Extracellular vesicle gel with high anti-inflammatory, anti-fibrotic activity

Abstract

The application provides a stem cell exosome gel with high anti-inflammatory and anti-fibrosis activity and a preparation method thereof, which comprises a polymer gel, nano calcium oxide, nano silver and stem cell exosomes dispersed in the polymer gel; and the polymer gel is a light-induced polymer gel of methacrylated polylysine (epsilon-PL-MA), carboxymethyl chitosan (CMCS) and itaconic acid. The stem cell exosome gel with high anti-inflammatory and anti-fibrosis activity is prepared by light-induced polymerization of methacrylated polylysine (epsilon-PL-MA), carboxymethyl chitosan (CMCS) and itaconic acid to form a polymer gel, and then nano calcium oxide, nano silver and epidermal stem cell exosomes are dispersed in the polymer gel. The stem cell exosome gel with high anti-inflammatory and anti-fibrosis activity is coated on the epidermis, can inhibit epidermal cell fibrosis, and has an anti-inflammatory effect.

Description

Technical Field

[0001] This invention relates to the application of stem cell secretions, specifically to a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity. Background Technology

[0002] Epidermal cell fibrosis disrupts the normal structure and function of the epidermis. A normal epidermis forms a physical barrier through the stratum corneum (multiple layers of flattened keratinocytes and intercellular lipids), preventing moisture loss and the invasion of external pathogens and irritants. In epidermal cell fibrosis: The stratum corneum becomes abnormal: Disordered differentiation of keratinocytes may lead to hyperkeratosis (thickening and scaling of the stratum corneum) or parakeratosis (accumulation of immature keratinocytes), resulting in a loosened barrier structure, increased transepidermal water loss (TEWL), and dry, flaky, and sensitive skin. Epidermal cell fibrosis can also disrupt cell junctions: The intercellular junctions (such as desmosomes and tight junctions) are damaged due to fibrosis-related EMT (epithelial-mesenchymal transition) (e.g., reduced E-cadherin), increasing intercellular spaces. This allows external allergens (such as pollen and chemicals) or pathogens to more easily penetrate the epidermis, inducing inflammation or infection (such as recurrent eczema and folliculitis).

[0003] Epidermal fibrosis is often accompanied by a chronic inflammatory state: activated keratinocytes excessively secrete pro-inflammatory factors (such as L-1β, TNF-α, and TGF-β), continuously stimulating dermal immune cells (such as mast cells and lymphocytes), triggering an inflammatory response manifested as erythema and swelling. Inflammation further drives keratinocyte activation, exacerbating fibrosis and forming a vicious cycle of "inflammation-fibrosis." At the same time, inflammatory mediators (such as histamine and neuropeptides) stimulate nerve endings in the skin, leading to intense itching. Repeated scratching mechanically damages the epidermis, exacerbating fibrosis and inflammation, and severely impacting quality of life. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity, wherein the stem cell extravesicular gel comprises a polymer gel, nano-calcium oxide, nano-silver and stem cell extravesicular vesicles dispersed in the polymer gel;

[0006] The polymer gel is a photoinitiated polymer gel composed of methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS), and itaconic acid.

[0007] The molecular structure of the polymer gel is as follows:

[0008]

[0009] Where a and b are the amounts of polymerizable monomers of methacrylamide polylysine (ε-PL-MA) and itaconic acid, respectively, with 15≤a≤50 and 20≤b≤80.

[0010] x represents the amount of lysine monomer in methacrylated polylysine (ε-PL-MA), where 20 ≤ x ≤ 40.

[0011] y represents the amount of six-membered ring monomer in carboxylated chitosan (CMCS), where 85 ≤ x ≤ 150.

[0012] The aforementioned stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is formed by photoinitiation of methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS), and itaconic acid to form a polymer gel. Nano-calcium oxide, nano-silver, and epidermal stem cell extravesicular vesicles are dispersed in the polymer gel. When the aforementioned stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is coated on the epidermis, it can inhibit epidermal cell fibrosis and has an anti-inflammatory effect.

[0013] Preferably, the content of stem cell extravesicles in the stem cell extravesicle gel is 20-200 mg / mL.

[0014] Preferably, the content of stem cell extravesicles in the stem cell extravesicle gel is 50-80 mg / mL.

[0015] Through research, the inventors discovered that stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity, when the content of stem cell extravesicular vesicles is 50-80 mg / mL, has a better effect on inhibiting epidermal cell fibrosis and anti-inflammation.

[0016] Preferably, the content of nano-calcium oxide in the stem cell extravesicular gel is 5-20 mg / mL.

[0017] Preferably, the content of nano-silver in the stem cell extravesicular gel is 3-15 mg / mL.

[0018] Through research, the inventors discovered that stem cell extravesicular gels with high anti-inflammatory and anti-fibrotic activity, when containing 3–15 mg / mL of nano-silver, have a better effect on inhibiting epidermal cell fibrosis and anti-inflammation.

[0019] Preferably, the stem cell vesicles are human epidermal stem cell vesicles; the preparation method of human epidermal stem cell vesicles includes the following steps: (a) human epidermal stem cells are cultured in a cell culture incubator at 36-38°C and 4.5-6% CO2 using DMEM basal medium, with 0.8%-1.2% penicillin and 8%-12% serum added; (2) when the confluence of human epidermal stem cell vesicles (MSCs) reaches 70%-80%, the cells and cell debris are removed by centrifugation, the precipitate is resuspended, and the human epidermal stem cell vesicle precipitate is collected.

[0020] In the preferred method for preparing human epidermal stem cell vesicles, (2) when the confluence of human epidermal stem cell vesicles (MSCs) reaches 70% to 80%, the complete culture medium is replaced with a vesicle-free special culture medium. After 48 hours, the cell supernatant is collected and centrifuged at 300g for 10 min and 2000g for 10 min at 4°C to remove cells and cell debris. Then, the supernatant is centrifuged at 10,000g for 30 min to remove impurities such as large vesicles. Subsequently, the supernatant is centrifuged at 100,000g at 4°C for 70 min, and the supernatant is completely discarded. Finally, the precipitate is resuspended with PBS and centrifuged at 100,000g at 4°C for 70 min to obtain human epidermal stem cell vesicle precipitate. The precipitate is resuspended with an appropriate amount of PBS according to its size and stored in a -80°C refrigerator for later use.

[0021] This invention also provides a method for preparing the stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity as described above, the preparation method comprising the following steps:

[0022] (1) Methacrylated polylysine (ε-PL-MA), carboxylated chitosan (CMCS), itaconic acid, nano-calcium oxide, nano-silver, and phosphate buffer solution of stem cell vesicles were passed into the droplet microfluidic aqueous phase channel to obtain solidified micron-sized hydrogel microspheres.

[0023] (2) The solidified micron-sized hydrogel microspheres were photo-initiated at 360nm~410nm for 10~50s to obtain stem cell extravesicle gel with high anti-inflammatory and anti-fibrotic activity.

[0024] Preferably, in step (1), methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS), itaconic acid, nano-calcium oxide, nano-silver, and stem cell vesicles are dispersed in a phosphate buffer solution in a weight ratio of 10–50 mmol, and the pH of the phosphate buffer solution is 7.0–7.5.

[0025] Preferably, the silver nanoparticles are formed by reducing silver ions to silver nanoparticles using ethanol or glucose as a reducing agent, and the particle size of the silver nanoparticles is 5–50 nm.

[0026] Preferably, the particle size of nano-calcium oxide is 10–50 nm.

[0027] The beneficial effects of this invention are as follows: This invention provides a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity. This stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is formed by photoinitiation of methacryloyl-polylysine (ε-PL-MA), carboxylated chitosan (CMCS), and itaconic acid to form a polymeric gel. Nano-calcium oxide, nano-silver, and epidermal stem cell extravesicular vesicles are dispersed in the polymeric gel. When this stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is coated on the epidermis, it can inhibit epidermal cell fibrosis and also has an anti-inflammatory effect. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the chemical structure of the polymer gel in the stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to an embodiment of the present invention.

[0029] Figure 2 The infrared spectrum of the polymer gel in the stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is shown in the embodiment of the present invention.

[0030] Figure 3 The infrared spectrum of the polymer gel in the stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is shown in the comparative example of the present invention. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Example 1

[0033] As an embodiment of the present invention, a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is provided, wherein the stem cell extravesicular gel comprises a polymer gel, nano-calcium oxide, nano-silver and stem cell extravesicular vesicles dispersed in the polymer gel;

[0034] The polymer gel is a photoinitiated polymer gel composed of methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS), and itaconic acid.

[0035] The molecular structure of the polymer gel is as follows:

[0036]

[0037] Where a and b are the amounts of polymerizable monomers of methacrylamide polylysine (ε-PL-MA) and itaconic acid, respectively, with 15≤a≤50 and 20≤b≤80.

[0038] x represents the amount of lysine monomer in methacrylated polylysine (ε-PL-MA), where 20 ≤ x ≤ 40. The methacrylated polylysine was purchased from Aladdin and has a molecular weight of 5000 Da.

[0039] y represents the amount of six-membered ring monomer in carboxylated chitosan (CMCS), 85≤x≤150. Carboxylated chitosan (CMCS) is N-carboxymethyl chitosan, purchased from Aladdin with a molecular weight of 50000 Da.

[0040] The stem cell extravesicular gel contains 50 mg / mL of stem cell extravesicular vesicles, 10 mg / mL of nano-calcium oxide, and 8 mg / mL of nano-silver. The nano-silver particles have a diameter of 10–15 nm, and the nano-calcium oxide particles have a diameter of 15–20 nm.

[0041] The stem cell vesicles are human epidermal stem cell vesicles; the preparation method of human epidermal stem cell vesicles includes the following steps: (a) human epidermal stem cells are prepared using DMEM basal culture medium, with 1.0% penicillin and streptomycin, 10% serum, at 37°C and 5%... (2) When the confluence of human epidermal stem cell extravesicular MSCs reaches 70% to 80%, the complete culture medium is replaced with a special culture medium for devesicles. After 48 hours, the cell supernatant is collected and centrifuged at 300g for 10 min and 2000g for 10 min at 4℃ respectively to remove cells and cell debris. Then, the supernatant is centrifuged at 10,000g for 30 min to remove impurities such as large vesicles. Subsequently, the supernatant is centrifuged at 100,000g at 4℃ for 70 min and the supernatant is completely aspirated. Finally, the precipitate is resuspended with PBS and centrifuged at 100,000g at 4℃ for 70 min to obtain human epidermal stem cell extravesicular precipitate. The precipitate is resuspended with an appropriate amount of PBS according to its size and stored in a -80℃ refrigerator for later use.

[0042] The preparation method of the stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity in this embodiment includes the following steps:

[0043] (1) Methacrylated polylysine (10g), carboxylated chitosan (5g), itaconic acid (0.65g), nano-calcium oxide (155mg), nano-silver (124mg), and stem cell extravesicles (775mg) were dispersed in a phosphate buffer solution (15mL, 20mmol, pH7.4) and passed into the droplet microfluidic aqueous phase channel to obtain 10-15μm solidified micron-sized hydrogel microspheres (which can also be extruded using an extruder); the concentration of nano-calcium oxide was 10mg / mL, the concentration of nano-silver was 8mg / mL, and the content of stem cell extravesicles was 50mg / mL;

[0044] (2) A stem cell extravesicle gel with high anti-inflammatory and anti-fibrotic activity was obtained by photo-initiating a 20s reaction of solidified micron-sized hydrogel microspheres at 365nm.

[0045] Example 2

[0046] As an embodiment of the present invention, this stem cell extravesicular gel has high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of stem cell extravesicular vesicles is 20 mg / mL.

[0047] Example 3

[0048] As an embodiment of the present invention, this is a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of stem cell extravesicular vesicles is 35 mg / mL.

[0049] Example 4

[0050] As an embodiment of the present invention, this is a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of stem cell extravesicular vesicles is 80 mg / mL.

[0051] Example 5

[0052] As an embodiment of the present invention, this stem cell extravesicular gel has high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of stem cell extravesicular vesicles is 150 mg / mL.

[0053] Example 6

[0054] As an embodiment of the present invention, this stem cell extravesicular gel has high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of nano-silver in the stem cell extravesicular gel is 3 mg / mL.

[0055] Example 7

[0056] As an embodiment of the present invention, this stem cell extravesicular gel has high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of nano-silver in the stem cell extravesicular gel is 5 mg / mL.

[0057] Example 8

[0058] As an embodiment of the present invention, this stem cell extravesicular gel has high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of nano-silver in the stem cell extravesicular gel is 12 mg / mL.

[0059] Example 9

[0060] As an embodiment of the present invention, this stem cell extravesicular gel has high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of nano-silver in the stem cell extravesicular gel is 15 mg / mL.

[0061] Example 10

[0062] As an embodiment of the present invention, this stem cell extravesicular gel has high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that the content of nano-calcium oxide is 15 mg / mL.

[0063] Comparative Example 1

[0064] As a comparative example of the present invention, a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity is shown. The only difference between this comparative example and Example 1 is that it does not contain nano-calcium oxide.

[0065] Comparative Example 2

[0066] As a comparative example of the present invention, this embodiment is a stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity. The only difference between this embodiment and Example 1 is that it does not contain nano-silver.

[0067] Comparative Example 3

[0068] As a comparative example of the present invention, this stem cell extravesicular gel exhibits high anti-inflammatory and anti-fibrotic activity. The only difference between this comparative example and Comparative Example 1 is:

[0069] The polymer gel is a photoinitiated polymer gel of methacrylamide polylysine (ε-PL-MA) and itaconic acid.

[0070] The molecular structure of the polymer gel is as follows:

[0071]

[0072] The preparation method of the stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity in this embodiment is the same as in Example 1, but without the addition of carboxylated chitosan (CMCS).

[0073] Experimental methods

[0074] I. Material Characterization

[0075] The photoinitiated polymer gel of methacrylated polylysine (ε-PL-MA), carboxylated chitosan (CMCS), and itaconic acid (Example) compared with the photoinitiated polymer gel of methacrylated polylysine (ε-PL-MA) and itaconic acid in Comparative Example 1 had a 1020-1080 cm⁻¹ length. -1The significantly enhanced response on the left and right sides indicates that the photoinitiated polymer gel (example) of methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS) and itaconic acid contains the stretching vibration response of glycosidic bonds (COC), and the photoinitiated polymerization of methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS) and itaconic acid is achieved.

[0076] II. Anti-fibrosis test

[0077] (I) Samples to be tested

[0078] Examples 1-10 and Comparative Examples 1-3: stem cell extravesicular gels with high anti-inflammatory and anti-fibrotic activity.

[0079] (II) Testing Methods

[0080] 1. Human epidermal stem cells (hEpSCs) are cultured at 37°C and 5% CO2, with the medium changed every 2-3 days. Cells with strong adhesion are selected for later use.

[0081] 2. Selected human epidermal stem cells were processed at a ratio of 1×10⁻⁶. 5 cells / cm 2 The concentration was inoculated into K-SFM (Keratinocyte-SFM, Gibco / Thermo Fisher) medium (10 mL), and 15 ng / mL transforming growth factor-β1 (TGF-β1) was added.

[0082] 3. Divide into 16 groups, with 3 replicates in each group, and take the average value. The experimental groups are Examples 1-10, Comparative Examples 1-3, and the control and blank groups, respectively. In the experimental groups, 1 ml of stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity was added and evenly dispersed. In the control group, 50 mg of stem cell extravesicular gel, 10 mg of nano-calcium oxide, and 8 mg of nano-silver were dispersed in 1 mL of K-SFM (Keratinocyte-SFM, Gibco / Thermo Fisher) medium and mixed with the above human epidermal stem cells.

[0083] 4. After culturing at 37℃ and 5% CO2 for 96 hours, the cell culture supernatant was collected by centrifugation and the concentrations of Type I collagen and fibronectin were detected using a LiankeBio kit.

[0084] 5. Anti-fibrosis rate (%) = (blank group - experimental group) / blank group × 100%.

[0085] The experimental results are shown in Table 1.

[0086] III. Anti-inflammatory detection methods

[0087] 1. Selected human epidermal stem cells were processed at a ratio of 1×10⁻⁶. 5 cells / cm 2 The concentration was inoculated into K-SFM (Keratinocyte-SFM, Gibco / Thermo Fisher) medium (10 mL).

[0088] 2. Divide into 16 groups, with 3 replicates in each group, and take the average value. The experimental groups are Examples 1-10, Comparative Examples 1-3, and the control and blank groups, respectively. In the experimental groups, 1 ml of stem cell extracellular vesicle gel with high anti-inflammatory and anti-fibrotic activity was added and evenly dispersed. In the control group, 50 mg of stem cell extracellular vesicles + 10 mg of nano-calcium oxide + 8 mg of nano-silver were dispersed in 1 mL of K-SFM (Keratinocyte-SFM, Gibco / Thermo Fisher) medium and mixed with the above human epidermal stem cells.

[0089] Each group was supplemented with 5 μg / mL lipopolysaccharide and cultured at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected by centrifugation, and the IL-6 concentration was detected by ELISA (R&D Systems kit).

[0090] Anti-inflammatory rate (%) = (blank group - experimental group) / blank group × 100%.

[0091]

[0092]

[0093] As shown in Table 1, the stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity was formed by photoinitiation of methacryloyl-polylysine (ε-PL-MA), carboxylated chitosan (CMCS), and itaconic acid into a polymeric gel. Nano-calcium oxide, nano-silver, and epidermal stem cell extravesicular vesicles were dispersed within the polymeric gel. When this stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity was coated onto the epidermis, it could inhibit epidermal cell fibrosis and also exhibit anti-inflammatory effects. The photoinitiated polymeric gel of methacryloyl-polylysine (ε-PL-MA) and itaconic acid, as demonstrated in the examples and comparative examples, is beneficial in improving the anti-fibrotic rate.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity, characterized in that, The stem cell extravesicular gel comprises a polymer gel, nano-calcium oxide, nano-silver, and stem cell extravesicular vesicles dispersed in the polymer gel; The polymer gel is a photoinitiated polymer gel composed of methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS), and itaconic acid. The molecular structure of the polymer gel is as follows: Where a and b are the amounts of polymerized monomers of methacrylamide polylysine (ε-PL-MA) and itaconic acid, respectively, with 15≤a≤50 and 20≤b≤80. x represents the amount of lysine monomer in methacrylated polylysine (ε-PL-MA), where 20 ≤ x ≤ 40. y represents the amount of six-membered ring monomer in carboxylated chitosan (CMCS), where 85 ≤ x ≤ 150; The stem cell extravesicular gel contains 20-200 mg / mL of stem cell extravesicular vesicles.

2. The stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 1, characterized in that, The stem cell extravesicular gel contains 50-200 mg / mL of stem cell extravesicular vesicles.

3. The stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 1, characterized in that, The stem cell extravesicular gel contains 50-80 mg / mL of stem cell extravesicular vesicles.

4. The stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 1, characterized in that, The content of nano-calcium oxide in the stem cell extravesicular gel is 5~20 mg / mL.

5. The stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 1, characterized in that, The content of nano-silver in the stem cell extravesicular gel is 3~15mg / mL.

6. The stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 1, characterized in that, The preparation method of human epidermal stem cell vesicles includes the following steps: (a) Human epidermal stem cells are cultured in DMEM basal medium with 0.8%~1.2% penicillin and streptomycin and 8%~12% serum in a cell culture incubator at 36~38℃ and 4.5%~6% CO2; (2) When the confluence of human epidermal stem cell vesicles (MSCs) reaches 70%~80%, the cells and cell debris are removed by centrifugation, the precipitate is resuspended, and the human epidermal stem cell vesicle precipitate is collected.

7. The method for preparing stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity as described in any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Methacrylated polylysine (ε-PL-MA), carboxylated chitosan (CMCS), itaconic acid, nano-calcium oxide, nano-silver, and phosphate buffer solution of stem cell vesicles were passed into the droplet microfluidic aqueous phase channel to obtain solidified micron-sized hydrogel microspheres. (2) Solidified micron-sized hydrogel microspheres were photo-initiated at 360nm~410nm for 10~50s to obtain stem cell extravesicle gel with high anti-inflammatory and anti-fibrotic activity.

8. The method for preparing stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 7, characterized in that, In step (1), methacrylamide polylysine (ε-PL-MA), carboxylated chitosan (CMCS), itaconic acid, nano-calcium oxide, nano-silver, and stem cell vesicles are dispersed in a phosphate buffer solution in a weight ratio of 10~50 mmol and the pH of the phosphate buffer solution is 7.0~7.

5.

9. The method for preparing stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 7, characterized in that, Silver nanoparticles are produced by reducing silver ions to silver nanoparticles using ethanol or glucose as a reducing agent. The particle size of silver nanoparticles is 5~50nm.

10. The method for preparing stem cell extravesicular gel with high anti-inflammatory and anti-fibrotic activity according to claim 7, characterized in that, The particle size of nano-calcium oxide is 10~50nm.

Images