A composite material containing umbilical cord mesenchymal stromal cell exosome combined with ECM and a preparation method and application thereof

Abstract

The embodiment of the application discloses a composite material containing umbilical cord mesenchymal stromal cell exosome combined with ECM and a preparation method and application thereof, and belongs to the technical field of biological medicine. The composite material comprises the following raw materials in parts by weight: 15-25 parts of umbilical cord mesenchymal stromal cell exosome, 50-75 parts of ECM, and 10-20 parts of stabilizer. The composite material provided by the application has excellent wound repair activity, can significantly promote the transformation of a chronic wound from an inflammation period to a proliferation period, accelerate the processes of angiogenesis, granulation tissue formation and epithelialization, and can be applied to the repair of chronic skin wounds such as diabetic foot ulcers, venous ulcers and pressure injuries.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM, its preparation method, and its application in skin wound repair. Background Technology

[0002] As the largest organ in the human body, the skin plays vital roles in protecting the body, regulating body temperature, and sensing external stimuli. Skin wounds, especially chronic wounds such as diabetic foot ulcers, venous ulcers, and pressure injuries, not only affect appearance but can also lead to infection, scarring, and even functional impairment, seriously threatening human health.

[0003] Traditional wound repair methods include surgery, dressing, and drug therapy. However, these methods suffer from problems such as long repair cycles, high scar formation rates, susceptibility to infection, and poor biocompatibility, making it difficult to meet the clinical demand for efficient and safe wound repair materials. In recent years, although some bioactive dressings based on growth factors (such as recombinant human epidermal growth factor and recombinant human basic fibroblast growth factor) have emerged, their clinical efficacy remains unsatisfactory due to the short half-life of growth factors in vivo, their susceptibility to protease degradation, and the difficulty of simulating the complex wound repair microenvironment with a single factor.

[0004] With the development of regenerative medicine, the application of mesenchymal stem cells and their derivatives in tissue repair has attracted much attention. Umbilical cord mesenchymal stromal cells (UC-MSCs) are widely available, easily obtained, and have low immunogenicity. Their secreted exosomes (UC-MSCs-Exos) contain various growth factors (such as EGF, VEGF, FGF, etc.), immunomodulatory factors, and bioactive substances, which can regulate inflammatory responses, promote cell proliferation and migration, and induce angiogenesis, showing good application potential in wound repair. At the same time, the extracellular matrix (ECM), as the microenvironment for cell survival, provides physical support and nutrition for cells, regulates cell proliferation, differentiation, and migration, and plays an important role in wound repair.

[0005] However, the following technical problems exist in the existing technology: (1) The content of repair-related factors in the exosomes secreted by UC-MSCs under conventional culture conditions is limited, which is difficult to meet the repair needs of severe wounds; (2) Exosomes have a short local retention time in the wound and are easily diluted and cleared by body fluids, resulting in low bioavailability; (3) The existing exosome-ECM composite material formulation and preparation process lack systematic optimization, and the synergistic effect of exosomes and ECM has not been fully utilized. Summary of the Invention

[0006] Therefore, embodiments of the present invention provide a composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM, its preparation method and application.

[0007] This invention scientifically combines umbilical cord mesenchymal matrix cell exosomes with ECM, enhances the repair function of exosomes through induced culture, and achieves synergistic effects between the two by optimizing the ratio and preparation process. The resulting wound repair material effectively promotes the transformation of chronic wounds from the inflammatory phase to the proliferative phase, accelerates angiogenesis, granulation tissue formation and epithelialization, significantly shortens healing time, and reduces scar formation rate. It is particularly suitable for chronic wounds such as diabetic foot ulcers, venous ulcers, and pressure injuries.

[0008] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0009] According to a first aspect of the present invention, the present invention provides a composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM, comprising the following raw materials in parts by weight: 15-25 parts of umbilical cord mesenchymal matrix cell exosomes, 50-75 parts of ECM, and 10-20 parts of stabilizer.

[0010] Furthermore, the mass ratio of the umbilical cord mesenchymal matrix cell exosomes to ECM is 1:4-5.

[0011] Furthermore, the method for preparing the umbilical cord mesenchymal matrix cell exosomes includes:

[0012] (1) Take P3-P5 generation umbilical cord mesenchymal stromal cells and expand them in DMEM complete medium containing 10% fetal bovine serum. When the cell confluence reaches 80%-90%, replace it with DMEM complete medium containing 1-2 μM dimethyl oxaloyl glycine, 1-2 μM dexamethasone, and 50-80 μg / mL L-ascorbic acid-2-phosphate for induction culture.

[0013] (2) Collect the supernatant after induction culture and centrifuge it in sequence as follows: centrifuge at 4℃ and 450g for 15 min, centrifuge at 4℃ and 2000g for 20 min, and centrifuge at 4℃ and 10000g for 30 min; filter the supernatant after centrifugation through a 0.45μm sterile filter membrane to obtain the pretreated supernatant.

[0014] (3) Centrifuge the pretreated supernatant at 4°C and 100,000g for 2 hours, discard the supernatant, resuspend the precipitate in sterile PBS, centrifuge again at 4°C and 100,000g for 2 hours, and finally resuspend the precipitate in sterile PBS at a volume ratio of 1:100-200 to obtain the umbilical cord mesenchymal matrix cell exosomes.

[0015] Furthermore, the conditions for induction culture are: static culture at 37℃ in a 5% CO2 incubator for 48-72 hours.

[0016] Furthermore, the method for preparing the ECM includes:

[0017] (1) Take human amniotic membrane tissue, mechanically separate it, remove blood stains and cut it into small pieces, and then immerse it in PBS solution containing 0.1-0.2% (w / v) SDS and 5-10mM EDTA for tissue lysis;

[0018] (2) Discard the lysis buffer, transfer the tissue into a PBS solution containing 1-1.5% (v / v) Triton X-100, shake, and then rinse the tissue repeatedly with sterile PBS until no SDS residue remains in the elution buffer.

[0019] (3) Immerse the tissue in a PBS solution containing 0.1-0.2% (v / v) peracetic acid and 4-5% (v / v) ethanol, shake, and then perform terminal sterilization;

[0020] (4) The tissue was repeatedly rinsed with sterile endotoxin-free PBS to remove residual peracetic acid, and then freeze-dried to obtain the ECM.

[0021] Furthermore, the stabilizer is selected from one or more of trehalose, mannitol, and sucrose.

[0022] Furthermore, the stabilizer is composed of trehalose, mannitol, and sucrose in a mass ratio of 4-8:1:2-3. Studies have found that when the stabilizer is a compound of trehalose, mannitol, and sucrose in the above mass ratio, the three components work synergistically to maintain the stability of the composite material for more than 6 months at 4°C, with an exosome bioactivity retention rate of more than 90%.

[0023] According to a second aspect of the present invention, the present invention provides a method for preparing a composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM as described in any of the preceding claims, the method comprising:

[0024] (1) Dissolve the exosomes of umbilical cord mesenchymal matrix cells in sterile physiological saline at a volume ratio of 1:20-30, and disperse them by ultrasonication under ice bath conditions to obtain an exosome suspension;

[0025] (2) Add ECM to 0.01-0.05M sterile acetic acid solution, mix at 4℃ to prepare 5-15mg / mL ECM solution, add stabilizer, mix at 4℃ to obtain premixed solution;

[0026] (3) Slowly add the exosome suspension obtained in step (1) to the premixed solution obtained in step (2), and mix at 4°C to obtain a mixed solution;

[0027] (4) Adjust the pH of the mixture obtained in step (3) to 7.2-7.4, and filter it through a 0.45μm sterile filter membrane to obtain the composite material.

[0028] According to a third aspect of the present invention, the present invention provides the application of the composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM as described above in the preparation of skin wound repair products.

[0029] Furthermore, the skin wounds include diabetic foot ulcers, venous ulcers, and pressure injuries.

[0030] The embodiments of the present invention have the following advantages:

[0031] 1. This invention uses a culture medium containing dimethyloxaloylglycine (DMOG), dexamethasone, and L-ascorbic acid-2-phosphate to induce the culture of UC-MSCs. DMOG, a prolyl hydroxylase inhibitor, can simulate a hypoxic environment, effectively promoting HIF-1α expression levels and thus upregulating the secretion of angiogenic factors such as VEGF. Dexamethasone can regulate cell differentiation and promote the loading of exosome active payloads. L-ascorbic acid-2-phosphate, as a collagen synthesis cofactor, can improve the secretion quality of exosomes. Under the synergistic effect of these three components, the content of key repair factors such as VEGF, FGF-2, and TGF-β1 in the resulting exosomes is significantly increased, greatly enhancing the pro-angiogenic and cell proliferation-promoting biological activities of exosomes.

[0032] 2. The three-dimensional network structure of ECM provides stable carrier support for exosomes, effectively prolonging the retention time of exosomes at the wound site. Bioactive factors in exosomes can activate the integrin signaling pathway in ECM, promoting cell adhesion and migration to ECM. This invention found that when the mass ratio of exosomes to ECM is 1:4-5, the prepared composite material balances good bioactivity and carrier support, thus exhibiting a better repair effect.

[0033] 3. The preparation method of this invention is highly operable, suitable for industrial production, and has good application prospects. Detailed Implementation

[0034] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Preparation and identification of umbilical cord mesenchymal stromal cells:

[0036] Fresh umbilical cord tissue was rinsed thoroughly with PBS solution, cut into 1cm pieces, and cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The tissue was incubated statically at 37°C with 5% CO2, with the medium changed every 24 hours thereafter, and every 2-3 days thereafter. When cell confluence reached 80%, cells were digested with 0.3% trypsin, centrifuged, and passaged in a 1:3 ratio to new flasks. P3 cells were selected for subsequent experiments.

[0037] Under an optical microscope, the cells showed adherent growth, resembling fibroblasts. Flow cytometry analysis of cell surface markers revealed positive expression of CD29 and CD90 (positive rate >95%), and negative expression of CD34 and CD45 (positive rate <2%). These findings indicate that the extracted cells are umbilical cord mesenchymal matrix cells.

[0038] Example 1

[0039] This embodiment provides a composite material, the raw materials of which are: 15 parts of umbilical cord mesenchymal matrix cell exosomes, 60 parts of ECM, and 15 parts of stabilizer. The stabilizer is composed of trehalose, mannitol and sucrose in a mass ratio of 6:1:2.

[0040] The methods for preparing umbilical cord mesenchymal matrix cell exosomes include:

[0041] (1) Take P3 generation umbilical cord mesenchymal stromal cells and expand them using DMEM complete medium containing 10% fetal bovine serum. When the cell confluence reaches 80%-90%, replace it with DMEM complete medium containing 1 μM dimethyl oxaloyl glycine, 2 μM dexamethasone, and 60 μg / mL L-ascorbic acid-2-phosphate. Incubate statically at 37℃ and 5% CO2 for 60 h.

[0042] (2) Collect the supernatant after induction culture and perform the following treatments in sequence: centrifuge at 4℃ and 450g for 15 min, centrifuge at 4℃ and 2000g for 20 min, and centrifuge at 4℃ and 10000g for 30 min; filter the supernatant after centrifugation through a 0.45μm sterile filter membrane to obtain the pretreated supernatant.

[0043] (3) Centrifuge the pretreated supernatant at 4℃ and 100,000g for 2 hours, discard the supernatant, resuspend the precipitate in an appropriate amount of sterile PBS, centrifuge again at 4℃ and 100,000g for 2 hours, and finally resuspend the precipitate in sterile PBS at a volume ratio of 1:100 to obtain umbilical cord mesenchymal matrix cell exosomes.

[0044] Methods for preparing ECM include:

[0045] (1) Human amniotic membrane tissue obtained by cesarean section was mechanically separated to remove the chorion membrane and blood stains and cut into 1mm pieces.3 After shaving, the fragments were immersed in a PBS solution containing 0.1% (w / v) SDS and 8 mM EDTA and lysed at room temperature with shaking for 24 h.

[0046] (2) Discard the lysis buffer, transfer the tissue into a PBS solution containing 1% (v / v) Triton X-100, and treat with shaking at room temperature for 12 h. Rinse the tissue repeatedly with sterile PBS until there is no SDS residue in the elution buffer.

[0047] (3) Immerse the tissue in a PBS solution containing 0.1% (v / v) peracetic acid and 5% (v / v) ethanol, shake at room temperature for 4 hours, and then perform terminal sterilization.

[0048] (4) The tissue was repeatedly rinsed 6 times with sterile endotoxin-free PBS to remove residual peracetic acid (residual peracetic acid detection <10ppm), and then freeze-dried at -50℃ for 48h to obtain ECM powder, which was then sealed and stored at -20℃.

[0049] The preparation methods of the above composite materials include:

[0050] (1) Dissolve the exosomes of umbilical cord mesenchymal matrix cells in sterile physiological saline at a volume ratio of 1:25, and disperse them by sonication at 40 kHz for 8 min under ice bath conditions to obtain a uniform exosome suspension.

[0051] (2) Add ECM powder to 0.03M sterile acetic acid solution, stir at 4℃ for 1h to prepare 10mg / mL ECM solution, add stabilizer, stir at 4℃ for 0.5h to obtain premixed solution;

[0052] (3) The exosome suspension obtained in step (1) is slowly added dropwise to the premixed solution obtained in step (2) at a rate of 2 drops per second, and stirred at 4°C for 1.5 h to obtain a mixture;

[0053] (4) Adjust the pH of the mixture obtained in step (3) to 7.3 using 0.1M NaOH solution, filter it through a 0.45μm sterile filter membrane to obtain the composite material, and store it at 4℃ after dispensing.

[0054] Example 2

[0055] This embodiment provides a composite material, which differs from Embodiment 1 in that:

[0056] 1. The amount of raw materials used is different. In this example, there are 15 parts of umbilical cord mesenchymal matrix cell exosomes, 75 parts of ECM, and 10 parts of stabilizer. The stabilizer is composed of trehalose, mannitol and sucrose in a mass ratio of 8:1:3.

[0057] 2. The preparation methods for umbilical cord mesenchymal matrix cell exosomes are different. In this example, when the cell confluence rate reaches 80%-90%, the medium is replaced with DMEM complete medium containing 2 μM dimethyl oxaloyl glycine, 1.5 μM dexamethasone, and 80 μg / mL L-ascorbic acid-2-phosphate, and then statically cultured in a 37℃, 5% CO2 incubator for 60 h.

[0058] The remaining operations are the same as in Example 1.

[0059] Example 3

[0060] This embodiment provides a composite material, which differs from Embodiment 1 in that:

[0061] 1. The amount of raw materials used is different. In this example, there are 25 parts of umbilical cord mesenchymal matrix cell exosomes, 50 parts of ECM, and 20 parts of stabilizer. The stabilizer is composed of trehalose, mannitol and sucrose in a mass ratio of 4:1:2.

[0062] 2. The preparation methods for umbilical cord mesenchymal matrix cell exosomes are different. In this example, when the cell confluence rate reaches 80%-90%, the medium is replaced with DMEM complete medium containing 1.5 μM dimethyl oxaloyl glycine, 1 μM dexamethasone, and 50 μg / mL L-ascorbic acid-2-phosphate, and the medium is incubated statically at 37°C and 5% CO2 for 60 h.

[0063] The remaining operations are the same as in Example 1.

[0064] Comparative Example 1

[0065] This comparative example provides a composite material, which differs from Example 1 in that the umbilical cord mesenchymal matrix cell exosomes used were not induced and cultured. That is, in step (1), when the cell confluence rate reaches 80-90%, it is directly replaced with DMEM complete culture medium, and the remaining operations are the same as in Example 1.

[0066] Comparative Example 2

[0067] This comparative example provides a composite material, which differs from Example 1 only in the preparation method of umbilical cord mesenchymal matrix cell exosomes. In this comparative example, when the cell confluence rate reaches 80%-90%, it is replaced with DMEM complete culture medium containing 1 μM dimethyl oxaloyl glycine and 2 μM dexamethasone. The remaining operations are the same as in Example 1.

[0068] Comparative Example 3

[0069] This comparative example provides a composite material, which differs from Example 1 only in the preparation method of umbilical cord mesenchymal matrix cell exosomes. In this comparative example, when the cell confluence rate reaches 80%-90%, it is replaced with DMEM complete culture medium containing 2 μM dexamethasone and 60 μg / mL L-ascorbic acid-2-phosphate. The remaining operations are the same as in Example 1.

[0070] Comparative Example 4

[0071] This comparative example provides a composite material, which differs from Example 1 only in the preparation method of umbilical cord mesenchymal matrix cell exosomes. In this comparative example, when the cell confluence rate reaches 80%-90%, it is replaced with DMEM complete culture medium containing 1 μM dimethyl oxaloyl glycine and 60 μg / mL L-ascorbic acid-2-phosphate. The remaining operations are the same as in Example 1.

[0072] Comparative Example 5

[0073] This comparative example provides a composite material, which differs from Example 1 only in the preparation method of umbilical cord mesenchymal matrix cell exosomes. In this comparative example, when the cell confluence rate reaches 80%-90%, it is replaced with DMEM complete culture medium containing 0.5 μM dimethyl oxaloyl glycine, 2 μM dexamethasone, and 100 μg / mL L-ascorbic acid-2-phosphate. The remaining operations are the same as in Example 1.

[0074] Test Example 1: Exosome Performance Testing

[0075] 1. Morphology and particle size analysis

[0076] The morphology of exosomes was observed using transmission electron microscopy (TEM), and the particle size distribution was detected using nanoparticle tracking analysis (NTA). The results showed that the exosomes prepared in Examples 1-3 exhibited a typical cup-shaped structure under TEM, with a diameter of approximately 30-150 nm; the main peak of the particle size distribution was located at 110-130 nm, and the average particle sizes were 118±12 nm, 122±15 nm, and 125±14 nm, respectively.

[0077] 2. Detection of marker proteins

[0078] Western blotting was used to detect exosome marker proteins. The results showed that the exosomes in Examples 1-3 all expressed exosome-specific markers such as CD9, CD63, CD81, and TSG101.

[0079] 3. Detection of functional factor content

[0080] The contents of VEGF, FGF-2, and TGF-β1 in the exosomes of umbilical cord mesenchymal stromal cells obtained in Examples 1-3 and Comparative Examples 1-5 were detected using an ELISA kit (calculated per mg of exosome protein). The results are shown in Table 1 below.

[0081] Table 1. Content of exosome functional factors (pg / mg exosome protein, n=6) )

[0082]

[0083] The results showed that after induction culture with a specific ratio of DMOG, dexamethasone and L-ascorbic acid-2-phosphate, the contents of VEGF, FGF-2 and TGF-β1 in the exosomes prepared in Examples 1-3 were significantly higher than those in Comparative Examples 1-5 (P<0.01).

[0084] Test Example 2

[0085] The effects of the composite material on the proliferation activity of human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVEC) were detected using the CCK-8 assay.

[0086] HSF and HUVEC were seeded into 96-well plates (5 × 10⁻⁶ m²). 3 After culturing for 24 hours, the composite materials from Examples 1-3 and Comparative Examples 1-5 (final concentration of 10 μg / mL containing exosome protein) were added to each well. After further culturing for 48 hours, 10 μL of LCK-8 solution was added to each well, and the mixture was incubated at 37°C for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the proliferation rate was calculated. The culture medium group without the added material served as a blank control (proliferation rate set at 100%). The results are shown in Table 2 below.

[0087] Table 2. Effects of composite materials on the proliferation activity of HSF and HUVEC (n=6, )

[0088]

[0089] The results showed that the composite materials of Examples 1-3 had a significantly better promoting effect on the proliferation of HSF and HUVEC than the comparative examples.

[0090] Test Example 3: Rat Skin Wound Healing Experiment

[0091] 1. Laboratory animals and grouping

[0092] Eight-week-old male SD rats (weighing 200-220g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in an SPF-grade environment (12h / 12h light / dark cycle, temperature 25±2℃, relative humidity 55±10%) with free access to food and water. The experimental protocol was approved by the animal ethics committee.

[0093] Rats were randomly divided into 9 groups (n=8): treatment groups 1-3, control groups 1-5, and blank control group.

[0094] 2. Preparation and treatment of wound models

[0095] Rats were anesthetized with isoflurane, their back hair was shaved, and after disinfection, a circular full-thickness skin (including subcutaneous fascia) with a diameter of 1.5 cm was removed from the midline of the back to create an acute open wound. After wound preparation, 200 μL of the corresponding material was applied to each group (treatment groups 1-3 were treated with the composite material of Examples 1-3, control groups 1-5 were treated with the composite material of Comparative Examples 1-5, and the blank control group was treated with physiological saline), and then covered with sterile gauze and bandaged. The dressing was changed every 3 days, and the wound healing was observed.

[0096] 3. Evaluation Indicators

[0097] Wound healing rate: Photos were taken on postoperative days 0, 3, 7, 14, and 21. The wound area was calculated using ImageJ software. Healing rate = (original area - unhealed area) / original area × 100%.

[0098] 4. Experimental Results

[0099] (1) Wound healing rate

[0100] The wound healing rate of each group changed over time as shown in Table 3 below.

[0101] Table 3. Wound healing rate of rats in each group (%, n=8). )

[0102]

[0103] The results showed that the composite materials of Examples 1-3 significantly accelerated wound healing, with a healing rate of 85-93% by day 14 and almost complete healing (94-99%) by day 21, which was significantly better than the comparative examples and the blank control (P<0.01). Among them, Examples 1 and 2 showed better results.

[0104] Test Example 4: Diabetic Foot Ulcer Wound Healing Experiment

[0105] 1. Model Preparation

[0106] A type 2 diabetic rat model was induced using a high-fat diet combined with streptozotocin (STZ). Eight-week-old SD rats were fed a high-fat diet for four weeks, followed by an intraperitoneal injection of STZ 35 mg / kg. Successful establishment of the type 2 diabetic rat model was indicated by a fasting blood glucose level >16.7 mmol / L 72 hours later. Rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (3 mL / kg). The sole of the right hind limb was shaved and disinfected. A 1.5 cm diameter full-thickness skin defect (reaching the fascia layer to avoid damage to blood vessels and nerves) was prepared using a sterile punch. The wound was not sutured but only gently covered with sterile gauze for protection, thus establishing a diabetic foot ulcer animal model.

[0107] 2. Experimental Grouping and Treatment

[0108] Rats with successfully induced diabetic foot ulcers were randomly divided into three groups of eight rats each (n=8): Example 1 group, Comparative Example 1 group, and blank control group. Wound management was performed in all groups according to the procedures outlined in Example 3, with daily wound treatment and regular observation of wound healing.

[0109] 3. Results

[0110] Wound healing rate 21 days post-operation: 85.6±3.5% in Example 1 group, 68.3±4.2% in Comparative Example 1 group, and 52.5±3.8% in the blank control group. Example 1 group was significantly better than Comparative Example 1 group and blank control group (P<0.01), demonstrating that the composite material of the present invention has a significant repair effect on refractory wounds such as diabetic foot ulcers.

[0111] Histological examination 14 days postoperatively showed that: in Example 1 group, the epidermal regeneration of the wound was relatively complete, with abundant granulation tissue and regular collagen arrangement; in Comparative Example 1 group, the epidermal regeneration was incomplete, with less granulation tissue; and in the blank control group, the wound healing was poor, with a large number of inflammatory cells infiltrating.

[0112] Test Example 5: Stability Investigation of Composite Materials

[0113] The composite material prepared in Example 1 was stored at 4°C and samples were taken at 0, 1, 2, 3 and 6 months to detect exosome particle size and cell proliferation activity.

[0114] Table 4. Results of stability study of composite materials (n=3, )

[0115]

[0116] The results showed that the composite material provided by this invention maintained stable exosome particle size and bioactivity (activity retention rate >90%) after being stored at 4℃ for 6 months. The stability test results of the composite materials in Examples 2-3 were not significantly different from those in Example 1.

[0117] Test Example 6: Safety Evaluation of Composite Materials

[0118] 1. Cytotoxicity assay

[0119] The MTT assay was used to detect the toxicity of the composite material to L929 mouse fibroblasts. The composite material from Example 1 was diluted with DMEM medium to concentrations of 100%, 50%, 25%, 12.5%, and 6.25%, and treated with L929 cells for 24 hours, and cell viability was detected.

[0120] Results: Cell survival rate was >95% in all concentration groups, and cytotoxicity was graded from 0 to 1, which meets the GB / T 16886.5-2017 standard for biological evaluation of medical devices, proving that the composite material of the present invention is non-cytotoxic.

[0121] 2. Acute systemic toxicity test for subcutaneous implantation

[0122] Twenty healthy Kunming mice, weighing 18-22g, were randomly divided into an experimental group and a control group (n=10). The experimental group was intraperitoneally injected with 50mL / kg of the extract of the composite material from Example 1, while the control group was injected with physiological saline. The general condition, weight changes, and mortality of the mice were observed over 72 hours.

[0123] Results: Both groups of mice were in good condition during the observation period, with no deaths and no significant difference in weight gain (P>0.05), proving that the composite material of Example 1 of the present invention has no acute systemic toxicity.

[0124] The safety evaluation results of the composite materials in Examples 2-3 were not significantly different from those in Example 1.

[0125] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A composite material combining umbilical cord mesenchymal matrix cell exosomes and ECM, characterized in that, The raw materials include the following parts by weight: 15-25 parts of umbilical cord mesenchymal matrix cell exosomes, 50-75 parts of ECM, and 10-20 parts of stabilizer.

2. The composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM according to claim 1, characterized in that, The mass ratio of umbilical cord mesenchymal matrix cell exosomes to ECM is 1:4-5.

3. The composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM according to claim 1, characterized in that, The method for preparing the umbilical cord mesenchymal matrix cell exosomes includes: (1) Take P3-P5 generation umbilical cord mesenchymal stromal cells and expand them in DMEM complete medium containing 10% fetal bovine serum. When the cell confluence reaches 80%-90%, replace it with DMEM complete medium containing 1-2 μM dimethyl oxaloyl glycine, 1-2 μM dexamethasone, and 50-80 μg / mL L-ascorbic acid-2-phosphate for induction culture. (2) Collect the supernatant after induction culture and process it in sequence as follows: centrifuge at 4℃ and 450g for 15min, centrifuge at 4℃ and 2000g for 20min, centrifuge at 4℃ and 10000g for 30min, and filter the supernatant after centrifugation through a 0.45μm sterile filter membrane to obtain the pretreated supernatant. (3) Centrifuge the pretreated supernatant at 4°C and 100,000g for 2 hours, discard the supernatant, resuspend the precipitate in sterile PBS, centrifuge again at 4°C and 100,000g for 2 hours, and finally resuspend the precipitate in sterile PBS at a volume ratio of 1:100-200 to obtain the umbilical cord mesenchymal matrix cell exosomes.

4. The composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM according to claim 3, characterized in that, The induction culture conditions are: static culture at 37℃ in a 5% CO2 incubator for 48-72 hours.

5. The composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM according to claim 1, characterized in that, The method for preparing the ECM includes: (1) Take human amniotic membrane tissue, mechanically separate it, remove blood stains and cut it into small pieces, and then immerse it in PBS solution containing 0.1-0.2% (w / v) SDS and 5-10 mM EDTA for tissue lysis; (2) Discard the lysis buffer, transfer the tissue into a PBS solution containing 1-1.5% (v / v) Triton X-100, shake, and then rinse the tissue repeatedly with sterile PBS until no SDS residue remains in the elution buffer. (3) Immerse the tissue in a PBS solution containing 0.1-0.2% (v / v) peracetic acid and 4-5% (v / v) ethanol, shake, and then perform terminal sterilization; (4) The tissue was repeatedly rinsed with sterile endotoxin-free PBS to remove residual peracetic acid, and then freeze-dried to obtain the ECM.

6. The composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM according to claim 1, characterized in that, The stabilizer is selected from one or more of trehalose, mannitol, and sucrose.

7. The composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM according to claim 6, characterized in that, The stabilizer is composed of trehalose, mannitol, and sucrose in a mass ratio of 4-8:1:2-3.

8. A method for preparing the composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM according to any one of claims 1-7, characterized in that, The method includes: (1) Dissolve the exosomes of umbilical cord mesenchymal matrix cells in sterile physiological saline at a volume ratio of 1:20-30, and disperse them by ultrasonication under ice bath conditions to obtain an exosome suspension; (2) Add ECM to 0.01-0.05M sterile acetic acid solution, mix at 4℃ to prepare 5-15mg / mL ECM solution, add stabilizer, mix at 4℃ to obtain premixed solution; (3) Slowly add the exosome suspension obtained in step (1) to the premixed solution obtained in step (2), and mix at 4°C to obtain a mixed solution; (4) Adjust the pH of the mixture obtained in step (3) to 7.2-7.4, and filter it through a 0.45μm sterile filter membrane to obtain the composite material.

9. The application of the composite material of umbilical cord mesenchymal matrix cell exosomes combined with ECM as described in claim 1 in the preparation of skin wound repair products.

10. The application according to claim 9, characterized in that, The skin wounds include diabetic foot ulcers, venous ulcers, and pressure injuries.

Images