Human umbilical cord mesenchymal stem cell exosome-copper metal organic framework-tricine modified chitosan composite dressing for skin wound repair and preparation method thereof

Abstract

The present application provides a composite dressing for skin wound repair, which is mainly composed of human umbilical cord mesenchymal stem cell exosomes (hUMSC-Exos), copper metal organic framework (CuMOFs) and tris(hydroxymethyl) methyl glycine modified chitosan derivative (CTMG). The ratio of the composite gel sponge dressing is hUMSCs:CuMOFs:CTMG is 1:50:1500. CTMG as a carrier has good biocompatibility and biodegradability, can effectively release Cu 2+ , has antibacterial properties, reduces the risk of infection, and promotes angiogenesis. The slow release of hUMSC-Exos carries a variety of bioactive molecules, enhances immune regulation function, and promotes wound healing. The composite dressing significantly improves the speed and quality of wound healing, providing a new theoretical basis and treatment method for clinical treatment of skin trauma.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of materials science and tissue engineering, specifically to a human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair and its preparation method. Background Technology

[0002] Poor wound healing is a major clinical challenge. Uncontrolled inflammation, frequent infections, and restricted angiogenesis hinder repair, and existing dressings are insufficient to meet the standards for comprehensively regulating inflammation, preventing infection, and promoting angiogenesis to improve healing. Therefore, novel treatment strategies have become a hot topic in medicine.

[0003] Currently, there are various treatment methods for skin wounds. Traditional gauze dressings, while inexpensive, readily available, and possessing some exudate absorption capacity, tend to stick to the wound during dressing changes, causing pain and secondary damage, and lack antibacterial and healing-promoting effects. For biomaterial dressings, hydrocolloid dressings can create a moist environment to promote debridement and analgesia, but they are lacking in infection control and breathability; hydrogel dressings can moisturize, promote healing, and relieve pain, but their mechanical properties are fragile, they dry out easily, and their ability to handle large amounts of exudate is limited; alginate dressings can absorb water and stop bleeding, but their degradation products can trigger inflammatory reactions, and their antibacterial properties are also weak. Platelet-rich plasma therapy has a complex composition, but its mechanism of action is unclear, its treatment effects fluctuate significantly, and it lacks standardized procedures. When constructing skin substitutes using tissue-engineered skin, challenges arise in vascularization and tissue integration, there is a risk of immune rejection, and production costs are high, greatly limiting its widespread clinical application. Therefore, developing a comprehensive and highly effective wound-healing dressing is of significant practical importance.

[0004] In recent years, the rapid development of the intersection of materials science and tissue engineering has provided new ideas for solving this problem. Some novel composite dressings, such as AgMOF hydrogels, gallium / chitosan / silk / umbilical cord mesenchymal stem cell exosome gel sponges, and CuMOF grapefruit peel gel sponges, have shown great potential in treating open skin wounds due to their unique physicochemical properties. However, most existing skin wound dressings only have a single therapeutic effect (such as antibacterial, anti-inflammatory, and hemostatic effects), which is difficult to meet the clinical need for rapid and effective healing of skin wounds. In order to achieve more effective promotion of open wound healing and reduce the occurrence of complications, it is urgent to develop a new type of dressing with comprehensive performance and high efficiency in wound repair.

[0005] Human umbilical cord mesenchymal stem cell exosomes-copper-metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressings for skin wound repair have broad application prospects in biomedical engineering, cell therapy and regenerative medicine, and wound repair medicine. For example, hUMSC-Exos contains bioactive components such as proteins, nucleic acids (e.g., miRNAs, mRNAs), and lipids, which can be taken up by surrounding cells to regulate cellular physiological functions, such as promoting cell proliferation, migration, angiogenesis, and regulating immune responses. Metal-organic frameworks (MOFs) are porous materials with various characteristics and broad application prospects. CuMOFs possess the general characteristics of MOFs, as well as unique biological functions, good antibacterial properties, and biocompatibility, showing potential value in biomedicine, especially in wound repair. Tris(hydroxymethyl)methylglycine-modified chitosan derivatives (CTMG) are novel biomaterials. Chitosan has good biocompatibility and biodegradability, providing new methods for clinical diagnosis and treatment.

[0006] Human umbilical cord mesenchymal stem cell exosomes-copper-metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair offers new ideas and methods for solving the problem of open skin wound healing in clinical treatment. With the continuous deepening of research, it is believed that this new composite dressing will play an important role in more fields. Summary of the Invention

[0007] The purpose of this invention is to provide a composite dressing of human umbilical cord mesenchymal stem cell exosomes-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan for skin wound repair, aiming to solve the problems of the single, limited and unsatisfactory treatment effects of existing wound dressings for skin wound healing.

[0008] Specific objectives include:

[0009] 1. To develop a dressing composed of human umbilical cord mesenchymal stem cell exosomes, copper-based metal-organic frameworks and tris(hydroxymethyl)methylglycine-modified chitosan, which can precisely and efficiently promote skin wound repair, accelerate wound healing rate, improve healing quality, reduce scar formation, and help skin wounds recover well.

[0010] 2. This composite dressing possesses strong antibacterial properties. With the copper ions released by the copper-based metal-organic framework and the inherent antibacterial properties of chitosan, it effectively resists the invasion of common bacteria in wounds, controls the risk of infection, creates a favorable microenvironment for wound healing, and reduces the incidence of complications.

[0011] 3. Optimize the biocompatibility of the composite dressing to ensure that when human umbilical cord mesenchymal stem cell exosomes, copper-based metal-organic frameworks and modified chitosan work synergistically, they have no toxic side effects or immune rejection on human cells and tissues, thus ensuring their safety and effectiveness throughout the wound repair process and promoting cell proliferation, migration and tissue regeneration.

[0012] 4. This dressing is designed with ideal physical properties, such as appropriate flexibility, breathability and absorbency, to adapt to the dynamic changes of the wound, maintain appropriate humidity and oxygen exchange, prevent exudate accumulation and wound maceration, promote the removal of necrotic tissue, accelerate granulation tissue growth and epithelialization, and improve the repair effect.

[0013] 5. The preparation process of this composite dressing is simple, cost-controllable, and of stable quality, which facilitates large-scale production and clinical application, providing an innovative, high-quality, and economical solution for skin wound repair, alleviating patient suffering and medical burden, and promoting the innovative development of wound repair technology.

[0014] To achieve the above objectives, the present invention adopts the following technical solution: a composite dressing for skin wound repair using human umbilical cord mesenchymal stem cell exosomes-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan, wherein the material is a composite dressing synthesized with hUMSC-Exos, CuMOFs and CTMG as the main components.

[0015] More preferably, the mass ratio of hUMSC-Exos, CuMOFs and CTMG is 1:50:1500 - 1.5:80:1700.

[0016] Another technical solution of the present invention is: a method for preparing a composite dressing of human umbilical cord mesenchymal stem cell exosomes-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine modified chitosan for skin wound repair, comprising the following steps:

[0017] Step 1: Accurately weigh a certain amount of chitosan (CS) and place it in a suitable container. Under stirring conditions, slowly add 1% acetic acid solution to the chitosan and continue stirring until the chitosan is completely dissolved to obtain solution A.

[0018] Step 2: Add a certain amount of crosslinking agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) to solution A in sequence. Stir continuously during the addition process to ensure that the reactants are fully mixed to obtain solution B.

[0019] Step 3: Stir solution B continuously for 10-60 minutes to allow the reactants to react fully, resulting in solution C.

[0020] Step 4: Add an appropriate amount of tris(hydroxymethyl)methylglycine (TMG) to solution C, stir for 12-48 h to obtain solution D, pour it into a special mold to make CTMG hydrogel.

[0021] Step 5: Accurately weigh a certain amount of anhydrous copper chloride (CuCl2) into a round-bottom flask, add an appropriate amount of deionized water and stir thoroughly to dissolve, thus obtaining solution E.

[0022] Step 6: Add an appropriate amount of 5-9 mol / L KOH solution to adjust the pH of solution E to 7.5, and heat the mixed solution in a reactor at 140℃ for 12-48 h to obtain solution F.

[0023] Step 7: Centrifuge solution F at 8000-12000 rpm for 10-30 min at 4℃ to separate the light gray solid.

[0024] Step 8: Wash the light gray solid with deionized water 1-3 times and dry it in a vacuum drying oven at 37℃ for 12-48 h to obtain CuMOFs powder.

[0025] Step 9: Slowly add the prepared CuMOFs powder dropwise into a certain amount of CTMG hydrogel using a dropper or syringe, ensuring that the CuMOFs powder is fully dissolved in the CTMG hydrogel. The mass ratio of CuMOFs to CTMG hydrogel is 1:1250 - 1:2000, resulting in solution G.

[0026] Step 10: Select 3rd to 5th generation human umbilical cord mesenchymal stem cells for culture. When the cells grow to the logarithmic growth phase, collect 100-2500 mL of culture medium into a sterile centrifuge tube.

[0027] Step 11: Centrifuge the collected culture medium at 4°C at 200-500 g for 5-15 min, 1000-2000 g for 5-15 min, and 8000-15000 g for 20-30 min to remove cell debris and large particles.

[0028] Step 12: Take the supernatant and centrifuge at 80,000 - 100,000 g for 40 - 80 min at 4℃. Resuspend the precipitate in PBS buffer. Centrifuge the resuspended solution again at 80,000 - 100,000 g for 40 - 80 min at 4℃. The resulting precipitate is hUMSC-Exos. Store it in a -80℃ freezer for later use.

[0029] Step 13: Take the hUMSC-Exos solution and slowly add it to solution G (the mass ratio of hUMSCs, CuMOFs and CTMG is 1:50:1500 - 1.5:80:1700). Add it dropwise using a pipette. Place the mixed solution in a 4°C environment and stir continuously for 2 hours to ensure that the exosomes are evenly dispersed in the composite solution, thus obtaining solution H.

[0030] Step 14: Dispense the H solution into specific molds, freeze at -20℃ for 12-48 h, then transfer to -80℃ for 12-24 h, and finally dry in a vacuum freeze dryer for 24-72 h to obtain CuMOFs / CTMG / Exos sponge gel.

[0031] The molar mass ratio of hUMSC-Exos, CuMOFs and CTMG in step 1 is 1:50:1500 - 1.5:80:1700.

[0032] In step 1, the raw material selection and dosage are as follows: the amount of chitosan (CS) is 6-20 g, and the chitosan is dissolved in 100-400 mL of 1% acetic acid solution.

[0033] In step 2, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is 0.1-0.3 g and the amount of N-hydroxy-succinimide (NHS) is 0.1-0.2 g.

[0034] The stirring time in step 3 is 10-60 min (preferably 30 min).

[0035] In step 4, the amount of tris(hydroxymethyl)methylglycine (TMG) used is 0.4-0.8 g, and the stirring time is 24 h-72 h.

[0036] In step 5, the amount of anhydrous copper chloride (CuCl2) used is 10-15 g, the volume of deionization is 100-1000 mL, the temperature of the reaction vessel is 100-200℃, and the heating time is 12-48 h.

[0037] In step 6, solution F is centrifuged at 5000-12000 rpm for 10-30 min at 4℃.

[0038] In step 7, the light gray solid is washed with deionized water 1-3 times and dried in a vacuum drying oven at 37°C for 12-48 h to obtain CuMOFs powder.

[0039] In step 8, the concentration of the KOH solution is 4-10 mol / L, and the volume is 1-10 mL.

[0040] In step 9, the mass ratio of CuMOFs to CTMG is 1:1250 - 1:2000, the stirring speed is 100 - 500 r / min, and the time is 1 - 2 h.

[0041] In step 10, the human umbilical cord mesenchymal stem cell culture medium is the 3rd to 5th generation culture medium, and the volume of culture medium used is 100 to 2500 mL.

[0042] In step 11, the parameters for each stage of the culture medium centrifugation process are precisely set. The first stage is 4℃, 200-500 g, 5-15 min; the second stage is 4℃, 1000-2000 g, 5-15 min for further clarification; and the third stage is 4℃, 8000-15000 g, 20-30 min.

[0043] In step 12, the ultracentrifugation is performed twice at 4°C, at 80,000 - 100,000 g for 40 - 80 min.

[0044] In step 13, the mass ratio of hUMSC-Exos, CuMOFs and CTMG is 1:50:1500 - 1.5:80:1700, and the stirring time is 1-2 h at 4℃.

[0045] In step 14, the freeze-drying conditions are: freezing at -20℃ for 1-48 h; refreezing at -80℃ for 1-48 h; vacuum degree of 0.08-0.1 MPa; and drying time of 1-72 h.

[0046] Modification and Functionalization of Chitosan: This invention utilizes a chemical cross-linking method, adding cross-linking agents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) to chitosan to form a cross-linked structure with uniform pore size, providing a slow release space for CuMOFs and hUMSC-Exos and maintaining their bioactivity; adding the adhesive tris(hydroxymethyl)methylglycine (TMG) to improve the adhesion of the gel sponge dressing, thereby better adhering to the skin wound; adding CuMOFs to exert the general characteristics of MOFs, as well as unique biological functions, good antibacterial properties and biocompatibility, synergistically promoting skin wound healing; adding hUMSC-Exos, which mainly plays a therapeutic role, contains bioactive components such as proteins, nucleic acids (such as miRNAs, mRNAs, etc.) and lipids, which can be taken up by surrounding cells and regulate cellular physiological functions, such as promoting cell proliferation, migration, angiogenesis and regulating immune responses. The combination of these three factors efficiently and rapidly promotes wound healing.

[0047] Material characterization: The prepared material was characterized using characterization method 1, such as 1H NMR and Fourier transform infrared spectroscopy (FT-IR), to determine the composition and structure of the material.

[0048] The morphology and size of the material are observed using characterization methods 2, such as scanning electron microscopy (SEM) and particle size analyzer.

[0049] Material performance testing of CuMOFs / CTMG: The performance of CuMOFs / CTMG carrier materials as wound dressings was evaluated by testing rheology, compressive strength, swelling rate, water retention, adhesiveness, burst pressure, enzyme degradation and antibacterial properties.

[0050] Cellular-level assessment of CuMOFs / CTMG and hUMSC-Exos binding:

[0051] In vitro cell assays of CuMOFs / CTMG:

[0052] Cytotoxicity and hemolytic activity were assessed in CuMOFs / CTMG carrier materials.

[0053] In vitro cell assays of CuMOFs / CTMG / Exos:

[0054] The bioactivity of CuMOFs / CTMG / Exos composite dressing was evaluated using live / dead cell assays, angiogenesis, cell scratch assays, and Transwell assays.

[0055] Antimicrobial profile assessment of CuMOFs / CTMG and hUMSC-Exos conjugate:

[0056] The antibacterial properties of CuMOFs / CTMG and CuMOFs / CTMG / Exos carrier materials against Staphylococcus aureus and Escherichia coli were evaluated by co-culturing the materials with bacteria after UV sterilization, and the antibacterial performance was assessed by counting and photographing, and the antibacterial mechanism was verified by SEM.

[0057] Animal-level assessment of CuMOFs / CTMG and hUMSC-Exos conjugate:

[0058] In vivo animal evaluation of CuMOFs / CTMG:

[0059] The CuMOFs / CTMG carrier material was evaluated using a rat liver hemostasis experiment.

[0060] In vitro animal evaluation of CuMOFs / CTMG / Exos:

[0061] A full-thickness skin defect model was established using CuMOFs / CTMG / Exos composite dressing, and its histological, inflammatory response, and immunofluorescence were evaluated.

[0062] Compared with the prior art, the present invention has the following advantages:

[0063] The present invention provides a human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair. Combining multiple functions such as high-efficiency antibacterial, hemostatic, anti-inflammatory, and promotion of angiogenesis and nerve cell generation, it has the following significant advantages.

[0064] 1. Multifunctional Integration: This invention utilizes a novel composite dressing combining human umbilical cord mesenchymal stem cell exosomes with CuMOFs, CTMG, and hUMSC-Exos to achieve integrated functions such as antibacterial, cell proliferation promotion, anti-inflammatory, and angiogenesis promotion. Compared to existing single-function wound repair materials, this dressing more comprehensively promotes the skin wound repair process and improves the repair effect.

[0065] 2. Synergistic Effect: The components work together to achieve superior performance compared to a single material. For example, the antibacterial and angiogenesis-promoting effects of CuMOFs, combined with the cell affinity and biocompatibility of CTMG, along with the regulatory effects of hUMSC-Exos on cell physiological functions, work together to promote wound healing and reduce the occurrence of complications.

[0066] 3. Excellent biocompatibility: CTMG exhibits excellent biocompatibility, and its interaction with cells is further enhanced after modification. Meanwhile, hUMSC-Exos, derived from human cells, has low immunogenicity and will not cause significant immune rejection. Therefore, the sponge gel dressing of this invention possesses excellent biocompatibility in vivo, which is beneficial for the smooth progress of wound repair.

[0067] 4. Controllable Preparation: By precisely controlling the proportions of each component and the preparation process parameters, CuMOFs / CTMG / Exos sponge gels with consistent performance can be stably prepared. This ensures quality control in clinical applications and helps improve the reliability of treatment effects.

[0068] 5. Potential Clinical Application Value: The sponge gel of this invention has significant advantages in skin wound repair and is expected to provide a new and effective material option for the clinical treatment of skin trauma. Its synergistic effects of multiple functions and good biocompatibility may help shorten wound healing time, reduce scar formation, and improve patients' quality of life. Attached Figure Description

[0069] To more clearly illustrate the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0070] Figure 1 , Figure 2 SEM images of CuMOFs / CTMG sponge gel

[0072] Figure 3 Schematic diagram showing the effects of different concentrations of CuMOFs / CTMG / Exos sponge gel on cell proliferation.

[0073] Figure 4 Schematic diagram of the antibacterial performance test results of CuMOFs / CTMG / Exos sponge gel Figure 5 Schematic diagram of experimental results showing that CuMOFs / CTMG / Exos sponge gel promotes cell migration. Detailed Implementation

[0074] The following are specific implementation examples provided by the inventors. It should be noted that these implementation examples are preferred examples of the present invention and are intended for those skilled in the art to understand the present invention. However, the present invention is not limited to these implementation examples.

[0075] Example 1

[0076] A composite dressing of human umbilical cord mesenchymal stem cell exosomes-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan for skin wound repair was prepared by dissolution and freeze-drying methods. Appropriate process parameters were set, and CuMOFs and CTMG were mixed at a molar mass ratio of 1:20-1:30. Based on different CTMG contents in the samples, CuMOFs / CTMG-1, CuMOFs / CTMG-3, CuMOFs / CTMG-5, CuMOFs / CTMG-7, and CuMOFs / CTMG-9 were obtained, and commercially available chitosan hemostatic gel sponge (CS) was used as a comparison.

[0077] SEM observation was used to obtain information on the surface morphology and microstructure of the samples. Observation of the CS surface revealed that it had many defects with numerous pores or high roughness, and the pores were unevenly distributed. Compared with the CS surface, the surfaces of CuMOFs / CTMG-1 to CuMOFs / CTMG-9 became smoother, flatter, and had smaller and more uniformly distributed pores. The surface of CuMOFs / CTMG-9 was the smoothest, flattest, and had the smallest and most uniformly distributed pores.

[0078] The above results clearly indicate that the interconnected network structure of hydrogels is beneficial to wound healing due to their moisturizing properties and oxygen transport capacity. The microstructure and pore size of CTMG gel sponges at different concentrations were compared with those of CS sponges. The results showed that the pore size range of CTMG sponges (30 to 110 μm) was smaller than that of CS sponges (approximately 170 μm). With increasing CTMG concentration, the porous structure became smaller due to stronger cross-linking, resulting in tighter connections within the gel and a smaller pore size. This porous structure allows CTMG gel sponges to absorb large amounts of water and create a stable, moist environment around the wound area. The presence of moisture supports cell growth and proliferation at the wound site, thereby promoting the healing process. SEM images of CS, CuMOFs / CTMG-1, and CuMOFs / CTMG-3 samples are shown below. Figure 1 As shown, the SEM spectra of CuMOFs / CTMG-5, CuMOFs / CTMG-7, and CuMOFs / CTMG-9 samples are as follows: Figure 2 As shown.

[0079] Example 2

[0080] A composite dressing of human umbilical cord mesenchymal stem cell exosomes-copper-metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan for skin wound repair was prepared by a dissolution method and a freeze-drying method. With appropriate process parameters, CuMOFs and CTMG were mixed at a molar ratio of 1:20 to 1:30 to obtain CuMOFs / CTMG. CuMOFs / CTMG was then mixed with hUMSC-Exos at a mass ratio of hUMSC-Exos, CuMOFs, and CTMG of 1:50:1500 to 1.5:80:1700 to obtain CuMOFs / CTMG / Exos. Cell compatibility was assessed by evaluating proliferation on the scaffold.

[0081] MTT assay results showed that, compared with the control group, both the CuMOFs / CTMG and CuMOFs / CTMG / Exos groups increased HUVEC proliferation, while the CuMOFs / CTMG / Exos group showed a more significant increase in HUVEC proliferation.

[0082] The above results clearly indicate that the CuMOFs / CTMG / Exos group significantly promoted the proliferation of HUVECs. The biocompatibility of CuMOFs / CTMG / Exos showed no cytotoxicity to cells, but rather promoted cell proliferation, thereby accelerating skin wound healing. A schematic diagram of cell proliferation in the Control group, CuMOFs / CTMG group, and CuMOFs / CTMG / Exos group is shown below. Figure 3 As shown.

[0083] Example 3

[0084] A composite dressing of human umbilical cord mesenchymal stem cell exosomes-copper-metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan for skin wound repair was prepared by a dissolution method and a freeze-drying method. With appropriate process parameters, CTMG was mixed with deionized water at a ratio of 0.75 g: 5.00 mL to obtain CTMG; then, CuMOFs / CTMG was mixed at a mass ratio of 1:20 to 1:30 to obtain CuMOFs / CTMG; CuMOFs / CTMG was mixed with hUMSC-Exos at a mass ratio of 1:50:1500 to 1.5:80:1700 to obtain CuMOFs / CTMG / Exos; the antibacterial properties of CuMOFs / CTMG / Exos and CuMOFs / CTMG sponge scaffolds against Escherichia coli and Staphylococcus aureus were evaluated using the direct contact method.

[0085] The antibacterial properties of CuMOFs / CTMG / Exos and CuMOFs / CTMG sponge scaffolds against *Escherichia coli* and *Staphylococcus aureus* were evaluated using the direct contact method. Inhibition zone measurements showed that the CTMG, CuMOFs / CTMG, and CuMOFs / CTMG / Exos groups exhibited significant antibacterial activity compared to the control group. Furthermore, no significant differences were observed between the CuMOFs / CTMG and CuMOFs / CTMG / Exos groups, but the CuMOFs / CTMG / Exos group showed stronger antibacterial activity compared to the CTMG group.

[0086] The above results clearly indicate that, in the antibacterial experiment, CuMOFs / CTMG / Exos exhibited significantly higher antibacterial activity than CTMG; and slightly higher antibacterial activity than CuMOFs / CTMG. Schematic diagrams of the antibacterial experiments for the Control, CTMG, CuMOFs / CTMG, and CuMOFs / CTMG / Exos groups are shown below. Figure 4 As shown.

[0087] Example 4

[0088] A composite dressing of human umbilical cord mesenchymal stem cell exosomes-copper-metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan for skin wound repair was prepared by a dissolution method and a freeze-drying method. With appropriate process parameters, CuMOFs and CTMG were mixed at a molar ratio of 1:20 to 1:30 to obtain CuMOFs / CTMG. CuMOFs / CTMG was then mixed with hUMSC-Exos at a molar ratio of hUMSC-Exos, CuMOFs, and CTMG of 1:50:1500 to 1.5:80:1700 to obtain CuMOFs / CTMG / Exos. The lateral mobility of HUVECs induced by the CuMOFs / CTMG-7 and CuMOFs / CTMG / Exos sponge scaffolds was evaluated using a scratch assay.

[0089] Angiogenesis plays a crucial role in skin wound healing. Compared to the control group, both the CuMOFs / CTMG and CuMOFs / CTMG / Exos groups increased HUVEC migration, while the CuMOFs / CTMG / Exos group showed a more significant increase in HUVEC proliferation.

[0090] The above results clearly indicate that CuMOFs / CTMG / Exos significantly promote angiogenesis in open wound healing, thus more efficiently promoting skin wound healing. The cell migration diagrams for the Control group, CuMOFs / CTMG group, and CuMOFs / CTMG / Exos group are shown below. Figure 5As shown.

Claims

1. A composite dressing for skin wound repair using human umbilical cord mesenchymal stem cell exosomes, a copper-based metal-organic framework, and tris(hydroxymethyl)methylglycine-modified chitosan, characterized in that... This dressing is a composite dressing made primarily of CuMOFs, CTMG, and hUMSC-Exos.

2. The human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 1, characterized in that, in, The mass ratio of hUMSCs-Exos, CuMOFs and CTMG is 1:50:1500 - 1.5:80:1700.

3. A method for preparing a composite dressing of human umbilical cord mesenchymal stem cell exosomes-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan for skin wound repair, characterized in that... Includes the following steps: Step 1: Accurately weigh a certain amount of chitosan (CS) and place it in a suitable container. Under stirring conditions, slowly add 1% acetic acid solution to the chitosan and continue stirring until the chitosan is completely dissolved to obtain solution A. Step 2: Add a certain amount of crosslinking agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) to solution A in sequence, stirring continuously during the addition process to ensure that the reactants are fully mixed, thus obtaining solution B; Step 3: Stir solution B continuously for 10-60 minutes to allow the reactants to react completely, resulting in solution C; Step 4: Add an appropriate amount of tris(hydroxymethyl)methylglycine (TMG) to solution C, stir for 12-48 h to obtain solution D, pour it into a special mold to make CTMG hydrogel; Step 5: Accurately weigh a certain amount of anhydrous copper chloride (CuCl2) into a round-bottom flask, add an appropriate amount of deionized water and stir thoroughly to dissolve, to obtain solution E; Step 6: Add an appropriate amount of 5-9 mol / L KOH solution to adjust the pH of solution E to 7.5, and heat the mixed solution in a reactor at 140℃ for 12-48 h to obtain solution F; Step 7: Centrifuge solution F at 8000-12000 rpm for 10-30 min at 4℃ to separate the light gray solid; Step 8: Wash the light gray solid with deionized water 1-3 times and dry it in a vacuum drying oven at 37℃ for 12-48h to obtain CuMOFs powder; Step 9: Slowly add the prepared CuMOFs powder to a certain amount of CTMG hydrogel using a dropper or syringe, so that the CuMOFs powder is fully dissolved in the CTMG hydrogel. The mass ratio of the two is 1:1250 - 1:2000, to obtain solution G. Step 10: Select 3rd to 5th generation human umbilical cord mesenchymal stem cells for culture. When the cells grow to the logarithmic growth phase, collect 100-2500 mL of culture medium into a sterile centrifuge tube. Step 11: Centrifuge the collected culture medium at 4°C at 200-500 g for 5-15 min, 1000-2000 g for 5-15 min, and 8000-15000 g for 20-30 min to remove cell debris and large particles. Step 12: Take the supernatant and centrifuge at 80,000 - 100,000 g for 40 - 80 min at 4℃. Resuspend the precipitate in PBS buffer. Centrifuge the resuspended solution again at 80,000 - 100,000 g for 40 - 80 min at 4℃. The resulting precipitate is hUMSC-Exos. Store it in a -80℃ freezer for later use. Step 13: Take the hUMSC-Exos solution and slowly add it to solution G (the mass ratio of hUMSC-Exos, CuMOFs and CTMG is 1:50:1500 - 1.5:80:1700). Add it dropwise using a pipette. Place the mixed solution in a 4°C environment and stir continuously for 2 hours to ensure that the exosomes are evenly dispersed in the composite solution, thus obtaining solution H. Step 14: Dispense the H solution into specific molds, freeze at -20℃ for 12-48 h, then transfer to -80℃ for 12-24 h, and finally dry in a vacuum freeze dryer for 24-72 h to obtain CuMOFs / CTMG / Exos sponge gel.

4. The preparation method of the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 2, characterized in that, In step 1, the raw material selection and dosage are as follows: the amount of chitosan (CS) is 6-20 g, and the chitosan is dissolved in 100-400 mL of 1% acetic acid solution.

5. The preparation method of the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 3, characterized in that, In step 2, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is 0.1-0.3 g and the amount of N-hydroxy-succinimide (NHS) is 0.1-0.2 g.

6. The preparation method of the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 3, as described in claim 4, is characterized in that... The stirring time in step 3 is 10-60 min.

7. The preparation method of the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 4, as described in claim 5, is characterized in that... In step 4, the amount of tris(hydroxymethyl)methylglycine (TMG) used is 0.4-0.8 g, and the stirring time is 24-72 h.

8. The preparation method of the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 5, as described in claim 6, is characterized in that... In step 5, the amount of anhydrous copper chloride (CuCl2) used is 10-15 g, the volume of deionization is 100-1000 mL, the temperature of the reactor is 100-200℃, and the heating time is 12-48 h.

9. The preparation method of the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 6, as described in claim 7, is characterized in that... In step 6, solution F is centrifuged at 5000-12000 rpm for 10-30 min at 4℃.

10. The preparation method of the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 7, as described in claim 8, is characterized in that... In step 7, the light gray solid is washed with deionized water 1-3 times and dried in a vacuum drying oven at 37°C for 12-48 h to obtain CuMOFs powder.

11. The method for preparing the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 8, as described in claim 9, is characterized in that... In step 8, the concentration of the KOH solution is 4-10 mol / L, and the volume is 1-10 mL.

12. The method for preparing the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 9, as described in claim 10, is characterized in that... In step 9, the mass ratio of CuMOFs to CTMG is 1:1250 - 1:2000, the stirring speed is 100 - 500 r / min, and the time is 1 - 2 h.

13. The method for preparing the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 10, as described in claim 11, is characterized in that... In step 10, the human umbilical cord mesenchymal stem cell culture medium is the 3rd to 5th generation culture medium, and the volume of culture medium used is 100 to 2500 mL.

14. The method for preparing the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 12, characterized in that, In step 11, the parameters for each stage of the culture medium centrifugation process are precisely set. The first stage is 4℃, 200-500 g, 5-15 min; the second stage is 4℃, 1000-2000 g, 5-15 min for further clarification; and the third stage is 4℃, 8000-15000 g, 20-30 min.

15. The method for preparing the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 13 and claim 12, characterized in that, In step 12, the ultracentrifugation is performed twice at 4°C, at 80,000 - 100,000 g for 40 - 80 min.

16. The method for preparing the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 14, characterized in that, In step 13, the mass ratio of hUMSC-Exos, CuMOFs and CTMG is 1:50:1500-1.5:80:1700, and the stirring time is 1-2 h at 4℃.

17. The method for preparing the human umbilical cord mesenchymal stem cell exosome-copper-based metal-organic framework-tris(hydroxymethyl)methylglycine-modified chitosan composite dressing for skin wound repair according to claim 14, as described in claim 15, is characterized in that... In step 14, the freeze-drying conditions are: freezing at -20℃ for 1-48 h; refreezing at -80℃ for 1-48 h; vacuum degree of 0.08-0.1 MPa; and drying time of 1-72 h.

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