after degradation in the PBS solution at different time intervals; Wb represents the initial weight of the hydrogel.

[0123] The degradation rates of the chitosan-giant salamander skin secretion hydrogels CSAD-6, CSAD-12, CSAD-18 prepared in Examples 2-4 and giant salamander skin secretion (SSAD) are shown in Table 2. Figure 9 The results show that the SSAD hydrogel has a high network density, and the degradation rate after 60h is only 53.94%, while the degradation rate of the CSAD hydrogel can be regulated by adjusting the ratio of CS solution to SSAD solution, so that its degradation behavior is more in line with the physiological wound repair rate Figure 9 ). CSAD-12 has the lowest degradation rate (47.12%) after 60h due to its uniform network structure. In addition, the VC in the CSAD hydrogel during the degradation process can help reduce oxidative stress damage and further promote tissue repair.

[0124] 3. Tissue adhesion performance

[0125] Tissue adhesion is an important indicator for evaluating hydrogel as a wound dressing, which directly affects the stability of the hydrogel on the wound surface and the effectiveness of biomedical applications. To evaluate the tissue adhesion capacity of the hydrogel, the hydrogel prepared in Examples 2-4 is injected onto the surface of pig skin, and its adhesion performance is observed under different physical action conditions (such as bending, pasting and pulling), and the specific steps are as follows:

[0126] (1) Fresh pigskin was soaked in physiological saline for 4h after removing excess fat, and then cut into 3cm x 1cm rectangular samples;

[0127] (2) 200μL hydrogel was applied to the inner surface of the pigskin, and the two pieces of pigskin were overlapped together with an overlapping area of 1cm x 1cm;

[0128] (3) The overlapped pigskin was subjected to tensile test using Texture Analyzer with a stretching speed of 20mm / min, and the maximum tensile force F (unit: Newton, N) at peeling was recorded;

[0129] (4) The adhesive strength was calculated according to formula 3:

[0130]

[0131] In the formula, F (N): the force when the overlapped skin is peeled; A (m2): the overlapping area (n = 4).

[0132] (5) The hydrogel was applied to fresh pigskin, and the changes after twisting and bending were tested.

[0133] The results are shown in Figure 10 . Figure 10 A in the results shows that the CSAD hydrogel can maintain a good adhesion state during deformation and stress, and exhibits excellent tissue adhesion performance. The results of tensile force test by Texture Analyzer are shown in B and C in the Figure 10 . The results of tensile force test by Texture Analyzer show that the hydrogel has strong adhesion with pigskin tissue, especially the CSAD-12 sample, which has a higher maximum tensile force than other samples, indicating that it has the optimal tissue adhesion capacity. This excellent adhesion property helps the hydrogel to be fixed in the dynamic environment of the wound, maintaining a moist healing environment, thereby accelerating tissue repair.

[0134] 4. Self-healing performance

[0135] During wound treatment, the self-healing ability of hydrogel can maintain the integrity of the dressing, enhance mechanical stability, and reduce the risk of infection. Hydrogels lacking self-healing performance are difficult to adapt to complex and irregularly shaped wounds, and are prone to damage due to external forces, thereby increasing the risk of bacterial invasion and delaying the healing process. Therefore, hemostatic hydrogels with excellent self-healing ability not only can effectively respond to damage by external forces, but also can provide a stable physical barrier during the entire repair process, promoting tissue regeneration. The self-healing performance of the hydrogel was tested according to the following steps:

[0136] (1) The CSAD-12 hydrogel was prepared according to the method of Example 3, and red ink was added for dyeing. The white undyed hydrogel was cut open and placed together with the dyed hydrogel of different color for 30 min, and then picked up with forceps and stretched to observe the self-healing.

[0137] (2) 2 The prepared sample was placed on a slide, and another dyed hydrogel was placed on the hydrogel for 10 min and then pulled up to observe the self-healing.

[0138] (3) Underwater self-healing performance: The prepared hydrogel was placed in PBS, and another hydrogel was placed on the hydrogel in water for 1 min and then picked up to observe the healing of the two hydrogels under water. The results are shown in Figure 11 . The results show that the cut hydrogel samples can be healed after being in contact with each other for 30 min (A in Figure 11 . In another set of experiments, good self-healing phenomenon was also observed after placing the hydrogel sample from above for 10 min (B in Figure 11 .

[0139] Since the surface of the wound is usually in a wet state, the performance of self-healing in underwater environment is particularly crucial. The underwater self-healing performance test results show that the CSAD hydrogel can quickly restore structural integrity after being in contact under water for 60 s (C in Figure 11 , indicating that it can still maintain the function of adhering to the wound in a high-humidity or bleeding environment.

[0140] Experimental Example 4. Antibacterial performance of chitosan-giant salamander skin secretion hydrogel

[0141] Antibacterial performance is crucial for wound dressings, especially in open wounds, where bacterial infection can significantly delay the wound healing process and can cause serious infections such as sepsis or chronic wounds. Therefore, the hydrogels prepared in Examples 2-4 were tested for antibacterial performance:

[0142] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were selected for the antibacterial activity study of the hydrogels.

[0143] (1) Prepare liquid medium: Tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (10 g / L), in a high-pressure steam sterilization pot, sterilize at 121°C for 20 min. After sterilization, cool and store in a 4°C refrigerator;

[0144] (2) Prepare solid medium: Tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (10 g / L), agar powder (15 g / L), in a high-pressure steam sterilization pot, sterilize at 121°C for 20 min. After sterilization, store in a 60°C oven;

[0145] (3) Take the frozen S. aureus and E. coli strains out of the freezer and place them in a clean bench. Inoculate a small amount of the strain into 5 mL of liquid medium and incubate in a 37°C, 120 rpm / min constant temperature incubator at 37°C. Activate the strain to the logarithmic growth phase (12-16 h). After the incubation is complete, dilute the bacterial solution with sterile physiological saline to a final concentration of 1 x 10 6 CFU / mL for standby;

[0146] (4) After ultraviolet sterilization of the hydrogels prepared in Examples 2-4, SSAD, CS, and VC, add them to the medium at a concentration of 60 mg / mL. Place the bacterial solution in a 37°C, 120 rpm / min constant temperature incubator for 12 h. The control group is added with an equal amount of sterile deionized water;

[0147] (5) After co-culturing, dilute the bacterial solution by gradient dilution, and then take an appropriate amount of the diluted bacterial solution and evenly spread it on the solid medium. Then invert it and incubate it in a 37°C constant temperature incubator for 12 h. Observe and record the growth of colonies on the plate, and the results are shown in Figure 12

[0148] The results show that SSAD itself also has certain antibacterial effect, which may be related to the special polypeptide contained therein. Due to the introduction of CS, the antibacterial performance of the hydrogel is significantly improved due to its cationic characteristics, and is enhanced with the increase of the proportion of CS. CSAD-6 and CSAD-12 hydrogels have better inhibitory effect on S. aureus.

[0149] Experimental Example 4. In vitro hemostatic performance of chitosan-Andrias davidian skin secretion hydrogel

[0150] 1. Hemolysis experiment

[0151] In a wound environment, when a biological material comes into contact with blood, if there is strong physical or chemical stimulation on its surface, it may cause the red blood cell membrane to rupture, triggering a hemolytic reaction. Therefore, hemolysis test is an important indicator for evaluating the compatibility of biological materials with red blood cells, which can directly reflect the safety and application potential of materials in the blood environment. The hemolysis performance of the hydrogels prepared in Examples 2-4 was systematically evaluated:

[0152] (1) Add each group of hydrogels to PBS for 24 h of pretreatment;

[0153] (2) Add PBS and anticoagulated rabbit blood in a ratio of 9:1, respectively;

[0154] (3) Centrifuge the mixture (1500 x g, 5 min) to remove the supernatant, and add PBS again, repeating three times. Resuspend the red blood cells with PBS to prepare a 10% red blood cell suspension;​

[0155] (4) The pretreated hydrogel was added to the red blood cell suspension, pure red blood cells were added to deionized water as a positive control, and the red blood cell suspension was added to PBS as a negative control;

[0156] (5) The above treated samples were placed in a 37°C incubator for 30 min, and the centrifuge tubes were removed and centrifuged (1500 x g, 5 min);

[0157] (6) The supernatant of each group was added to a 96-well plate, and the OD value at 540 nm was determined by an enzyme marker (n = 3);

[0158] (7) The hemolysis rate was calculated by formula 4:

[0159] Hemolysis rate = (OD sample - OD negative ) / (OD positive - OD negative ) x 100% (Formula 4)

[0160] In the formula, OD sample is the OD value of the sample group; OD negative is the OD value of the negative control; and OD positive is the OD value of the positive control.

[0161] The results are shown in Table 1. Figure 13 The results show that the hemolysis rate of the hydrogel is less than 5%, which meets the safety standards of medical biological materials, indicating that the CSAD hydrogel has excellent blood compatibility in the blood environment and does not destroy red blood cells.

[0162] 2. Coagulation experiment

[0163] (1) Clean test tubes were taken, and 100 mg of hydrogel samples of Examples 2-4 were added, and a blank test tube was used as a negative control;

[0164] (2) 37°C water bath for 10 min, add fresh anticoagulant rabbit blood (1 mL) to the above test tube, continue water bath for 10 min;

[0165] (3) After adding calcium chloride solution (100 μL, 0.25 mol / L) to activate the blood coagulation process, immediately start timing, and during the timing process, slowly tilt each group of test tubes (<30°), and when the blood in the test tube begins to coagulate, stop timing immediately, and record the coagulation time of each group (n = 3).

[0166] Good coagulation performance can accelerate blood coagulation and reduce bleeding time, thereby playing a key role in wound repair and surgical operation. The lower the coagulation time, the better the coagulation ability of the material. Figure 14It can be seen that the blood clotting time of the CSAD-12 hydrogel is 354.33 s, which is significantly lower than that of the blank control group of 503.33 s. It shows that the CSAD hydrogel of the application can significantly reduce the blood clotting time.

[0167] 3. Blood clotting index

[0168] The blood clotting index is to evaluate the efficiency of blood clotting on the surface of the material, and reflects the promotion or inhibition of the material to the blood clotting process. The ideal properties of hemostatic materials include rapid absorption of blood, promotion of blood clotting factor aggregation and acceleration of platelet adhesion and activation, thereby shortening the blood clotting time and improving the blood clotting efficiency. The blood clotting time of the hydrogel prepared in Examples 2-4 is determined, and the blood clotting index is evaluated to evaluate the hemostatic performance:

[0169] (1) Add CaCl2 solution (1 mL, 0.25 mol / L) to the anticoagulated rabbit blood (9 mL) to activate the blood;

[0170] (2) Place 50 μL of the activated blood on the surface of the hydrogel, and after 5 min, dissolve the uncoagulated blood with 5 mL of deionized water, and the solution of 50 μL of blood in 5 mL of deionized water is used as a reference (n = 3);

[0171] (3) The OD value at 540 nm is determined by an enzyme marker;

[0172] (4) The blood clotting index (BCI) is calculated by formula 5:

[0173] Blood clotting index (%) = OD sample / OD blank x 100% (Formula 5)

[0174] In the formula, OD sample is the OD value of the sample group; and OD blank is the OD value of the deionized water reference.

[0175] The results are shown in Table 1. Figure 15 The results show that the blood clotting time of all hydrogel groups is lower than that of the control group, and the blood clotting index is also lower than that of the control group. The CSAD-12 hydrogel exhibits the lowest blood clotting index of 79.86%, indicating that it can effectively promote blood clotting.

[0176] Platelet adhesion and activation on the surface of hydrogel is a key step in the hemostatic response, and its adhesion efficiency is regulated by the surface roughness, porous structure and active groups of the material. In the process of hemostasis, moderate red blood cell adhesion helps to form a preliminary thrombus, providing a basis for subsequent platelet aggregation and coagulation factor activation. Porous hydrogels with high specific surface area can adsorb more platelets and induce platelet aggregation and degranulation through interaction with plasma proteins and coagulation factors, further accelerating thrombus formation. The present invention evaluates the interaction of hydrogel with blood by detecting the adhesion of platelets and red blood cells on the surface of hydrogel.

[0177] 4. Red blood cell adhesion

[0178] (1) Add 100 μL of hydrogel sample to a 96-well plate, then add 50 μL of anticoagulated whole blood to the surface of the hydrogel;

[0179] (2) Incubate at 37°C for 10 min to ensure that the red blood cells are in full contact with the hydrogel;

[0180] (3) Gently wash the surface of the sample with PBS solution to remove unattached red blood cells;

[0181] (4) Subsequently, transfer the hydrogel to a centrifuge tube containing 3 mL of deionized water and incubate at 37°C for 30 min to completely lyse the adsorbed red blood cells and release hemoglobin;

[0182] (5) Measure the OD value at 540 nm by a microplate reader, and at the same time, prepare a reference control group: add 50 μL of whole blood directly to 3 mL of deionized water, and measure the OD value after incubation at 37°C for 30 min as a reference (n = 3);

[0183] (6) Calculate the red blood cell adsorption rate by formula 6:

[0184] Red blood cell adsorption rate (%) = OD sample / OD negative × 100% (Formula 6)

[0185] In the formula, OD sample is the red blood cell OD value of the sample group; and OD negative is the OD value of the deionized water reference.

[0186] 5. Platelet adhesion

[0187] (1) Centrifuge the whole blood at 4000 rpm for 10 min to separate the platelets (PRP);

[0188] (2) Add 50 μL of PRP to the surface of 100 μL of hydrogel and incubate at 37°C for 10 min;

[0189] (3) Unstuck platelets were washed with PBS, and then the hydrogel was immersed in 1 mL deionized water and incubated at 37 °C for 1 h to lyse the platelets;

[0190] (4) The OD value at 490 nm was determined by a microplate reader, with 50 μL PRP without treatment added to 1 mL deionized water as a reference (n = 3);

[0191] (5) The platelet adhesion rate was calculated by Formula 7:

[0192] Platelet adhesion rate = OD sample / OD positive x 100% (Formula 7)

[0193] In the formula, OD sample is the platelet OD value of the sample group; and OD positive is the OD value of the deionized water reference.

[0194] The results of red blood cell adhesion and platelet adhesion are shown in Figure 16 . The results show that the CSAD hydrogel prepared in the application can effectively promote the adhesion and aggregation of platelets and red blood cells, enhance the local blood clotting process, and thus accelerate the hemostatic effect.

[0195] Example 5. In vivo hemostatic performance

[0196] 1. Mouse tail amputation hemostasis

[0197] (1) Healthy mice (male, 25-27 g) were adaptively fed for 7 d before the experiment, and then the mice were anesthetized by intraperitoneal injection of urethane (2%, 100 mg / mL);

[0198] (2) A mouse tail amputation model was used, and the mouse tail was cut at 1 / 3 of the tail, and 1 mL of each group of mucous hydrogel was injected into the amputated tail for hemostasis, and the blank control was not treated.

[0199] (3) The bleeding conditions and bleeding amount of each group were recorded (n = 3).

[0200] The results are shown in Figure 17 . The results show that within 1 min after tail amputation, the bleeding amount of each group treated with the hydrogel is lower than that of the control group, indicating that the hydrogel can effectively promote blood clotting and reduce bleeding. Among them, CSAD-12 shows the best hemostatic effect, with a bleeding amount of only 3 mg. At this time, due to its suitable pore structure and surface properties, it can quickly adsorb blood, concentrate blood clotting factors, and enhance platelet adhesion and aggregation, thereby accelerating the blood clotting reaction.

[0201] 2. Mouse liver hemostasis

[0202] (1) Mouse (male, 25-27 g) liver hemorrhage model was used, the mice were anesthetized by intraperitoneal injection of urethane (2%, 100 mg / mL), the abdomen was incised to expose the liver, a 5 mm diameter wound was made on the liver, and the weighed filter paper was placed under the liver;

[0203] (2) 1 mL of hydrogel was injected into the bleeding site, and the blank control was not treated, and then the bleeding condition and amount of each group were recorded (n=3).

[0204] The results are shown in Figure 18 . The results show that the CSAD hydrogel can effectively stop bleeding, and the hydrogel of CSAD-12 (21 mg) has the best hemostatic effect. This is because the appropriate pore size and surface properties of the hydrogel can provide more attachment sites for platelets, promote their aggregation and activation, and accelerate thrombus formation.

[0205] Example 6. Tissue compatibility

[0206] 1. Cell compatibility

[0207] Cell compatibility is an important indicator for evaluating the biocompatibility of biomedical materials, reflecting the interaction between the material and the cell and its influence on cell growth. In the present application, the toxicity of CSAD hydrogel on L929 mouse fibroblasts was evaluated by CCK-8 method.

[0208] (1) Material extract preparation: after sterilization of each group of hydrogels and their samples, 10 mg / mL of each was added to 1640 culture medium containing 10% fetal bovine serum and incubated for 24 h to obtain the extract.

[0209] (2) Cell culture

[0210] After thawing the cells, add 1640 culture medium (containing 10% fetal bovine serum), centrifuge at 1000 rpm for 4 min, discard the supernatant; resuspend the cells with culture medium and transfer to a new culture bottle, supplement 6 mL of culture medium, and incubate in a 37°C, 5% CO2 incubator. The culture medium is slightly yellow, and the culture medium is replaced. When the cells grow to about 85-90%, they are in the logarithmic growth phase, and are subcultured. The old culture medium is pumped out, the cells are washed twice with PBS, and the cell fragments are digested with 1-2 mL of trypsin until the cells are rounded and detached, then 3 mL of complete culture medium is added to terminate digestion; collect the cells at 1000 rpm for 4 min, discard the supernatant, resuspend and count, then divide into new culture bottles, supplement 6 mL of culture medium, and incubate in the incubator.

[0211] (3) CCK-8 treatment

[0212] After cell resuscitation, cells were passaged twice to ensure stable cell growth. L929 cells were seeded into 96-well plates at a density of 200 μL per well, resulting in a cell concentration of 6 × 10⁻⁶ cells / well. 3 / well; incubate in a 5% CO2 cell culture incubator at 37℃ for 24h. After the cells adhere, discard the original culture medium and add the extract of each sample (the extract is filtered through a 0.22μm filter to remove bacteria) to replace the culture medium and incubate for 24h. Add 10μL CCK-8 reagent and 100μL complete culture medium to each well and incubate for 2h. Measure the absorbance (OD value) at 450nm using a microplate reader and calculate the cell viability using Equation 8.

[0213] Cell viability = (OD) s -OD o ) / (OD c -OD o )×100% (Equation 8)

[0214] In the formula, OD s The OD value represents the experimental group sample; OD o The OD value represents the background group sample; OD c This represents the OD value of the control group sample.

[0215] The results are as follows Figure 19 As shown in the figure. The results showed that after 24 h and 48 h of incubation, none of the hydrogels and their components affected the growth of L929 cells, and the cell viability was over 90%. This result indicates that the prepared CSAD hydrogel and its components have no significant toxicity to L929 cells, exhibit good cell compatibility, and can effectively support cell survival and proliferation, meeting the requirements for use as a medical dressing material.

[0216] 2. Staining of live and dead cells

[0217] (1) L929 cells were seeded into 48-well plates at a concentration of 2 × 10⁻⁶ cells / well. 4 / well. After incubation for 24 hours, replace the culture medium with hydrogel extract and incubate for another 24 hours;

[0218] (2) Prepare the reaction buffer, Calcein-AM / propidium iodide (PI), staining reagent according to the manufacturer's instructions. Remove the hydrogel extract and rinse the cells three times with the reaction buffer;

[0219] (3) Add 100 μL of staining reagent and 200 μL of reaction buffer to each well, incubate at 37°C for 15 min, and then rinse the cells 3 times with reaction buffer.

[0220] (4) Live cells and dead cells were observed under an inverted fluorescence microscope at 490±10nm and 545nm, respectively.

[0221] Live cells showed green fluorescence after staining, while dead cells showed red fluorescence. The staining results showed that almost all L929 cells cultured in CSAD hydrogel extract showed green fluorescence, indicating a high cell viability. Figure 20 This further demonstrates that the prepared CSAD hydrogel has good biocompatibility in cell culture.

[0222] 3. Effects of hydrogels on cell migration ability

[0223] (1) L929 cells were seeded into 12-well plates at a concentration of 3 × 10⁻⁶. 6 / well, incubated in a 5% CO2 cell culture incubator at 37°C for 24 hours;

[0224] (2) After the cells adhered to the well, a 20 μL pipette tip was used to create scratches in the well. Then, the cell debris was washed away with PBS and photographed with a microscope.

[0225] (3) Add the extract of each sample separately, and add the control group to the basal culture medium and incubate for 24 h. Continue to incubate in the culture medium containing 1% serum with the added extract for 24 h. After removing the culture medium and extract, wash with PBS and add the basal culture medium to take pictures. Calculate the scratch area using ImageJ.

[0226] (4) Calculate cell migration rate using Equation 9.

[0227] Cell migration rate = (A o -A i ) / A o ×100% (Equation 9)

[0228] In the formula: A o A represents the initial scratch area. i The scratch area after 24 hours of cultivation.

[0229] The cell scratch assay is an in vitro experimental method used to assess cell migration ability and has important biological significance for studying cell behavior during wound healing. Cell migration is a key step in promoting wound healing; therefore, hydrogels used as wound dressings should possess the ability to promote cell migration. The results showed that the CSAD hydrogels prepared in Examples 2–4 all promoted cell migration, and within 24 hours, the cell migration rate of all hydrogel groups exceeded 80%. Figure 21 This indicates that the CSAD hydrogel and its components prepared in this invention can effectively accelerate the cell migration process and have the potential to promote wound healing.

[0230] 4. Effects of CSAD hydrogel on cell oxidative damage repair

[0231] (1) L929 cells were fed at a rate of 6 × 10⁻⁶ 3 / wells were seeded into 96-well plates and incubated in a 5% CO2 cell culture incubator at 37°C for 24 hours;

[0232] (2) Replace with fresh culture medium and add 200 μM H2O2 and incubate for 2 h to induce oxidative damage in cells;

[0233] (3) Add the extract of each sample (the extract was sterilized by filtration through a 0.22 μm filter membrane) to replace the culture medium and incubate for 24 h, then remove the extract;

[0234] (4) Add 10 μL of CCK-8 reagent and 100 μL of complete culture medium to each well and incubate for 2 h. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay reader.

[0235] (5) Calculate cell viability according to Equation 10:

[0236] Cell viability (%) = (OD) s -OD o ) / (OD c -OD o )×100% (Formula 10)

[0237] In the formula, OD s The OD value represents the experimental group sample; OD o The OD value represents the background group sample; OD c This represents the OD value of the control group sample.

[0238] Cellular oxidative damage is caused by the accumulation of reactive oxygen species (ROS), resulting in damage to biomacromolecules, including lipid peroxidation, protein denaturation, and DNA damage, which in turn leads to cell dysfunction and even apoptosis or necrosis. Studying the ability of hydrogels to repair cellular oxidative damage allows us to observe whether they promote cell repair and accelerate tissue regeneration. The results showed that the cell viability of the CSAD hydrogel extracts prepared in Examples 2-4 of this invention exceeded 60%, significantly higher than the 20% of the control group. Figure 22 This indicates that hydrogels have a good repair and protection effect on oxidative damage to cells.

[0239] Example 7. Hydrogel promotes deep wound healing

[0240] The healing of deep wounds involves complex biological processes, including inflammation regulation, angiogenesis, cell migration, and tissue remodeling. Furthermore, the integrity of dressings can be affected by muscle contraction or joint movement at the wound site. Traditional dressings are prone to displacement or detachment, leading to disruption of the wound microenvironment's stability, which in turn results in a persistent inflammatory response and delays the healing process. This embodiment evaluates the impact of CSAD hydrogel on deep wound healing, following these steps:

[0241] 1. Animal Model Construction

[0242] (1) Allow the experimental animals to acclimatize for one week;

[0243] (2) Anesthetize the experimental animals with isoflurane, shave them, use hair removal cream to remove hair from their backs, disinfect the surgical site with povidone-iodine, and establish a deep wound model on the back of rats: 15 mm long and 2 mm deep.

[0244] The experiment was divided into three groups: a control group (no sutures, no hydrogel added), a CASD hydrogel group, and a suture group (the epidermal wound was sutured). The control group did not receive any hydrogel injections or have their wounds sutured. The CASD hydrogel group received CASD-12 hydrogel injections at the wound site but did not have their wounds sutured. The suture group did not receive any hydrogel injections but had their epidermal wounds sutured.

[0245] 2. Performance Testing

[0246] (1) Self-healing performance: 3 days after molding, the CSAD-12 hydrogel (prepared according to the method of Example 3) was physically manufactured to break, and the self-healing of the hydrogel was observed after 1 minute.

[0247] (2) Wound healing: The healing time of the three groups of deep wounds was compared. The size of the wounds was recorded by taking pictures. The wound area was calculated using ImageJ, and the wound healing rate was calculated according to Equation 11.

[0248] Wound healing rate = (Wb - Wo) / Wb × 100% (Equation 11)

[0249] In the formula, Wb: the original wound area; Wo: the wound area after healing at different times.

[0250] Wound healing in the three experimental groups was monitored at 0, 3, 7, 10 and 14 days, and the wound closure rate (percentage of closed wound area) was quantitatively calculated using the imageJ digital image analysis system. Figure 23 Figure A shows the wound healing outcomes of different treatments. Compared to the control group, both the suture group and the CSAD hydrogel group promoted wound healing to varying degrees. At 10 days, the CSAD hydrogel group achieved near-complete epithelialization (wound closure rate of 98.2 ± 1.5%). Quantitative analysis showed that the wound closure rate of the CSAD hydrogel group was higher than that of the other two groups at all time points (P < 0.05), especially during the critical transition from the inflammatory phase to the proliferative phase (3-7 days). Figure 23 (B) When hydrogel dressings are applied to a wound, they can break due to external factors. Broken hydrogels easily lose their ability to adhere to the wound. However, CSAD hydrogel can self-heal within 1 minute of breaking, maintaining wound closure and continuously exerting its effect on the wound. Figure 23 (C in the middle).

[0251] From the above examples, the application provides a viscosity-controllable chitosan-and-giant salamander-skin-secretion hydrogel as well as a preparation method and application thereof.

[0252] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a viscosity-controllable chitosan-giant salamander skin secretion hydrogel, characterized in that, Includes the following steps: (1) Collect the skin secretions of the giant salamander, freeze-dry them, and obtain freeze-dried powder of the skin secretions of the giant salamander; (2) Dissolve the freeze-dried powder of giant salamander skin secretions in acetic acid solution, stir to obtain a mixed solution, centrifuge the mixed solution, take the supernatant, place the supernatant in a dialysis bag for dialysis for 70-75 hours, freeze-dry the solution in the dialysis bag to obtain purified freeze-dried powder of giant salamander skin secretions; (3) The purified freeze-dried powder of giant salamander skin secretions and chitosan were mixed and stirred with ascorbic acid solution to obtain giant salamander skin secretion solution and chitosan solution. (4) Mix the giant salamander skin secretion solution and the chitosan solution at a mass ratio of 6 to 18:1 and stir to obtain the chitosan-giant salamander skin secretion hydrogel with controllable viscosity.

2. The preparation method according to claim 1, characterized in that, The freeze-drying temperature is -50 to -40°C, the vacuum degree is 10 to 20 Pa, and the freeze-drying time is 22 to 26 hours.

3. The preparation method according to claim 1, characterized in that, The acetic acid solution has a molar concentration of 0.4–0.6 M; the mixed solution contains 30–50 mg / mL of the giant salamander skin secretions.

4. The preparation method according to claim 1, characterized in that, The centrifugal force is 4500–5500 g, and the centrifugation time is 8–12 min.

5. The preparation method according to claim 1, characterized in that, The molecular cutoff of the dialysis bag is 280–320 Da.

6. The preparation method according to claim 1, characterized in that, The molar concentration of the ascorbic acid solution is 0.6–0.9 M.

7. The preparation method according to claim 1, characterized in that, The salamander skin secretion solution has a mass concentration of 50-70 mg / mL; the chitosan solution has a mass concentration of 50-70 mg / mL.

8. The preparation method according to claim 1, characterized in that, The degree of polymerization of the chitosan is 100 to 300.

9. A chitosan-giant salamander skin secretion hydrogel with controllable viscosity prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the viscosity-controllable chitosan-giant salamander skin secretion hydrogel of claim 9 in the preparation of dressings that promote skin healing.

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