Divalent or trivalent anionic salt cross-linked cryogel, preparation method therefor, and use thereof

Abstract

The present invention provides a divalent or trivalent anionic salt cross-linked cryogel, a preparation method therefor, and use thereof, and belongs to the technical field of biomedical materials. The cross-linked cryogel is prepared by cross-linking a polysaccharide and / or a protein polymer cryogel scaffold with a divalent or trivalent anionic salt. The cross-linked cryogel has good mechanical properties, morphological memory properties, hemostatic properties, and the effect of promoting wound healing. The cross-linked cryogel, as a wound dressing, is environmentally friendly and economical, and has characteristics enabling large-scale production, fatigue resistance, breathability, anti-adhesion function, rapid shape memory function after extreme compression, injectability, and exudate absorption capacity, providing a new approach and option for wound repair.

Description

A negative di- or trivalent anion salt crosslinked cryogel and a preparation method and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a negative di- or trivalent anion salt crosslinked cryogel and a preparation method and use thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, and is one of the most vulnerable tissues of the human body due to its direct contact with the external environment. After skin damage, its ability to resist bacterial invasion and maintain internal environment stability is weakened. Severe skin defects are usually accompanied by the formation of scar tissue and the loss of skin appendages such as hair, sweat glands and sebaceous glands. Effective management of wound healing has always been an important clinical problem faced by clinicians. There are many factors that affect wound healing, including the patient's physical condition, accurate assessment of wound condition, selection of appropriate wound dressing, etc. Among them, the selection of appropriate wound dressing is an important factor for the successful healing of skin. However, most traditional and inexpensive wound dressings, such as gauze and bandage, only have hemostatic function. Other biological, synthetic or biological-synthetic dressings, such as allogeneic skin, pigskin, functional hydrogel, nanoparticle-based dressing, also have disadvantages such as limited supply, high price, antigenicity, etc. The repair of skin wounds is a complex biological process involving hemostasis, inflammation, proliferation and remodeling. An ideal wound dressing needs to maintain a moist healing environment for the wound, allow gas exchange, act as a microbial barrier, absorb and remove excess exudates, while also being non-toxic, not causing allergies, not adhering to soft tissues and easily removed without causing secondary trauma. Therefore, it is of great significance to develop a low-cost, high-biological safety wound dressing that can not only control wound bleeding, but also promote wound healing and skin appendage regeneration.

[0003] In recent years, cryogel has attracted widespread attention from many scholars, and it is an economical and inexpensive porous material with potential hemostatic and wound healing-promoting effects. Cryogel has highly connected macropores and elastic structure, forms a sponge-like morphology with high mechanical stability, can quickly absorb blood, expand on the wound to form a physical barrier, increase the adhesion of blood cells and platelets, and ultimately achieve the purpose of hemostasis. In addition, cryogel can also maintain a moist environment for the wound and allow cells to migrate into the porous network of cryogel, promoting various life activities of cells and ultimately promoting wound healing.

[0004] Materials for preparing cryogels are numerous, and can be mainly divided into three categories of natural polysaccharides, proteins and synthetic polymers. Polysaccharide and protein components are similar to human extracellular base components, have relatively superior biocompatibility and biomimetic characteristics, and have a certain promoting effect on tissue wound healing. Among them, chitosan is the only known natural cationic polysaccharide, which comes from natural crustaceans, and has been widely concerned due to its hemostatic ability and wound healing performance. Protein polymers such as collagen, silk fibroin, mussel adhesive protein and gelatin are widely used for wound repair and have achieved good therapeutic effect. In recent years, chitosan (CS) and protein-based high molecular weight components have been used for wound repair. These materials can provide a moist healing environment and accelerate wound closure. However, traditional chitosan and / or protein cryogels are often crosslinked with glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) / N-hydroxysuccinimide (NHS), genipin, and other chemical crosslinking agents. The cryogels prepared by these chemical crosslinking agents have low mechanical strength, poor stability and certain biological toxicity. In addition, a small number of studies use sodium hydroxide + ethanol as a crosslinking agent for polysaccharide cryogels, but the introduction of sodium hydroxide may change the acid-base properties of the material, thereby affecting its biological safety.

[0005] Therefore, it is urgent to develop a new crosslinking method to obtain an anti-adhesion cryogel with excellent mechanical properties, biological safety, biological activity, air permeability, rapid hemostasis and exudate management ability, so as to provide a new choice for clinical wound repair. SUMMARY

[0006] The purpose of the present application is to provide a negative bivalent or negative trivalent anion salt crosslinked cryogel and a preparation method and use thereof.

[0007] The present application provides a crosslinked cryogel, which is prepared by freeze-drying of a polymer cryogel scaffold and a negative bivalent or negative trivalent anion salt, and the polymer is a polysaccharide and / or a protein-based high molecular weight.

[0008] Further, the polysaccharide is low molecular weight chitosan, medium molecular weight chitosan, high molecular weight chitosan or carboxymethyl chitosan; and the protein-based high molecular weight is gelatin, collagen, mussel adhesive protein or silk fibroin.

[0009] The average molecular weight of the low molecular weight chitosan is greater than or equal to 10,000 daltons and less than 50,000 daltons, the average molecular weight of the medium molecular weight chitosan is greater than or equal to 50,000 daltons and less than 150,000 daltons, and the average molecular weight of the high molecular weight chitosan is greater than or equal to 150,000 daltons and less than 500,000 daltons.

[0010] Further, the method for preparing the polymer cryogel scaffold comprises the following steps: configuring 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] Further, the solvent in the polysaccharide solution, the protein polymer solution or the mixed solution of polysaccharide and protein polymer is water or an acetic acid aqueous solution.

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

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

[0014] Further, the concentration of polysaccharide in the polysaccharide solution or the mixed solution of polysaccharide and protein polymer is 20 mg / ml; the concentration of protein polymer in the protein polymer solution or the mixed solution of polysaccharide and protein polymer is 1-100 mg / ml; and the freeze-drying condition is: pre-freezing at -20 ℃ for 2 hours, and then continuing to freeze at 20 mbar and -20 ℃ for 24 hours.

[0015] The application also provides a method for preparing the cross-linked cryogel as described above, which comprises the following steps: soaking the polymer cryogel scaffold in a negative bivalent or negative trivalent anion salt solution, taking out, washing, and freeze-drying to obtain the cross-linked cryogel; and the freeze-drying condition is the same as the freeze-drying condition as described above.

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

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

[0018] The application also provides the use of the cross-linked cryogel as described above in the preparation of a wound dressing.

[0019] Further, the wound dressing is a hemostatic, anti-inflammatory, anti-adhesion and / or skin healing-promoting wound dressing.

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

[0021] (1) The present application uses a negative bivalent or negative trivalent anion salt solution to soak the freeze-dried polysaccharide and / or protein-based frozen gel support, so that the negative charge group of the anion is coordinated with the high molecular amino group to obtain a stable ionic cross-linked complex. The frozen gel has good mechanical properties, shape memory properties, hemostatic properties and effects of promoting wound healing. As a wound dressing, the frozen gel has the characteristics of green safety, economy, large-scale preparation, anti-fatigue, air permeability, anti-adhesion, rapid shape memory after extreme compression, injectability and exudate absorption capacity, which provides a new idea and choice for wound repair.

[0022] (2) Compared with the traditional chemically cross-linked frozen gel, the mechanical properties, biodegradability, biocompatibility, in vivo and in vitro hemostatic properties, effects of promoting skin wound healing, promoting vascularization and hair regeneration of the anion cross-linked frozen gel of the present application are obviously improved.

[0023] (3) Compared with the traditional cross-linking method, the cross-linking method of the negative bivalent or negative trivalent anion salt cross-linked frozen gel of the present application does not involve the use of organic solvents, the main ingredient degradation product used is a human metabolite, and the biological safety is higher, without potential carcinogenic and toxic side effects; the method is simple, economical, environmentally friendly and safe, and can be produced on a large scale, and the prepared material has good uniformity.

[0024] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present application.

[0025] The above content of the present application will be further described in detail through specific embodiments. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a general diagram, microstructure and chemical composition analysis diagram of CA-CS and GA-CS frozen gel. (A) CA-CS and CA-CS macroscopic picture; (B, C) material microstructure and void statistical diagram; (D-H) material chemical composition and structure analysis.

[0027] Figure 2 is a graph of the water absorption performance, mechanical performance, shape memory ability and anti-fatigue test of the frozen gel. (A-C) Rapid water absorption shape memory function of CA-CS frozen gel; (D) Compression process graph of CA-CS and GA-CS frozen gel; (E) Stress-strain curve graph of CA-CS and GA-CS frozen gel; (F) and (G) Cycle compression stress-strain curve graph of CA-CS and GA-CS frozen gel.

[0028] Figure 3 is a graph of the results of cell compatibility and the influence of RAW 264.7 phenotype polarization of each group. (A) Live and dead staining results of L929 cells treated with CA-CS and GA-CS frozen gel for 48h; (B) Statistical analysis of CCK-8 results of L929 cells treated with CA-CS and GA-CS frozen gel for 0-7d; (C) Flow cytometry analysis results of RAW264.7 cells after treatment with CA-CS and GA-CS frozen gel.

[0029] Figure 4 is a graph of the results of the in vitro coagulation experiment of the frozen gel. (A-C) In vitro hemostasis results, hemostasis index and in vitro hemolysis statistical analysis of CA-CS and GA-CS frozen gel; (D-H) Hemostasis results of CA-CS and GA-CS frozen gel on mouse liver surface defect and mouse liver hole defect.

[0030] Figure 5 is a graph of the detection results of the frozen gel promoting the healing of acute full-thickness skin defects in mice. (A) Representative pictures of the healing of skin defects in mice treated with different materials at different time points; (B) Continuous change graph of the healing process of the wound in each group for 0-14d; (C) Statistical analysis results of the change of the wound area in each group within 2 weeks; (D) Statistical analysis results of the change of the wound perimeter in each group within 2 weeks; (E) Anti-adhesion results of CA-CS; (F) Representative pictures of H&E and MASSON staining of the skin defects in the back of mice in each treatment group for 7d. DETAILED DESCRIPTION

[0031] The raw materials and equipment used in the present application are known products, which can be obtained by purchasing commercially available products.

[0032] The chitosan used in the embodiments of the present application has an average molecular weight of 400,000 Dalton, which belongs to high molecular weight chitosan.

[0033] Sodium citrate is trisodium citrate.

[0034] Example 1, preparation of a sodium citrate cross-linked chitosan-based frozen gel

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

[0036] First, 1 ml of glacial acetic acid was added to 49 ml of deionized water to obtain a 2% (v / v) acetic acid solution. 1 g of chitosan was added to 50 ml of the 2% (v / v) acetic acid solution, and stirring was performed at 600 r / min in an 80°C oil bath until the chitosan was completely dissolved. After 2 h of constant temperature standing at 80°C, the bubbles were removed to obtain a uniform chitosan solution. Subsequently, the uniform chitosan solution was injected into a 24-well plate using a template method, and after being frozen at -20°C for 24 h, the frozen chitosan was transferred into a freeze dryer, and after being freeze-dried at -20°C for 24 h under 20 mbar, the freeze-dried chitosan was taken out and reserved.

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

[0038] First, a sodium citrate saturated solution (concentration of about 2M) was prepared, and the freeze-dried chitosan was soaked in 10 times the volume of the sodium citrate solution for 30 min. After soaking, the sample was washed with ultrasonic oscillation in 20 times the volume of deionized water for 30 min to remove the uncross-linked sodium citrate. Finally, the washed sample was frozen at -20°C for 24 h, and then transferred into a freeze dryer, and after being freeze-dried at -20°C for 24 h under 20 mbar, the freeze-dried sodium citrate cross-linked chitosan-based frozen gel (CA-CS) was taken out and reserved under vacuum at -20°C.

[0039] Preparation of glutaraldehyde cross-linked chitosan-based frozen gel (GA-CS) of Comparative Example 1

[0040] First, 1 ml of glacial acetic acid was added to 49 ml of deionized water to obtain a 2% (v / v) acetic acid solution. 1 g of chitosan was added to 50 ml of the 2% (v / v) acetic acid solution, and stirring was performed at 600 r / min in an 80°C oil bath until the chitosan was completely dissolved. After 2 h of constant temperature standing at 80°C, the bubbles were removed to obtain a uniform chitosan solution. Subsequently, the uniform chitosan solution was injected into a 24-well plate using a template method, and after being frozen at -20°C for 24 h, the frozen chitosan was transferred into a freeze dryer, and after being freeze-dried at -20°C for 24 h under 20 mbar, the freeze-dried chitosan was taken out and reserved.

[0041] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0042] In the following experiment, the CA-CS and GA-CS frozen gel extraction solution was prepared as follows: the frozen gel CA-CS (20 mg) or GA-CS (20 mg) was immersed in a PBS solution (2 mL), and the extraction solution was taken out after 3 days of incubation at 37°C on a shaking table.

[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 and images were collected by scanning electron microscope (SEM) for analysis of the pore structure. The SEM images of CA-CS and GA-CS cryogels are shown in FIG. IB, and the statistical analysis of the size and number of pores of CA-CS and GA-CS cryogels (FIG. 1C) showed that the pores of CA-CS cryogels were larger, more numerous and more evenly distributed than those of GA-CS.

[0046] 2. Evaluation of material plasticity

[0047] CA-CS and GA-CS solutions were placed in different 3D molds for freezing, lyophilization, and molding treatment, and then removed to obtain cryogels with different 3D shapes. The results are shown in FIG. 1A, and both CA-CS and GA-CS can be prepared into different 3D shapes, but the integrity of GA-CS is easily damaged after being removed from the mold and repeatedly compressed, while the mechanical strength of CA-CS is greater and the mechanical properties are more stable.

[0048] 3. FT-IR, XRD and XPS characterization analysis

[0049] (1) FT-IR characterization analysis

[0050] The FT-IR results are shown in FIG. ID, and chitosan shows characteristic peaks at 1643, 1436, 1403 and 1376 cm -1 , corresponding to C=O stretching vibration (amide I), N-H bending vibration (amide II), C-H and O-H bending vibration, respectively. The asymmetric stretching characteristic peak of C-O-C appears at 1152 cm -1 . The FT-IR results of CA-CS cryogels crosslinked by citrate show that the N-H bending peak at 1536 cm -1 shifts to the left to 1558 cm -1 , indicating that the addition of sodium citrate enhances the ionization of the amino group. In addition, two new peaks appear near 1297 cm -1 and 1258 cm -1 , 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 that the hydrogen bond characteristics (length and direction) are more diverse, and the shift of the peak segment indicates that hydrogen bonds are formed between the carboxylate groups of citrate and the amino groups of the polymer in chitosan.

[0051] (2) XRD characterization analysis

[0052] XRD results are shown in Figure 1E. The intensity of the characteristic diffraction peaks of XRD in CA-CS is lower than that of pure chitosan, indicating that ion cross-linking occurs between citric acid and chitosan, and the addition of citrate destroys the hydrogen bonds between chitosan molecules and the regularity of the molecular chain, resulting in a decrease in the crystallinity of CA-CS. Within a certain range, the adsorption capacity of chitosan increases with the decrease of the crystallinity of chitosan. The adsorption capacity of exudates is one of the important characteristics of wound dressings.

[0053] (3) XPS characterization analysis

[0054] XPS results are shown in Figures 1F-H. Compared with pure chitosan, a new Na 1s signal peak appears in CA-CS. In addition, high-resolution XPS spectral results show that compared with pure chitosan and sodium citrate, the peak intensity of -C=O (530.89 eV) and NH-C=O (287.83 eV) in CA-CS is increased, while the peak intensity of -C-OH (532.40 eV) and -C-N (284.53 eV) is decreased. The results show that a large number of carboxyl groups in sodium citrate are ionically coordinated with the amino groups in the chitosan network.

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

[0056] 4. Material liquid absorption capacity and shape memory ability evaluation

[0057] The results are shown in Figures 2A-C. After the CA-CS cryogel is compressed to the extreme, it is placed in deionized water, and the CA-CS cryogel can quickly recover its original shape.

[0058] Experimental Example 2, mechanical property test

[0059] 1. Test method

[0060] A universal mechanical testing machine was used to detect the compression performance of CA-CS and GA-CS cryogels (cylindrical, diameter 8.5 mm, height 20 mm). The maximum strain of compression-strain experiment was 80%, and the speed was 100 μm / s. The cyclic compression test was performed by adding a drop of water around the sample on the platform, and 80% compression strain was preset before the test started. First, the compression strain was applied to the preset strain, and then released to 0% strain at a constant compression release strain rate of 100 μm / s for 4 cycles.

[0061] 2. Test results

[0062] The test results are shown in FIGS. 2D-G. In the compression stress-strain test, the CA-CS did not break down until the compression strain reached 80%, while the GA-CS broke down when the compression stress reached 76.09 kPa (strain 50.29%). In addition, a dynamic compression stress-strain test was performed with 80% strain for 4 cycles. The CA-CS cryogel did not have significant recovery loss until the fourth compression cycle, and still had good elasticity and maintained the initial shape (FIG. 2F). However, the GA-CS cryogel showed significant recovery loss and broke down in the first compression cycle, and completely broke down in the third cycle, so the experiment could not continue (FIG. 2G).

[0063] Experimental Example 3, Cell Compatibility Test of Cryogel

[0064] 1. Test Method

[0065] The CCK-8 (Cell Counting Kit-8) cell proliferation toxicity kit was used to detect the effect of the CA-CS and GA-CS cryogels on cell proliferation. The Calcein-AM / PI live / dead cell double staining kit was used to detect the effect of the blank control group (Control group), CA-CS cryogel group, and GA-CS cryogel group on cell activity (wherein the inverted fluorescence microscope observed the green fluorescence emitted by the living cells under an excitation wavelength of 490 nm, and observed the red fluorescence emitted by the dead cells under an excitation wavelength of 545 nm).

[0066] 2. Test Results

[0067] The test results are shown in FIGS. 3A-B. The CA-CS had good biocompatibility and essentially no cytotoxicity, while the GA-CS had significant cytotoxicity.

[0068] Experimental Example 4, In Vitro Immune Regulation Test of Cryogel

[0069] 1. Test Method

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

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

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

[0073] (4) After the establishment of RAW 264.7 inflammatory cell model, the old culture solution was removed, 2ml of CA-CS and GA-CS frozen gel extract was added to the CA-CS+LPS group and the GA-CS+LPS group respectively; 20U / ml of IL-4 was added to the IL-4 group; 2ml of CA-CS and GA-CS frozen gel extract was added to the CA-CS group and the GA-CS group respectively, and placed in a 37℃, 5% CO2 incubator for induction and culture for 24h;

[0074] (5) The supernatant was collected at 12h induction and culture and stored at -20℃ for subsequent ELISA detection (detection of RAW 264.7 cell IL-10, IL-1β, TNF-α protein secretion), and the cells in each group were gently scraped off after 24h using a cell scraper, centrifuged at 3000r / min for 5min in a refrigerated centrifuge, and the cell precipitate was collected for flow cytometry analysis (identification of RAW 264.7 cell M1 and M2 phenotype).

[0075] (6) A part of the cells in the blank control group were not stained for subsequent blank test by flow cytometry, and the rest of the cells in each group were divided into two parts and stained with CD80 and CD163 respectively, incubated at room temperature for 30min in the dark, washed with PBS to remove excess antibody, resuspended with 200μl of PBS, and detected for cell phenotype by flow cytometry;

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

[0077] 2. Test results

[0078] The results are shown in Figure 3C. When CA-CS frozen gel extract was used alone for induction, the CD163 expression of RAW264.7 cells was close to that of the IL-4 group and significantly higher than that of the GA-CS group and the blank control group, indicating that CA-CS frozen gel could promote the polarization of RAW264.7 cells to M2 phenotype. After 12h induction and culture with E. coli LPS, the mRNA expression levels of pro-inflammatory factors TNF-α and IL-1β were significantly increased compared with the blank control, indicating that the in vitro inflammatory cell model was successfully constructed. Compared with the LPS group, the CA-CS frozen gel treatment group could significantly inhibit the expression of TNF-α and IL-1β, while significantly up-regulate the expression of anti-inflammatory factor IL-10.

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

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

[0081] 1. Test method

[0082] (1) Isoflurane gas was used to anesthetize the mice. The left thumb, index finger and middle finger of the hand were used to hold the mouse's neck skin, and the little finger and ring finger were used to fix the tail. The skin of the eye to be removed was gently pressed to make the eyeball congested and prominent. Surgical scissors were used to cut the mouse's beard to prevent hemolysis caused by blood remaining in the beard. The eyeball was clamped with tweezers and quickly removed, and the blood was allowed to flow from the eye socket into an anticoagulant tube. When the blood drop rate slowed down, the mouse's heart area was gently pressed to speed up the heart pump to obtain more blood, and then the mouse was executed by cervical dislocation.

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

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

[0085] (4) 10 ml of double distilled water (DD H2O) was added gently, and the uncoagulated blood was evenly dispersed in the DD H2O by gently shaking to avoid dissolving the coagulated blood. Carefully collect 1 ml of each group of sample washing solution in a 1.5 ml EP tube, and take a photo to record the color of each group of solution.

[0086] (5) 200 microliters of each group of sample washing solution was placed in a 96-well plate, and the absorbance of each group of sample at 540 nm was recorded using an enzyme marker. 10 repeats were performed.

[0087] The calculation formula of the in vitro blood clotting index (BCI) is as follows: BCI (%) = Is / Ib x 100%

[0088] In the formula, Is is the absorbance value of the sample group, and Ib is the negative control group (DD H2O).

[0089] 2. Test results

[0090] As shown in FIGS. 4A and B, the BCI of the CA-CS cryogel was significantly lower than that of the gauze group and the gelatin sponge group, and had good in vitro blood clotting promoting ability.

[0091] Experimental Example 6, in vitro hemolysis performance test of the freeze gel

[0092] 1. Test method

[0093] (1) Mix PBS with the GA-CS freeze gel group, the CA-CS freeze gel group, the gauze group (Gauze group), and the gelatin sponge group (Gelatin sponge group) at a ratio of 1:10, respectively. Place the PBS group and the above four groups in a 37°C incubator for 24 hours, and extract the leaching liquid.

[0094] (2) Take the eyeball blood as before.

[0095] (3) Divide into six groups: GA-CS freeze gel, CA-CS freeze gel, gauze (Gauze), gelatin sponge (Gelatin sponge) leaching liquid, deionized water positive control group, and PBS negative control group.

[0096] (4) Centrifuge the whole blood of Kunming mice at 1500 rpm for 10 min to obtain red blood cells. Wash with PBS for 3 times to obtain purified red blood cells. Dilute the purified red blood cells to a concentration of 5% (v / v).

[0097] (5) Add 500 μL of sample leaching liquid and 500 μL of red blood cell diluent to a 2 ml EP tube, and mix gently. Place in a 37°C incubator for 1 h.

[0098] (6) Centrifuge at 1000 rpm for 10 min twice to completely remove red blood cells and cell debris.

[0099] (7) Place 200 microliters of each group of sample supernatant in a 96-well plate, and use an enzyme marker to record the absorbance of each group at 540 nm. Repeat 10 times.

[0100] 2. Test results

[0101] The results are shown in Figure 4C. The optical density value (OD value) of the CA-CS freeze gel group is the lowest, which is significantly lower than that of the GA-CS freeze gel group. The OD value of the CA-CS freeze gel group has no significant difference compared with the gelatin sponge group, the gauze group and the PBS group. It shows that the CA-CS freeze gel has low hemolysis rate.

[0102] Experimental Example 7, in vivo hemostatic performance test of the freeze gel

[0103] 1. Test method

[0104] 1.1 Mouse liver surface defect model

[0105] (1) After KM mice were anesthetized with pentobarbital, they were fixed in a supine position on the operation board;

[0106] (2) The mice were randomly divided into 5 groups, 6 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 to the same size (14 mm in diameter and 4 mm in height) and were weighed in advance;

[0107] (3) The mouse liver was exposed through an abdominal incision, and the serous fluid around the liver was carefully removed with a cotton roll. A paraffin film was placed under the liver, and a filter paper was placed on top of the paraffin film, which was weighed in advance;

[0108] (4) A 1-cm-long incision was made on the surface of the liver with a scalpel, and different materials were used to cover the wound under slight pressure until the bleeding stopped;

[0109] (5) The bleeding time and bleeding volume of each group were recorded.

[0110] 1.2 Mouse liver hole defect model

[0111] (1) The KM mouse anesthesia and fixation method was the same as that of the mouse surface defect model;

[0112] (2) The mouse random grouping method was the same as that of the mouse surface defect model;

[0113] (3) The mouse liver exposure and cleaning, and the placement of paraffin film and filter paper were the same as those of the mouse surface defect model;

[0114] (4) The surgical board was tilted at 30°, a 1-cm-deep defect was made on the liver with a 20G syringe needle, and different materials were used to cover the wound under slight pressure until the bleeding stopped;

[0115] (5) The bleeding time and bleeding volume of each group were recorded.

[0116] 2. Test results

[0117] The results are shown in FIGS. 4D-H. Compared with the gelatin sponge and medical gauze, the CA-CS cryogel had a significantly faster hemostatic speed and a significantly smaller bleeding volume. Compared with the GA-CS cryogel, the CA-CS cryogel had a significantly smaller bleeding volume. This indicates that the CA-CS cryogel has excellent hemostatic performance.

[0118] Experimental Example 8: Detection of cryogel promoting healing of acute full-thickness skin defects in mice

[0119] 1. Test method

[0120] (1) KM mice were inhaled anesthetized with isoflurane. The mouse was observed to breathe steadily and have weak limbs, proving that the anesthesia was stable.

[0121] (2) Mouse back shaver, depilatory cream to remove the remaining fluff, fixed in a prone position on the operating table, the skin iodophor disinfection, through 6mm punch on both sides of the midline of the back of each mouse to establish full-thickness skin defects, wound interval of about 2cm.

[0122] (3) The mice were randomly divided into 4 groups: blank control, 3M dressing, CA-CS cryogel and GA-CS cryogel, 9 in each group. The blank control group was directly covered with transparent dressing, and the other groups were covered with the corresponding materials after covering the wound area, and then fixed and bandaged with transparent dressing. The wound dressing was replaced every two days, and the adhesion of the dressing and soft tissue was recorded when the dressing was replaced.

[0123] (4) At 1, 3, 7 and 14 days after operation, isoflurane anesthetized mice were used to observe and record the healing of rat skin defects; and at 14 days, the hair regeneration in the defect area was recorded by taking pictures.

[0124] 2. Test results

[0125] The results are shown in Figure 5, CA-CS cryogel can accelerate the healing of full-thickness skin defects in mice, in addition to significantly faster wound healing than the blank control group, 3M dressing group and GA-CS cryogel group, it can also promote granulation tissue and collagen production, promote vascularization and hair regeneration.

[0126] The above experiments show that CA-CS cryogel is a porous bioactive material with good plasticity, flexibility, shape memory ability, liquid absorption capacity, mechanical stability and compressibility, which can promote hemostasis in vivo and in vitro, and has excellent immunomodulatory ability, which can effectively promote skin wound healing.

[0127] In summary, the present application provides a cross-linked cryogel and its preparation method and use. The cross-linked cryogel has good mechanical properties, shape memory properties, hemostatic properties and effects of promoting wound healing. As a wound dressing, the cross-linked cryogel has the characteristics of green safety, economy, large-scale preparation, anti-fatigue, air permeability, anti-adhesion, rapid shape memory after extreme compression, injectability and exudate absorption, providing a new idea and choice for wound repair.

Claims

1. A crosslinked cryogel, characterized in that: The cross-linked cryogel is prepared by freeze-drying of a polymer cryogel scaffold and a negative divalent or negative trivalent anion salt, wherein the polymer is a polysaccharide and / or a protein polymer.

2. The crosslinked frozen gel of claim 1, wherein: 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 myeloprotein or silk fibroin.

3. The crosslinked frozen gel according to claim 1 or 2, characterized in that: 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.

4. The crosslinked frozen gel of claim 3, wherein: The concentration of the polysaccharide in the polysaccharide solution or the mixed solution of polysaccharide and protein polymer is 1-50 mg / ml; the concentration of the protein polymer in the protein polymer solution or the mixed solution of polysaccharide and protein polymer is 0.1-200 mg / ml; and the freeze-drying conditions are: pre-freezing at 0 to -196 ℃ for more than 1 hour, and then continuously freezing at 0.01-20 mbar and -10 to -80 ℃ for more than 4 hours.

5. The crosslinked cryogel of claim 4, wherein: The concentration of the polysaccharide in the polysaccharide solution or the mixed solution of polysaccharide and protein polymer is 20 mg / ml; the concentration of the protein polymer in the protein polymer solution or the mixed solution of polysaccharide and protein polymer is 1-100 mg / ml; and the freeze-drying conditions are: pre-freezing at -20 ℃ for 2 hours, and then continuously freezing at 20 mbar and -20 ℃ for 24 hours.

6. A method of preparing the crosslinked cryogel according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: immersing the polymer cryogel scaffold in a negative divalent or negative trivalent anion salt solution, taking out, washing, and freeze-drying to obtain the cross-linked cryogel; and the freeze-drying conditions are the same as those in any one of claims 1-5.

7. The method of claim 6, wherein: The salt solution is an aqueous solution of sodium salt, lithium salt, ammonium salt or potassium salt.

8. The method of claim 6, wherein: The sodium salt includes disodium carbonate, disodium sulfate, disodium hydrogen phosphate, disodium silicate, disodium succinate, disodium alpha-ketoglutarate, disodium malate, disodium fumarate and trisodium citrate.

9. Use of the cross-linked cryogel according to any one of claims 1-5 in the preparation of a wound dressing.

10. Use according to claim 9, characterized in that: The wound dressing is a hemostatic, anti-inflammatory, anti-adhesion and / or skin healing-promoting wound dressing.

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