Mesenchymal stem cell-based skin injury repair gel and preparation method and application thereof

Abstract

This invention belongs to the field of biomedical technology and discloses a skin injury repair gel based on mesenchymal stem cells, its preparation method, and its application. By weight, it comprises 1.0-1.5 parts of stem cell exosomes, 2-4 parts of methacrylamide sodium hyaluronate, 8-10 parts of methacrylamide gelatin, 0.4-0.6 parts of ε-polylysine hydrochloride, 0.2-0.4 parts of asiaticoside, 0.1-0.3 parts of LAP photoinitiator, and phosphate buffer. This invention prepares highly active exosomes through a three-stage gradient synergistic induction system, and achieves long-term sustained release of exosomes using a hydrogel carrier. The gel can be cured in situ by 405nm blue light, perfectly adhering to irregular wound surfaces. It possesses antibacterial, anti-inflammatory, regenerative, and anti-scarring full-cycle repair effects, and can be widely used in the preparation of drugs for repairing mechanical trauma, burns, and hypertrophic scars. It has high safety, strong process controllability, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a skin damage repair gel based on mesenchymal stem cells, its preparation method and application. Background Technology

[0002] As the largest barrier organ in the human body, the skin is easily damaged by mechanical trauma, burns, scalds, chemical irritants and other factors. If the damage does not heal well, it can easily lead to wound infection, delayed healing, chronic and difficult-to-heal ulcers, and even hypertrophic scars, which seriously affect the patient's physiological functions and quality of life.

[0003] Currently, commonly used clinical methods for skin damage repair include medical dressings, growth factor preparations, glucocorticoids, and stem cell transplantation, but all have significant limitations: medical dressings can only provide physical isolation and moisturization, without any repair effect; growth factor preparations have problems such as short half-life, easy inactivation, need for frequent administration, and high doses can easily cause scar hyperplasia; glucocorticoids can only relieve scar hyperplasia, without promoting repair, and long-term use has side effects; living mesenchymal stem cell transplantation has safety risks such as immune rejection, tumorigenesis risk, harsh storage and transportation conditions, and poor batch stability, which limits its clinical application.

[0004] Mesenchymal stem cell exosomes are nanoscale extracellular vesicles secreted by mesenchymal stem cells. They carry active substances such as lipids, mRNA, and functional proteins from the mother cell. They can regulate the wound microenvironment through paracrine effects, exerting anti-inflammatory effects, promoting the proliferation and migration of keratinocytes / fibroblasts, promoting angiogenesis, and inhibiting excessive collagen deposition. At the same time, they reduce the safety risks of live cell preparations, making them a research hotspot in the field of skin damage repair. However, existing skin repair preparations based on mesenchymal stem cell exosomes suffer from insufficient exosome activity and limited function. They can only promote basic healing of acute wounds and have no significant intervention effect on chronic, difficult-to-heal wounds or scar hyperplasia after burns and scalds. In fact, some exosomes overexpress the pro-fibrotic factor TGF-β1, which can easily exacerbate scar formation. Furthermore, the low purity of exosome extraction and poor batch stability fail to meet the quality control requirements for large-scale production and clinical use. Additionally, the gel carrier system has poor compatibility with exosomes; existing exosome gels mostly use a single hydrogel matrix, resulting in poor mechanical properties, inability to adhere to irregular wounds, a mismatch between degradation rate and wound healing cycle, and severe exosome burst release. Moreover, they lack synergistic antibacterial and anti-scarring effects, failing to achieve full-cycle wound repair. Therefore, developing a highly active mesenchymal stem cell exosome repair gel with comprehensive antibacterial, anti-inflammatory, regenerative, and anti-scarring effects, and high safety, has significant clinical value and importance. Summary of the Invention

[0005] To address the aforementioned technical issues, this invention proposes a skin damage repair gel based on mesenchymal stem cells, its preparation method, and its application. Highly active exosomes are prepared through a three-stage dual-factor gradient induction process, and combined with a basic hydrogel to achieve long-term sustained release of exosomes. The compounded functional components provide synergistic effects. The resulting gel can be photocured in situ and possesses antibacterial, anti-inflammatory, regenerative, and anti-scarring effects throughout the entire repair cycle. The process is controllable and highly safe.

[0006] To achieve the above objectives, the present invention provides a skin damage repair gel based on mesenchymal stem cells, comprising, by weight parts: 1.0-1.5 parts stem cell exosomes, 2-4 parts methacrylamide sodium hyaluronate, 8-10 parts methacrylamide gelatin, 0.4-0.6 parts ε-polylysine hydrochloride, 0.2-0.4 parts asiaticoside, 0.1-0.3 parts LAP photoinitiator, and phosphate buffer to a total of 100 parts.

[0007] Preferably, the concentration of stem cell exosome protein is 1.0-1.5 mg / mL.

[0008] A method for preparing a skin damage repair gel is also provided, comprising the following steps: S1. Isolation and purification of mesenchymal stem cells: Human umbilical cord blood mesenchymal stem cells were seeded in culture medium and cultured. After three generations of cell fusion, the cells were digested and collected. S2, Three-stage synergistic induction culture: After the purified cells are cultured to the point of adhesion, they are cultured in stages using the first, second and third induction media in sequence, and then cultured for a period of time and the supernatant is collected. S3. Preparation of high-purity exosomes: The supernatant was subjected to differential centrifugation, tangential flow ultrafiltration, size exclusion chromatography, and sterile filtration to obtain an exosome solution; S4. Preparation of basic hydrogel: Sodium methacrylamide hyaluronic acid, methacrylamide gelatin, ε-polylysine hydrochloride, asiaticoside, and LAP photoinitiator are dissolved in phosphate buffer, stirred and dissolved, and filtered under sterile conditions to obtain basic hydrogel. S5. Final gel preparation: Under sterile conditions, the exosome solution is mixed with the basic hydrogel to obtain the skin damage repair gel.

[0009] Preferably, in step S1, the culture medium is DMEM / F12 medium; the mixture is fused to 85-95%; and digestion is performed with 0.25% trypsin for 1 min.

[0010] Preferably, in step S2, the first induction medium is DMEM / F12 medium supplemented with 2.5-10 ng / mL TNF-α, 5-20 ng / mL IFN-γ, 15-60 μg / mL vitamin C, 5-20 μmol / L melatonin, and 2.5-10 μg / mL low molecular weight HA, and cultured for 10-14 h; the second induction medium is DMEM / F12 medium supplemented with 5-20 ng / mL TNF-α, 10-40 ng / mL IFN-γ, 30-90 μg / mL vitamin C, 10-30 μmol / L melatonin, and 5-15 μg / mL low molecular weight HA, and cultured for 10-14 h; the third induction medium is DMEM / F12 medium supplemented with 10-40 ng / mL TNF-α and 20-80 ng / mL IFN-γ. Incubate in DMEM / F12 medium containing IFN-γ, 45-120 μg / mL vitamin C, 15-40 μmol / L melatonin, and 10-20 μg / mL low molecular weight HA for 22-26 h; then continue culturing in DMEM / F12 medium for another 22-26 h.

[0011] Preferably, in step S3, differential centrifugation is performed at 4°C, centrifuging at 300×g for 10 min, followed by centrifugation at 2000×g for 20 min, and finally centrifugation at 10000×g for 30 min; the molecular weight cutoff for tangential flow ultrafiltration is 100 kDa; size exclusion chromatography collects the elution peaks from tubes 3-5 and sterilizes them with a 0.22 μm filter membrane.

[0012] Preferably, in step S4, the stirring and dissolving process is carried out at 37°C in the dark for 1.5-3 hours, and the sterilization filtration is carried out using a 0.22μm sterile filter membrane.

[0013] Preferably, the prepared skin damage repair gel can be directly applied to the wound or cured by irradiation with 405nm blue light for 20-40 seconds.

[0014] It also provides the application of skin damage repair gel in the preparation of drugs for repairing mechanical trauma, burns, scalds, chronic refractory ulcers, and hypertrophic scars.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention constructs a dual photosensitive crosslinked hydrogel carrier system by compounding sodium hyaluronate (HAMA) and gelatin (GelMA) in a mass ratio of 1:3. It has excellent mechanical properties and adhesion, and can match the mechanical properties of skin soft tissue. It can be cured in situ by short-term irradiation with 405nm blue light, perfectly adhering to various irregular wounds, forming a stable physical barrier to isolate external bacterial invasion. The degradation cycle is precisely controlled at about 21 days, which is completely matched with the healing cycle of human skin wounds, reducing the problem of drug efficacy loss caused by excessively rapid degradation of the matrix, or the problem of excessively slow degradation hindering the epithelialization of the wound.

[0016] (2) This invention sets up a three-stage synergistic induction system with a gradient increase of TNF-α / IFN-γ dual factors, and with the synergistic effect of vitamin C, melatonin and low molecular weight HA, it realizes the targeted and precise regulation of the phenotype of mesenchymal stem cell exosomes. Compared with conventionally cultured exosomes, this invention reduces the defects of traditional exosomes in promoting fibrosis and easily aggravating scar hyperplasia. At the same time, cell experiments have verified that the exosomes of this invention can increase the proliferation rate of keratinocytes to 218.56% after 48 hours of treatment, which is 1.7 times that of conventionally cultured exosomes. It has excellent cell proliferation and wound regeneration capabilities, and provides core pharmacological support for skin damage repair.

[0017] (3) This invention achieves efficient separation and purification of exosomes through a three-step purification process of differential centrifugation, tangential flow ultrafiltration and size exclusion chromatography. The exosomes have uniform particle size, which solves the problems of low purity, many impurities, poor batch stability and inability to scale up in traditional exosome preparation processes. At the same time, this process does not require high-end ultra-high speed centrifugation equipment, the process parameters are controllable throughout the process, and the repeatability is good. It can be directly adapted to large-scale industrial production and meets the quality control requirements of clinical drug use.

[0018] (4) Based on exosomes, this invention combines ε-polylysine hydrochloride and asiaticoside functional components to form a synergistic effect with exosomes. Specifically, ε-polylysine hydrochloride is a broad-spectrum antibacterial agent that has an inhibitory effect on common pathogenic bacteria in wounds such as Staphylococcus aureus and Escherichia coli, which can effectively prevent wound infection and has no risk of antibiotic resistance. asiaticoside can work synergistically with exosomes to inhibit excessive collagen deposition, regulate the orderly arrangement of collagen, further enhance the anti-scar hyperplasia effect, and exert antioxidant, soothing and anti-inflammatory effects. The gel of this invention integrates five major functions: physical barrier, antibacterial and anti-infection, anti-inflammatory regulation, tissue regeneration promotion, and scar hyperplasia inhibition. It can meet the repair needs of skin injuries such as mechanical trauma, burns, scalds, chronic ulcers, and hyperplastic scars.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 The bar graph shows the test results of the skin damage repair gels prepared in Examples 1-3 and Comparative Examples 1-5 of this invention on the proliferation capacity of keratinocytes (HaCaT). Figure 2 This is a bar chart showing the wound healing rate of each experimental group in the rat deep second-degree burn model at 21 days, as described in this embodiment of the invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0024] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0025] Human umbilical cord blood mesenchymal stem cells were purchased from Wuhan Pronosai Life Technology Co., Ltd., product number: CP-CL11.

[0026] Example 1 I. Preparation of mesenchymal stem cell exosomes.

[0027] (1) Isolation and purification of mesenchymal stem cells: Human umbilical cord blood mesenchymal stem cells were seeded in DMEM / F12 medium for passage culture at a seeding density of 1×10⁻⁶. 4 Cells / ml were cultured under conditions of 5% CO2 and 37°C. Then, third-generation mesenchymal stem cells were selected and cultured again in DMEM / F12 medium. When the cells reached 90% confluence, they were digested with 0.25% trypsin for 1 min, and the cells were collected by pipetting after adding stop solution.

[0028] (2) Induction culture of mesenchymal stem cells: Select cells that are in good condition after digestion, and culture them at a concentration of 1×10⁻⁶ cells / year. 4Inoculate in DMEM / F12 medium and culture for 12 hours until cells are fully adherent. Discard the original medium and complete induction in the following three stages. Then, place the cells in a 37°C, 5% CO2 environment: First stage: Add the first induction medium (DMEM / F12 medium supplemented with 5 ng / mL TNF-α, 10 ng / mL IFN-γ, 30 μg / mL vitamin C, 10 μmol / L melatonin, and 5 μg / mL low molecular weight HA) and incubate for 12 h to complete pre-activation; Second stage: Discard the first induction medium and add the second induction medium (DMEM / F12 medium containing 10 ng / mL TNF-α, 20 ng / mL IFN-γ, 45 μg / mL vitamin C, 15 μmol / L melatonin, and 10 μg / mL low molecular weight HA), and continue culturing for 12 hours to complete the enhancement. Third stage: Discard the second induction medium and add the third induction medium (DMEM / F12 medium containing 20 ng / mL TNF-α, 40 ng / mL IFN-γ, 60 μg / mL vitamin C, 20 μmol / L melatonin, and 15 μg / mL low molecular weight HA), and continue culturing for 24 h.

[0029] After induction culture is completed, discard the induction medium, wash gently twice with sterile PBS, replace with fresh DMEM / F12 medium, and continue culturing for 24 hours. After the culture is completed, collect the cell culture supernatant for subsequent exosome extraction.

[0030] (3) Extraction of mesenchymal stem cell exosomes: ① Differential centrifugation: After filtration, the supernatant was centrifuged at 300×g for 10 min at 4℃ to remove live cells, and the supernatant was retained; then centrifuged at 2000×g for 20 min to remove cell debris, and the supernatant was retained; finally, centrifuged at 10000×g for 30 min to remove apoptotic bodies and the supernatant was collected.

[0031] ②Tangential flow ultrafiltration: The supernatant is concentrated 60 times by a tangential flow ultrafiltration membrane with a molecular weight cutoff of 100kDa, and then the solution is changed into sterile PBS to obtain exosome concentrate; ③ Size exclusion chromatography: The concentrated solution was loaded onto an agarose gel SEC column, eluted with sterile PBS, and the characteristic elution peaks of exosomes in tubes 3-5 were collected. The solution was then sterilized using a 0.22 μm filter membrane to obtain a high-purity exosome solution.

[0032] II. Preparation of skin damage repair gel.

[0033] (1) Formulation of skin damage repair gel: Take 10 mL of the exosome solution prepared in Example 1 (protein concentration 1.2 mg / mL, 1.2 parts by mass), 3 g of sodium hyaluronate (HAMA), 9 g of gelatin (GelMA), 0.5 g of ε-polylysine hydrochloride, 0.3 g of asiaticoside, 0.2 g of LAP photoinitiator, and make up to 100 g with sterile pH 7.4 phosphate buffer.

[0034] (2) Preparation of skin damage repair gel: ①According to the above ratio, take HAMA, GelMA, ε-polylysine hydrochloride, asiaticoside, and LAP photoinitiator, add them to sterile pH 7.4 phosphate buffer, stir at 37℃ in the dark for 2 hours until completely dissolved, filter through a 0.22μm sterile filter membrane to remove bacteria, and store at 4℃ in the dark. ② Under sterile conditions, the exosome solution prepared in Example 1 was thoroughly mixed with the basic hydrogel to obtain a skin damage repair gel; When using, apply the skin damage repair gel evenly to the damaged area of ​​the skin, or apply it and then irradiate it with 405nm blue light for 30 seconds for in-situ photocuring.

[0035] Example 2 The preparation method and steps are the same as in Example 1, except that: First stage: Add the first induction medium (DMEM / F12 medium supplemented with 2.5 ng / mL TNF-α, 5 ng / mL IFN-γ, 15 μg / mL vitamin C, 5 μmol / L melatonin, and 2.5 μg / mL low molecular weight HA) and incubate for 12 h to complete pre-activation; Second stage: Discard the first induction medium and add the second induction medium (DMEM / F12 medium containing 5 ng / mL TNF-α, 10 ng / mL IFN-γ, 30 μg / mL vitamin C, 10 μmol / L melatonin, and 5 μg / mL low molecular weight HA), and continue culturing for 12 hours to complete the enhancement. Third stage: Discard the second induction medium and add the third induction medium (DMEM / F12 medium containing 10 ng / mL TNF-α, 20 ng / mL IFN-γ, 45 μg / mL vitamin C, 15 μmol / L melatonin, and 10 μg / mL low molecular weight HA), and continue culturing for 24 h.

[0036] Example 3 The preparation method is the same as in Example 1, except that: First stage: Add the first induction medium (DMEM / F12 medium supplemented with 10 ng / mL TNF-α, 20 ng / mL IFN-γ, 60 μg / mL vitamin C, 20 μmol / L melatonin, and 10 μg / mL low molecular weight HA) and incubate for 12 h to complete pre-activation; Second stage: Discard the first induction medium and add the second induction medium (DMEM / F12 medium containing 20 ng / mL TNF-α, 40 ng / mL IFN-γ, 90 μg / mL vitamin C, 30 μmol / L melatonin, and 15 μg / mL low molecular weight HA), and continue culturing for 12 hours to complete the enhancement. Third stage: Discard the second induction medium and add the third induction medium (DMEM / F12 medium containing 40 ng / mL TNF-α, 80 ng / mL IFN-γ, 120 μg / mL vitamin C, 40 μmol / L melatonin, and 20 μg / mL low molecular weight HA), and continue culturing for 24 h.

[0037] Comparative Example 1 The preparation method was the same as in Example 1, except that there were no first, second, or third stage inductions. Specifically, after mesenchymal stem cells were seeded, they were cultured in DMEM / F12 medium for 72 hours, and the supernatant was directly collected to extract exosomes without the addition of any inducing agents.

[0038] Comparative Example 2 The preparation method was the same as in Example 1, except that only TNF-α was used for induction. Specifically, the first, second, and third induction media were DMEM / F12 media supplemented only with TNF-α, vitamin C, melatonin, and low molecular weight hyaluronic acid, without the addition of IFN-γ.

[0039] Comparative Example 3 The preparation method is the same as in Example 1, except that the concentrations of TNF-α and IFN-γ added in the first, second and third induction media are as follows: 10 ng / mL TNF-α + 5 ng / mL IFN-γ in the first stage, 20 ng / mL TNF-α + 10 ng / mL IFN-γ in the second stage, and 40 ng / mL TNF-α + 20 ng / mL IFN-γ in the third stage.

[0040] Comparative Example 4 The preparation method is the same as in Example 1, except that a single GelMA matrix is ​​used. In the formulation of the skin damage repair gel, 12g of GelMA is used as the hydrogel matrix, and no HAMA is used. The other components are the same as in Example 1.

[0041] Comparative Example 5 The preparation method is the same as in Example 1, except that the mass ratio of HAMA to GelMA is 1:1, and the proportion of HAMA and GelMA in the skin damage repair gel is 6g and 6g respectively, with the remaining components being the same as in Example 1.

[0042] Experimental Example 1 The effect of skin damage repair gel on the proliferative capacity of keratinocytes (HaCaT).

[0043] (1) Take HaCaT cells in the logarithmic growth phase and use 5×10 3 The cells were seeded at a density of 100 μL of culture medium per well in a 96-well plate and incubated in a cell culture incubator for 24 h until the cells adhered to the plate. (2) Discard the original culture medium and divide into 8 groups, with 6 replicates in each group: blank control group (only DMEM culture medium added); the drug administration groups are: Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group; the final concentration of exosomes in the drug administration groups is 100 μg / mL. (3) After culturing for 24h and 48h respectively, add 10μL of CCK-8 reagent to each well, incubate at 37℃ for 2h, and detect the absorbance value at 450nm with an ELISA reader to calculate the relative cell proliferation rate.

[0044] The results are as follows Figure 1 As shown, the exosomes in Examples 1-3 significantly promoted HaCaT cell proliferation. Compared with the blank control group, the proliferation rate of Examples 1-3 reached a maximum of 218.56 ± 8.74% at 48 h. P <0.01%, or 1.7 times that of conventionally cultured exosomes in Comparative Example 1 ( P <0.01); while the exosomes in comparative examples 1-4 had no significant proliferative effect, proving that the synergistic induction system of the present invention can significantly enhance the cell proliferation activity of exosomes, providing core efficacy support for wound treatment.

[0045] Experiment Example 2 Verification of the repair effect in a rat model of deep second-degree burns.

[0046] (1) Experimental animals and grouping: 100 SPF-grade SD rats (weighing 200-250g), half male and half female, were randomly divided into 10 groups after 7 days of acclimatization feeding, with 10 rats in each group: blank control group (wounds were treated with sterile PBS only), commercially available positive control group (using commercially available recombinant human epidermal growth factor gel), Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group.

[0047] (2) Modeling method: 25% pentobarbital was injected into the peritoneum for anesthesia (35mg / kg), hair was removed, and the same method was used for anesthesia the next day. The back was placed in 80℃ water for 6 seconds, covering an area of ​​about 10% of the total body surface area (TBSA) to create a deep second-degree burn model. Immediately after modeling, 5ml of lactated Ringer's solution was injected into the peritoneum, and routine anti-infection treatment was performed.

[0048] (3) Administration method: The drug was administered immediately after modeling. For groups 1-3 and comparative groups 1-5, the drug was administered once a day. Each time, 0.5g of gel was evenly applied to the wound and cured by 405nm blue light irradiation. For the blank control group and the commercially available positive control group, the drug was administered once a day. All groups were administered the drug continuously for 21 days.

[0049] (4) Detection indicators: Recorded at 7, 14 and 21 days after modeling, and the wound healing rate was calculated using ImageJ software.

[0050] (5) Experimental results: The results are shown in Table 1 and Figure 2 As shown, the healing speed and healing rate of the gel in Example 1 are significantly better than those of commercially available products and comparative examples. The healing rate reached 97.2% in 21 days, with no obvious redness, swelling, infection, or scar hyperplasia on the wound surface, demonstrating the best skin damage repair effect.

[0051] Table 1. Wound healing rate at 7, 14, and 21 days post-modeling.

[0052] In Examples 1-3, the wound healing rates at 7d, 14d, and 21d were significantly better than those of the blank control group and Comparative Example 1. P <0.01), among which, the first group of examples showed the best results, with significantly faster wound healing speed and higher final healing rate.

[0053] In summary, the skin damage repair gel based on mesenchymal stem cells disclosed in this invention prepares highly active and low-scarring mesenchymal stem cell exosomes through a three-stage synergistic induction culture, combined with a photocurable basic hydrogel matrix of HAMA and GelMA in a 1:3 mass ratio, and compounded with functional ingredients such as ε-polylysine hydrochloride and asiaticoside. From the three dimensions of active ingredient preparation, carrier system construction, and functional component synergy, it solves the core technical defects of existing skin damage repair preparations, such as insufficient exosome activity, low extraction purity, poor batch stability, poor gel carrier compatibility, exosome burst release, and single function.

[0054] This invention relates to a skin injury repair gel based on mesenchymal stem cells. All raw materials are commercially available and compliant products. The preparation process employs standardized cell culture, purification, and gel preparation techniques, requiring no special high-end equipment. Process parameters are controllable and highly reproducible, enabling large-scale industrial production. This gel can be applied in hospital wound care departments, burn departments, dermatology departments, and cosmetic surgery institutions, showing broad clinical application prospects and significant industrial applicability.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A mesenchymal stem cell based skin lesion repair gel, characterized in that, By weight, the following components are included: 1.0-1.5 parts stem cell exosomes, 2-4 parts sodium methacrylamide hyaluronic acid, 8-10 parts methacrylamide gelatin, 0.4-0.6 parts ε-polylysine hydrochloride, 0.2-0.4 parts asiaticoside, 0.1-0.3 parts LAP photoinitiator, and phosphate buffer to bring the total to 100 parts.

2. The mesenchymal stem cell-based skin lesion repair gel according to claim 1, wherein, The concentration of stem cell exosome protein is 1.0-1.5 mg / mL.

3. A method of preparing a skin lesion repair gel according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Isolation and purification of mesenchymal stem cells: Human umbilical cord blood mesenchymal stem cells are seeded in culture medium and cultured. After three generations of cell fusion, the cells are digested and collected. S2, Three-stage synergistic induction culture: After the purified cells are cultured to the point of adhesion, they are cultured in stages using the first, second and third induction media in sequence, and then cultured for a period of time and the supernatant is collected. S3. Preparation of high-purity exosomes: The supernatant was subjected to differential centrifugation, tangential flow ultrafiltration, size exclusion chromatography, and sterile filtration to obtain an exosome solution; S4. Preparation of basic hydrogel: Sodium methacrylamide hyaluronic acid, methacrylamide gelatin, ε-polylysine hydrochloride, asiaticoside, and LAP photoinitiator are dissolved in phosphate buffer, stirred and dissolved, and filtered under sterile conditions to obtain basic hydrogel. S5. Final gel preparation: Under aseptic conditions, the exosome solution is mixed with the basic hydrogel to obtain the skin damage repair gel.

4. The production method according to claim 3, characterized by, In step S1, the culture medium is DMEM / F12 medium; the confluence is increased to 85-95%; digestion is performed with 0.25% trypsin for 1 min.

5. The production method according to claim 3, wherein In step S2, the first induction medium was DMEM / F12 medium supplemented with 2.5-10 ng / mL TNF-α, 5-20 ng / mL IFN-γ, 15-60 μg / mL vitamin C, 5-20 μmol / L melatonin, and 2.5-10 μg / mL low molecular weight HA, and cultured for 10-14 h; the second induction medium was DMEM / F12 medium supplemented with 5-20 ng / mL TNF-α, 10-40 ng / mL IFN-γ, 30-90 μg / mL vitamin C, 10-30 μmol / L melatonin, and 5-15 μg / mL low molecular weight HA, and cultured for 10-14 h; the third induction medium was supplemented with 10-40 ng / mL TNF-α, 10-40 ng / mL IFN-γ, 30-90 μg / mL vitamin C, 10-30 μmol / L melatonin, and 5-15 μg / mL low molecular weight HA, and cultured for 10-14 h; Incubate in DMEM / F12 medium containing TNF-α, 20-80 ng / mL IFN-γ, 45-120 μg / mL vitamin C, 15-40 μmol / L melatonin, and 10-20 μg / mL low molecular weight HA for 22-26 h; then continue culturing in DMEM / F12 medium for another 22-26 h.

6. The preparation method according to claim 3, characterized in that, In step S3, differential centrifugation was performed at 4°C, centrifuging at 300×g for 10 min, followed by centrifugation at 2000×g for 20 min, and finally centrifugation at 10000×g for 30 min; the molecular weight cutoff for tangential flow ultrafiltration was 100 kDa; size exclusion chromatography was used to collect the elution peaks from tubes 3-5 and sterilized them using a 0.22 μm filter membrane.

7. The preparation method according to claim 3, characterized in that, In step S4, the stirring and dissolving process is carried out at 37°C in the dark for 1.5-3 hours, and the sterilization filtration is carried out through a 0.22μm sterile filter membrane.

8. The preparation method according to claim 3, characterized in that, The prepared skin damage repair gel can be applied directly to the wound or cured by irradiation with 405nm blue light for 20-40 seconds.

9. The use of the skin damage repair gel according to any one of claims 1-2 in the preparation of drugs for repairing mechanical trauma, burns, scalds, chronic refractory ulcers, and hypertrophic scars.

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