Sponge patch as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a sponge patch as well as a preparation method and application thereof. The platelet vesicles are combined with absorbable gelatin, collagen or collagen peptide sponge, and the formation of keratin and the activation of inflammatory cells are promoted by virtue of a thrombin cascade effect after platelet activation and a neutrophil anti-inflammatory axis effect, so that conditions are provided for cell proliferation and migration, fibroblast proliferation and matrix deposition processes in the middle and later periods of healing; according to the present invention, with the application of the compound, the intercellular messenger effect is performed, the intercellular regulation is amplified, the functional proteins (CD47, CD55 and GPVI) on the platelets are retained, and the blood coagulation cascade reaction and the endothelial tissue repair are promoted; besides, the absorbable gelatin sponge is of a compact porous structure, is rich in a large amount of collagen, can neutralize excessive matrix protease on damaged wounds as a natural extracellular matrix, has an RGD amino acid sequence, and can promote cell adhesion, reduce inflammatory response, up-regulate growth factor level and accelerate wound healing.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a sponge patch and a preparation method and application thereof. Background Art

[0002] The oral ulcer or trauma hemostatic patches available on the market mainly exert their therapeutic effects by suppressing the immune system, anti-bacterial and anti-inflammatory, and anti-allergic effects, and lack the ability to heal and repair the wound surface.

[0003] Patent CN202410585810.1 discloses a thermosensitive gel spray loaded with platelet extracellular vesicles, including platelet extracellular vesicles, thermosensitive hydrogel poly(d,L-lactide)-poly(ethylene glycol)-poly(d,L-lactide) and solvent. The platelet extracellular vesicles use the thermosensitive gel as a carrier and are sprayed on the body surface to form a solid gel film, which is beneficial to the continuous and stable release of growth factors from platelet extracellular vesicles, effectively solving the problems of poor localization, easy burst release and easy degradation of growth factors in platelets.

[0004] Patent CN202311007015.6 discloses a hemostatic material, in which non-steroidal drug-polylysine hemostatic microparticles are cross-linked with platelets and then added to a gelatin solution, and cross-linked together to obtain a hemostatic gelatin sponge with hemostatic microparticles.

[0005] How to simultaneously solve problems such as bleeding, inflammation and functional damage is of great significance for the treatment of traumatic diseases such as uncontrollable bleeding and tissue damage. Summary of the invention

[0006] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a sponge patch and a preparation method and application thereof, which innovatively combines platelet vesicles with absorbable gelatin (collagen, collagen peptide) sponges, relies on the thrombin cascade effect after platelet activation and the anti-inflammatory axis effect of neutrophils to promote the formation of keratin and the activation of inflammatory cells, provides conditions for cell proliferation and migration, fibroblast proliferation and matrix deposition in the middle and late stages of healing, and plays a role as an intercellular messenger.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The first object of the present invention is to provide a method for preparing a sponge patch, wherein platelet vesicles are loaded on a sponge structure substrate, wherein the sponge structure is prepared from gelatin, collagen or collagen peptide, and the preparation method comprises the following steps: S1. Centrifuge whole blood, extract platelets using a platelet extraction kit, ultra-low temperature freezing stimulation, then use a cell disruptor to sonicate, centrifuge at low temperature, retain the supernatant, and obtain platelet vesicles; the whole blood is porcine whole blood or mouse whole blood; S2. Grind the raw materials of the sponge structure, add into ultrapure water, swell to obtain a sponge-based solution; heat to dissolve and defoam, add a cross-linking agent, change the rotation speed, add platelet vesicles, stir evenly, quickly transfer into a mold, shape, freeze-dry, and obtain a sponge patch.

[0008] Furthermore, the cross-linking agent is any one of glutaraldehyde, formaldehyde or carbodiimide.

[0009] Furthermore, the mass concentration of the cross-linking agent is 1% to 20%.

[0010] Furthermore, the mass concentration of the sponge-based solution is 10% to 15%.

[0011] Further, the rotation speed is changed to 800~3000rpm.

[0012] Furthermore, the shaping temperature is between -20°C and -80°C, and the shaping time is between 0.5 and 48 hours.

[0013] The second object of the present invention is to provide a sponge patch prepared by the above preparation method.

[0014] The third object of the present invention is to provide the use of the sponge patch in the preparation of hemostatic products.

[0015] The fourth object of the present invention is to provide the use of the sponge patch in the preparation of a medicine for treating wound repair.

[0016] The fifth object of the present invention is to provide the use of the sponge patch in the preparation of a medicine for treating oral ulcers.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a method for preparing a sponge patch, which utilizes the natural pig skin-derived gelatin polymer itself, which has the characteristics of low immunogenicity, plasticity, gelation and degradability, good affinity with biological tissues, macrophage activation and hemostasis functions, and better simulates the ECM required for tissue repair. On this basis, the AGS formed by formaldehyde cross-linking treatment can quickly induce platelets in the blood to aggregate in its internal porous structure, which is very beneficial to the aggregation of platelets and red blood cells, and acts as a "scaffold" for platelet aggregation, so that platelets gather in large quantities on its surface and internal gaps, adhere to each other, and release a variety of coagulation factors, promote platelet adhesion and thrombin generation. At the same time, the gelatin sponge will quickly absorb water in the blood, resulting in a sharp increase in the concentration of coagulation factors and thrombin in the blood, and promote the large-scale generation of thrombin; thrombin induces the fibrinogen in the blood to convert into fibrin, and the porous structure of the gelatin sponge is used to quickly form a bionic scab repair layer.

[0018] (2) The sponge patch prepared by the present invention utilizes the characteristics of platelet membrane vesicles as intercellular messengers to carry out receptor-mediated signal transduction, thereby promoting collagen remodeling and extracellular matrix deposition to form a durable and removable scar; in addition, it can also improve the cellular processes necessary for wound healing, including proliferation, angiogenesis and immune regulation. It can rescue abnormal fibroblast and endothelial cell behavior and restart the healing process, and is the main mediator of the therapeutic effect of platelet therapy. After platelet activation, platelets release growth factors, cytokines and extracellular matrix regulators stored in the cells. These factors can be released as free proteins, but can also be released in an enriched form in extracellular vesicles, including transforming growth factor (TGF-β), one of the most critical growth factors in tissue repair. The platelet-neutrophil axis is a regulator during inflammation. Neutrophils reach the damaged site, proliferate in large numbers to defend against cellular infection, and are then phagocytosed by macrophages. Macrophages release growth factors, cytokines and angiogenic factors at the damaged site to promote the formation of granulation tissue and angiogenesis. They can also secrete pro-inflammatory factors such as tumor necrosis factor TNF-α, interleukin IL-1β and IL-6. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a normal distribution diagram of the particle size of the platelet vesicles prepared by the present invention; Figure 2 The zata potential diagram of the platelet vesicles prepared by the present invention; Figure 3 This is a graph showing the dispersion stability of platelet vesicles prepared by the present invention; Figure 4 This is an upright microscope observation picture of the platelet vesicle prepared by the present invention; Figure 5 The morphology and electron microscope images of sponge patches with different compositions; Figure 6 This is the infrared spectrum of red blood cells in the red blood cell aggregation test. In the figure, PMVs-AGS is platelet vesicle gelatin sponge, and AGS is gelatin sponge; Figure 7 This is the result of ultraviolet absorption in the red blood cell aggregation test; Figure 8 It is the infrared spectrum of platelets in platelet aggregation test; Fig. 9 The morphological observation diagram of the treatment process of oral ulcer in mice treated with model group, gelatin sponge group, platelet vesicle gelatin sponge group and dexamethasone acetate group; Fig.10 Comparison of the ulcer area in the model group, gelatin sponge group, platelet vesicle gelatin sponge group and mice treated with dexamethasone acetate for 2d, 5d and 9d after oral ulceration; Fig.11Comparison of ulcer areas in the model group, gelatin sponge group, platelet vesicle gelatin sponge group and dexamethasone acetate group after 2 days, 5 days and 9 days respectively; Fig.12 Comparison of the ulcer areas of oral ulcers in mice treated with the model group, gelatin sponge group, platelet vesicle gelatin sponge group and dexamethasone acetate group at 2d, 5d and 9d. Fig.13 This is the result of platelet-loaded gelatin sponge promoting AST, a liver function index after liver resection; Fig.14 This is the result of platelet-loaded gelatin sponge promoting the liver function index ALT after liver resection; Fig.15 This is the result of the third day of the pro-inflammatory factor TNF-α level in the platelet-loaded collagen sponge; Fig.16 The results of the pro-inflammatory factor IL-6 level in platelet-loaded collagen sponge on the third day; Fig.17 This is the result graph of the level of pro-inflammatory factor TNF-α in platelet-loaded collagen sponge on the seventh day; Fig.18 This is the result of the seventh day increase in the level of pro-inflammatory factor IL-6 in platelet-loaded collagen sponge. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific test method, instrument or condition is specified in the examples, it is carried out according to the technology or condition described in the literature in this field or according to the product instructions. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0021] Example 1 This example provides a method for preparing platelet vesicle-loaded gelatin sponge (PMVs-AGS).

[0022] (1) Preparation of platelet vesicles from pig blood The whole blood of the pig was centrifuged at 500g for 20min, and platelets were extracted using a platelet extraction kit. The platelets were stimulated by ultra-low temperature freezing at -80℃ refrigerator for 7 times (1 time / 30s), and then ultrasonicated with a cell disruptor for 20min, centrifuged at 6000g for 30min, and the supernatant was retained as platelet vesicles (PMVs).

[0023] like Figure 1 and Figure 2As shown, it is the normal distribution diagram of particle size and zeta potential diagram of the platelet vesicles prepared by the present invention, the average particle size is 185nm, and the average Zeta potential is: -20mV.

[0024] like Figure 3 As shown in the figure, the dispersion index stability result diagram of the platelet vesicles prepared by the present invention shows that within 30 days, the particle size of the platelet vesicles hardly changes and remains at 180-200 nm, the dispersion index is 0.20-0.25, and the dispersion index stability is good. The 30-day stability experiment of samples from the same batch (storage condition: -20°C) shows that the maximum fluctuation of the particle size is 30 nm, which meets the requirements for long-term storage and use after large-scale extraction in the experiment. The polymer dispersion index is 0.20-0.25, and the dispersion is relatively good, which is easy to be distributed into the dense porous gelatin sponge at a later time.

[0025] like Figure 4 As shown, it is an upright microscope observation picture of the platelet vesicles prepared by the present invention, and the platelet vesicles are mostly uniformly distributed spherical vacuolar structures.

[0026] (2) Preparation of platelet vesicle-loaded gelatin sponge (PMVs-AGS) Prepare gelatin solution with a concentration of 10%: Grind medical gelatin, put it into ultrapure water, and swell it at 30℃ for 30min. Place it in a 40℃ water bath, foam it at 600rpm, and stir it for about 40min. After heating to 40℃, add glutaraldehyde crosslinker (5%), change the speed to about 1000rpm, then add platelet membrane vesicles, and stir for 30min. Quickly transfer the solution into a mold, place it at -20℃ for shaping for 24h, and freeze-dry it. The platelet vesicle-loaded gelatin sponge is obtained.

[0027] Example 2 This embodiment provides a method for preparing platelet vesicle-loaded collagen sponge (PMVs-Co).

[0028] (1) Preparation of platelet vesicles from pig blood The whole blood of the pig was centrifuged at 500g for 20min, and platelets were extracted using a platelet extraction kit. The platelets were stimulated by ultra-low temperature freezing at -80℃ refrigerator for 7 times (1 time / 30s), and then ultrasonicated with a cell disruptor for 20min, centrifuged at 6000g for 30min, and the supernatant was retained as platelet vesicles.

[0029] (2) Preparation of platelet vesicle-loaded collagen sponge (PMVs-Co) Prepare collagen solution with a concentration of 15%, select commercially available porcine collagen, grind it, put it into ultrapure water, and swell it at 37℃ for 20min. Place it in a 30℃ water bath, foam it at 1000rpm, and stir it for about 30min. After heating to 37℃, add formaldehyde crosslinker (1%), change the speed to about 800rpm, then add platelet membrane vesicles, and stir for 30min. Quickly transfer it into a mold, place it at -20℃ for shaping for 24h, and freeze-dry it. You will get a platelet vesicle-loaded collagen sponge.

[0030] Example 3 This embodiment provides a method for preparing a platelet vesicle-loaded collagen peptide sponge (PMVs-Cop).

[0031] (1) Preparation of platelet vesicles from pig blood The whole blood of the pig was centrifuged at 500g for 20min, and platelets were extracted using a platelet extraction kit. The platelets were stimulated by ultra-low temperature freezing at -80℃ refrigerator for 7 times (1 time / 30s), and then ultrasonicated with a cell disruptor for 20min, centrifuged at 6000g for 30min, and the supernatant was retained as platelet vesicles.

[0032] (2) Preparation of platelet vesicle-loaded collagen peptide sponge (PMVs-Cop) Prepare collagen peptide solution with a concentration of 15%, put it into ultrapure water, and swell it at 37°C for 30 minutes. Place it in a 37°C water bath, foam it at 400 rpm, and stir it for about 20 minutes. After heating to 37°C, add carbodiimide crosslinker (20%), change the speed to about 5000 rpm, then add platelet membrane vesicles, and stir for 30 minutes. Quickly transfer it into a mold, place it at -80°C for 48 hours, and freeze-dry it.

[0033] Collagen peptides are a mixture, including: Gly-Pro-Leu (GPL), Gly-Phe-Ala (GFA), Gly-Ala-Phe (GAF), Gly-Pro-Lys (GPK), Gly-Pro-Arg (GPR), Gly-Phe-Asn (GFN).

[0034] like Figure 5 As shown, the morphology and electron microscope images of sponge patches with different compositions show that the platelet vesicles are obviously loaded onto the sponge structure skeleton.

[0035] Example 4 To investigate the procoagulant effect of platelet vesicle gelatin sponge in vitro.

[0036] (1) Red blood cell aggregation test: Platelet vesicle gelatin sponge samples were cut into a size of 1 cm × 0.5 cm × 0.5 cm in length, width and height, and placed in a centrifuge tube. 5 ml of 5% red blood cell suspension was added. At the same time, a PBS group was set up as a control (also with 5% red blood cell suspension). The samples were then placed in a water bath and incubated at 37°C for 15 min. 2 ml of the suspension was taken and the absorbance value (A520) was detected by a UV spectrophotometer. Another 20 μl of the suspension was pressed with potassium bromide tablets for infrared detection.

[0037] (2) Platelet aggregation test: Platelet vesicle gelatin sponge samples were cut into a size of 1.5 cm × 1 cm × 0.5 cm in length, width and height. 5 ml of sodium citrate anticoagulated blood was taken and centrifuged at 1000 rpm for 10 min at room temperature. The upper layer of platelet-rich plasma (PRP) was retained and diluted with PBS for later use. A 10 ml centrifuge tube was added with the cut sample, and each tube was filled with 3 ml of diluted platelet plasma. At the same time, a PBS group was set up as a control (PRP was also added). The samples were incubated at 37°C for 15 min. 20 μl of the suspension was taken and pressed with potassium bromide for infrared detection.

[0038] like Figure 6 As shown in the infrared spectrum of red blood cells, at 2962cm -1 At the wavelength (CH3 antisymmetric stretching vibration of proteins, lipids and DNA), the peak of PMVs-AGS group increased by 1% compared with that of AGS alone. The carrier itself is rich in amino acids and polypeptide structures; at 1402cm -1 Wavelength (protein) and 1289cm -1 The peak value of protein in the AGS group increased at 1101 cm -1 At the wavelength (hemoglobin C=O or CNC stretching vibration), the spectral peak value also increased compared with the AGS group. The loaded PMVs helped the aggregation of red blood cells, and the peak values ​​showed an upward trend with the increase of concentration, which showed that a certain PMVs concentration could easily enrich red blood cells and promote the formation of blood clots.

[0039] like Figure 7 As shown, based on the UV results, judging by the low absorbance promoting aggregation, the absorbance values ​​of the AGS group and the 250μg / mlPMVs-AGS group decreased to a certain extent compared with the control group, indicating that both are conducive to red blood cell aggregation, and the red blood cell aggregation in the PMVs-loaded group is slightly more; in addition, compared with the 1mg / mlPMVs-AGS group, the 250μg / ml group accounted for 56% of the control group and the 1mg / ml group accounted for 50%, and the absorbance value of the high concentration group decreased more significantly. Overall, the dense and porous characteristics of the carrier gelatin sponge and the activated platelets loaded to form a fibrin network make it easier for red blood cells to aggregate.

[0040] like Figure 8As shown in the infrared spectrum of platelets, there is an absorption peak of characteristic functional groups at a wavelength of 1550cm -1 In the form of hydrogen single bond bending vibration, at a wavelength of 3300cm -1 It appears in the form of single bond stretching vibration; compared with the AGS group, the 1mg / ml PMVs-AGS group has a wavelength of 3300cm -1 and wavelength 1550cm -1 The absorption peak increased and platelet aggregation occurred. Compared with the control group (PBS), the absorption peaks of the AGS group and the 1mg / mlPMVs-AGS group both floated upward, indicating that the material carrier itself also has the ability to enrich platelets.

[0041] Example 5 To investigate the therapeutic effect of platelet vesicle gelatin sponge oral ulcer patch on oral ulcer in rats.

[0042] The rats were divided into model group, gelatin sponge group (AGS), platelet vesicle gelatin sponge group (PMVs-AGS) and dexamethasone acetate oral ulcer tablet group (DAAT). Cotton balls soaked in 60% glacial acetic acid were placed on the mucosa of the lower lip of the rats and gently pressed for 35 seconds. After the model was successfully established, the treatment was started. The maximum and minimum cross-sections were measured on the 2nd, 5th and 9th days of treatment to calculate the ulcer area, and the oral ulcer tissue was taken for pathological section detection.

[0043] like Figure 9-12 As shown in the figure, the morphological observation of the oral ulcer treatment process and the statistics of wound repair area are shown. It can be seen that the ulcer healing effect of PMVs-AGS is better than that of the positive control DAAT. In the model group (Model), from the second day of successful modeling to the end of treatment, there were still signs of white pseudomembrane, redness, swelling and congestion in the ulcer area. At the end of treatment, the local redness and swelling in the AGS group subsided, but there was some white pseudomembrane. In contrast, the ulcer area in the PMVs-AGS group and the DAAT group had recovered by naked eye observation on the 5th day of treatment; ImageJ was used to display and analyze the ulcer area in two dimensions. As the treatment time prolonged, the area of ​​the PMVs-AGS group and the DAAT group gradually decreased. Comparing the treatment effects of 5d and 9d, both groups could play a role in wound repair and the effects were comparable, meeting our treatment expectations; finally, the oral repair area of ​​each group was compared horizontally and vertically ( Figure 11-12 As shown in the figure, the ulcer areas in the AGS group, PMVs-AGS group and DAAT group showed a significant downward trend during the treatment. At the end of the treatment, the ulcer areas decreased by 51.71%, 93.74% and 89.38%, respectively. This indicates that the prepared PMVs-AGS adhesive sheet helps to repair the ulcer wound surface. It can also be seen that the carrier (AGS) itself also promotes the treatment of oral ulcers.

[0044] Example 6 To investigate the wound repair effect of platelet vesicle gelatin sponge in liver resection model.

[0045] Babl / c mice were randomly divided into a common sponge group, a gelatin sponge group, and a platelet vesicle gelatin sponge group. The mice were anesthetized, the abdomen was cut open, part of the liver was removed, and then hemostasis and repair treatment were performed. Two weeks later, the mice were killed by cervical dislocation, and the liver was taken for embedding, sectioning, staining, and observation, and the liver function (ALT, AST) was measured.

[0046] The results are as follows Figure 13-14 As shown, the platelet vesicle gelatin sponge group can significantly reduce the level of transaminase, and the effect is better than that of the ordinary sponge group and the gelatin sponge group.

[0047] Example 7 To investigate the wound repair effect of platelet vesicle collagen sponge in liver resection model.

[0048] Babl / c mice were randomly divided into a common sponge group, a gelatin sponge group, and a platelet vesicle collagen sponge group. The mice were anesthetized, the abdomen was cut open, part of the liver was removed, and then hemostasis and repair treatment were performed. Samples were left in the mice, and indicators such as TNF-α, IL-6, and SOD were measured on day 1, day 3, and day 7.

[0049] The results are as follows Figure 15-16 As shown in Figure 2, on the third day, the levels of pro-inflammatory cytokines (TNF-α, IFN-γ) in the platelet vesicle collagen sponge group were higher than those in the ordinary sponge group and the gelatin sponge group. Figure 17-Figure 18 As shown in the figure, on the seventh day, the levels of pro-inflammatory cytokines (TNF-α, IFN-γ) in the platelet vesicle collagen sponge group were significantly lower than those in the common sponge group and the gelatin sponge group, indicating that the platelet vesicle collagen sponge group can significantly reduce the levels of pro-inflammatory cytokines.

[0050] Example 8 To investigate the wound repair effect of platelet vesicle collagen peptide sponge in liver resection model.

[0051] Babl / c mice were randomly divided into a common sponge group, a common sponge + platelet vesicle group, and a platelet vesicle collagen peptide sponge group. The mice were anesthetized, the abdomen was cut open, part of the liver was removed, and then hemostasis and repair treatment were performed. Two weeks later, the mice were killed by dislocation of the neck, and the liver was taken for embedding, sectioning, staining and observation, and liver function (ALT, AST) was measured. The results showed that the platelet vesicle collagen peptide sponge group could significantly reduce the level of transaminase, and the effect was better than that of the common sponge group and the common sponge + platelet vesicle group.

[0052] Platelet vesicles with negative charge characteristics can be loaded onto the sponge structure skeleton through charge action, utilize intercellular messenger function, amplify intercellular regulation, retain functional proteins on platelets (CD47, CD55 and GPVI), promote the occurrence of coagulation cascade reaction and the repair of endothelial tissue; in addition, the dense porous structure of absorbable gelatin sponge is rich in collagen. As a natural extracellular matrix, it can neutralize excessive matrix proteases in damaged wounds. It has an RGD amino acid sequence that can promote cell adhesion, reduce inflammatory response, upregulate growth factor levels, and accelerate wound healing.

[0053] For matters not mentioned above, the prior art applies.

[0054] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a sponge patch, characterized in that: Platelet vesicles are loaded on a sponge structure substrate, wherein the sponge structure is prepared from gelatin, collagen or collagen peptide, and the preparation method comprises the following steps: S1. Centrifuge whole blood, extract platelets using a platelet extraction kit, ultra-low temperature freezing stimulation, then use a cell disruptor to sonicate, centrifuge at low temperature, retain the supernatant, and obtain platelet vesicles; the whole blood is porcine whole blood or mouse whole blood; S2. Grind the raw materials of the sponge structure, add into ultrapure water, swell to obtain a sponge-based solution; heat to dissolve and defoam, add a cross-linking agent, change the rotation speed, add platelet vesicles, stir evenly, quickly transfer into a mold, shape, freeze-dry, and obtain a sponge patch.

2. The preparation method according to claim 1, characterized in that The cross-linking agent is any one of glutaraldehyde, formaldehyde or carbodiimide.

3. The preparation method according to claim 2, characterized in that The mass concentration of the cross-linking agent is 1% to 20%.

4. The preparation method according to claim 3, characterized in that: The mass concentration of the sponge-based solution is 10% to 15%.

5. The preparation method according to claim 4, characterized in that: Change the speed to 800~3000rpm.

6. The preparation method according to claim 4, characterized in that: The shaping temperature is between -20°C and -80°C, and the shaping time is between 0.5 and 48 hours.

7. A sponge patch prepared by the preparation method as described in any one of claims 1 to 6.

8. Use of the sponge patch as claimed in claim 7 in the preparation of hemostatic products.

9. Use of the sponge patch as claimed in claim 7 in preparing a medicine for treating wound repair.

10. Use of the sponge patch as claimed in claim 7 in preparing a medicine for treating oral ulcers.

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