CN118924956A - A hyaluronic acid oligosaccharide-chitosan biomimetic thermosensitive gel encapsulating extracellular vesicles derived from hemangioma stem cells - Google Patents

Abstract

The present invention discloses a hyaluronic acid oligosaccharide-chitosan biomimetic thermosensitive gel EVs@oHA/CS ^gel encapsulating extracellular vesicles (EVs) derived from hemangioma stem cells (HemSC). By extracting EVs derived from abnormally proliferating vascular endothelial cells HemSC as therapeutic active substances that promote angiogenesis, and encapsulating them in hydrogels as wound dressings, it can effectively promote ischemic tissue repair, angiogenesis, and local microcirculation reconstruction. The hyaluronic acid oligosaccharides used in the present invention perform biomimetic modification on the chitosan thermosensitive gel, making it softer and having higher elastic deformability, effectively improving its biocompatibility, optimizing its sustained release performance and EVs protection ability when used as an EVs carrier, and enhancing the angiogenic activity of EVs.

Description

Hyaluronic acid oligosaccharide-chitosan bionic temperature-sensitive gel for encapsulating hemangioma stem cell-derived extracellular vesicles

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to hyaluronic acid oligosaccharide-chitosan bionic temperature-sensitive gel for encapsulating hemangioma stem cell-derived extracellular vesicles, which is used for promoting tissue repair, in particular angiogenesis and microcirculation reconstruction of ischemic tissues.

Background

Ischemic tissue repair and regeneration are a critical area of recent studies and are also in the treatment of many diseases, such as acute vascular injury caused by trauma, thrombosis, cerebral apoplexy, chronic wound difficult to heal caused by diabetes, etc. Among them, diabetic wounds, also known as diabetic ulcers or diabetic feet, are a typical complication of diabetes, which occurs in about 20% of the diabetic population, and many patients eventually require amputation, which is a major cause of diabetes disability. In the complex pathological mechanism of this disease, microcirculation disturbance caused by vascular lesions is an important factor, which is related to wound oxygen supply, effector cell chemotaxis, cytokine production and transportation, and is also closely related to inflammation and oxidative stress. In current clinical practice, the treatment of diabetic ulcers is mainly based on traditional debridement and antibacterial treatment, and the wound dressing used has the main functions of bacteriostasis and absorbing wound exudates. Therefore, there is a need to design and develop a wound dressing which can effectively promote angiogenesis and microcirculation reconstruction and can comprehensively improve the wound environment.

Extracellular Vesicles (EVs) are nanovesicles secreted by cells and having a phospholipid bilayer structure, which contain various proteins and mirnas, and can be used as a main medium for intercellular communication, and simultaneously regulate various cell functions. In recent years, EVs have been used in various medical fields such as diagnosis and treatment of diseases. Infantile hemangioma is a benign tumor common in infants and is characterized by abnormal proliferation of vascular endothelial cells. Hemangio stem cells (HemSC) are a class of CD133 positive cells isolated from infant hemangio that promote cellular angiogenic differentiation and peripheral vascular proliferation by autocrine and paracrine action, thought to play a key regulatory role in the progression of the hemangio disease.

Chitosan (CS) is a natural polymer material with wide sources and has the functions of antibiosis and anti-inflammatory. The hydrogel constructed by CS and beta-sodium glycerophosphate (beta-GP) is a classical temperature-sensitive hydrogel, has the advantages of low toxicity, neutral pH, mild phase change condition and the like, but also has the defects of overhigh osmotic pressure, overhigh cationic charge and the like.

Based on the current state of the art, the invention aims to provide oHA/CS biochemical temperature-sensitive gel for entrapping HemSC-EVs, and the Hyaluronic Acid (HA) is an important component of extracellular matrix (ECM), HAs good biocompatibility and moisturizing and lubricating capabilities, and is widely applied to the fields of medical treatment and cosmetics. Hyaluronic acid oligosaccharides (oHA) are degradation products of HA. oHA also HAs some additional biological functions compared to the macromolecular HA (molecular weight typically up to several hundred thousand Da). Studies have shown that oHA can activate Src, FAK, ERK-1/2 and other receptors, thereby promoting angiogenesis and wound healing.

Disclosure of Invention

The invention aims to provide oHA/CS biochemical-imitating temperature-sensitive gel EVs@oHA/CS ^gel with HemSC-EVs entrapped therein, and the invention effectively promotes the microcirculation reconstruction of diabetic wounds by skillfully utilizing HemSC EVs which are derived from pathological tissues as angiogenesis-promoting therapeutic active substances. In order to further improve the curative effect of EVs in treating diabetic wounds, the invention develops a novel chitosan-based thermosensitive gel which is biomimetically modified by hyaluronic acid oligosaccharide (oHA) and is used as an administration carrier.

In the invention, a strategy of utilizing the interaction of negative charges on oHA and positive charges on HA to modify chitosan temperature-sensitive gel is provided. oHA addition shields the CS from excessive positive charge, and the hydrogels exhibit a variety of properties that are more conducive to EVs administration. Simultaneously oHA can be used as a mimic of ECM, further improves the biocompatibility of the hydrogel and promotes the adhesion and spreading of cells. In addition, we also screened and optimized the basic prescription of hydrogels, such as adding hydroxyethyl cellulose (HEC) to adjust the gel phase transition point, adding sodium bicarbonate (NaHCO ₃) to adjust the gel osmotic pressure and pH, adding gelatin to adjust the gel degradation rate, etc.

Comprehensively, hemSC-EVs are extracted as angiogenesis promoting therapeutic active substances, and are entrapped in the well-designed and optimized oHA biomimetic modified chitosan hydrogel, so that a brand-new therapeutic system EVs@oHA/CSgel is developed for tissue repair, in particular to microcirculation reconstruction of ischemic tissues.

The invention thoroughly evaluates the physicochemical property and in-vitro angiogenesis promoting capacity of the hydrogel treatment system, and further verifies the angiogenesis promoting and wound healing treatment effects in a rat diabetes wound model.

The invention uses differential centrifugation method to obtain EVs from HemSC in vitro cell culture supernatant, and the specific obtaining method is as follows: ① HemSC culturing until about 80% of the culture bottle bottom is fully paved, discarding the original culture medium, washing twice by using Phosphate Buffer Solution (PBS), and adding a serum-free culture medium according to a certain proportion; ② Placing the cells in a cell incubator, and continuing to culture for a certain time; ③ Collecting cell culture supernatant; ④ Centrifuging the cell culture supernatant at 4 ℃ and 200g for 5min to remove floating cells; ⑤ Centrifuging 10000g of supernatant obtained in the previous step at 4 ℃ for 30min to remove cell debris; ⑥ Centrifuging the supernatant obtained in the previous step at 4deg.C and 100000g for 70min, and collecting EVs; ⑦ The precipitate in the previous step is resuspended with a proper amount of PBS, and centrifuged for 70min at 100000g at 4 ℃ again, and the precipitate is the purified EVs.

In the above operation: the ratio of the serum-free culture medium added in the step ① is 0.05-0.2 mL/square centimeter culture area, preferably 0.12-0.15 mL/square centimeter culture area; the time for collecting EVs in serum-free culture in step ② is 24-72 hours, preferably 48 hours.

The method for preparing EVs@oHA/CS ^gel comprises the following steps: ① Dissolving CS, HEC, oHA of prescription amount and gelatin in dilute acid to obtain solution A; ② Dissolving the beta-GP and NaHCO ₃ with the prescription amount in water to obtain solution B; ③ Resuspending the prescribed amount of EVs in PBS to obtain solution C; ④ Dropwise adding the solution B into the solution A at a certain temperature and a certain stirring speed; ⑤ Adding the solution C into the mixture to obtain hydrogel (hydrogel precursor solution) to be formed; ⑥ Placing the hydrogel precursor in a 37 ℃ incubator, and waiting for about 3min to form the hydrogel.

In the above operation: the diluted acid in the step ① is 0.05-0.2 mol/L hydrochloric acid or acetic acid, preferably 0.1mol/L hydrochloric acid; the temperature in step ④ is 0 to 20 ℃, preferably 10 ℃; the stirring speed in step ④ is 50 to 1000rpm, preferably 200rpm. The contents of each substance in the prescription in the final hydrogel are as follows: CS is added in an amount of 1 to 3 percent, preferably 1.5 to 2 percent; HEC is added in an amount of 0 to 0.2%, preferably 0.05 to 0.1%; oHA is added in an amount of 0 to 0.5%, preferably 0.1 to 0.25%; the addition amount of gelatin is 0-1%, preferably 0.5-0.75%; the addition amount of beta-GP is 1-6%, preferably 2-3%; the addition amount of NaHCO ₃ is 0-0.5%, preferably 0.05-0.2%; henSC-EV is added in an amount of 0 to 100. Mu.g/mL in terms of protein content and 0 to 1X 10 ¹¹/mL in terms of particle count, preferably 40 to 60. Mu.g/mL in terms of protein content and 4X 10 ¹⁰～6×10¹⁰/mL in terms of particle count.

The invention has the beneficial effects that

The invention obtains the EVs from HemSC, and the morphology, the particle size and the characteristic protein expression of the EVs accord with the general characteristics of the EVs. In one embodiment HemSC-EVs exhibit good pro-angiogenic activity and can be used as pro-angiogenic therapeutically active substances.

The invention obtains oHA bionic modified chitosan-based temperature-sensitive hydrogel. In one embodiment, oHA modified hydrogels exhibit softer, more elastically deformable properties. In another embodiment, oHA modified hydrogels exhibit better biocompatibility.

The invention obtains a hyaluronic acid-chitosan bionic temperature-sensitive gel treatment system EVs@oHA/CS ^gel for encapsulating hemangioma stem cell-derived extracellular vesicles. In one embodiment, the therapeutic system exhibits good wound healing and angiogenesis promoting effects.

In summary, the invention obtains EVs from HemSC as a therapeutic active substance for promoting angiogenesis, encapsulates the EVs in oHA biomimetic modified chitosan-based hydrogel, and constructs a therapeutic system EVs@oHA/CS ^gel. The invention evaluates the characteristics and the drug effect of EVs@oHA/CS ^gel through a plurality of in-vivo and in-vitro experiments, and particularly evaluates the effects of promoting wound healing and angiogenesis of the treatment system through a rat diabetes wound model. The result shows that EVs@oHA/CS ^gel can be used as a tissue repair material with application prospect, and is particularly suitable for recovery and microcirculation reconstruction of ischemic damaged tissues.

Drawings

FIG. 1 characterization and identification of HemSC-EVs

(A) Transmitting an electron microscope photograph;

(B) NTA particle size distribution;

(C) And (5) detecting a Western-blot protein.

FIG. 2 preparation of EVs@oHA/CS ^gel

(A) A temperature-sensitive forming process of the gel;

(B) The modulus-temperature scan curve of CS ^gel,oHA/CS^gel,EVs@CS^gel,EVs@oHA/CS^gel during the temperature programming at 25-40 ℃.

FIG. 3 characterization of EVs@oHA/CS ^gel

(A) Modulus-time scan curve of hydrogels at 10,25,32,37 ℃;

(B) Rheological frequency sweep curves after hydrogel formation;

(C) Rheological strain sweep curves after hydrogel formation;

(D) Osmotic pressure of the hydrogel leach solution;

(E) The pH of the hydrogel leach;

(F) Scanning electron microscope photographs of hydrogels.

FIG. 4 in vitro proangiogenic Activity Studies of EVs@oHA/CS ^gel

(A) Scratch test representative pictures;

(B) Schematic diagram of scratch experiment device;

(C) Reducing the quantitative result of the scratch area;

(D) Representative pictures of hydrogel-cell co-culture experiments;

(E) Quantitative results of the number of living cells in the co-culture experiment;

(F) Quantitative results of cell viability in co-culture experiments.

FIG. 5 study of efficacy of EVs@oHA/CS ^gel to promote wound healing and microcirculation reconstruction in rats

(A) Representative photographs of wounds;

(B) Trend of wound shape and area variation;

(C) A wound area profile over time;

(D) Wound area reduction values on day 7 and day 14;

(E) Laser doppler flow imaging photographs of the wound area;

(F) Quantitative results of blood perfusion index on day 17.

Detailed Description

In order to facilitate the understanding of the present disclosure by a person skilled in the art, the technical solutions of the present disclosure and the corresponding evaluation characterization solutions will be further described below in connection with specific examples, but the following should not be construed as limiting the scope of the claimed invention in any way.

Example 1 extraction, characterization and identification of HemSC-EVs

The method comprises the following steps: hemSC when the culture was carried out to about 80% of the total culture flask bottom, the original culture medium was discarded, rinsed twice with Phosphate Buffer (PBS), and 0.135mL of serum-free medium was added per square centimeter of culture area. The cells were placed in a cell incubator and cultured for a further 48 hours, and the cell culture supernatant was collected. The cell culture supernatant was centrifuged at 200g for 5min at 4℃to remove floating cells. The supernatant was centrifuged at 10000g for 30min at 4℃to remove cell debris. The supernatant was centrifuged at 100000g for 70min at 4℃and EVs were collected. The precipitate was resuspended in an appropriate amount of PBS, centrifuged again at 100000g for 70min at 4deg.C, and the precipitate was the purified EVs.

The protein content of HemSC-EVs was determined using BCA kit, its morphology was photographed using a Transmission Electron Microscope (TEM), and its particle size distribution and particle density were analyzed using a Nanoparticle Tracking Analyzer (NTA). In addition, the characteristic proteins are identified by using a Western-blot technology, wherein CD9, CD63 and TSG101 are positive expression proteins, and Calnexin is a negative expression protein.

Results: when the amount of the serum-free medium for extracting EVs is 0.135mL per square centimeter of the culture area, the amount of protein contained in the EVs extracted from 100mL of the culture supernatant is 151.3 mug, and the number of EVs particles is 1.47×10 ¹¹. TEM results indicate that HemSC-EVs have the typical appearance of vesicles of the tea-cup-holder-like vesicles, with particle sizes of about 50-150 nm (FIG. 1A). Particle size measurement showed that it had a peak of 106nm in particle size distribution (FIG. 1B). Western-blot results show that the HemSC-EVs express CD9, CD63 and TSG101 proteins at high level, and Calnexin proteins at low level (shown in figure 1C), which shows that the extracted HemSC-EVs have higher purity and less impurity proteins.

Example 2 preparation of EVs@oHA/CS ^gel

The method comprises the following steps: taking 15mL of hydrogel as an example, CS, HEC, oHA of the prescription amount and gelatin are stirred and dissolved in 12mL of 0.1mol/L hydrochloric acid, and the mixture is marked as solution A; the prescribed amounts of beta-GP and NaHCO3 were dissolved in 2mL of purified water, designated solution B; the prescribed amount of HemSC-EVs was resuspended in 1mL PBS and noted as solution C. Slowly dropping the solution B into the solution A under the magnetic stirring at 10 ℃ and 200rpm, and then dropping the solution C into the mixture. The hydrogel precursor is placed in a 37 ℃ water bath to form a gel within 5 minutes.

To further compare the effect of oHA and EVs addition on hydrogel phase transition temperature, we performed temperature-rising scans on four hydrogel precursors, CS ^gel、oHA/CS^gel、EVs@CS^gel and evs@oha/CS ^gel, using a rotarheometer. The rheometer used a parallel plate clamp 20mm in diameter, set Gap Size at 3000 μm, sweep frequency at 2.0rad/s, strain at 2%, programmed temperature range from 25℃to 40℃with a temperature ramp rate of 0.5℃per minute.

Results: as shown in FIG. 2A, the hydrogel precursor liquid is in a liquid state and can rapidly form a gel after being placed at 37 ℃. As shown in fig. 2B, the sol-gel phase transition temperatures of CS ^gel、oHA/CS^gel、EVs@CS^gel and evs@oha/CS ^gel under the heating conditions are 31.4 ℃, 34.6 ℃, 33.3 ℃ and 35.1 ℃ respectively, which meet the use requirements in actual administration.

Example 3 characterization of EVs@oHA/CS ^gel

The method comprises the following steps: hydrogel precursors of EVs@oHA/CS ^gel were prepared according to the method in example 2, and were time scanned using a rotarheometer at four temperatures of 10 ℃, 25 ℃, 32 ℃, 37 ℃ to plot the modulus versus time curve. The rheometer used a parallel plate clamp 20mm in diameter, set Gap Size at 3000 μm, sweep frequency at 2.0rad/s, strain at 2%.

The EVs@oHA/CS ^gel hydrogel precursor was incubated at 37℃for 12h to allow the hydrogel to fully form. It is frequency swept and strain swept using a rotational rheometer. The rheometer used a parallel plate fixture 20mm in diameter and set Gap Size to 3000 μm. The fixed strain was 2% during the frequency sweep, and the set frequency was increased from 0.1rad/s to 20rad/s. The fixed frequency at strain sweep was 2.0rad/s, setting the strain to rise from 0.1% to 1000%.

Hydrogel leach liquor acquisition and osmolality and pH determination: four hydrogel precursors, CS ^gel、oHA/CS^gel、EVs@CS^gel and EVs@oHA/CS ^gel, were placed in 6-well cell culture plates to form a liquid surface of approximately 5mm in height, and placed in a 37℃incubator until they formed hydrogels. Physiological saline was added to each well in an amount of 0.5mL per square cm of culture area, and the well plate was returned to the incubator and incubated for 24 hours to obtain a hydrogel extract. Osmolarity and pH were determined using a freezing point osmometer and pH meter.

Taking the formed hydrogel, pre-freezing the hydrogel at the temperature of-20 ℃ for 24 hours, and freeze-drying the hydrogel in a freeze dryer. And (3) brittle breaking the freeze-dried gel block by liquid nitrogen, spraying metal on the section, and observing the microstructure by using a scanning electron microscope.

Results: as shown in FIG. 3A, EVs@oHA/CS ^gel can maintain a liquid state for more than 20 minutes at 10 ℃ and 25 ℃, and can quickly form gel at 32 ℃ and 37 ℃, so that the method is convenient for practical application. The results of frequency scanning and strain scanning of EVs@oHA/CS ^gel are shown in FIG. 3B and FIG. 3C, respectively, which show that the gel treatment system has the characteristics of softness and high elastic deformability. The osmotic pressure and pH of the hydrogel leach solution are shown in FIGS. 3D and 3E, respectively, indicating that the osmotic pressure and pH of the hydrogel are physiologically safe. An SEM photograph of EVs@oHA/CS ^gel is shown in FIG. 3F, which shows a typical three-dimensional network structure.

Example 4 in vitro proangiogenic Activity Studies of EVs@oHA/CS ^gel

The method comprises the following steps: four hydrogel precursors of CS ^gel、oHA/CS^gel、EVs@CS^gel and EVs@oHA/CS ^gel were prepared according to the procedure described in example 2.

Scratch experiments on Human Umbilical Vascular Endothelial Cells (HUVEC): using 24mm diameter with 0.4 μm pore sizeThe device, to which 3mL of the hydrogel precursor solution was added, was placed in an incubator at 37℃until it was molded. HUVECs were inoculated into 6-well cell culture plates and incubated until approximately 95% confluent with the bottom of the well plate. Scratches were made straight on the bottom surface using a 200. Mu.L tip and ruler, then the original medium was discarded, and washed twice with PBS to remove floating cells, then the reduced serum medium (containing 5% FBS) and hydrogel-loaded were addedThe device is put back into the incubator for continuous cultivation. Scratch photographs were taken at 0,12,24,36,48h and scratch areas were quantified using image J software.

HUVEC Co-culture experiments on hydrogel surface: to each well of a 12-well cell culture plate, 0.6mL of the hydrogel precursor solution was added, and the mixture was placed in an incubator at 37℃until it was molded. HUVEC cells were seeded into the hydrogel-plated 12-well cell culture plates at a number of 1X10 ⁵ cells per well and cultured in a 37℃incubator. At 12,24,48,72h, calceinAM/PI live/dead cell staining reagents were added to the well plate, photographed with a fluorescence microscope, and the cells in the field of view were counted.

Results: fig. 4B is a schematic diagram of HUVEC scratch experiments. The scratch photograph is shown in FIG. 4A, and the quantitative result is shown in FIG. 4C. The results show that both oHA and EVs released from the EVs@oHA/CS ^gel therapeutic system have certain angiogenesis promoting effect, wherein the EVs have stronger effect. FIG. 4D is a representative photograph of the growth of HUVECs on the surface of hydrogels, wherein the viable cell count at each time point of each group is shown in FIG. 4E and the cell viability is shown in FIG. 4F. The result shows that the chitosan gel modified by oHA can greatly improve the biocompatibility, and the cells on the surface of the chitosan gel have higher survival rate. The EVs@oHA/CS ^gel treatment system can effectively promote the proliferation, spreading and growth of HUVEC on the surface of the HUVEC.

Example 5. Methods for efficacy study of evs@oha/CS ^gel to promote wound healing and microcirculation reconstruction in rats: the SD rats are subjected to type I diabetes modeling by injecting streptozotocin in the abdominal cavity at a rate of 55mg/kg after 12h of empty stomach. Rats with fasting blood glucose values above 11.1mmol/L were considered successful in molding, both at day 3 and day 7 after molding. Shaving the hair of the rat back modeling area, suturing a silica gel fixing ring with the outer diameter of 30mm, the inner diameter of 20mm and the thickness of 2mm by using a surgical thread, and cutting off the whole skin in the area with the diameter of 16mm to obtain the SD rat diabetes wound model.

Each rat in the EVs@oHA/CS ^gel group and oHA/CS ^gel group is respectively smeared with 1.5mL of corresponding hydrogel, 1.5mL of physiological saline containing the same concentration of EVs is dripped into the wound by the EVs solution group, and 1.5mL of physiological saline is dripped into the wound by the control group (untreated group). The above treatments were repeated 2 times per week, photographs of the wound were taken at each administration, and wound area was quantified using image J software. On days 10 and 17, wound blood perfusion was assessed using a laser doppler blood flow imager.

Results: the present example successfully constructed an SD rat diabetic wound model. A typical photograph of a wound of each group during treatment is shown in fig. 5A, and the shape and size of the wound vary as shown in fig. 5B. The average wound area over time curves for each group are shown in fig. 5C, and the wound area reduction values on days 7 and 14 are shown in fig. 5D. The results showed that at day 14, the average wound area of the EVs solution group was only 58.5% of the untreated group, while the average wound area of the EVs@oHA/CS ^gel group was only 26.9% of the untreated group. The results indicate that HemSC-EVs are effective in promoting wound healing, and EVs@oHA/CS ^gel encapsulating EVs have better wound healing promoting effect, probably because the treatment system can be used as a reservoir of EVs to slowly release EVs. Furthermore, by the wound area change profile, it was found that the two treatment groups with EVs added exhibited better healing rates at the second week (day 7 to day 14). This suggests that the pro-angiogenic effect of HemSC-EVs is more potent in promoting overall tissue repair in the mid-to-late stages of wound healing (migration and remodeling phases), consistent with previous literature reports. The laser doppler blood flow imaging pictures are shown in fig. 5E, and the quantitative results of the blood flow perfusion index on day 17 are shown in fig. 5F, which shows that the blood flow perfusion level of the EVs aqueous solution group is 63.1% higher than that of the untreated group, while the blood flow perfusion level of the evs@oha/CS ^gel group is 126.8% higher than that of the untreated group. This result further demonstrates the pro-angiogenic effect of HemSC-EVs, and that the oHA/CS ^gel hydrogel carrier enhances the therapeutic effect of EVs.

Claims (8)

1. A biological dressing, characterized in that the dressing comprises extracellular vesicles derived from hemangioma stem cells (HemSC-EVs) and biomimetic modified chitosan thermosensitive gel (oHA/CS ^gel ). 2. The biological dressing according to claim 1 is characterized in that the hemangioma stem cell-derived extracellular vesicles (HemSC-EVs) are extracted from the serum-free cell culture supernatant of HemSC cultured in vitro, and the cell surface expresses CD9, CD63, and TSG101 proteins and does not express Calnexin protein. 3. The biological dressing according to claim 1, characterized in that the biomimetic modified chitosan thermosensitive gel (oHA/CS ^gel ) is prepared from purified water, chitosan (CS), hyaluronic acid oligosaccharide (oHA), gelatin, hydroxyethyl cellulose (HEC), sodium β-glycerophosphate (β-GP) and sodium bicarbonate (NaHCO ₃ ). 4. The biological dressing according to claim 3 is characterized in that the preparation process comprises stirring and mixing the three aqueous phases in a constant temperature water bath to form a hydrogel pre-liquid after mixing. 5. The biological dressing according to claim 4 is characterized in that, during the preparation process, the amount of CS added is 1-3%, preferably 1.5-2%; the amount of HEC added is 0-0.2%, preferably 0.05-0.1%; the amount of oHA added is 0-0.5%, preferably 0.1-0.25%; the amount of gelatin added is 0-1%, preferably 0.5-0.75%; the amount of β-GP added is 1-6%, preferably 2-3%; the amount of _NaHCO3 added is 0-0.5%, preferably 0.05-0.2%. 6. The biological dressing according to any one of claims 3 to 5, characterized in that, during the preparation process, HemSC-EVs are uniformly dispersed in the hydrogel system, and the amount of HenSC-EV added is 0 to 100 μg/mL in terms of protein content and 0 to 1×10 ¹¹ particles/mL in terms of particle number, preferably 40 to 60 μg/mL in terms of protein content and 4×10 ¹⁰ to 6×10 ¹⁰ particles/mL in terms of particle number. 7. Use of the biological dressing according to any one of claims 1 to 6 in preparing a material for accelerating vascular reconstruction and promoting wound repair. 8. The use according to claim 7, characterized in that the material is used for microcirculation reconstruction of diabetic wounds.