Cell membrane and corresponding medical repair material as well as preparation method and application of cell membrane and corresponding medical repair material

Abstract

The invention relates to a cell membrane and a corresponding medical repair material as well as a preparation method and application thereof. The extraction method of the mesenchymal stem cell membrane comprises the following steps: carrying out cracking, ultrasonication and centrifugation on mesenchymal stem cells to obtain the mesenchymal stem cell membrane. The mesenchymal stem cell membrane can promote wound healing and damage repair of skin barriers, and can be used for preparing skin dressings, medicines for treating wound healing and cosmetics. The obtained mesenchymal stem cell membrane can be used as an effective component for treating wounds and repairing skin, a new situation that the cell membrane is applied to wound healing treatment and skin repairing is developed, and a new choice is provided for skin dressing, wound healing drugs and cosmetics.

Description

Technical Field

[0001] The present invention relates to a cell membrane and a corresponding medical repair material, as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. Background Art

[0002] The skin, as the largest, most widely exposed, and highly sensitive defense barrier of the human body, has the core function of protecting internal organs from external aggression, preventing the invasion of pathogens, and maintaining the dynamic balance of the body's microenvironment. However, once the skin is damaged, its self-healing process is often long and accompanied by multiple complications, including inflammatory response, secondary tissue damage, and a high risk of bacterial infection. Wound healing is an intricate and finely regulated physiological process. When faced with delayed healing conditions such as chronic diabetic wounds or leg venous ulcers, the economic burden and medical challenges they bring are particularly significant. This process can be divided into four closely linked stages: hemostasis, inflammation, proliferation, and remodeling.

[0003] After skin damage, the hemostatic mechanism is immediately triggered. Platelets quickly aggregate and cooperate with blood coagulation to effectively block damaged blood vessels and form a wound scab (eschar) as a preliminary protective barrier. Subsequently, within a few hours, neutrophils are recruited to the scab area. Their main task is to remove invading microorganisms and cell debris, thereby promoting the development of the inflammatory response. In addition, M1 inflammatory macrophages exacerbate the early inflammatory response.

[0004] Entering the proliferation stage, keratinocytes at the edge of the wound begin to actively proliferate and migrate, aiming to rebuild the epidermis of the skin (i.e., re-epithelialization). At the same time, new blood vessels sprout from the existing vascular network (angiogenesis), providing the necessary nutrients and oxygen to the wound area. Fibroblasts also flock to the wound at this stage and differentiate into myofibroblasts, which not only promote collagen synthesis, but also participate in the contraction process of the wound. It is worth noting that as healing progresses, the role of macrophages changes from M1 to M2 with anti-inflammatory function, further assisting various repair mechanisms including angiogenesis.

[0005] Finally, during the remodeling phase, the wound is completely closed and the skin's integrity is restored through the delicate reorganization of collagen fibers within the scar tissue. Particularly noteworthy is that the use of appropriate moist wound dressings has been shown to effectively prevent scab formation and significantly promote wound healing by accelerating re-epithelialization, reducing inflammation, increasing collagen content, and reducing scar hyperplasia.

[0006] The wound healing process is delicate and complex, following four closely interconnected and partially overlapping phases. These intertwined stages form one of the most intricate chapters in the human body's self-repair mechanism. This process not only demonstrates the body's ability to adapt to injury, but also, due to its dynamic nature, presents numerous challenges for wound management. To effectively address these challenges, precisely understanding the characteristics of each wound healing phase and implementing targeted intervention strategies are essential for skin wound treatment. In the initial inflammatory phase, timely anti-inflammatory measures aim to reduce tissue damage and lay a good foundation for subsequent repair. Subsequently, in the proliferative phase, promoting the growth of granulation tissue and accelerating the construction of blood vessels and extracellular matrix are key to accelerating the healing process. Following this, in the re-epithelialization phase, rapid closure of the wound surface is achieved by promoting the migration and proliferation of epidermal cells. Finally, in the remodeling phase, the deposition and arrangement of collagen fibers are optimized, enhancing the strength and elasticity of the wound site, ensuring complete healing and functionality. Therefore, a deep understanding of the wound healing process and precise intervention are crucial for improving the efficiency and quality of wound treatment and promoting rapid patient recovery.

[0007] Although a variety of treatments are currently available for skin wound repair, including traditional skin dressings, vacuum-assisted closure, artificial skin, growth factor therapy, and physical therapies such as photoelectromagnetic therapy, these methods face challenges in their widespread application due to limitations such as high cost, cumbersome procedures, low patient compliance, and potential immune rejection. However, new skin dressings developed in conjunction with new technologies have become an effective method for wound healing due to their ease of use and significant effectiveness.

[0008] The functions of traditional dressings are relatively simple, mainly limited to providing a basic physical barrier, and their limitations are becoming increasingly prominent: insufficient moisturizing properties make it difficult to maintain an appropriate humidity in the wound environment; easy adhesion to new granulation tissue, which may cause secondary trauma when the dressing is changed; lack of sufficient elasticity, which restricts the patient's daily activities; limited ability to absorb exudate, and once soaked, it loses its effective barrier function. These problems not only affect the healing efficiency of the wound, but may also increase the pain and inconvenience of the patient. In view of this, the development of new wound dressings that combine moisturizing properties, excellent elasticity and multifunctionality has become a key issue that urgently needs to be broken through in clinical practice. This type of innovative dressing aims to overcome the various shortcomings of traditional dressings and, through technological empowerment, provide a more ideal microenvironment for wounds that promotes healing, thereby accelerating the patient's recovery process and improving the treatment experience and quality.

[0009] A search revealed that the invention patent application with application number 202410567531.2 and publication number CN118415970A discloses a process for preparing an umbilical cord-derived stem cell exosome skin repair agent. The process comprises the following steps: primary extraction and culture of umbilical cord mesenchymal stem cells; preparation of chitosan modified with a catechol derivative; preparation of oxidized dextran; preparation of black phosphorus nanosheets; preparation of a multi-walled carbon nanotube / polyethyleneimine composite; preparation of a black phosphorus nanosheet composite thermosensitive gel; and loading the black phosphorus nanosheet composite thermosensitive gel with exosomes to produce the skin repair agent. The research results of the present invention are significantly different from those of the present invention, and the preparation process is much simpler. Summary of the Invention

[0010] The primary objective of the present invention is to overcome the problems of the prior art by providing a method for extracting mesenchymal stem cell membranes and the resulting mesenchymal stem cell membranes, which can promote wound healing and repair of skin barrier damage and can be used to prepare skin dressings, wound healing medications, and cosmetics. Also provided are applications of the mesenchymal stem cell membranes and products containing the same.

[0011] The technical solution of the present invention to solve the technical problem is as follows:

[0012] A method for extracting cell membranes of mesenchymal stem cells comprises the following steps:

[0013] Add cell lysis buffer to mesenchymal stem cells and lyse them according to preset lysis conditions; disrupt the cells by ultrasound; centrifuge at 18,000-20,000g for the first time to collect the supernatant, and then centrifuge at 120,000-125,000g for a second time to collect the solids to obtain mesenchymal stem cell membranes.

[0014] This method uses mesenchymal stem cells as raw materials, successfully extracts and obtains mesenchymal stem cell cell membranes, and is simple to operate and easy to implement.

[0015] Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose-derived mesenchymal stem cells, bovine umbilical cord mesenchymal stem cells, bovine placental mesenchymal stem cells, bovine adipose-derived mesenchymal stem cells, sheep umbilical cord mesenchymal stem cells, sheep placental mesenchymal stem cells, or sheep adipose-derived mesenchymal stem cells. This extraction method is suitable for extracting cell membranes from a variety of mesenchymal stem cells and is not limited to the aforementioned specific mesenchymal stem cells.

[0016] Preferably, the number of mesenchymal stem cells: the volume of cell lysate = 1 ± 0.2 × 10 7The method comprises the following steps: preparing 5±1 mL of cell lysate containing 225±25 mM mannitol, 75±10 mM sucrose, 0.5±0.1% bovine serum albumin, 0.5±0.1 mM ethylene glycol ditetraacetic acid, and 1× protease inhibitor in 30±5 mM Tris buffer; performing lysis at 0-4°C for 8-10 hours; ultrasonically disrupting the cells using a cell ultrasonic disruptor; performing the first centrifugation for 20-25 minutes; and performing the second centrifugation at 0-4°C for 50-60 minutes. The obtained mesenchymal stem cell membranes are resuspended in a buffer solution (e.g., PBS) to obtain a mesenchymal stem cell membrane dispersion, which is then stored below -20°C for later use.

[0017] After adopting the above preferred scheme, the specific technical details of the extraction method can be further optimized.

[0018] The mesenchymal stem cell membrane was prepared by the mesenchymal stem cell membrane extraction method described above.

[0019] The mesenchymal stem cell membrane can promote wound healing and repair of skin barrier damage, and can be used to prepare skin dressings, drugs for treating wound healing, and cosmetics.

[0020] The mesenchymal stem cell membrane described above can be used to prepare skin dressings, or to prepare drugs for treating wound healing, or to prepare cosmetics.

[0021] In addition, the present invention also provides:

[0022] A product containing the above-mentioned mesenchymal stem cell membrane is a skin dressing, a medicine, or a cosmetic.

[0023] Preferably, the dosage form of the drug is an external preparation, including creams, tinctures, patches, ointments, cream preparations, gels, sprays, and transdermal absorbents; the specific functions of the drug are to promote wound healing, reduce wound area and healing time, promote angiogenesis at the wound, and restore skin structure; the cosmetics are skin repair or moisturizing cosmetics.

[0024] More preferably, the product is a mesenchymal stem cell membrane hydrogel, and is prepared by the following steps: gelatin and chitosan are added to an acetic acid aqueous solution, heated and stirred to dissolve, to obtain a gelatin solution and a chitosan solution; the gelatin solution and the chitosan solution are mixed, and a mesenchymal stem cell membrane dispersion is added, and after stirring evenly, the mixed solution is placed at ambient temperature to solidify to obtain a preliminary hydrogel; the preliminary hydrogel is immersed in a trisodium citrate aqueous solution for cross-linking, and finally a mesenchymal stem cell membrane hydrogel is obtained.

[0025] The concentration of the acetic acid aqueous solution is 1±0.2% by weight and volume; the mixture is heated and stirred in a water bath magnetic stirrer at 60±1°C to dissolve; the concentration of the gelatin solution is 1±0.2% by weight and volume, the concentration of the chitosan solution is 0.3±0.05% by weight and volume, and the concentration of the mesenchymal stem cell membrane dispersion is 1±0.1 g / mL; the volume ratio of the gelatin solution, chitosan solution, and mesenchymal stem cell membrane dispersion is 50:50±2:5±1; the mixed solution is poured into a sterile culture dish and placed at ambient temperature for 12-18 hours to obtain a preliminary hydrogel; a trisodium citrate aqueous solution with a mass concentration of 20±1% is used and the soaking time is 72-84 hours.

[0026] By adopting this preferred solution, the specific technical details of preparing the mesenchymal stem cell membrane hydrogel can be further optimized.

[0027] More preferably, the product is a mesenchymal stem cell membrane cream, and is prepared by the following steps: white vaseline, liquid paraffin, isopropyl myristate, and stearyl alcohol are mixed and added to a beaker as the oil phase, heated and melted, sodium lauryl sulfate, glycerin, ethylparaben, and distilled water are mixed and poured into the oil phase, stirred and mixed, allowed to cool, and then the mesenchymal stem cell membrane dispersion is added, and stirring is continued until completely emulsified to finally obtain the mesenchymal stem cell membrane cream.

[0028] Among them, the heating and melting temperature is 70±1°C; the stirring and mixing time is 15-20 minutes; the temperature is cooled to 30±1°C; the concentration of the mesenchymal stem cell membrane dispersion is 1±0.1g / mL; the ratio of the mesenchymal stem cell membrane dispersion, white vaseline, liquid paraffin, sodium lauryl sulfate, isopropyl myristate, glycerin, stearyl alcohol, ethylparaben, and distilled water is 5±0.5mL: 2±0.5g: 1.5±0.3g: 0.1±0.05g: 0.5±0.1g: 2±0.5g: 2±0.5g: 0.02±0.01g: 11.8±2g.

[0029] By adopting this preferred solution, the specific technical details of preparing the mesenchymal stem cell membrane cream can be further optimized.

[0030] More preferably, the product is a mesenchymal stem cell membrane spray, and is prepared by the following steps: taking Tween 80, adding sorbitol and anhydrous ethanol, adding mesenchymal stem cell membrane dispersion, and then adding an appropriate amount of distilled water to a preset volume, mixing evenly, stirring with a disperser homogenizer, filtering with a filter, and bottling to obtain the mesenchymal stem cell membrane spray.

[0031] Among them, in the mixed solution after constant volume, the concentration of Tween 80 is 0.005±0.001% by weight, the concentration of sorbitol is 0.5±0.1% by weight, the concentration of anhydrous ethanol is 3±0.5% by weight, and the concentration of mesenchymal stem cell membrane is 10±2% by weight; the preset volume is 50-2000mL; the disperser homogenizer uses a high-speed disperser homogenizer, the stirring speed is 15000-20000r / min, and the stirring time is at least 5 minutes; the pore size of the filter is less than 0.22μm.

[0032] By adopting this preferred solution, the specific technical details for preparing the mesenchymal stem cell membrane spray can be further optimized.

[0033] Compared with the existing technology, the mesenchymal stem cell membrane obtained by the present invention can be used as an effective ingredient for treating wounds and repairing skin, opening up a new situation for the application of cell membranes in treating wound healing and repairing skin, and providing new options for skin dressings, wound healing drugs, and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the tensile stress results of Example 2 of the present invention. In the figure, CM-Gel is a CM-Gel-CS-T composite hydrogel, and Gel is a blank gel.

[0035] Figure 2 This is a graph showing the elongation at break results of Example 2 of the present invention. In the figure, CM-Gel is a CM-Gel-CS-T composite hydrogel, and Gel is a blank gel.

[0036] Figure 3 This is a graph showing the compressive stress results of Example 2 of the present invention. In the figure, CM-Gel is a CM-Gel-CS-T composite hydrogel, and Gel is a blank gel.

[0037] Figure 4 This is a graph showing the compression strain results of Example 2 of the present invention. In the figure, CM-Gel is a CM-Gel-CS-T composite hydrogel, and Gel is a blank gel.

[0038] Figure 5 This is a graph showing the swelling rate results of Example 2 of the present invention. In the figure, CM-Gel is a CM-Gel-CS-T composite hydrogel, and Gel is a blank gel.

[0039] Figure 6 This is a graph showing the diameter of the inhibition zone of Example 2 of the present invention. In the graph, S. aureus is Staphylococcus aureus and E. coli is Escherichia coli.

[0040] Figure 7This is a graph showing the relative cell viability results of Example 2 of the present invention. In the graph, Con is the negative control group, M-Gel is the CM-Gel-CS-T composite hydrogel, and Gel is the blank gel.

[0041] Figure 8 This is a graph showing the inhibition zone diameter results of Example 3 of the present invention. In the graph, S. aureus is Staphylococcus aureus and E. coli is Escherichia coli.

[0042] Figure 9 This is a graph showing the diameter of the inhibition zone of Example 4 of the present invention. In the graph, S. aureus is Staphylococcus aureus and E. coli is Escherichia coli.

[0043] Figure 10 This is a general diagram of the efficacy of the hydrogel in Example 5 of the present invention in treating wounds and ulcers.

[0044] Figure 11 This is a fitting diagram of the hydrogel in Example 5 of the present invention for treating wounds and ulcers.

[0045] Figure 12 These are the H&E and Masson staining results of the wound treated with the hydrogel in Example 5 of the present invention.

[0046] Figure 13 1 is a graph showing the cell survival rate results of Example 6 of the present invention. In the graph, Con is the negative control group, M-Gel is the CM-Gel-CS-T composite hydrogel, and Gel is the blank gel. DETAILED DESCRIPTION

[0047] The method for extracting cell membranes of mesenchymal stem cells specifically implemented in the present invention comprises the following steps:

[0048] Add cell lysis buffer to mesenchymal stem cells and lyse them according to preset lysis conditions; disrupt the cells by ultrasound; centrifuge at 18,000-20,000g for the first time to collect the supernatant, and then centrifuge at 120,000-125,000g for a second time to collect the solids to obtain mesenchymal stem cell membranes.

[0049] Mesenchymal stem cells include human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose-derived mesenchymal stem cells, bovine umbilical cord mesenchymal stem cells, bovine placental mesenchymal stem cells, bovine adipose-derived mesenchymal stem cells, sheep umbilical cord mesenchymal stem cells, sheep placental mesenchymal stem cells, or sheep adipose-derived mesenchymal stem cells. The number of mesenchymal stem cells is as follows: the volume of cell lysate = 1 ± 0.2 × 10 7The method comprises the following steps: preparing 5±1 mL of cell lysate containing 225±25 mM mannitol, 75±10 mM sucrose, 0.5±0.1% bovine serum albumin, 0.5±0.1 mM ethylene glycol ditetraacetic acid, and 1× protease inhibitor in 30±5 mM Tris buffer; performing lysis at 0-4°C for 8-10 hours; ultrasonically disrupting the cells using a cell ultrasonic disruptor; performing the first centrifugation for 20-25 minutes; and performing the second centrifugation at 0-4°C for 50-60 minutes. The obtained mesenchymal stem cell membranes are resuspended in a buffer solution (e.g., PBS) to obtain a mesenchymal stem cell membrane dispersion, which is then stored below -20°C for later use.

[0050] The mesenchymal stem cell membrane of the present invention obtained by the above extraction method can be used to prepare skin dressings, or to prepare medicines for treating wound healing, or to prepare cosmetics.

[0051] The drugs are available in topical formulations, including creams, tinctures, patches, ointments, creams, gels, sprays, and transdermal formulations. Their specific functions include promoting wound healing, reducing wound area and healing time, promoting angiogenesis at the wound site, and restoring skin structure. Cosmetics are skin repair and moisturizing cosmetics.

[0052] The mesenchymal stem cell membrane hydrogel specifically implemented in the present invention is prepared by the following steps:

[0053] Gelatin and chitosan are respectively added to an acetic acid aqueous solution, heated and stirred to dissolve, thereby obtaining a gelatin solution and a chitosan solution; the gelatin solution and the chitosan solution are mixed, and a mesenchymal stem cell membrane dispersion is added thereto. After stirring evenly, the mixed solution is placed at ambient temperature to solidify to obtain a preliminary hydrogel; the preliminary hydrogel is immersed in a trisodium citrate aqueous solution for cross-linking, thereby finally obtaining a mesenchymal stem cell membrane hydrogel.

[0054] The concentration of the acetic acid aqueous solution is 1±0.2% by weight and volume; the mixture is heated and stirred in a water bath magnetic stirrer at 60±1°C to dissolve; the concentration of the gelatin solution is 1±0.2% by weight and volume, the concentration of the chitosan solution is 0.3±0.05% by weight and volume, and the concentration of the mesenchymal stem cell membrane dispersion is 1±0.1 g / mL; the volume ratio of the gelatin solution, chitosan solution, and mesenchymal stem cell membrane dispersion is 50:50±2:5±1; the mixed solution is poured into a sterile culture dish and placed at ambient temperature for 12-18 hours to obtain a preliminary hydrogel; a trisodium citrate aqueous solution with a mass concentration of 20±1% is used and the soaking time is 72-84 hours.

[0055] Note: The above “weight-to-volume ratio” means the number of grams of solute per milliliter of solution. For example, a weight-to-volume ratio of 1% means that every 100 mL of solution contains 1 g of solute, and so on.

[0056] The above preparation process uses natural materials gelatin (Gel) and chitosan (CS) as raw materials, adds mesenchymal stem cell membranes, and adopts an immersion cross-linking strategy to prepare gelatin / chitosan drug-loaded hydrogels with good biocompatibility. The immersion cross-linking strategy enables the amino groups on the gelatin and chitosan molecules to interact electrostatically with citrate ions, forming an intertwined network structure. This makes the hydrogel more compact, has higher tensile strength, significantly improves its compressive strength, and has a higher swelling rate. The addition of mesenchymal stem cell membranes effectively enhances the antibacterial activity of the hydrogel.

[0057] The mesenchymal stem cell membrane hydrogel of the present invention can be used as a skin dressing or as a medicine for treating wound healing.

[0058] The mesenchymal stem cell membrane cream specifically implemented in the present invention is prepared by the following steps:

[0059] White petrolatum, liquid paraffin, isopropyl myristate, and stearyl alcohol were mixed and added to a beaker as the oil phase. After heating and melting, sodium lauryl sulfate, glycerin, ethylparaben, and distilled water were mixed and poured into the oil phase. The mixture was stirred and allowed to cool. The mesenchymal stem cell membrane dispersion was then added and stirred until completely emulsified to obtain a mesenchymal stem cell membrane cream.

[0060] Among them, the heating and melting temperature is 70±1°C; the stirring and mixing time is 15-20 minutes; the temperature is cooled to 30±1°C; the concentration of the mesenchymal stem cell membrane dispersion is 1±0.1g / mL; the ratio of the mesenchymal stem cell membrane dispersion, white vaseline, liquid paraffin, sodium lauryl sulfate, isopropyl myristate, glycerin, stearyl alcohol, ethylparaben, and distilled water is 5±0.5mL: 2±0.5g: 1.5±0.3g: 0.1±0.05g: 0.5±0.1g: 2±0.5g: 2±0.5g: 0.02±0.01g: 11.8±2g.

[0061] The mesenchymal stem cell membrane cream of the present invention can be used as a skin dressing and also as a medicine for treating wound healing.

[0062] The mesenchymal stem cell membrane spray specifically implemented in the present invention is prepared by the following steps:

[0063] Take Tween 80, add sorbitol and anhydrous ethanol, add mesenchymal stem cell membrane dispersion, and then add appropriate amount of distilled water to the preset volume, mix evenly, stir with a disperser homogenizer, filter with a filter, and bottle to obtain the mesenchymal stem cell membrane spray.

[0064] Among them, in the mixed solution after constant volume, the concentration of Tween 80 is 0.005±0.001% by weight, the concentration of sorbitol is 0.5±0.1% by weight, the concentration of anhydrous ethanol is 3±0.5% by weight, and the concentration of mesenchymal stem cell membrane is 10±2% by weight; the preset volume is 50-2000mL; the disperser homogenizer uses a high-speed disperser homogenizer, the stirring speed is 15000-20000r / min, and the stirring time is at least 5 minutes; the pore size of the filter is less than 0.22μm.

[0065] Note: The above “weight-to-volume ratio” means the number of grams of solute per milliliter of solution. For example, a weight-to-volume ratio of 1% means that every 100 mL of solution contains 1 g of solute, and so on.

[0066] The mesenchymal stem cell membrane spray of the present invention can be used as a skin dressing or as a medicine for treating wound healing.

[0067] The present invention will be described in further detail below with reference to the following examples, but the present invention is not limited to the examples given.

[0068] Example 1

[0069] This example is about the extraction and identification of mesenchymal stem cell membranes.

[0070] The specific contents of this embodiment are as follows:

[0071] 1. Extraction of Mesenchymal Stem Cell Membranes

[0072] Human umbilical cord mesenchymal stem cells, human placenta mesenchymal stem cells, human adipose mesenchymal stem cells, bovine umbilical cord mesenchymal stem cells, bovine placenta mesenchymal stem cells, bovine adipose mesenchymal stem cells, sheep umbilical cord mesenchymal stem cells, sheep placenta mesenchymal stem cells, and sheep adipose mesenchymal stem cells were used, and the number of each mesenchymal stem cell was counted as 1×10 7, and were placed in 5 mL of Tris buffer (30 mM) containing mannitol (225 mM), sucrose (75 mM), bovine serum albumin (0.5%), ethylene glycol ditetraacetic acid (0.5 mM) and protease inhibitors (1×), and lysed overnight at 4°C (8-10 h). The cells were ultrasonically disrupted using a cell ultrasonic disruptor. After ultrasonic treatment, the cells were centrifuged at 20,000 g for 20 minutes and the supernatant was collected. The resulting supernatant solution was placed in an ultracentrifuge and centrifuged at 120,000 g for 50 minutes at 4°C to obtain a cell membrane pellet, which is the pure mesenchymal stem cell membrane. It was resuspended in PBS to obtain a mesenchymal stem cell membrane dispersion.

[0073] 2. Particle size analysis of mesenchymal stem cell membrane

[0074] Freshly prepared mesenchymal stem cell membrane dispersion was diluted with double-distilled water and purified using a 400 nm filter to obtain nanoparticles. Subsequently, the size and size distribution of the nanoparticles were analyzed using a laser diffraction (DLS) particle size analyzer.

[0075] 3. Experimental results

[0076] To verify the physical characteristics of the extracted mesenchymal stem cell membranes, particle size and polydispersity coefficient analysis were performed in this example. The results are shown in Table 1 below. As can be seen from the results, the cell membranes obtained from mesenchymal stem cells in this example using the above method had a particle size of approximately 350 nm and a polydispersity coefficient of approximately 0.3.

[0077] Table 1. Cell membrane size and PDI of mesenchymal stem cells

[0078]

[0079] Example 2

[0080] This example describes the preparation and characterization of a cell membrane hydrogel. This example uses a human umbilical cord mesenchymal stem cell membrane hydrogel as an example.

[0081] The specific contents of this embodiment are as follows:

[0082] 1. Preparation of human umbilical cord mesenchymal stem cell membrane hydrogel

[0083] Gelatin and chitosan were added to 50 mL of 1% (w / v) acetic acid aqueous solution, respectively, and placed on a 60°C water bath magnetic stirrer and stirred to dissolve to obtain a viscous solution, forming a final concentration of 1% (w / v) gelatin solution and a final concentration of 0.3% (w / v) chitosan solution. Subsequently, the two solutions were mixed, and 5 mL of human umbilical cord mesenchymal stem cell membrane dispersion with a concentration of 1 g / mL freshly prepared according to Example 1 was added and stirred evenly. The dissolved mixed solution was poured into a sterile square culture dish and placed at room temperature for 12 hours to obtain a CM-Gel-CS composite hydrogel. Finally, the hydrogel was soaked in a 20% trisodium citrate aqueous solution for 72 hours for immersion crosslinking to obtain a human umbilical cord mesenchymal stem cell membrane hydrogel, recorded as CM-Gel-CS-T composite hydrogel.

[0084] Meanwhile, a blank gel group (ie, Gel group) was prepared according to the above preparation process without adding the human umbilical cord mesenchymal stem cell membrane dispersion.

[0085] 2. Characterization Experiment

[0086] 2-1. Mechanical properties test

[0087] (1) Tensile properties test

[0088] The tensile properties test was performed on an EZ-Test series single-column electronic universal testing machine. Before the test, the hydrogel samples were cut into 10 cm × 0.5 cm strips. The thickness of the samples was measured with a micrometer. The clamp spacing was set to 50 mm, and the tensile speed was set to 5 mm / min.

[0089] (2) Compression performance test

[0090] An electronic universal materials testing machine was used to study the effects of varying chitosan and human umbilical cord mesenchymal stem cell membrane content on the compressive properties of hydrogels. The samples were shaped into cylindrical tubes with a diameter of 20 mm and a height of 15 mm. The compression speed was set to 5 mm / min, and the pressure sensor was set to 100 kN.

[0091] 2-2. Swelling performance test

[0092] Liquid absorption capacity is an important indicator for wound dressing evaluation; good liquid absorption capacity facilitates the absorption of wound exudate. 8mm diameter cylindrical samples were cut into cylinders approximately 5mm in height, and their initial weights were measured. The hydrogel samples were immersed in 10mL of PBS at room temperature. The samples were removed from the PBS at regular intervals, dried with filter paper to remove excess moisture, and reweighed.

[0093] 2-3. Antibacterial properties of hydrogels

[0094] Staphylococcus aureus and Escherichia coli were inoculated into nutrient broth and cultured in a constant temperature incubator at 37°C for 24 hours to obtain a bacterial suspension of a certain concentration. The two bacterial suspensions were diluted with sterile PBS to obtain a test bacterial suspension. 1 mL of the test bacterial suspension was added to 150 mL of sterile agar medium and mixed evenly. The agar containing the bacterial suspension was then poured into the upper layer of the solidified agar culture dish. After solidification, a double-layer agar medium containing bacteria was obtained. The hydrogel sample was punched into a disc with a diameter of 15 mm, placed in the center of the double-layer agar medium, and cultured in a constant temperature incubator at 37°C for 24 hours. The antibacterial activity of the hydrogel was qualitatively evaluated by measuring the diameter of the inhibition zone.

[0095] 2-4. Cytotoxicity of Hydrogels

[0096] The cytotoxicity of the hydrogels was studied using the CCK-8 assay, and the effects of different sample extracts on cell viability and proliferation were examined. The hydrogel samples were sterilized under UV irradiation for 30 minutes and then added with 1 mL of complete culture medium for 24 hours to obtain the sample extracts. NIH 3T3 fibroblasts were seeded in a 96-well plate, with 100 μL of cell suspension added to each well. The cell concentration was 5 × 10 3 After cells adhere, replace 100 μL of culture medium with the sample extract (add an equal volume of complete culture medium to the negative control group). After 24 hours of co-culture, remove the extract and add 10 μL of CCK-8 reagent and 100 μL of complete culture medium to each well. Incubate at 37°C in an incubator with 5% CO2 for 2 hours. Place the 96-well plate in a microplate reader and measure its absorbance at 450 nm.

[0097] 3. Experimental results

[0098] The hydrogel was cross-linked using an immersion strategy, which allowed the amino groups on the gelatin and chitosan molecules to interact electrostatically with the citrate ions, forming an entangled network structure, making the hydrogel structure more compact and having a higher tensile strength ( Figures 1 to 2 ), and its compressive strength is significantly improved (increase of 15%-50%) ( Figures 3 and 4 The hydrogel with uniform porous structure is obtained by cross-linking gelatin and chitosan, and the pore size increases with the increase of chitosan content. The swelling rate of the hydrogel can reach 152% ( Figure 5 ).

[0099] The addition of human umbilical cord mesenchymal stem cell membrane effectively improved the antibacterial activity of the hydrogel against Staphylococcus aureus. The human umbilical cord mesenchymal stem cell membrane and the hydrogel had a synergistic antibacterial effect against Staphylococcus aureus and also had a good antibacterial effect against Escherichia coli. The maximum inhibition zone diameter can reach 2.1mm ( Figure 6 ).

[0100] The cytotoxicity test results after co-culture of the hydrogel with NIH T3T cells showed that the cell viability value could reach up to 1.59, indicating that the composite hydrogel would not have adverse effects on cell activity and had good biocompatibility ( Figure 7 ).

[0101] Example 3

[0102] This example is the preparation and characterization of a cell membrane cream. This example uses a bovine placenta mesenchymal stem cell membrane cream as an example.

[0103] The specific contents of this embodiment are as follows:

[0104] 1. Preparation of bovine placenta mesenchymal stem cell membrane cream

[0105] The prescription is: 1 mL of bovine placental mesenchymal stem cell membrane dispersion with a concentration of 1 g / mL prepared according to Example 1, 2 g of white petrolatum, 1.5 g of liquid paraffin, 0.1 g of sodium lauryl sulfate, 0.5 g of isopropyl myristate, 2 g of glycerin, 2 g of stearyl alcohol, 0.02 g of ethylparaben, and 11.8 g of distilled water.

[0106] According to the above prescription, white vaseline, liquid paraffin, isopropyl myristate, and stearyl alcohol are mixed and added to a beaker as the oil phase, and heated to 70°C until melted. At this time, sodium lauryl sulfate, glycerin, ethylparaben, and distilled water are mixed and poured into the oil phase. The mixture is quickly and thoroughly stirred for 15 minutes. After cooling to 30°C, bovine placental mesenchymal stem cell membranes are added, and stirring is continued until completely emulsified to obtain a bovine placental mesenchymal stem cell membrane cream.

[0107] 2. Characterization Experiment

[0108] 2-1. Appearance and shape

[0109] Observe the appearance, color and shape of the cream obtained above with the naked eye and record them.

[0110] 2-2. Viscosity measurement

[0111] The cream obtained above was measured using a NDJ-8S digital rotational viscometer, with the rotation speed adjusted to 15 rpm, the opening angle stabilized at 15%-85%, and the reading was taken after the data stabilized.

[0112] 2-3. pH determination

[0113] Weigh 1 g of the cream obtained above, add 5 mL of double-distilled water, stir evenly until a white emulsion is formed, and measure the pH value using a pH meter.

[0114] 2-4. Antibacterial properties

[0115] Staphylococcus aureus and Escherichia coli were inoculated into nutrient broth and cultured in a constant temperature incubator at 37°C for 24 hours to obtain a bacterial suspension of a certain concentration. The two bacterial suspensions were diluted with sterile PBS to obtain a test bacterial suspension. 1 mL of the test bacterial suspension was added to 150 mL of sterile agar medium and mixed evenly. The agar containing the bacterial suspension was then poured into the upper layer of the solidified agar culture dish. After solidification, a double-layer agar medium containing bacteria was obtained. The above-obtained cream was taken as a sample and smeared on the center of the double-layer agar medium and cultured in a constant temperature incubator at 37°C for 24 hours. The antibacterial activity of the above-obtained cream was qualitatively evaluated by measuring the diameter of the inhibition zone.

[0116] 3. Experimental results

[0117] After three batches of reproducibility experiments, the bovine placenta mesenchymal stem cell membrane cream prepared in this example was milky white, semi-solid in viscosity, with a pH close to neutral, and a stable formulation and process (Table 2). The bovine placenta mesenchymal stem cell membrane cream had a synergistic antibacterial effect against Staphylococcus aureus and also had a good antibacterial effect against Escherichia coli, with a maximum inhibition zone diameter of 1.8 mm ( Figure 8 ).

[0118] Table 2. Properties, viscosity, and pH of bovine placenta mesenchymal stem cell membrane cream

[0119]

[0120] Example 4

[0121] This example describes the preparation and characterization of a cell membrane spray. This example uses a sheep adipose-derived mesenchymal stem cell membrane spray as an example.

[0122] The specific contents of this embodiment are as follows:

[0123] 1. Preparation of Sheep Adipose-Derived Mesenchymal Stem Cell Membrane Spray

[0124] 0.005% of the prescribed amount of Tween 80 was measured, 0.5% of the prescribed amount of sorbitol and 3% of the prescribed amount of anhydrous ethanol were added, 5 mL of the sheep adipose mesenchymal stem cell membrane dispersion with a concentration of 1 g / mL prepared according to Example 1 was added, and then an appropriate amount of distilled water was added to make the volume 50 mL. The above formula was thoroughly mixed, stirred at a speed of 15000 r / min for 5 minutes using a high-speed disperser homogenizer, filtered using a 0.22 μm filter, and bottled to obtain a sheep adipose mesenchymal stem cell membrane spray.

[0125] 2. Characterization Experiment

[0126] 2-1. Observation of the properties of the spray

[0127] After the spray obtained above was packaged, its properties, solubility and viscosity were observed and recorded.

[0128] 2-2. Centrifugal stability of spray

[0129] Take an appropriate amount of the spray obtained above into a centrifuge tube, centrifuge at 10000 r / min for 30 min, and observe its properties.

[0130] 2-3. pH determination of spray

[0131] 1 mL of the spray obtained above was placed in a beaker, and the pH value was measured using a pH meter.

[0132] 2-4. Antibacterial properties of sprays

[0133] Staphylococcus aureus and Escherichia coli were inoculated into nutrient broth and cultured in a constant temperature incubator at 37°C for 24 hours to obtain a bacterial suspension of a certain concentration. The two bacterial suspensions were diluted with sterile PBS to obtain a test bacterial suspension. 1 mL of the test bacterial suspension was added to 150 mL of sterile agar medium and mixed evenly. The agar containing the bacterial suspension was then poured into the upper layer of the solidified agar culture dish. After solidification, a double-layer agar medium containing bacteria was obtained. The spray obtained above was applied as a sample to the center of the double-layer agar medium and cultured in a constant temperature incubator at 37°C for 24 hours. The antibacterial activity of the spray obtained above was qualitatively evaluated by measuring the diameter of the inhibition zone.

[0134] 3. Experimental results

[0135] After three batches of reproducibility experiments, the sheep adipose-derived mesenchymal stem cell membrane spray prepared in this example showed good appearance, solubility, viscosity and centrifugal stability, and the pH value was close to neutral (partial results are shown in Table 3). The sheep adipose-derived mesenchymal stem cell membrane spray had a synergistic antibacterial effect against Staphylococcus aureus and also had a good antibacterial effect against Escherichia coli, with the maximum inhibition zone diameter reaching 2 mm ( Figure 9 ).

[0136] Table 3. Properties, solubility, viscosity, and pH of sheep adipose-derived mesenchymal stem cell membrane spray

[0137]

[0138] Example 5

[0139] This example is an animal study on the efficacy of mesenchymal stem cell membranes in treating wound healing. This example uses human umbilical cord mesenchymal stem cell membrane hydrogels prepared according to the method of Example 2 as an example.

[0140] The specific contents of this embodiment are as follows:

[0141] 1. Experimental Animals

[0142] The experimental animals were male C57BL / 6J mice, 6-7 weeks old and weighing 18-20 g, purchased from the Comparative Medical Center of Yangzhou University. C57BL / 6J mice were housed under normal conditions: a temperature of 25±2°C, a humidity of 50%-70%, and a 12-hour light and dark cycle. They had free access to food and water, and their bedding was changed every two days. Experiments began after 7 days of acclimatization.

[0143] 2. Evaluation of the therapeutic effect of human umbilical cord mesenchymal stem cell membrane hydrogel on diabetic foot ulcer model mice

[0144] After anesthesia, the mice were fixed on the operating table. The wound was first disinfected with an alcohol cotton ball, and a 10mm diameter wound was cut on the back of the mouse using a punch. The treatment group was treated with human umbilical cord mesenchymal stem cell membrane hydrogel every day, while the control group was treated with a blank gel. The mice were then housed in an SPF-grade clean animal room, and the recovery of the wound was observed daily. The size of the wound was measured with a ruler and photographed. After the wound was fully recovered, the mice were killed by cervical dislocation, and the new tissue was removed and placed in a 4% paraformaldehyde solution at 4°C for storage.

[0145] 3. Pathological testing

[0146] 3-1. Paraffin-embedded sections

[0147] After sampling, the skin tissues were washed three times with PBS buffer and fixed in 4% paraformaldehyde solution at 25°C for 48 h.

[0148] 1) Dehydration: Soak the tissue in 75% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II, respectively, for 10 min each.

[0149] 2) Transparency: Soak the dehydrated tissue in 1 / 2 xylene II, xylene I, and xylene II for 10 minutes in sequence.

[0150] 3) Wax embedding: Immerse the sample fully in liquid paraffin at 60℃-65℃ (2h×2 times). Pay attention to controlling the temperature to prevent the loss of tissue protein. At this time, the sample can be embedded into a cube.

[0151] 4) Sectioning: Freeze the paraffin block at -20°C for 30 min, cut the tissue in the paraffin block into 5 μm thick slices using a microtome, develop the slices in a 50°C water bath, and fish out the slices.

[0152] 5) Baking: Place the slices in a 60°C oven for 4 hours and then take them out for later use.

[0153] 3-2. Pathological staining

[0154] The dried sections were placed in a xylene solution and soaked for 15 minutes twice. After being taken out, they were placed in anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol solutions for 5 minutes, and then rinsed with distilled water for 3 minutes to dewax. The sections were placed in a boiling 0.01M sodium citrate solution (pH 6.0) and soaked for 2 minutes. The sections were taken out and rinsed with distilled water twice, each time for 3 minutes to complete antigen retrieval. After cooling, they were rinsed with PBS buffer, the PBS solution was discarded, and 1 drop (50μL) of 3% H2O2 was added to each section and incubated at room temperature for 10 minutes to block the endogenous peroxidase activity.

[0155] The sections were rinsed with PBS for 5 min, and then the subsequent antigen-antibody incubation process was carried out according to the instructions of the HE and MASSON staining kits. Finally, after decolorization and sealing, the results were observed under a microscope and photographed for record.

[0156] 4. Experimental results

[0157] The wounds were treated with human umbilical cord mesenchymal stem cell membrane hydrogel. The wound recovery was observed daily, and the wound size was measured with a ruler and photographed. Statistical analysis of the data showed that the wounds in the cell membrane treatment group healed completely in about 14 days. Meanwhile, the blank gel group and the PBS group had larger wounds ( Figure 10 The wound surface was measured and photographed daily, and the area was calculated using ImageJ software ( Figure 11 ), the cell membrane treatment group can greatly shorten the wound healing time of animals compared with the control group, and significantly accelerate the healing speed of diabetic foot wounds. According to the results of H&E staining, it can be observed that the cell membrane treatment group can restore the structure of the skin and reduce the diameter of the wound. According to the results of Masson staining, it can be observed that the cell membrane treatment group can promote the accumulation of collagen in the wound site and promote wound healing ( Figure 12 ).

[0158] 5. Based on the contents of this embodiment and embodiment 2, it can be seen that: (1) In order to construct a hydrogel dressing with antibacterial effect and biocompatibility, the present invention uses natural materials gelatin (Gel) and chitosan (CS) as raw materials, adds human umbilical cord mesenchymal stem cell cell membrane, and adopts an immersion cross-linking strategy to prepare a gelatin / chitosan drug-loaded hydrogel antibacterial dressing. (2) The hydrogel of the present invention has a uniform porous structure. The mechanical property test results show that the immersion cross-linking strategy increases the compressive strength of the hydrogel by 15%-50%, the swelling rate is as high as 152%, and the maximum cell viability value is 1.59, with good biocompatibility and blood compatibility. The hydrogel of the present invention has a strong antibacterial ability against Staphylococcus aureus and Escherichia coli. The results of the mouse wound model healing experiment show that the hydrogel of the present invention helps to accelerate wound healing. Therefore, the hydrogel dressing of the present invention has great application potential in wound healing and drug release.

[0159] Example 6

[0160] This example is a study on the promotion of HaCat cell proliferation by mesenchymal stem cell membranes. This example uses the human umbilical cord mesenchymal stem cell membrane dispersion prepared according to the method of Example 1 as an example.

[0161] 1. CCK-8 detection of HaCaT cell proliferation

[0162] HaCaT cells were plated at 1×10 4 Cells were seeded at a density of 100 μL of H-DMEM medium containing 10% FBS per well in a 96-well plate. 100 μL of H-DMEM medium containing 10% FBS was added to each well and the plate was placed in an incubator and cultured until the cells adhered. 1 μL of the 1 g / mL human umbilical cord mesenchymal stem cell membrane dispersion prepared according to Example 1 was added to each well and the plate was cultured in an incubator for 24 hours. The supernatant was removed and 10 μL of CCK-8 reagent and 100 μL of complete culture medium were added to each well. The plate was incubated at 37°C in an incubator containing 5% CO2 for 2 hours. The 96-well plate was placed in a microplate reader and the absorbance at 450 nm was measured.

[0163] 2. Experimental results

[0164] Keratinocytes are the main component of the outer layer of the skin. They are tightly connected and intertwined, forming a physical barrier that effectively prevents harmful substances from entering the skin. When the skin is damaged, such as by ultraviolet rays, chemicals or physical stimulation, the protective barrier function of the stratum corneum may be damaged, leading to dryness, scaling, redness and other phenomena. Figure 13 As shown, human umbilical cord mesenchymal stem cell membranes can promote the proliferation of HaCaT cells, which indicates that human umbilical cord mesenchymal stem cell membranes can promote the repair of skin barrier damage to a certain extent and have application value in cosmetics and skin care products.

[0165] Based on the above specific implementation contents:

[0166] (1) The present invention extracts mesenchymal stem cell membranes from mesenchymal stem cells of various animal and tissue sources. The cell membranes are used as active ingredients to prepare dressings in the form of hydrogels, creams, sprays, etc., and their physical and chemical properties are characterized. The results of a wound healing experiment using a mouse model show that the dressing containing the cell membranes helps accelerate wound healing. The results of an in vitro co-culture experiment with HaCaT cells show that the dressing containing the cell membranes helps promote the repair of HaCaT cell damage. Therefore, the cell membranes can be used as an active ingredient for treating wound healing and in cosmetics.

[0167] (2) The dressing prepared using the mesenchymal stem cell membrane of the present invention is applied to the wound surface. First, it can play a protective role as a barrier, isolating the wound surface from contact with the external environment and preventing dust and bacterial infection. Second, it can combine with matrix molecules such as collagen and fibrin to form a temporary scaffold that supports cell migration and adhesion, promote collagen production and tissue repair. At the same time, it can also partially penetrate the wound tissue to form a supporting medium between the cells of the damaged tissue, fill the gaps between the damaged cells, restore the dense interstitial tissue, prevent the penetration of damaged cell tissue fluid, and accelerate wound healing. Therefore, the dressing prepared using the mesenchymal stem cell membrane of the present invention can effectively treat wound healing; and has a skin repair effect and can be used in the field of cosmetics.

[0168] (3) The key technical points of the present invention are:

[0169] i) In animal experiments, the cell membrane-containing dressing of the present invention showed significant promotion of wound healing, restoration of normal skin structure, and a positive effect on the treatment of wound healing.

[0170] ii) The cell membrane-containing dressing of the present invention has good morphology, excellent mechanical properties and other physical properties.

[0171] iii) The cell membrane-containing dressing of the present invention has good in vitro biocompatibility and can effectively inhibit bacterial proliferation.

[0172] iv) The cell membrane-containing dressing of the present invention can promote the damage repair of HaCaT and can be applied in the field of cosmetics.

[0173] In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the present invention.

Claims

1. A method for extracting cell membranes of mesenchymal stem cells, characterized in that: The following steps are involved: Add cell lysis buffer to mesenchymal stem cells and lyse them according to preset lysis conditions; disrupt the cells by ultrasound; centrifuge at 18,000-20,000g for the first time to collect the supernatant, and then centrifuge at 120,000-125,000g for a second time to collect the solids to obtain mesenchymal stem cell membranes.

2. The method for extracting cell membranes of mesenchymal stem cells according to claim 1, wherein: The mesenchymal stem cells are human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose mesenchymal stem cells, bovine umbilical cord mesenchymal stem cells, bovine placental mesenchymal stem cells, bovine adipose mesenchymal stem cells, sheep umbilical cord mesenchymal stem cells, sheep placental mesenchymal stem cells, or sheep adipose mesenchymal stem cells.

3. The method for extracting cell membranes of mesenchymal stem cells according to claim 1, wherein: The number of mesenchymal stem cells: the volume of cell lysate = 1 ± 0.2 × 10 7 The method comprises the following steps: adding 5±1 mL of the cell lysate containing 225±25 mM mannitol, 75±10 mM sucrose, 0.5±0.1% bovine serum albumin, 0.5±0.1 mM ethylene glycol ditetraacetic acid, and 1× protease inhibitor in 30±5 mM Tris buffer; the preset lysis conditions are lysis at 0-4°C for 8-10 hours; ultrasonic cell disruption is performed using a cell ultrasonic disruptor; the first centrifugation is performed for 20-25 minutes; the second centrifugation is performed at a temperature of 0-4°C and for 50-60 minutes; the obtained mesenchymal stem cell membranes are resuspended in the buffer to obtain a mesenchymal stem cell membrane dispersion, which is stored below -20°C for later use. 4 . The mesenchymal stem cell membrane prepared by the method for extracting mesenchymal stem cell membrane according to claim 1 .

5. Use of the mesenchymal stem cell membrane according to claim 4 in preparing skin dressings, or in preparing wound healing medicines, or in preparing cosmetics.

6. A product containing the mesenchymal stem cell membrane according to claim 4, wherein the product is a skin dressing, a medicine, or a cosmetic.

7. The product according to claim 6, characterized in that The dosage form of the drug is an external preparation, including ointment, tincture, patch, ointment, cream preparation, gel, spray, and transdermal absorbent; the specific function of the drug is to promote wound healing, reduce wound area and healing time, promote angiogenesis at the wound, and restore skin structure; the cosmetic is a skin repair or moisturizing cosmetic.

8. The product according to claim 7, characterized in that: The product is a mesenchymal stem cell membrane hydrogel, and is prepared by the following steps: adding gelatin and chitosan to an acetic acid aqueous solution, heating and stirring to dissolve, to obtain a gelatin solution and a chitosan solution; mixing the gelatin solution and the chitosan solution, adding a mesenchymal stem cell membrane dispersion, stirring evenly, and placing the mixed solution at ambient temperature to solidify to obtain a preliminary hydrogel; immersing the preliminary hydrogel in a trisodium citrate aqueous solution for cross-linking, and finally obtaining a mesenchymal stem cell membrane hydrogel; The concentration of the acetic acid aqueous solution is 1±0.2% by weight to volume; the mixture is placed on a water bath magnetic stirrer at 60±1°C for heating, stirring and dissolution; the concentration of the gelatin solution is 1±0.2% by weight to volume, the concentration of the chitosan solution is 0.3±0.05% by weight to volume, and the concentration of the mesenchymal stem cell membrane dispersion is 1±0.1 g / mL; the volume ratio of the gelatin solution, chitosan solution and mesenchymal stem cell membrane dispersion is 50:50±2:5±1; the mixed solution is poured into a sterile culture dish and placed at ambient temperature for 12-18 hours to obtain a preliminary hydrogel; a trisodium citrate aqueous solution with a mass concentration of 20±1% is used and the soaking time is 72-84 hours.

9. The product according to claim 7, characterized in that: The product is a mesenchymal stem cell membrane cream, and is prepared by the following steps: white vaseline, liquid paraffin, isopropyl myristate, and stearyl alcohol are mixed and added to a beaker as an oil phase; after heating and melting, sodium lauryl sulfate, glycerin, ethyl hydroxybenzoate, and distilled water are mixed and poured into the oil phase; the mixture is stirred and evenly mixed; after cooling, a mesenchymal stem cell membrane dispersion is added; and stirring is continued until completely emulsified, thereby obtaining the mesenchymal stem cell membrane cream; The heating and melting temperature is 70±1°C; the stirring and mixing time is 15-20 minutes; the mixture is cooled to 30±1°C; the concentration of the mesenchymal stem cell membrane dispersion is 1±0.1g / mL; the ratio of the mesenchymal stem cell membrane dispersion, white vaseline, liquid paraffin, sodium lauryl sulfate, isopropyl myristate, glycerin, stearyl alcohol, ethylparaben, and distilled water is 5±0.5mL: 2±0.5g: 1.5±0.3g: 0.1±0.05g: 0.5±0.1g: 2±0.5g: 2±0.5g: 0.02±0.01g: 11.8±2g.

10. The product according to claim 7, characterized in that: The product is a mesenchymal stem cell membrane spray, and is prepared by the following steps: taking Tween 80, adding sorbitol and anhydrous ethanol, adding a mesenchymal stem cell membrane dispersion, and then adding an appropriate amount of distilled water to a preset volume, mixing evenly, stirring with a disperser homogenizer, filtering with a filter, and bottling to obtain the mesenchymal stem cell membrane spray; In which, in the fixed volume mixed solution, the concentration of Tween 80 is 0.005±0.001% by weight-volume ratio, the concentration of sorbitol is 0.5±0.1% by weight-volume ratio, the concentration of anhydrous ethanol is 3±0.5% by weight-volume ratio, and the concentration of mesenchymal stem cell membrane is 10±2% by weight-volume ratio; the preset volume is 50-2000 mL; the dispersing homogenizer adopts a high-speed dispersing homogenizer, the stirring speed is 15000-20000 r / min, and the stirring time is at least 5 minutes; the pore size of the filter is less than 0.22 μm.

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