A negative divalent or trivalent anion salt cross-linked cryogel and its preparation method and use

Abstract

The present invention provides a cross-linked cryogel of a negative divalent or negative trivalent anion salt, and a preparation method and use thereof, belonging to the technical field of biomedical materials. The cross-linked cryogel is prepared by cross-linking a polysaccharide and / or protein polymer cryogel scaffold with a negative divalent or negative trivalent anion salt. The cross-linked cryogel has good mechanical properties, morphological memory properties, hemostatic properties and the effect of promoting wound healing. As a wound dressing, the cross-linked cryogel is green and safe, economical, can be prepared on a large scale, has anti-fatigue properties, and is breathable. It has anti-adhesion function, rapid shape memory function after extreme compression, injectability and exudate adsorption capacity, providing new ideas and options for wound repair.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a negative divalent or negative trivalent anion salt cross-linked cryogel, a preparation method and application thereof. Background Art

[0002] The skin is the largest organ in the human body and, due to its direct contact with the external environment, one of its most vulnerable tissues. Following skin injury, its ability to resist bacterial invasion and maintain a stable internal environment is weakened. Severe skin defects are often accompanied by scar tissue formation and loss of skin appendages such as hair, sweat glands, and sebaceous glands. Effective wound healing management remains a critical clinical challenge for clinicians. Numerous factors influence wound healing, including the patient's physical condition, accurate wound assessment, and the selection of appropriate wound dressings. Among these, selecting the right wound dressing is crucial for successful skin healing. However, most traditional and inexpensive wound dressings, such as gauze and bandages, only provide hemostasis. Other biological, synthetic, or bio-synthetic dressings, such as allogeneic skin, porcine skin, functional hydrogels, and nanoparticle-based dressings, also have drawbacks such as limited supply, high cost, and antigenicity. Skin wound repair is a complex biological process involving hemostasis, inflammation, proliferation, and remodeling. An ideal wound dressing needs to maintain a moist wound healing environment, allow for gas exchange, act as a microbial barrier, and absorb and remove excess exudate. It should also be non-toxic, non-allergenic, non-adhesive to soft tissue, and easily removable without causing secondary trauma. Therefore, developing a low-cost, biosafe wound dressing that can control wound bleeding while promoting wound healing and skin appendage regeneration is of great significance.

[0003] In recent years, cryogels have garnered widespread attention from researchers. These inexpensive, porous materials have the potential to stop bleeding and promote wound healing. Cryogels possess highly interconnected macropores and an elastic structure, forming a sponge-like morphology with high mechanical stability. These materials rapidly absorb blood, expand over wounds to form a physical barrier, and enhance the adhesion of blood cells and platelets, ultimately achieving hemostasis. Furthermore, cryogels maintain a moist environment within wounds and allow cells to migrate into their porous network, promoting various cellular activities and ultimately promoting wound healing.

[0004] Numerous materials are used to prepare cryogels, which can be primarily categorized into three main groups: natural polysaccharides, proteins, and synthetic polymers. Polysaccharide and protein components are similar to the basic extracellular components of the human body, possessing superior biocompatibility and biomimetic properties, and have a certain promoting effect on tissue wound healing. Chitosan, the only known natural cationic polysaccharide, originates from crustaceans and has garnered widespread attention for its hemostatic and wound-healing properties. Protein polymers such as collagen, silk fibroin, mussel mucin, and gelatin are widely used in wound repair and have achieved excellent therapeutic effects. In recent years, cryogels based on chitosan (CS) and protein polymers have been used in wound repair. These materials provide a moist healing environment and accelerate wound closure. However, traditional chitosan and / or protein cryogels are often cross-linked with glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) / N-hydroxysuccinimide (NHS), or genipin. Cryogels prepared with these chemical cross-linkers exhibit low mechanical strength, poor stability, and certain biotoxicity. A small number of studies have used sodium hydroxide and ethanol as cross-linkers for polysaccharide cryogels, but the introduction of sodium hydroxide may alter the material's acidity and alkalinity, thereby affecting its biosafety.

[0005] Therefore, there is an urgent need to develop a new cross-linking method to obtain anti-adhesion cryogels with excellent mechanical properties, biosafety, bioactivity, breathability, rapid hemostasis, and exudate management capabilities, providing new options for clinical wound repair. Summary of the Invention

[0006] The present invention aims to provide a negative divalent or negative trivalent anion salt cross-linked cryogel and a preparation method and application thereof.

[0007] The present invention provides a cross-linked cryogel, which is a product prepared by freeze-drying a polymer cryogel scaffold and a negative divalent or trivalent anion salt as raw materials, wherein the polymer is a polysaccharide and / or protein macromolecule.

[0008] Furthermore, the polysaccharide is low molecular weight chitosan, medium molecular weight chitosan, high molecular weight chitosan or carboxymethyl chitosan; and the protein polymer is gelatin, collagen, mussel mucin or silk fibroin.

[0009] The low molecular weight chitosan of the present invention has an average molecular weight greater than or equal to 10,000 Daltons and less than 50,000 Daltons, the medium molecular weight chitosan has an average molecular weight greater than or equal to 50,000 Daltons and less than 150,000 Daltons, and the high molecular weight chitosan has an average molecular weight greater than or equal to 150,000 Daltons and less than 500,000 Daltons.

[0010] Furthermore, the preparation method of the polymer cryogel scaffold comprises the following steps: preparing a polysaccharide solution, a protein polymer solution or a mixed solution of polysaccharide and protein polymer, and freeze-drying to obtain a polysaccharide cryogel scaffold, a protein polymer cryogel scaffold or a polysaccharide / protein composite cryogel scaffold.

[0011] Furthermore, in the polysaccharide solution, protein polymer solution or mixed solution of polysaccharide and protein polymer, the solvent is water or acetic acid aqueous solution.

[0012] Furthermore, the acetic acid aqueous solution is a 2% (v / v) acetic acid aqueous solution.

[0013] Furthermore, in the polysaccharide solution and the mixed solution of polysaccharide and protein polymer, the concentration of polysaccharide is 1-50 mg / ml; in the protein polymer solution and the mixed solution of polysaccharide and protein polymer, the concentration of protein polymer is 0.1-200 mg / ml; and the freeze-drying conditions are: first pre-freezing at 0 to -196°C for more than 1 hour, and then continuing freezing at 0.01-20 mbar and -10 to -80°C for more than 4 hours.

[0014] Furthermore, in the polysaccharide solution and the mixed solution of polysaccharide and protein polymer, the concentration of polysaccharide is 20 mg / ml; in the protein polymer solution and the mixed solution of polysaccharide and protein polymer, the concentration of protein polymer is 1-100 mg / ml; the freeze-drying conditions are: pre-freezing at -20°C for 2 hours, and then continuing freezing at 20 mbar and -20°C for 24 hours.

[0015] The present invention also provides a method for preparing the aforementioned cross-linked cryogel, which comprises the following steps: soaking the polymer cryogel scaffold in a negative divalent or negative trivalent anion salt solution, taking it out, washing it, and freeze-drying it to obtain the cross-linked cryogel; the freeze-drying conditions are the same as the aforementioned freeze-drying conditions.

[0016] Furthermore, the salt solution is an aqueous solution of sodium salt, lithium salt, ammonium salt or potassium.

[0017] Furthermore, the sodium salt includes disodium carbonate, disodium sulfate, disodium hydrogen phosphate, disodium silicate, disodium succinate, disodium α-ketoglutarate, disodium malate, disodium fumarate, and trisodium citrate.

[0018] The present invention also provides use of the cross-linked cryogel in preparing wound dressings.

[0019] Furthermore, the wound dressing is a wound dressing that stops bleeding, resists inflammation, prevents wound adhesion and / or promotes skin healing.

[0020] The present invention has the following advantages and beneficial effects:

[0021] (1) The present invention uses a negative divalent or trivalent anion salt solution to soak freeze-dried polysaccharide and (or) protein cryogel scaffold, so that the negatively charged groups of the anions coordinate with the polymer amino groups to obtain a stable ion cross-linked complex. The cryogel has good mechanical properties, shape memory properties, hemostatic properties and the effect of promoting wound healing. As a wound dressing, the cryogel is green and safe, economical, can be prepared on a large scale, has anti-fatigue properties, breathability, anti-adhesion function, rapid shape memory function after extreme compression, injectability and exudate adsorption capacity, providing new ideas and options for wound repair.

[0022] (2) Compared with traditional chemically cross-linked cryogels, the anionic cross-linked cryogels of the present invention have significantly improved mechanical properties, biodegradability, biocompatibility, in vitro and in vivo hemostatic properties, and the effects of promoting skin wound healing, vascularization, and hair regeneration.

[0023] (3) Compared with traditional cross-linking methods, the cross-linking method of the present invention for cross-linking cryogels with negative divalent or trivalent anionic salts does not involve the use of organic solvents. The degradation products of the main components used are human metabolites, which have high biosafety and no potential carcinogenicity or toxic side effects. The method is relatively simple, economical, environmentally friendly, and safe, can be mass-produced, and the prepared materials have good uniformity.

[0024] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0025] The following further details the above contents of the present invention through specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following embodiments. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Figures show the macroscopic image, microstructure, and chemical composition analysis of CA-CS and GA-CS cryogels. (A) Macroscopic image of CA-CS and GA-CS; (B) Scanning electron microscopy (SEM) images of CA-CS and GA-CS; (C) Average pore size and pore number statistics of CA-CS and GA-CS; (D) Fourier transform infrared spectroscopy (FT-IR) transmittance comparison of CA-CS and chitosan; (E) X-ray diffraction (XRD) characteristic diffraction peak intensity comparison of CA-CS and chitosan; (F) X-ray photoelectron spectroscopy (XPS) full spectra of CA-CS, chitosan, and citric acid; (G) XPS carbon elemental analysis of CA-CS, chitosan, and citric acid; (H) XPS oxygen elemental analysis of CA-CS, chitosan, and citric acid.

[0027] Figure 2 Figures showing the plasticity, liquid absorption capacity, shape memory, mechanical properties, and mechanical stability of CA-CS cryogels. (A) Schematic diagram showing the instantaneous return of CA-CS to its original state after absorbing liquid, forming regular 3D shapes such as circles, hearts, and hexagons. (B) Schematic diagram showing the return of CA-CS to its original state after load removal from its original state after load removal from its original state. (C) Schematic diagram showing the transformation of CA-CS from its initial state to its bent state, compressed state, and complete recovery to its original shape after load removal. (D) Schematic diagram showing the compression process of CA-CS and GA-CS cryogels using a universal testing machine. (E) Schematic diagram showing the stress-strain curves of CA-CS and GA-CS cryogels. (F) Schematic diagram showing the cyclic compression stress-strain curve of CA-CS cryogels. (G) Schematic diagram showing the cyclic compression stress-strain curve of GA-CS cryogels.

[0028] Figure 3 Figures show the effects of CA-CS and GA-CS cryogels on cytocompatibility and phenotypic polarization of RAW 264.7 cells. (A) Live / dead staining of L929 cells treated with CA-CS and GA-CS cryogels for 48 hours; (B) CCK-8 analysis of L929 cells treated with CA-CS and GA-CS cryogels for 0-7 days; (C) Flow cytometry analysis of RAW 264.7 cells treated with CA-CS and GA-CS cryogels; (D) mRNA expression levels of the pro-inflammatory cytokines TNF-α and IL-1β, and the anti-inflammatory cytokine IL-10, after CA-CS cryogels treatment.

[0029] Figure 4Results of cryogel in vitro coagulation experiments. (A) Visualization of the in vitro hemostatic effect of different materials within EP tubes; (B) Graph showing hemostasis time after treatment with different materials; (C) Statistical analysis of hemolysis after treatment with different materials; (D) Graph showing hemostasis results after treatment with different materials in surface and perforated defects in mouse livers; (E) Statistical analysis of hemostasis time after treatment with different materials in surface defects in mouse livers; (F) Statistical analysis of bleeding volume after treatment with different materials in surface defects in mouse livers; (G) Statistical analysis of hemostasis time after treatment with different materials in perforated defects in mouse livers; (H) Statistical analysis of bleeding volume after treatment with different materials in perforated defects in mouse livers.

[0030] Figure 5 Results of CA-CS and GA-CS cryogels promoting the healing of acute full-thickness skin defects in mice. (A) Graph showing the healing of skin defects in mice treated with different materials at different time points; (B) Graph showing the continuous changes in wound healing in each group from day 0 to day 14; (C) Statistical analysis of changes in wound area over 14 days in each group; (D) Statistical analysis of changes in wound perimeter over 14 days in each group; (E) Graph showing the results of CA-CS and GA-CS cryogels for preventing adhesion before and after dressing changes; (F) H&E and MASSON staining of skin defects on the back of mice in each treatment group after 7 days. DETAILED DESCRIPTION

[0031] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0032] The chitosan used in the embodiment of the present invention has an average molecular weight of 400,000 Daltons, which is a high molecular weight chitosan.

[0033] Sodium citrate is trisodium citrate.

[0034] Example 1: Preparation of a sodium citrate cross-linked chitosan-based cryogel

[0035] (1) Preparation of freeze-dried chitosan

[0036] First, add 1 ml of glacial acetic acid to 49 ml of deionized water to obtain a 2% (v / v) acetic acid solution. Then, add 1 g of chitosan to 50 ml of this 2% (v / v) acetic acid solution. Stir at 600 rpm in an 80°C oil bath until the chitosan is completely dissolved. The mixture is then allowed to stand at 80°C for 2 hours to eliminate all bubbles, resulting in a uniform chitosan solution. The homogenized chitosan solution is then poured into a 24-well plate using a template method. The plate is frozen at -20°C for 24 hours, then transferred to a freeze dryer and freeze-dried at -20°C at 20 mbar for 24 hours before being removed for later use.

[0037] (2) Preparation of sodium citrate cross-linked chitosan-based cryogel

[0038] First, a saturated sodium citrate solution (approximately 2M) was prepared. Freeze-dried chitosan was then immersed in 10 volumes of this solution for 30 minutes. The sample was then ultrasonically cleaned in 20 volumes of deionized water for 30 minutes to remove any uncross-linked sodium citrate. Finally, the cleaned sample was frozen at -20°C for 24 hours, then transferred to a freeze dryer and freeze-dried at -20°C for 24 hours under 20 mbar. This yielded a sodium citrate-cross-linked chitosan cryogel (CA-CS), which was then stored under vacuum at -20°C.

[0039] Comparative Example 1: Preparation of a glutaraldehyde-crosslinked chitosan-based cryogel (GA-CS)

[0040] First, add 1 ml of glacial acetic acid to 49 ml of deionized water to obtain a 2% (v / v) acetic acid solution. Then, add 1 g of CS to 50 ml of the 2% (v / v) acetic acid solution. Heat in a water bath at 65°C and stir until the powder is completely dissolved, resulting in a 2% (w / v) CS solution. Subsequently, add 250 µl of a 25% GA solution to 25 ml of the 2% CS solution and stir rapidly until the two are thoroughly mixed, resulting in a 2% GA-CS cross-linked solution. Finally, pour 25 ml of the GA-CS cross-linked solution into a 24-well plate, freeze at -20°C for 24 hours, and then freeze-dry in a freeze dryer for 24 hours before removing the GA-CS cryogel.

[0041] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0042] The CA-CS and GA-CS cryogel extracts used in the following experiments were prepared as follows: cryogel CA-CS (20 mg) or GA-CS (20 mg) was immersed in PBS solution (2 mL), incubated on a shaker at 37°C for 3 days, and then the extract was removed.

[0043] Experimental Example 1: Structural Characterization

[0044] 1. Morphological Characterization

[0045] The surface of CA-CS and GA-CS cryogels was sprayed with gold, and the surface morphology was observed using a scanning electron microscope (SEM) and images were collected to analyze their pore structure. Figure 1 B shows the statistical analysis of the size and number of pores in the combined CA-CS and GA-CS cryogels ( Figure 1C) It was found that the pores of CA-CS cryogel were larger, more numerous, and more evenly distributed than those of GA-CS.

[0046] 2. Material plasticity evaluation

[0047] CA-CS solution and GA-CS solution were placed in different 3D molds for freezing, freeze-drying, and molding, and then taken out to obtain frozen gels with different 3D shapes. Figure 1 As shown in Figure A, both CA-CS and GA-CS can be prepared into different 3D shapes, but the integrity of GA-CS is easily destroyed after being removed from the mold and repeatedly compressed. In comparison, CA-CS has greater mechanical strength and more stable mechanical properties.

[0048] 3. FT-IR, XRD, and XPS Characterization Analysis

[0049] (1) FT-IR characterization analysis

[0050] FT-IR results are as follows Figure 1 As shown in D, chitosan has the peaks at 1643, 1436, 1403 and 1376 cm −1 Characteristic peaks are shown at 1152 cm, corresponding to C=O stretching vibration (amide I), NH bending vibration (amide II), CH and OH bending vibration. The asymmetric stretching characteristic peak of COC appears at 1152 cm −1 The FT-IR results of the citrate cross-linked CA-CS cryogel showed that the −1 The NH bending peak at 1558 cm shifts to the left −1 , indicating that the addition of sodium citrate enhanced the ionization of amino groups. In addition, at 1297 cm −1 and 1258cm −1 Two new peaks appeared nearby, which can be attributed to the formation of new chemical bonds between sodium citrate and chitosan. In summary, the broadening of the peak segment indicates a more diverse range of hydrogen bond characteristics (length and direction), while the shift of the peak segment indicates the formation of hydrogen bonds between the carboxylic acid groups of citrate and the amino groups of the polymer in chitosan.

[0051] (2) XRD characterization analysis

[0052] XRD results are as follows Figure 1 As shown in Figure E, the XRD characteristic diffraction peak intensity of CA-CS is lower than that of pure chitosan, indicating that citric acid and chitosan undergo ionic crosslinking. Furthermore, the addition of citrate disrupts the hydrogen bonds between chitosan molecules and the regularity of the molecular chains, resulting in a decrease in the crystallinity of CA-CS. Within a certain range, as the crystallinity of chitosan decreases, its adsorption capacity increases. Exudate adsorption is a key characteristic of wound dressings.

[0053] (3) XPS characterization analysis

[0054] XPS results are as follows Figure 1 As shown in Figures FH, a new Na 1s signal peak appears in CA-CS compared to pure chitosan. Furthermore, high-resolution XPS spectroscopy reveals increased peak intensities for -C=O (530.89 eV) and NH-C=O (287.83 eV), while decreased peak intensities for -C-OH (532.40 eV) and -CN (284.53 eV) are observed in CA-CS compared to pure chitosan and sodium citrate. These results indicate that a large number of carboxyl groups in sodium citrate undergo ionic coordination with amino groups in the chitosan network.

[0055] In summary, FT-IR, XRD and XPS experiments proved that the carboxyl groups of sodium citrate have been successfully integrated into the polymer network of chitosan; scanning electron microscopy, plasticity, and material liquid absorption and shape memory evaluations proved that CA-CS has a three-dimensional porous and uniform structure with good plasticity, liquid absorption capacity and shape memory.

[0056] 4. Evaluation of the material's liquid absorption capacity and shape memory ability

[0057] The results are as follows Figure 2 As shown in Figures AC, after CA-CS cryogel is compressed to the extreme and then placed in deionized water, it can quickly restore its original shape.

[0058] Experimental Example 2: Mechanical Properties Test

[0059] 1. Test Method

[0060] The compressive properties of CA-CS and GA-CS cryogels (cylindrical, 8.5 mm diameter, 20 mm height) were tested using a universal mechanical testing machine. Compression-strain experiments were performed at a maximum strain of 80% and a velocity of 100 µm / s. Cyclic compression testing was performed by placing a drop of water around the sample on a platform and setting a preset compressive strain of 80% before the test began. Compressive strain was first applied to the preset strain and then released at a constant compression-release rate of 100 µm / s to 0% strain for four cycles.

[0061] 2. Test Results

[0062] The test results are as follows Figure 2As shown in DG, in the compressive stress-strain test, CA-CS did not suffer structural damage even when the compressive strain reached 80%, while GA-CS broke when the compressive stress reached 76.09 kPa (strain 50.29%). In addition, a dynamic compressive stress-strain test was performed with 80% strain for 4 cycles. The CA-CS cryogel showed no obvious recovery loss until the fourth compression cycle, and still had good elasticity and maintained its initial shape ( Figure 2 F). However, the GA-CS cryogel showed significant recovery loss and rupture in the first compression cycle. In the third cycle, the GA-CS cryogel completely ruptured and could not be used for further experiments ( Figure 2 G).

[0063] Experimental Example 3: Cytocompatibility test of cryogel

[0064] 1. Test Method

[0065] The effects of CA-CS and GA-CS cryogels on cell proliferation were assessed using the CCK-8 (Cell Counting Kit-8) cytotoxicity assay. The effects of the blank control group (control group), CA-CS cryogel group, and GA-CS cryogel group on cell viability were assessed using a Calcein-AM / PI live / dead cell double staining kit. Live cells emitted green fluorescence, while dead cells emitted red fluorescence, were observed under an inverted fluorescence microscope at an excitation wavelength of 490 nm, and 545 nm, respectively.

[0066] 2. Test Results

[0067] The test results are as follows Figure 3 As shown in Figure AB, CA-CS has good biocompatibility and is basically non-cytotoxic, while GA-CS has obvious cytotoxicity.

[0068] Experimental Example 4: In vitro immunomodulation assay of cryogel

[0069] 1. Test Method

[0070] (1) RAW264.7 cells were seeded into 6-well plates at a density of 1 × 105 cells per well. 2 ml of DMEM complete medium was added to each well and cultured in a 37°C, 5% CO2 incubator overnight until the cells adhered.

[0071] (2) Each well in the 6-well plate was grouped and numbered into blank control group (Control group), LPS group, IL-4 group, CA-CS + LPS group, GA-CS + LPS group, CA-CS group, and GA-CS group;

[0072] (3) Establishment of RAW 264.7 inflammatory cell model: 20 μl of 10 μg / ml E. coli LPS solution was added to the LPS group, CA-CS+LPS group, and GA-CS+LPS group to make the final concentration of E. coli LPS 100 ng / ml. After induction for 12 h, the RAW 264.7 inflammatory cell model was established.

[0073] (4) After the RAW 264.7 inflammatory cell model was established, the old culture medium was removed, and 2 ml of CA-CS and GA-CS cryogel extracts were added to the CA-CS+LPS group and the GA-CS+LPS group, respectively; IL-4 at a concentration of 20 U / ml was added to the IL-4 group; and 2 ml of CA-CS and GA-CS cryogel extracts were added to the CA-CS group and the GA-CS group, respectively. The cells were placed in an incubator at 37°C and 5% CO2 for 24 h of induction culture.

[0074] (5) After 12 hours of induction culture, the supernatant was collected and stored at -20°C for subsequent ELISA detection (to detect the secretion of IL-10, IL-1β, and TNF-α proteins in RAW264.7 cells). After 24 hours, the cells in each group were gently scraped off using a cell scraper and centrifuged at 3000 r / min for 5 minutes in a refrigerated centrifuge. The cell pellets were collected for flow cytometric analysis (to identify the M1 and M2 phenotypes of RAW 264.7 cells).

[0075] (6) A portion of cells in the blank control group was left unstained for blank testing in subsequent flow cytometry. The remaining cells in each group were divided into two parts and stained for CD80 and CD163, respectively. The cells were incubated in the dark at room temperature for 30 minutes, washed with PBS to remove excess antibodies, and fully resuspended in 200 μL PBS. The cells were then run on the flow cytometer to detect cell phenotypes.

[0076] (7) Use FlowJo software for data analysis.

[0077] 2. Test Results

[0078] The results are as follows Figure 3As shown in Figure C, when treated with CA-CS cryogel extract alone, CD163 expression in RAW264.7 cells was similar to that in the IL-4 group and significantly higher than in the GA-CS group and the blank control group, suggesting that CA-CS cryogel promotes polarization of RAW264.7 cells toward an M2 phenotype. After 12 hours of induction with E. coli LPS, mRNA expression levels of the proinflammatory cytokines TNF-α and IL-1β were significantly increased compared to the blank control, indicating the successful establishment of an in vitro inflammatory cell model. Compared with the LPS group, treatment with CA-CS cryogel significantly inhibited the expression of TNF-α and IL-1β, while significantly upregulating the expression of the anti-inflammatory cytokine IL-10.

[0079] The above experimental results show that the CA-CS cryogel of the present invention has excellent anti-inflammatory effect.

[0080] Experimental Example 5: In vitro hemostatic performance test of cryogel

[0081] 1. Test Method

[0082] (1) Anesthetize the mouse with isoflurane gas. Grab the mouse's neck and scalp with the thumb, index finger, and middle finger of the left hand. Fix the tail with the little finger and ring finger. Gently press the skin around the eye to be removed so that the eyeball becomes congested and protrudes. Use surgical scissors to cut the mouse's whiskers to prevent blood from accumulating in the whiskers and causing hemolysis. Use tweezers to grasp the eyeball and remove it quickly, allowing the blood to flow from the eye socket into the anticoagulant tube. When the blood dripping in slows down, gently press the mouse's heart to speed up the heart's pumping speed to obtain more blood. Then, kill the mouse by cervical dislocation.

[0083] (2) The mice were divided into five groups: GA-CS cryogel group, CA-CS cryogel group, gauze group, gelatin sponge group and blank control group. The materials in each group were prepared into equal volumes (14 mm in diameter and 4 mm in height) and preheated in a 37°C incubator for 30 min.

[0084] (3) 100 μl of mouse recalcified (0.2 mM calcium chloride) whole blood solution was added dropwise to each group of materials and incubated at 37°C for 10 min.

[0085] (4) Gently add 10 ml of double-distilled water (DD H2O) and shake gently to evenly disperse the uncoagulated blood in the DDH2O to avoid dissolving the coagulated blood. Carefully collect 1 ml of the washing solution from each group of samples in a 1.5 ml EP tube and take a photo to record the color of each group of solutions.

[0086] (5) Place 200 μl of each sample wash solution in a 96-well plate and use a microplate reader to record the absorbance of each sample at 540 nm. Repeat 10 times.

[0087] The calculation formula of in vitro blood coagulation index (BCI) is as follows:

[0088] BCI (%) = Is / Ib × 100%

[0089] In the formula, Is is the absorbance value of the sample group, and Ib is the negative control group (DD H2O).

[0090] 2. Test Results

[0091] like Figure 4 As shown in A and B, the BCI of CA-CS cryogel was significantly lower than that of the gauze group and gelatin sponge group, and it had a good ability to promote coagulation in vitro.

[0092] Experimental Example 6: In vitro hemolytic performance test of cryogel

[0093] 1. Test Method

[0094] (1) PBS was mixed with the GA-CS cryogel group, CA-CS cryogel group, gauze group, and gelatin sponge group at a ratio of 1:10. The PBS group and the above four groups were placed in an incubator at 37°C for 24 hours, and the extract was extracted.

[0095] (2) The procedure for collecting blood from the eyeball is the same as before.

[0096] (3) Divided into six groups: GA-CS cryogel, CA-CS cryogel, gauze, gelatin sponge extract, deionized water positive control group and PBS negative control group.

[0097] (4) Centrifuge whole blood from Kunming mice at 1500 rpm for 10 min to obtain red blood cells. Wash three times with PBS to obtain purified red blood cells. Dilute the purified red blood cells to a concentration of 5% (v / v).

[0098] (5) Add 500µL of sample extract and 500µL of red blood cell diluent to a 2ml EP tube and gently invert to mix. Place in a 37℃ incubator for 1 hour.

[0099] (6) Centrifuge twice at 1000 rpm for 10 min each time to fully remove red blood cells and cell debris.

[0100] (7) Place 200 μl of the supernatant from each sample into a 96-well plate and use a microplate reader to record the absorbance of each group at 540 nm. Repeat 10 times.

[0101] 2. Test Results

[0102] The results are as follows Figure 4 As shown in Figure C, the optical density (OD) of the CA-CS cryogel group was the lowest, significantly lower than that of the GA-CS cryogel group. The OD values of the CA-CS cryogel group were not significantly different from those of the gelatin sponge, gauze, and PBS groups, indicating that the CA-CS cryogel has a low hemolysis rate.

[0103] Experimental Example 7: In vivo hemostatic performance testing of cryogel

[0104] 1. Test Method

[0105] 1.1 Mouse liver surface defect model

[0106] (1) KM mice were anesthetized with pentobarbital and fixed on the operating board in the supine position;

[0107] (2) Mice were randomly divided into five groups, with six mice in each group: blank control group (Control group), gauze group (Gauze group), gelatin sponge group (Gelatin sponge group), GA-CS cryogel group, and CA-CS cryogel group. The materials in each group were prepared with the same size (14 mm in diameter and 4 mm in height) and weighed in advance;

[0108] (3) Expose the mouse liver through an abdominal incision and carefully remove the serous fluid surrounding the liver with a cotton roll. Place a paraffin film under the liver and a pre-weighed filter paper on top.

[0109] (4) Use a scalpel to make a 1 cm long incision on the surface of the liver, and use different materials to cover the wound under slight pressure until the bleeding stops;

[0110] (5) Record the bleeding time and amount of bleeding in each group.

[0111] 1.2 Mouse liver perforation defect model

[0112] (1) The anesthesia and fixation methods for KM mice are the same as those for the mouse surface defect model;

[0113] (2) Mice were randomly grouped in the same manner as the mouse surface defect model;

[0114] (3) Mouse liver exposure, cleaning, paraffin film and filter paper placement are the same as those in the mouse surface defect model;

[0115] (4) Tilt the surgical board 30° and create a 1 cm deep defect in the liver using a 20 G syringe needle. Cover the wound with different materials under slight pressure until the bleeding stops.

[0116] (5) Record the bleeding time and amount of each group

[0117] 2. Test Results

[0118] The results are as follows Figure 4 As shown in DH, compared with gelatin sponge and medical gauze, CA-CS cryogel stopped bleeding significantly faster and caused significantly less bleeding; compared with GA-CS cryogel, CA-CS cryogel also caused significantly less bleeding, indicating that CA-CS cryogel has excellent hemostatic properties.

[0119] Experimental Example 8: Detection of the effect of cryogel on the healing of acute full-thickness skin defects in mice

[0120] 1. Test Method

[0121] (1) KM mice were anesthetized with isoflurane. The mice were observed to have stable breathing and weak limbs, indicating that anesthesia was secure.

[0122] (2) The mouse's back was shaved with a shaver and the remaining hair was removed with depilatory cream. The mouse was placed in a prone position on the operating table and the back skin was disinfected with iodine. A full-thickness skin defect was created on both sides of the midline of the back of each mouse using a 6mm punch, with the wounds spaced approximately 2 cm apart.

[0123] (3) Mice were randomly divided into four groups: blank control, 3M dressing, CA-CS cryogel, and GA-CS cryogel, with 9 mice in each group. The blank control group was directly covered with a transparent dressing, while the other groups covered the wound area with the corresponding materials and then fixed with a transparent dressing. The wound dressing was changed every two days, and the adhesion between the dressing and the soft tissue was recorded during the dressing change.

[0124] (4) Mice were anesthetized with isoflurane ventilation on days 1, 3, 7, and 14 after surgery, and the healing of skin defects in the rats was observed and recorded; photos were taken on day 14 to record the hair regeneration in the defect area.

[0125] 2. Test Results

[0126] The results are as follows Figure 5 As shown in the results, CA-CS cryogel can accelerate the healing of full-thickness skin defects in mice. In addition to the wound healing speed being significantly faster than that of the blank control group, 3M dressing group and GA-CS cryogel group, it can also promote the production of granulation tissue and collagen, and promote vascularization and hair regeneration.

[0127] The above experiments show that CA-CS cryogel is a porous bioactive material with good plasticity, flexibility, shape memory, liquid absorption capacity, mechanical stability and compressibility. It can promote hemostasis in vivo and in vitro, and has excellent immunomodulatory ability, which can effectively promote skin wound healing.

[0128] In summary, the present invention provides a cross-linked cryogel, its preparation method, and use. The cross-linked cryogel exhibits excellent mechanical properties, shape memory properties, hemostatic properties, and the ability to promote wound healing. As a wound dressing, the cross-linked cryogel is environmentally friendly, safe, economical, scalable, fatigue-resistant, breathable, and possesses anti-adhesion properties, rapid shape memory after extreme compression, injectability, and exudate adsorption capacity, providing new ideas and options for wound repair.

Claims

1. Use of a cross-linked cryogel with stable and excellent mechanical properties in the preparation of a wound dressing for hemostasis, anti-inflammation, wound adhesion prevention, and skin healing promotion, characterized in that: The cross-linked cryogel is a product prepared by freeze-drying a polysaccharide cryogel scaffold and trisodium citrate as raw materials. The polysaccharide is high molecular weight chitosan; The preparation method of the polysaccharide cryogel scaffold comprises the following steps: preparing a polysaccharide solution, freeze-drying, and obtaining a polysaccharide cryogel scaffold; the polysaccharide concentration in the polysaccharide solution is 20 mg / mL; The preparation method of the cross-linked cryogel comprises the following steps: soaking the polysaccharide cryogel scaffold in a trisodium citrate solution, taking it out, washing it, and freeze-drying it to obtain the cross-linked cryogel.

2. The use according to claim 1, characterized in that: The freeze-drying conditions are: pre-freezing at 0 to -196°C for more than 1 hour, and then continuing to freeze at 0.01-20 mbar and -10 to -80°C for more than 4 hours.

3. The use according to claim 2, characterized in that: The freeze-drying conditions are: pre-freezing at -20°C for 2 hours, and then continuing to freeze at 20 mbar and -20°C for 24 hours.

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