Novel hydrogel for treating chronic wounds of old people as well as method and application of novel hydrogel

Abstract

The invention discloses novel hydrogel for treating chronic wounds of old people and a preparation method and application of the novel hydrogel, and the preparation method of the novel hydrogel comprises the following steps: S1, preparing an apoptotic extracellular vesicle solution; s2, preparing an acellular fat matrix hydrogel; s3, adding the apoptotic extracellular vesicle solution into the acellular fat matrix hydrogel to obtain novel hydrogel; the novel hydrogel provided by the invention can improve the wound surface microenvironment, further enhances the wound surface healing effect, and provides an innovative solution for treatment of refractory wound surfaces, especially senile chronic wound surfaces.

Description

Technical Field

[0001] The present invention relates to the technical field of wound healing drugs, and in particular to a novel hydrogel for treating senile chronic wounds, and its preparation method and application. Background Art

[0002] Wound healing is a complex biological process involving multiple stages such as inflammation, proliferation, and remodeling. The treatment of difficult-to-heal wounds such as chronic wounds and diabetic foot ulcers remains a huge challenge. Traditional treatment methods often have limited effects and are difficult to meet clinical needs.

[0003] Senile chronic wounds refer to skin or soft tissue injuries that occur in the elderly population and have a long duration and are difficult to heal. The main reasons affecting the healing of senile chronic wounds include malnutrition, total protein / calorie (marasmic) malnutrition, protein (edematous) malnutrition; peripheral vascular lesions, peripheral neuropathy, and infections caused by diabetes; the use of some therapeutic drugs that affect wound healing such as steroids, chemotherapy, or radiotherapy drugs, and senile neurological diseases such as dementia and epilepsy. In addition to chronic wound healing problems, there are other reasons for the occurrence of problematic wounds in the elderly. Pressure ulcers can occur at any age, but are common in the elderly; arterial insufficiency ulcers, simple arterial ulcers are rare and are mostly caused by insufficient blood supply to the lower extremities; venous stasis ulcers, such as deep vein thrombosis and varicose veins, are mainly caused by stasis of lower extremity venous blood; there are also other conditions such as tophus, uremia, malignant tumors, and immune diseases.

[0004] The treatment of senile chronic wounds occupies a large amount of medical resources, increases the burden on families and society, and seriously affects the quality of life of the elderly. The incidence of senile chronic wounds increases with age, the pathogenesis is complex, the course of disease is long, and multidisciplinary comprehensive treatment is required.

[0005] Therefore, it is of great significance to develop new drugs for promoting wound healing, especially for the healing of senile chronic wounds. Summary of the Invention

[0006] The present invention overcomes these problems and provides a novel hydrogel that can be used to promote wound healing, especially for the healing of senile chronic wounds, as well as the preparation method and application of the novel hydrogel.

[0007] To achieve this purpose, the present invention provides the following technical solutions:

[0008] In the first aspect of the present invention, a preparation method of a novel hydrogel is provided, comprising the following steps:

[0009] S1. Prepare an apoptotic extracellular vesicle solution;

[0010] S2. Prepare a decellularized adipose matrix hydrogel;

[0011] S3. Add the apoptotic cell-derived exosome solution to the decellularized adipose matrix hydrogel to obtain a novel hydrogel.

[0012] Preferably, in step S3, in the novel hydrogel, the concentration of apoptotic cell-derived exosomes is 100 - 300 μg / mL; more preferably, in step S3, in the novel hydrogel, the concentration of apoptotic cell-derived exosomes is 200 μg / mL.

[0013] Preferably, in step S1, the solute of the apoptotic cell-derived exosome solution includes water and PBS, the concentration of apoptotic cell-derived exosomes in the solution is 10 - 20 μg / mL, and the storage time of the prepared apoptotic cell-derived exosome solution is less than 24 hours.

[0014] The present invention has proven by experiments that apoptotic cell-derived exosomes can only maintain their morphology persistently in a specific solution. Apoptotic cell-derived exosomes are severely damaged after 24 hours in PBS. However, they can continuously maintain their morphology in the decellularized adipose matrix hydrogel of the present invention.

[0015] Preferably, step S1 includes:

[0016] S11. Provide adipose tissue raw materials;

[0017] S12. Cut the adipose tissue raw materials into pieces and induce them with a medium containing staurosporine to obtain adipose tissue with induced apoptosis;

[0018] S13. Centrifuge the cell culture solution of the apoptotic adipose tissue at high speed to obtain apoptotic cell-derived exosomes released by the apoptotic adipose tissue, and dilute them with sterile PBS to prepare an apoptotic cell-derived exosome - PBS mixture;

[0019] Preferably, step S2 includes:

[0020] S21. Perform freeze-thaw treatment on the adipose tissue raw materials to obtain adipose tissue after freeze-thaw treatment;

[0021] S22. Perform the first trypsin treatment on the adipose tissue after freeze-thaw treatment to remove cells, and obtain adipose tissue after the first trypsin treatment;

[0022] S23. Perform the first extraction treatment on the adipose tissue after the first trypsin treatment, wash and drain it to obtain adipose tissue after the first extraction treatment;

[0023] S24. Perform the second trypsin treatment on the adipose tissue after the first extraction treatment to further remove cells, wash and drain it to obtain adipose tissue after the second trypsin treatment;

[0024] S25. Hydrolyze the adipose tissue after the second trypsin treatment, wash and drain it to obtain the adipose tissue after hydrolysis treatment.

[0025] S26. Perform a second extraction treatment on the adipose tissue after hydrolysis treatment, wash and drain it to obtain acellular adipose matrix.

[0026] S27. After freeze-drying and grinding the acellular matrix, obtain acellular adipose matrix powder.

[0027] S28. Digest the acellular adipose matrix powder with pepsin, adjust the pH to obtain acellular adipose matrix hydrogel.

[0028] Preferably, in steps S23 - S26, the cleaning solution used for the cleaning treatment contains Na2HPO4, KH2PO4, phenylmethylsulfonyl fluoride, penicillin and streptomycin.

[0029] Preferably, in step S21, the freeze-thaw solution used for the freeze-thaw treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or; in steps S22 and S24, the trypsin treatment solution used for the first trypsin treatment and the second trypsin treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or; in steps S23 and S26, the extraction solution used for the first extraction treatment and the second extraction treatment contains: phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or; in step S25, the hydrolysis solution used for the hydrolysis treatment contains: phenylmethylsulfonyl fluoride, penicillin and streptomycin.

[0030] Preferably, in step S21, the freeze-thaw treatment includes: freezing at below -80°C for 1 - 2 h, then warming up to 25 - 37°C, and repeating 3 - 5 times.

[0031] Preferably, the first trypsin treatment and the second trypsin treatment are carried out at a temperature of 25 - 37°C for 8 - 16 h.

[0032] Preferably, the extraction solution used for the first extraction treatment and the second extraction treatment contains isopropanol.

[0033] Preferably, the first extraction treatment and the second extraction treatment are carried out at 25 - 37°C for 8 - 16 h.

[0034] Preferably, the hydrolysis solution used for the hydrolysis treatment contains deoxyribonuclease, ribonuclease and lipase.

[0035] Preferably, the hydrolysis treatment is carried out at 25 - 37°C for 16 - 24 h.

[0036] Preferably, the obtained acellular adipose matrix is subjected to freeze-drying treatment.

[0037] Preferably, to obtain the acellular adipose matrix powder after digestion treatment, the pH of the solution needs to be adjusted to 7.3.

[0038] In the second aspect of the present invention, a novel hydrogel prepared by the method of the present invention is provided.

[0039] In the third aspect of the present invention, an application of the novel hydrogel prepared by the method of the present invention in the preparation of a drug for treating wounds is provided.

[0040] Preferably, the wound is an elderly chronic wound.

[0041] Compared with the prior art, the beneficial effects and remarkable progress of the present invention are as follows:

[0042] 1. In the present invention, apoptotic extracellular vesicles are combined with an acellular adipose matrix hydrogel, and the apoptotic extracellular vesicles can maintain their morphology in the acellular adipose matrix hydrogel for a long time, and the vesicles are less likely to degrade, so that the drug effect of promoting wound healing is more persistent.

[0043] 2. The novel hydrogel provided by the present invention can improve the wound microenvironment, further enhance the wound healing effect, and provide an innovative solution for the treatment of refractory wounds, especially elderly chronic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] To more clearly illustrate the technical solutions of the present invention, the following briefly introduces the drawings required for the embodiments of the present invention.

[0045] Obviously, the drawings in the following description are only the drawings of some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings, but these other drawings also belong to the scope of the drawings required for the embodiments of the present invention.

[0046] Figure 1 It is the transmission electron microscope image and particle size diagram of apoptotic extracellular vesicles extracted from human adipose tissue in Example 1 of the present invention;

[0047] Figure 2 It is the appearance photos of acellular adipose matrix (DAT) and its freeze-dried product in Example 2 of the present invention;

[0048] Figure 3 It is the scanning electron microscope photo of acellular adipose matrix (DAT) in Example 2 of the present invention;

[0049] Figure 4 It is the appearance photo of the novel hydrogel in Example 3 of the present invention;

[0050] Figure 5 It is the scanning electron microscope photo of the novel hydrogel in Example 3 of the present invention;

[0051] Figure 6 It is the injectability detection experimental diagram of the novel hydrogel of Example 3 of the present invention;

[0052] Figure 7 It is the scanning electron microscope diagram of Comparative Example 1 of the present invention;

[0053] Figure 8 It is the appearance picture of the novel hydrogel of Example 4 of the present invention for wound healing. Detailed implementation manners

[0054] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following embodiments can be obtained from commercially available channels without special instructions.

[0055] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners of this application.

[0056] It should be noted that the terms "first", "second" and "third" (if any) in the specification, claims and drawings of the present invention are only used to distinguish different objects and not to describe a specific order. In addition, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products or devices.

[0057] Next, the technical solutions of the present invention will be described in detail with specific embodiments.

[0058] Example 1 Extraction of apoptotic extracellular vesicles

[0059] (1) Acquisition and preservation of adipose tissue raw materials

[0060] Adipose tissue needs to be obtained from people aged 18 - 50. Before obtaining adipose tissue, donors need to be screened for tuberculosis, hepatitis, syphilis, AIDS, tumors, malaria, and leprosy. At the same time, it is ensured that the donors have no large - area burn lesions, no autoimmune diseases, the application of steroid drugs does not exceed three months, no history of drug or other substance poisoning, and the donor area has not received radiotherapy. In the donor serum test report, HIV - 1 / 2 - Ab, HBs - Ag, HCV - Ab, RPR, and HBc - Ab need to be negative. After adipose resection surgery, adipose tissue is obtained in a clean environment and placed in sterile PBS supplemented with 1% bovine serum albumin (BSA) to minimize tissue loss, obtain a higher yield of acellular matrix, and the tissue is packaged using disinfected or sterilized tools or containers, and the acquisition process is registered. During transportation, the tissue is stored in a 4°C refrigerated transport box. When the tissue is received, the donor's medical history and test results need to be verified, and the tissue acquisition time, quantity, type, and transportation situation are registered. The tissue can be stored in a 4°C refrigerator for no more than 72 hours; the tissue can be stored in an - 80°C refrigerator for long - term preservation. This is the raw material of adipose tissue.

[0061] (2) Prepare DMEM medium containing 10% FBS. Take 20 g of adipose tissue, cut it into pieces, and add it to 100 mL of the medium, and the medium contains 1% penicillin - streptomycin solution.

[0062] (3) Add 250 nM of staurosporine to the medium. Under a sterile magnetic stirrer, in a 37°C incubator, stir the medium at a speed of 300 rpm for 2 days to induce apoptosis of adipose tissue.

[0063] (4) Discard the adipose tissue and leave the medium. Perform three centrifugation operations, which are to take the supernatant after centrifugation at 800 g for 10 min, take the supernatant after centrifugation at 2000 g for 15 min, and centrifuge at 13000 g for 1 h to obtain the apoptotic extracellular vesicles in the precipitate.

[0064] (5) Measure the weight of the apoptotic extracellular vesicles and prepare a 10 mg / mL apoptotic extracellular vesicle solution with PBS.

[0065] All of the above steps are operated in a laminar flow hood. See Figure 1 The electron microscope image shows an obvious bilayer membrane structure, indicating successful extraction. The particle size diagram shows that its average size is in the range of 445.1 ± 85.8 nm, which is at the nanometer level.

[0066] Example 2 Preparation of acellular adipose matrix

[0067] Prepare the solution

[0068] Prepare phenylmethylsulfonyl fluoride (PMSF) solution: Add 25 mL of isopropanol to 1 g of PMSF and stir it until it becomes transparent.

[0069] Prepare the freeze-thaw solution: Weigh 1.21 g of Tris and 1.46 g of EDTA, add deionized water to a final volume of 1 L and stir until dissolved, adjust the pH to 7.0, add 5 μL of PMSF solution and 1 mL of penicillin-streptomycin solution (100× double antibody).

[0070] Prepare the trypsin treatment solution: First prepare a 0.25% trypsin-EDTA solution, and then add 5 μL of PMSF solution and 1 mL of 100× double antibody solution to every 100 ml of trypsin-EDTA solution.

[0071] Prepare the extraction solution: Add 5 μL of PMSF solution and 1 mL of 100× double antibody solution to every 100 ml of isopropanol.

[0072] Prepare the cleaning solution: Weigh 19.6392 g of Na2HPO4·12H2O and 2.3 g of KH2PO4, add deionized water to a final volume of 1 L and stir until dissolved, adjust the pH to 7.0, add 5 μL of PMSF solution and 1 mL of 100× double antibody solution.

[0073] Prepare the NaCl-glycerol mixture: Weigh 0.175 g of NaCl and dissolve it in 10 mL of deionized water, adjust the pH to 7.3, add 10 mL of glycerol and mix well.

[0074] Prepare the deoxyribonuclease stock solution: Add 15 mL of the NaCl-glycerol mixture to 200,000 units of DNase and mix well.

[0075] Prepare the Tris-NaCl solution: Weigh 0.06 g of Tris, 0.44 g of NaCl and dissolve them in 50 mL of deionized water, adjust the pH to 7.5.

[0076] Prepare the ribonuclease stock solution: Add 40 mL of the Tris-NaCl solution to 500 mg of RNase.

[0077] Prepare the frozen CaCl2 solution: Weigh 0.02775 g of CaCl2 powder, add deionized water to a final volume of 50 mL, dissolve it completely, and cool to 4 °C.

[0078] Prepare the lipase stock solution: Add 50 mL of the frozen CaCl2 solution to 100,000 units of lipase.

[0079] Preparation of hydrolysis solution: Weigh 19.6392 g of Na2HPO4·12H2O, 0.58536 g of MgSO4 and 2.3 g of KH2PO4, add deionized water to a final volume of 1 L and stir until dissolved. Adjust the pH to 7.0. For every 200 ml of the liquid, add 1 mL of deoxyribonuclease stock solution, 1 mL of ribonuclease stock solution, 2 mL of lipase stock solution, 5 μL of PMSF solution and 1 mL of 100× double antibody solution.

[0080] (1) Obtaining raw materials of adipose tissue: The same as in Example 1.

[0081] (2) Freeze-thaw treatment

[0082] Use forceps and surgical scissors to take out sterile adipose tissue from the raw materials of adipose tissue obtained in step (1), transfer it to a new sterile specimen jar, add freeze-thaw solution at a volume ratio of 1:1, place it at -80 °C for freezing for 1 h, then warm it up to 25 °C, fully melt it and replace the freeze-thaw solution, repeat 3 times, and finally pour out the freeze-thaw solution to obtain adipose tissue after freeze-thaw treatment. The adipose tissue after freeze-thaw treatment melts and becomes soft.

[0083] Using this kind of freeze-thaw solution can more easily remove grease, reduce the protease activity released during cell lysis, and retain cytokines.

[0084] (3) First trypsin treatment

[0085] Add the adipose tissue after freeze-thaw treatment obtained in step (2) to trypsin treatment solution at a volume ratio of 1:1, and carry out stirring digestion: temperature 25 °C, frequency 50 rpm, time 8 h, remove the trypsin treatment solution to obtain adipose tissue after the first trypsin treatment.

[0086] Using this kind of trypsin treatment can remove cells while reducing the protease activity released during cell lysis and retaining cytokines.

[0087] (4) First extraction treatment

[0088] Add the adipose tissue after the first trypsin treatment obtained in step (3) to the extraction solution at a volume ratio of 1:1, continuously stir at 25 °C, frequency 50 rpm, and replace the extraction solution 2 times during this period, time 8 h.

[0089] After the extraction is completed, remove the extraction solution, then wash and drain to obtain adipose tissue after the first extraction treatment.

[0090] The specific steps of washing and draining are: add washing solution at a volume ratio of 1:1, continuously stir at 25 °C, frequency 50 rpm, time 20 min, repeat 3 times, and drain the water;

[0091] With this extraction treatment, isopropanol is miscible with water and oil, used to extract lipids from tissues, reduce the protease activity released during cell lysis, and retain cytokines.

[0092] (5) Second trypsin treatment

[0093] The adipose tissue after the first extraction treatment obtained in step (4) is subjected to a second trypsin treatment using the same method as in step (3), and then washed and drained using the same method as in step (4) to obtain adipose tissue after the second trypsin treatment.

[0094] (6) Hydrolysis treatment

[0095] The adipose tissue after the second trypsin treatment prepared in step (5) is added to the hydrolysis solution at a volume ratio of 1:1, continuously stirred at 25 °C, with a frequency of 50 rpm and a time of 16 h. The hydrolysis solution is removed, and then washed and drained using the same method as in step (4) to obtain adipose tissue after hydrolysis treatment.

[0096] With this hydrolysis treatment, fat and residual nucleic acids can be further removed, the protease activity released during cell lysis can be reduced, and cytokines can be retained.

[0097] (7) Second extraction treatment

[0098] The adipose tissue after hydrolysis treatment obtained in step (6) is subjected to an extraction treatment using the same method as in step (4), washed and drained, then rinsed in 60% ethanol for 20 min and repeated 3 times, and then freeze-dried to obtain acellular adipose matrix. The steps of the freeze-drying treatment are as follows: The sample is placed in a centrifuge tube and frozen overnight at -20 °C. The sample is freeze-dried for 48 h.

[0099] (8) Performance of the prepared acellular adipose matrix

[0100] Appearance observation of the prepared acellular adipose matrix

[0101] The appearance photo of the prepared acellular adipose matrix is as shown in the appendix Figure 2 As shown, it can be observed that the DAT tissue has a light yellowish-white appearance (a darker yellow or brown appearance indicates incomplete lipid and / or cell extraction), is clean and free of impurities. After taking it out, it is found to have excellent physical properties and good mechanical properties.

[0102] The scanning electron microscope photo of the prepared acellular adipose matrix (DAT) is as shown in Figure 3 As shown. It can be seen from the figure that there is no cell structure in DAT, presenting a porous structure with different pore sizes, and obvious collagen fiber entanglement can be seen.

[0103] Example 3 Preparation of a new hydrogel

[0104] (1) Freeze-drying, grinding and sterilization of acellular adipose matrix

[0105] Freeze-dry and store at -20°C. Cut the freeze-dried ADM (prepared in Example 2) into pieces of 5 mm * 5 mm size, add them to the grinding jar, cool with liquid nitrogen for 2 min, then add liquid nitrogen again until the liquid nitrogen stops boiling (about 3 - 5 min), start the machine, with a frequency of 30 and a grinding time of 2 min. Add liquid nitrogen for cooling again, start the machine, with a frequency of 30 and a grinding time of 2 min. White powder can be seen. Aliquot 200 mg and sterilize with ethylene oxide.

[0106] (2) Take 20 ml of 0.01 M dilute hydrochloric acid, add 20 mg of pepsin, and vortex. Sterilize the pepsin solution through a 0.22 μm filter, add 200 mg of sterilized DAT powder, and shake and digest at room temperature for 12 h to obtain a transparent and viscous solution. Dropwise add 10 M NaOH to the DAT solution until pH = 7.4, add 100 μL of 10X PBS to every 900 μL of ADM, mix evenly, and store at 4°C to obtain the acellular adipose matrix hydrogel liquid. A small amount of ADM can be taken and placed at 37°C, and it can be observed that gelation occurs in 30 min.

[0107] (3) Add the apoptotic cell-derived exosome solution (prepared in Example 1) to the acellular adipose matrix hydrogel liquid, and adjust the exosome concentration to 200 μg / mL, then the preparation is completed.

[0108] The appearance photo of the prepared novel hydrogel is as Figure 4 shown.

[0109] The scanning electron microscope photo of the prepared novel hydrogel is as Figure 5 shown. It can be seen from the figure that the collagen fiber structure of the novel hydrogel intersects, and obvious void structures can be observed, with a diameter of about 8 μm, providing a good environment for cell ingrowth in the wound surface.

[0110] Inject the prepared novel hydrogel into a syringe for injectability detection, and the results are as Figure 6 shown. It can be seen that the novel hydrogel can be used for injection.

[0111] Comparative Example 1

[0112] This comparative example is used to observe the morphology of apoptotic cell-derived exosomes in different solutions.

[0113] Add the apoptotic cell-derived exosome solution (prepared in Example 1) to the acellular adipose matrix hydrogel liquid (prepared in Example 3), and adjust the exosome concentration to 200 μg / mL. Label it as the DAT group.

[0114] Add the apoptotic cell-derived exosome solution (prepared in Example 1) to PBS liquid, and adjust the exosome concentration to 200 μg / mL. Label it as the PBS group.

[0115] The morphology of the two groups was observed under the electron microscope after 24 h, and the results were as Figure 7 shown. It can be seen that the apoptotic extracellular vesicles were severely damaged in PBS, while they were well preserved in the acellular adipose matrix hydrogel liquid.

[0116] This comparative example proves that apoptotic extracellular vesicles need to be in the specific solution (acellular adipose matrix hydrogel liquid) provided by the present invention to maintain an intact state. If in other solutions, the vesicles are more likely to degrade, and the bioactive components inside also disappear faster.

[0117] Example 4

[0118] Experimental mice

[0119] Construction of young wounds (Young / Y group): After anesthetizing 8-week-old (25 - 30 g) male black mice with tribromoethanol, a circular skin with a diameter of 5 mm was excised in the center of their backs using a puncher, scissors and forceps.

[0120] Construction of aged chronic wounds (Age / A group): 8-week-old C57 black mice were intraperitoneally injected with 100 mg / kg of D-galactose every day for 60 consecutive days, and a subacute aging model was successfully constructed. The subacute aging model was anesthetized with tribromoethanol, and a circular skin with a diameter of 5 mm was excised in the center of its back using a puncher, scissors and forceps.

[0121] Experimental drugs

[0122] DAT group: The acellular adipose matrix hydrogel liquid prepared in Example 3 was applied to the affected area.

[0123] DAT@Apo group: The novel hydrogel prepared in Example 3 was applied to the affected area.

[0124] The blank control group was not smeared with drugs.

[0125] Experimental results

[0126] As Figure 8 shown, DAT can promote wound healing, but the healing effect on aged chronic wounds is significantly worse. However, the novel hydrogel DAT@Apo of the present invention has obvious advantages in the healing effect on aged chronic wounds.

[0127] During the description of the above specification:

[0128] The descriptions of terms such as "this embodiment", "embodiments of the present invention", "as shown in...", "further", "further improved technical solution", etc. mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example, and moreover, the specific features, structures, materials or characteristics, etc. described can be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, on the premise of not generating contradictions, those of ordinary skill in the art can combine or combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0129] Finally, it should be noted that:

[0130] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them;

[0131] Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or equivalently replace some or all of the technical features, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or replacements made by those skilled in the art according to the content of this specification all fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a novel hydrogel, characterized in that: The following steps are involved: S1. Prepare apoptotic extracellular vesicle solution; S2, preparing acellular adipose matrix hydrogel; S3. Add the apoptotic extracellular vesicle solution into the decellularized fat matrix hydrogel to obtain a new type of hydrogel.

2. The method for preparing a novel hydrogel according to claim 1, characterized in that: In step S3, the concentration of apoptotic extracellular vesicles in the novel hydrogel is 100-300 ug / mL.

3. The method for preparing a novel hydrogel according to claim 2, characterized in that: In step S1, the solutes of the apoptotic extracellular vesicle solution include water and PBS, the concentration of the apoptotic extracellular vesicles in the solution is 10-20 ug / mL, and the prepared apoptotic extracellular vesicle solution is stored for less than 24 hours.

4. The method for preparing a novel hydrogel according to claim 1, characterized in that: Step S1 includes: S11, providing adipose tissue raw materials; S12, chopping the adipose tissue raw material, and inducing it with a culture medium containing staurosporine to obtain apoptosis-induced adipose tissue; S13. The cell culture medium of the apoptotic fat tissue is subjected to high-speed centrifugation to obtain the apoptotic extracellular vesicles released by the apoptotic fat tissue, which are then diluted with sterile PBS to prepare an apoptotic extracellular vesicle-PBS mixed solution.

5. The method for preparing a novel hydrogel according to claim 1, characterized in that: Step S2 includes: S21, performing freeze-thaw treatment on the adipose tissue raw material to obtain adipose tissue after freeze-thaw treatment; S22, performing a first trypsin treatment on the adipose tissue after the freeze-thaw treatment to remove cells, thereby obtaining adipose tissue after the first trypsin treatment; S23, subjecting the adipose tissue after the first trypsin treatment to a first extraction treatment, washing, and draining to obtain the adipose tissue after the first extraction treatment; S24, subjecting the adipose tissue after the first extraction treatment to a second trypsin treatment to further remove cells, washing, and draining to obtain adipose tissue after the second trypsin treatment; S25, hydrolyzing the adipose tissue after the second trypsin treatment, washing, and draining to obtain adipose tissue after hydrolysis treatment; S26, performing a second extraction process on the hydrolyzed fat tissue, washing, and draining to obtain a decellularized fat matrix; S27, freeze-drying and grinding the decellularized matrix to obtain decellularized fat matrix powder; S28. Digest the decellularized fat matrix powder with pepsin, adjust the acidity and base, and obtain the decellularized fat matrix hydrogel.

6. The method for preparing a novel hydrogel according to claim 5, characterized in that: In steps S23-S26, the cleaning solution used for the cleaning process includes Na2HPO4, KH2PO4, phenylmethylsulfonyl fluoride, penicillin and streptomycin.

7. The method for preparing a novel hydrogel according to claim 5, characterized in that: In step S21, the freeze-thaw solution used in the freeze-thaw treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or, In steps S22 and S24, the trypsin treatment solution used in the first trypsin treatment and the second trypsin treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or, In steps S23 and S26, the extracting solution used in the first extraction process and the second extraction process contains: phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or, In step S25, the hydrolysis solution used for the hydrolysis treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin.

8. A novel hydrogel prepared by the method according to any one of claims 1 to 7.

9. Use of the novel hydrogel prepared by the method according to any one of claims 1 to 7 in preparing a medicine for treating wounds.

10. The use according to claim 9, characterized in that The wound surface is a chronic wound surface of the elderly.

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