Umbilical cord mesenchymal stem cell exosome and GPT hydrogel combined composition, preparation method and application

Abstract

The invention provides a composition of umbilical cord mesenchymal stem cell exosome combined with GPT hydrogel, a preparation method and application, and belongs to the technical field of wound dressing. The invention solves the problems of fast degradation and low utilization rate in the existing mode of treating the diabetic foot wound surface by purely injecting exosome into the wound surface. The preparation method of the composition of the umbilical cord mesenchymal stem cell exosome and the GPT hydrogel comprises the following steps: S1, culturing to obtain the umbilical cord mesenchymal stem cell exosome; s2, preparing a 4-carboxyphenylboronic acid modified gelatin solution; s3, preparing a tannic acid solution; s4, mixing and stirring the 4-carboxyphenylboronic acid modified gelatin solution prepared in the step 2 and the tannic acid solution prepared in the step 3 to finally obtain the injectable hydrogel containing the exosome. The exosome containing umbilical cord mesenchymal stem cells is combined with the GPT hydrogel microspheres, so that the residence time of the exosome in the wound skin can be prolonged, and a better treatment effect can be achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of wound dressings and relates to a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel, a preparation method and application thereof. Background Art

[0002] Wound repair in diabetic foot is a challenging problem in clinical practice. Due to the influence of factors such as local tissue damage and regeneration failure, impaired vascular and neurotrophic wound repair pathways, and disorders of multiple cellular factors, traditional treatments often fail to achieve satisfactory therapeutic effects. Exosomes derived from stem cells have a good effect in improving the healing of damaged diabetic wounds, can enhance the proliferation and migration of diabetic fibroblasts and macrophages, and promote selective or M2 macrophage polarization. Wound healing can be accelerated by reducing inflammation, increasing wound cell proliferation, stimulating angiogenesis, and inducing M2 macrophage polarization. When the skin is damaged, it will go through stages such as hemostatic response, inflammatory response, angiogenesis, tissue regeneration and remodeling. Exosomes can promote angiogenesis, reduce inflammation, etc., and have great application potential in promoting wound repair.

[0003] Mesenchymal stem cells are multipotent precursor cells with the potential for self-renewal and differentiation into different types of specialized cells in different tissues. As a type of stem cell, mesenchymal stem cells have the advantages of easy separation, easy culture, low immunogenicity, and strong self-repair ability. But there are also potential disadvantages: it may cause excessive proliferation. The exosomes produced by stem cells can circumvent these problems. Exosomes are vesicles composed of lipid bilayers with a diameter of 30-120nm, containing a variety of lipids, proteins, nucleic acids and cytokines. Studies have shown that exosomes have potential advantages in wound repair: they can promote skin regeneration. However, the current method of simply injecting exosomes into the wound to treat diabetic foot wounds is generally used, but this method faces problems such as rapid degradation and low utilization rate. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned problems existing in the existing method of treating diabetic foot wounds by simply injecting exosomes into the wound surface, and to propose another composition, preparation and application of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel that can enhance the residence time of exosomes in the wound skin and thus exert a better therapeutic effect.

[0005] The purpose of the present invention can be achieved by the following technical solutions: A method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel, characterized in that it comprises the following method: S1. Culture and obtain exosomes of umbilical cord mesenchymal stem cells; S2, dissolving the 4-carboxyphenylboronic acid modified gelatin solid and the exosome solid obtained in step S1 in ultrapure water to prepare a 4-carboxyphenylboronic acid modified gelatin solution; S3, dissolving tannic acid powder in ultrapure water to prepare tannic acid solution; S4. The 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid solution prepared in step 2 and step 3 are mixed and stirred to finally obtain an injectable hydrogel composition containing exosomes.

[0006] Preferably, in the above step S1, the method for obtaining umbilical cord mesenchymal stem cell exosomes is as follows: a. Using the fetal umbilical cord discarded by the parturient to extract mesenchymal stem cell seeds; b. Collecting the culture medium supernatant of the fourth-generation umbilical cord mesenchymal stem cells through expansion culture; c. The supernatant is triple filtered, concentrated, and purified to remove dead cells and large cell debris; d. Ultracentrifugation was used to remove soluble proteins, protein aggregates and other impurities, and the umbilical cord mesenchymal stem cell exosomes were obtained by 20-fold concentration.

[0007] Preferably, the concentration of 4-carboxyphenylboric acid in the 4-carboxyphenylboric acid modified gelatin solution is 100-200 mg / mL, and the concentration of exosomes is 10-20 mg / mL.

[0008] Preferably, the concentration of 4-carboxyphenylboronic acid in the 4-carboxyphenylboronic acid modified gelatin solution is 150 mg / mL, and the concentration of exosomes is 15 mg / mL.

[0009] Preferably, the concentration of tannic acid in the tannic acid solution is 10-20 mg / mL.

[0010] Preferably, the concentration of tannic acid in the tannic acid solution is 15 mg / mL.

[0011] Preferably, in step S4, the mixing ratio of the 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid solution is 2:1.

[0012] A composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel prepared by the above method.

[0013] A use of the above composition for repairing diabetic foot wounds.

[0014] Compared with the prior art, the present invention can enhance the residence time of exosomes in the wound skin by combining microspheres containing umbilical cord mesenchymal stem cells with GPT hydrogel, thereby exerting a better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a characterization diagram of the morphology of GPT exosome hydrogel.

[0016] Figure 2 This is a graph showing the results of the exosome release behavior test.

[0017] Figure 3 Schematic diagram of cytotoxicity test results.

[0018] Figure 4 Schematic diagram of cell proliferation test results.

[0019] Figure 5 Schematic diagram of cell migration test results, where ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0020] Figure 6 Schematic diagram of angiogenesis experiment results, where * indicates p<0.05.

[0021] Figure 7 The figures are the wound healing status of mice in each group at 0d, 4d, 7d, and 14d, where * indicates p<0.05.

[0022] Figure 8 The results of H&E staining of wound tissues of rats in each group on the 7th and 14th days, where * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. DETAILED DESCRIPTION

[0023] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the accompanying drawings, but the present invention is not limited to these embodiments.

[0024] Reagents and materials: Male 6-week-old Sprague-Dawley (SD) rats (180-200 g), Youkang serum-free stem cell medium (Youkang Biotechnology Beijing Co., Ltd.), 4-carboxyphenylboronic acid modified gelatin solid, tannic acid, citric acid (Sinopharm Chemical Reagent Co., Ltd.), sodium citrate (Sinopharm Chemical Reagent Co., Ltd.). 89% α-MEM basal medium (Gibco), 10% FBS fetal bovine serum (Gibco), 1% double antibody (Gibco). HUVEC cell culture medium ratio: 89% DMEM basal medium (Gibco), 10% FBS fetal bovine serum (Gibco), 1% double antibody (Gibco). Streptozotocin (STZ, Sigma-Aldrich), DPBS buffer (Youkang Biotechnology Beijing Co., Ltd.), Bradford protein quantification kit (Biyuntian).

[0025] Example 1 A method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel, comprising the following steps:

[0026] S1. Using the fetal umbilical cord discarded by the parturient, processing the umbilical cord tissue, and extracting mesenchymal stem cell seeds from the umbilical cord; S2. The umbilical cord mesenchymal stem cells are cultured to the third generation through expansion culture, and then inoculated into Youkang serum-free medium, cultured to 90% density at 37°C and 5% CO2, and the supernatant is collected after 72 hours to obtain the culture supernatant of the fourth generation umbilical cord mesenchymal stem cells; S3. The supernatant obtained in step S3 was centrifuged at 500 g for 10 min, and the precipitate was discarded to obtain the supernatant. The supernatant was further concentrated and sterilized and filtered 20 times using the exosome harvesting and concentration system of Beijing Huakan to obtain umbilical cord mesenchymal stem cell exosomes. The obtained exosomes were quantified by Bradford method for protein.

[0027] S4. At room temperature, 30 mg of 4-carboxyphenylboronic acid-modified gelatin solid and 3 mg of exosome solid (pure exosome) were dissolved in 200 μL ultrapure water to prepare a 4-carboxyphenylboronic acid-modified gelatin solution with a concentration of 150 mg / mL. S5, dissolving 1.5 mg of tannic acid powder in 100 μL of ultrapure water to prepare a tannic acid solution with a concentration of 15 mg / mL; S6. The 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid solution prepared in steps S4 and S5, respectively, were mixed and stirred in a ratio of 2:1 to finally obtain a composition containing exosomes in the form of an injectable hydrogel, named GPT exosome hydrogel, and marked as GPT@Exos (exosome content of 1%).

[0028] The prepared hydrogel was freeze-dried and then soaked in liquid ammonia for a period of time before being taken out. The hydrogel became hard and brittle, and the hydrogel was broken at this time. The hydrogel was then glued to the conductive glue, sprayed with gold, and placed in the SEM instrument for measurement. The morphology of the GPT exosome hydrogel is characterized as follows: Figure 1 .

[0029] In order to demonstrate the efficacy of GPT exosome hydrogel containing exosomes (GPT@Exos), an injectable hydrogel (labeled as GPT, without exosomes) was prepared as a comparative example for comparison. The hydrogel (GPT) was prepared as follows: at room temperature, 30 mg of 4-carboxyphenylboronic acid modified gelatin solid was dissolved in 200 μL ultrapure water, 1.5 mg of tannic acid powder was dissolved in 100 μL ultrapure water, and then the 4-carboxyphenylboronic acid modified gelatin solution was mixed with 100 μL tannic acid solution and stirred for 30 seconds to finally obtain an injectable hydrogel labeled as GPT.

[0030] The exosome release behavior of GPT exosome hydrogel (GPT@Exos) was tested (e.g. Figure 2 As shown in the figure, the specific experimental steps are as follows: 300 μL of each of the two hydrogels, GPT and GPT@Exos, were placed in 500 μL of DPBS and incubated at 37°C. The supernatant was collected after 6, 12, 24, 36, 48, 60 and 72 hours, and fresh DPBS solution was added. The protein concentration of the collected supernatant was determined using a BCA protein concentration assay kit. Specifically, 100 μL of the supernatant and 100 μL of the BCA working solution were mixed and incubated at 60°C for 60 minutes. After 60 minutes, 100 μL was aspirated and the absorbance at 562 nm was measured using an enzyme reader. The value obtained by subtracting the OD of the GPT group from the OD of the GPT@Exos group was compared with the BCA protein concentration standard curve to obtain the amount of released exosomes.

[0031] from Figure 2 It can be seen that at 12h, the protein content detected in the supernatant was about 50%, indicating that at this time, about half of the exosomes were released into the supernatant. From 12h, the protein content in the supernatant was detected at 24h, 36h, and 48h, indicating that the release of exosomes gradually slowed down until 72h. The experiment showed that it takes about 3 days for GPT exosome hydrogel (GPT@Exos) to be completely released in vivo.

[0032] The GPT exosome hydrogel was subjected to a cytotoxicity test. The prepared GPT exosome hydrogel was co-cultured with cells and the cell growth was observed to verify whether the hydrogel was toxic.

[0033] like Figure 3As shown, the cells used in the test are L929 cells and HUVEC cells. The ratio of L929 cell culture medium is: 89% α-MEM basal medium (Gibco), 10% FBS fetal bovine serum (Gibco), 1% double antibody (Gibco). The ratio of HUVEC cell culture medium is: 89% DMEM basal medium (Gibco), 10% FBS fetal bovine serum (Gibco), 1% double antibody (Gibco). The hydrogel and exosomes were placed in the culture medium respectively (50 μL of hydrogel or 0.5 mg of exosomes were added to each 1 mL of culture medium, and 50 μL of DPBS was used instead of the control group). After 24 hours, the supernatant was taken to obtain the hydrogel extract. The L929 and HUVEC cell suspensions were respectively added at 5×10 3 The cells / well were added to a 96-well plate and cultured in a cell culture incubator for 24 hours. The culture medium was aspirated, 100 μL of the extract was added, and the cells were cultured in the incubator for another 24 hours, then washed three times with DPBS, and then stained with Alamar Blue Detection Reagent (Gibco) for 2 hours. Finally, the absorbance was tested with an ELISA reader, and the cell activity was calculated.

[0034] Based on the dosage of exosomes in animal experiments, cytotoxicity experiments were conducted. After browsing most of the literature, it was found that the concentration of exosomes used was generally low, between 10μg / mL-200μg / mL. In this experiment, we set up 4 concentration gradients, namely 1mg / mL, 5mg / mL, 10mg / mL, and 15mg / mL to test whether exosomes have an effect on cells. It was found that cell compatibility was still good at these concentrations.

[0035] The effect of GPT exosome hydrogel (GPT@Exos) on cell growth and proliferation was tested, and hydrogel (GPT) was added for comparative testing. The cells used in the test were HUVEC cells. The HUVEC cell culture medium ratio is: 89% DMEM basal medium (Gibco), 10% FBS fetal bovine serum (Gibco), 1% double antibody (Gibco). The hydrogel and exosomes were placed in the culture medium respectively (50 μL hydrogel or 0.5 mg exosomes were added to each 1 mL culture medium, and 50 μL DPBS was used instead for the control group). After 24 hours, the supernatant was taken to obtain the hydrogel extract. First, the cells were digested and then 3.0×10 3The cells were seeded at a density of 10 cells per well in a 96-well plate and cultured for 24 hours. Then, the culture medium in the well plate was aspirated and replaced with the hydrogel extract, and the control group was still treated with normal culture medium. After incubation in the cell culture incubator for 24, 48, and 72 hours, the well plate was taken out, and the culture medium in the well plate was aspirated, washed three times with DPBS, and then Alamar Blue detection reagent (Gibco) was added to the well plate and incubated in the incubator for 2 hours. Finally, the absorbance of the solution in the well plate was measured by a microplate reader. Cell live / dead staining was also used to demonstrate cytotoxicity. After 48 hours of incubation with the extract, the cells were stained with calcein-AM and propidium iodide (Gibco). Subsequently, fluorescent images were taken.

[0036] like Figure 4 As shown, a cell proliferation experiment was conducted using hydrogel and exosome extracts. Four groups were set up according to the different substances added to the cells, including a control group with DPBS (Control group), a group with a hydrogel and culture medium concentration ratio of 50μl / ml in the hydrogel extract (GPT group), an exosome extract concentration of 100μg / ml (Exos group), and a hydrogel containing exosomes at a concentration of 2mg / ml (GPT@Exos group) co-cultured with cells. The cells were stained with Alamar Blue detection reagent and their optical density was measured to determine cell activity. Finally, the cells were stained with calcein-AM and propidium iodide to determine the live or dead state of the cells. Figure 4 (a) is the fluorescence intensity diagram of cells in the four experimental groups at different time periods (1 day, 2 days, and 3 days). After staining, it can be seen that the living cells in the figure have fluorescence. The experimental results show that the exosome extract group has a certain promotion effect on the proliferation of endothelial cells. Compared with the pure exosome extract group, the hydrogel exosome group has a better proliferation effect, indicating that the bioavailability of exosomes is improved. Figure 4 (b) is the cell fluorescence image of the four experimental groups. It can be seen from the fluorescence image that the fluorescence intensity of the hydrogel exosome group is the strongest.

[0037] In order to test the effect of hydrogel on cell migration, an experiment was conducted. The cells used in the test were HUVEC, and the effect of hydrogel on cell migration was evaluated by cell scratch test. The specific experimental method is as follows: First, HUVEC cells were digested and 5.0×10 4The cells were planted at a density of 10 cells per well in a 24-well plate and cultured for 24 hours until they filled the well plate, and then cultured with a medium with a 2.5% serum concentration for 6 hours. A scratch was then made on the bottom of the 24-well plate with a 200μL pipette tip. Then, the culture medium in the well plate was aspirated, washed three times with DPBS, and replaced with normal culture medium, 0.1mM H2O2 culture medium, and 4g / mL glucose culture medium, and 0.5mg exosomes, 50μL GPT, and GPT@Exos hydrogel were added, respectively. Then, the culture was continued in the incubator, and the closure of the scratch was observed at 0 hours, 12 hours, and 20 hours, respectively.

[0038] GPT hydrogel is a microenvironment-responsive hydrogel that is responsive to H2O2 and glucose. Exosomes were loaded into GPT hydrogel and cell migration in H2O2 and glucose environments was tested. Figure 5 As shown, Figure 5 (a) shows the cell migration of the four experimental groups under different environments and at different time periods; Figure 5 (b) shows the cell migration rate of the four experimental groups under different environments and at different time periods. Exosome hydrogel can release exosomes to promote cell migration under normal conditions, and can respond to and improve the microenvironment in H2O2 and glucose environments to maintain cell migration effects.

[0039] In order to determine the angiogenesis-promoting effect of the hydrogel, angiogenesis experiments were performed using HUVECs cells. The specific experimental method is as follows: First, 200 μL of matrix gel was pre-coated on the bottom of a 24-well plate. After the matrix gel was evenly spread, the 24-well plate was transferred to a 37°C incubator for 30 minutes to stabilize the matrix gel. Then, the HUVECs cells were digested and incubated at 7.0×10 4 The cells were seeded on the matrix gel at a density of 100 cells per well. After waiting for 30 minutes, the cells were completely attached to the wall, the original culture medium was aspirated, and replaced with 1 mL of hydrogel and exosome extract, respectively. The culture was continued for 6 hours, and the angiogenesis of the cells was observed and photographed using a fluorescent microscope in bright field mode. Five areas were randomly selected from each group to calculate the number of tubes and nodes.

[0040] The angiogenesis experiment was carried out using hydrogel and exosome extracts. The concentration ratio of hydrogel to culture medium in the extract was 50 μL / mL, and the concentration of exosome extract was 100 μg / mL. Figure 6 As shown, Figure 6 (a) shows the cell vascular conditions of the four experimental groups. Figure 6 (b) in the figure shows the number of cell angiogenesis and vascular nodes in the four experimental groups. By counting the number of nodes and tubes, it can be seen that exosomes have good pro-angiogenic properties. The angiogenesis rate in the exosome hydrogel group is higher than that in the exosome group, indicating that the bioavailability of hydrogel-loaded exosomes is increased.

[0041] Animal experiment, modeling: In this experiment, streptozotocin (STZ, Sigma-Aldrich) solution was used to induce type 1 diabetes in SD rats. Specifically, 6-week-old SD rats were adaptively fed for one week before modeling. STZ was dissolved in a 2.1wt% citric acid solution and a 2.94wt% sodium citrate solution mixed in a 1:1 ratio, and then the STZ solution was intraperitoneally injected at a dose of 65mg / kg body weight. Blood glucose was monitored in SD rats three days after modeling. The blood glucose of SD rats was measured by blood drawing from the tail vein, and each measurement was separated by 2 days thereafter. When blood glucose was stable above 16mmol / L, it was considered hyperglycemia. When hyperglycemia was stable for one week, it was considered that the diabetic rat model was successfully constructed.

[0042] Experiment: After the modeling was successful, the rats were depilated with a razor and depilatory cream to reveal smooth skin. A circular blade was used to cut off the entire layer of skin with a diameter of 10 mm on the back of the rat. Two wounds were created in one rat. 50 μL of GPT hydrogel, 50 μL of GPT@Exos hydrogel, and 20 μL of exosome solution (exosome content of 0.5 mg) were added to the material group, and 50 μL of DPBS was used instead in the control group (Control group). After adding the materials, the dressing was applied. The wound healing of the rats on days 0, 4, 7, and 14 was recorded (e.g. Figure 7 ), Figure 7 (a) shows the inflammation of wound surface in 4 experimental groups. Figure 8 (b) is a comparison table of wound healing rates in the four experimental groups. On the 7th and 14th days, one SD rat from each group was sacrificed to collect wound tissue for H&E and Masson section staining.

[0043] like Figure 8 As shown, Figure 8 (a) shows the H&E staining results of wound tissues of rats in each group on the 7th and 14th days. Figure 8 (b) is a comparison of the relative collagen deposition areas of the four experimental groups. The H&E staining results show that at 7 days, there was a severe inflammatory reaction in the wound of the Control group, the inflammatory reaction in the wound of the GPT and Eoxs groups was mild, and there was almost no inflammatory reaction in the GPT@Exos group. At 14 days, the inflammatory reaction of each group was alleviated, and there was still mild inflammation in the Control group. The wound healing of the GPT@Exos group was the best, and at 14 days, the wound healing of the GPT@Exos group was the best. The experimental results show that there are significant differences in the wound healing rates of the control group (Control group) and the GPT@Exos group at 4d, 7d, and 14d. The H&E staining results show that GPT@Exos has the effect of promoting wound healing.

[0044] Example 2: The difference between this example and Example 1 is that the concentration of the 4-carboxyphenylboronic acid modified gelatin solution is 100 mg / mL, and the concentration of the tannic acid solution is 10 mg / mL.

[0045] Example 3: The difference between this example and Example 1 is that the concentration of the 4-carboxyphenylboronic acid modified gelatin solution is 200 mg / mL, and the concentration of the tannic acid solution is 20 mg / mL.

[0046] It should be understood that in the claims and description of the present invention, all "including..." should be understood as an open-ended meaning, that is, its meaning is equivalent to "at least contains...", and should not be understood as a closed meaning, that is, its meaning should not be understood as "only includes...".

[0047] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel, characterized in that: It includes the following methods: S1. Culture and obtain exosomes of umbilical cord mesenchymal stem cells; S2, dissolving the 4-carboxyphenylboronic acid modified gelatin solid and the exosome solid obtained in step S1 in ultrapure water to prepare a 4-carboxyphenylboronic acid modified gelatin solution; S3, dissolving tannic acid powder in ultrapure water to prepare tannic acid solution; S4. The 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid solution prepared in step 2 and step 3 are mixed and stirred to finally obtain an injectable hydrogel composition containing exosomes.

2. The method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to claim 1, characterized in that: In the above step S1, the method for obtaining umbilical cord mesenchymal stem cell exosomes is as follows: a. Using the fetal umbilical cord discarded by the parturient to extract mesenchymal stem cell seeds; b. Collecting the culture medium supernatant of the fourth-generation umbilical cord mesenchymal stem cells through expansion culture; c. The supernatant is triple filtered, concentrated, and purified to remove dead cells and large cell debris; d. Ultracentrifugation was used to remove soluble proteins, protein aggregates and other impurities, and the umbilical cord mesenchymal stem cell exosomes were obtained by 20-fold concentration.

3. The method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to claim 1, characterized in that: The concentration of 4-carboxyphenylboronic acid in the 4-carboxyphenylboronic acid modified gelatin solution is 100-200 mg / ml, and the concentration of exosomes is 10-20 mg / ml.

4. The method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to claim 1, characterized in that: The concentration of 4-carboxyphenylboronic acid in the 4-carboxyphenylboronic acid modified gelatin solution is 150 mg / ml, and the concentration of exosomes is 15 mg / ml.

5. The method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to claim 1, characterized in that: The concentration of tannic acid in the tannic acid solution is 10-20 mg / ml.

6. The method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to claim 1, characterized in that: The concentration of tannic acid in the tannic acid solution is 15 mg / ml.

7. The method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to claim 1, characterized in that: In the step S4, the mixing ratio of the 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid solution is 2:

1.

8. A composition obtained by the method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to any one of claims 1 to 7.

9. Use of a composition prepared by the method for preparing a composition of umbilical cord mesenchymal stem cell exosomes combined with GPT hydrogel according to claim 8 for repairing diabetic foot wounds.

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