An antibacterial and anti-scarring proliferative skin repair patch and a preparation method thereof

Abstract

The application discloses an antibacterial and anti-scarring skin repair patch and a preparation method thereof, and belongs to the technical field of medical dressings. The skin repair patch has a double-layer structure with functional zones, namely, a loose porous layer and a dense support layer, and is prepared from decellularized fat matrix, decellularized vascular matrix, antibacterial peptide and gluconic acid as raw materials through layered preparation and differential cross-linking. The loose porous layer contacts the skin, is rich in fat matrix components and loaded with antibacterial peptides, and is used for broad-spectrum antibacterial and anti-inflammatory in the early stage of a wound surface; the dense support layer is dense and stable, rich in vascular matrix components and loaded with gluconic acid, and is used for anti-scarring regulation and vascular reconstruction in the late healing stage. The skin repair patch is a double-layer biomimetic medical dressing with a hierarchical structure, and realizes innovative design of structural bionics and functional stratification.

Description

Technical Field

[0001] This invention belongs to the field of medical dressing technology, specifically relating to an antibacterial and anti-scar hyperplasia skin repair patch and its preparation method. Background Technology

[0002] Skin tissue defects are a major global clinical challenge caused by burns, trauma, surgery, and chronic diseases such as diabetes. These wounds, which disrupt the skin's physical barrier function, are highly susceptible to secondary bacterial infections, leading to delayed healing and even serious complications such as systemic infection. The underlying pathological mechanism lies in the significant loss of dermis and subcutaneous soft tissue (including fat and vascular networks) during the repair process, causing impaired local blood supply reconstruction and severely weakening the tissue's self-regenerative capacity. More importantly, the terminal stage of wound repair is often accompanied by the formation and contracture of pathological scars, which not only affect the patient's appearance but may also lead to functional impairment and lasting psychological trauma, becoming a clinical challenge that urgently needs to be overcome in the fields of plastic surgery and wound repair.

[0003] Currently, the main clinical treatments for complex skin wounds still involve debridement, infection control, and dressing. However, traditional dressings such as gauze, hydrocolloid dressings, and silver ion dressings have relatively limited functions, providing only basic barrier protection or short-term antibacterial effects. While silver ion dressings possess some antibacterial capabilities, the released silver ions are cytotoxic, inhibiting the activity of fibroblasts and keratinocytes, thus delaying the normal wound healing process.

[0004] In recent years, a series of functional hydrogel materials have emerged, such as self-healing hyaluronic acid nanocomposite hydrogels (Li, Shangzhi, et al. Self-Healing Hyaluronic Acid Nanocomposite Hydrogels with Platelet-Rich Plasma Impregnated for Skin Regeneration. ACS Nano, 2022, 16(7): 11346–11359.), injectable carboxymethyl cellulose / dopamine hydrogels (Cui, Longlong, et al. Injectable Multifunctional CMC / HA-DA Hydrogel for Repairing Skin Injury. Materials Today Bio, 2022, 14: 100257.), and responsive multifunctional hydrogels (Zhang, Ying, et al. Responsive Multifunctional Hydrogels Emulating the Chronic Wounds Healing Cascade for Skin Repair. Advanced Science, 2024, 11(2): 11346–11359.). Systems such as 2304567. have shown potential in the field of skin regeneration, promoting angiogenesis and epithelialization to some extent by regulating the wound microenvironment. However, existing hydrogels are mostly based on simple composites of synthetic polymers and bioactive factors, and their functional design is often focused on a certain stage of the healing process. It is difficult to achieve "temporal synergistic regulation" of early infection control and later scar inhibition, thus remaining significantly limited in dealing with deep contaminated wounds and chronic, difficult-to-heal wounds.

[0005] In terms of novel active ingredients, antimicrobial peptides, as natural immune molecules, have advantages such as broad-spectrum antibacterial activity, low susceptibility to inducing drug resistance, and rational designability. They can exert anti-infective effects by disrupting bacterial membrane structure and regulating local immune responses (Koehbach, Johannes, and David J. Craik. The Vast Structural Diversity of Antimicrobial Peptides. Trends in Pharmacological Sciences, 2019, 40(7): 517–528.). On the other hand, gluconic acid, as a small metabolic molecule, has recently been reported to effectively reduce excessive collagen cross-linking and α-SMA expression by binding to PLOD1, inhibiting the p-AKT signaling pathway, and activating autophagy, thereby inhibiting hypertrophic scar formation (Li, Jingyun, et al. Gluconic Acid Alleviates Hypertrophic Scar Formation through Binding PLOD1, Reducing p-AKT Signaling and Activating Autophagy. Phytomedicine, 2025, 143: 156825.), providing a new approach for anti-fibrotic treatment in the later stages of wound healing.

[0006] Decellularized matrix (dECM), as a class of natural biomimetic materials with excellent biocompatibility, low immunogenicity, and tissue-specific structure, has been widely used in the field of soft tissue repair. Decellularized adipose matrix (dAFM), rich in type III collagen, fibronectin, and hyaluronic acid, can provide a favorable regenerative microenvironment for adipose-derived stem cells and fibroblasts; decellularized vascular matrix (dVAM), rich in vascular-specific components such as elastin, type IV collagen, and laminin, can effectively promote endothelial cell adhesion, migration, and angiogenesis. Theoretically, combining dAFM and dVAM in a specific ratio can construct a biomimetic scaffold that combines soft tissue regeneration and angiogenesis induction functions, providing an ideal matrix for the repair of deep skin defects. However, existing modification strategies for dECM materials mostly focus on improving mechanical properties through cross-linking or composite polymer materials, or enhancing their bioactivity by adding growth factors, exosomes, etc., which still have the following key drawbacks: First, the added bioactive factors mostly adopt a single release mode, which cannot achieve precise temporal regulation of different stages of wound healing; second, the introduced cross-linking agents or polymer materials may interfere with the degradation behavior and biological signal release of dECM itself, which not only makes it difficult to effectively regulate the fibrosis process in the later stage of healing, but may even exacerbate the risk of scar formation; in addition, the material itself lacks strong and sustained antibacterial ability, and is prone to repair failure in contaminated wounds due to the imbalance of infection and inflammation.

[0007] In summary, no existing skin repair material can simultaneously achieve the following objectives: while maintaining the inherent bioactivity and good biocompatibility of dECM materials, constructing a biomimetic matrix with adjustable mechanical and biological properties through the rational compounding of dAFM / dVAM, and further integrating active ingredients such as antimicrobial peptides and gluconic acid to form an intelligent repair system with time-sequential release and multifunctional synergistic effects, so as to meet the clinical needs of full-cycle healing of complex skin wounds. Summary of the Invention

[0008] One object of the present invention is to provide a skin repair patch having a double-layer structure, loaded with antibacterial or anti-scarring factors, and with adjustable performance, prepared from decellularized adipose matrix and decellularized vascular matrix as the main materials; another object of the present invention is to provide a method for preparing the skin repair patch; yet another object of the present invention is to provide the application of the skin repair patch in the preparation of medical dressings for the repair of deep skin trauma, diabetic foot, and burns.

[0009] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a skin repair patch, the skin repair patch having a bilayer structure formed by a loose porous layer and a dense support layer, the loose porous layer being in contact with the skin.

[0010] The loose porous layer is formed by cross-linking decellularized adipose matrix (dAFM) and decellularized vascular matrix (dVAM) in a mass ratio of (2-9):1; the dense support layer is formed by cross-linking decellularized vascular matrix (dVAM) and decellularized adipose matrix (dAFM) in a mass ratio of (2-9):1, wherein the cross-linking method is selected from chemical cross-linking or enzymatic cross-linking.

[0011] In some embodiments of the present invention, the loose porous layer is formed by cross-linking decellularized adipose matrix (dAFM) and decellularized vascular matrix (dVAM) in a mass ratio of (2-9):1; the dense support layer is formed by cross-linking decellularized vascular matrix (dVAM) and decellularized adipose matrix (dAFM) in a mass ratio of (5-6):1.

[0012] In a more preferred embodiment of the present invention, the loose porous layer is formed by cross-linking decellularized adipose matrix (dAFM) and decellularized vascular matrix (dVAM) in a mass ratio of (2-7):1; the dense support layer is formed by cross-linking decellularized vascular matrix (dVAM) and decellularized adipose matrix (dAFM) in a mass ratio of 6:1.

[0013] In the most preferred embodiment of the present invention, the loose porous layer is formed by cross-linking decellularized adipose matrix (dAFM) and decellularized vascular matrix (dVAM) in a mass ratio of 2:1; the dense support layer is formed by cross-linking decellularized vascular matrix (dVAM) and decellularized adipose matrix (dAFM) in a mass ratio of 6:1.

[0014] In a specific embodiment of the present invention, the chemical crosslinking is selected from EDC / NHS crosslinking or glutaraldehyde crosslinking, and the enzymatic crosslinking is selected from gelatinase crosslinking.

[0015] In a preferred embodiment of the present invention, the crosslinking method for forming the loose porous layer is selected from EDC / NHS crosslinking or gelatinase crosslinking, preferably EDC / NHS crosslinking; the crosslinking method for forming the dense support layer is selected from glutaraldehyde crosslinking.

[0016] In some embodiments of the present invention, the thickness of the loose porous layer is 0.5-5 mm, and the thickness of the dense support layer is 0.1-2 mm.

[0017] Preferably, the loose porous layer is loaded with antimicrobial peptides, wherein the antimicrobial peptides are selected from ε-polylysine, nisin, LL-37, Pexiganan, PXL01 or their derivative fragments.

[0018] Preferably, the dense support layer is loaded with gluconic acid, which is selected from sodium gluconate or any sustained-release formulation with gluconic acid as the active molecule.

[0019] Furthermore, the skin repair patch of the present invention also includes a protective layer, which is attached to the loose porous layer to protect the active ingredients in the loose porous layer from contamination. When the skin repair patch is used, the protective layer is peeled off.

[0020] In some embodiments of the present invention, the protective layer is made of polyethylene or polyterephthalate plastic.

[0021] Furthermore, the skin repair patch of the present invention also includes a backing layer, which is on top of the dense support layer and is used to fix the skin repair patch to the skin surface. The backing layer is made of plastic, non-woven fabric, chemical fiber fabric, pure cotton fabric or silk fabric.

[0022] Unless otherwise specified, the decellularized adipose-derived matrix (dAFM) and decellularized vascular matrix (dVAM) described in this invention are prepared using methods well known to those skilled in the art. In a specific embodiment of this invention, the decellularized adipose-derived matrix (dAFM) and decellularized vascular matrix (dVAM) are prepared by the following method: adipose tissue and arterial vessels of mammals are taken, washed, and then subjected to decellularization treatment sequentially with a solution containing Triton X-100 and sodium deoxycholate, followed by freeze-drying and grinding to obtain dAFM and dVAM powders.

[0023] The mammals are selected from one or more of the following: pigs, sheep, rabbits, humans, and laboratory mice.

[0024] In a second aspect, the present invention provides a method for preparing a skin repair patch, the method comprising the following steps: (1) Mix dAFM and dVAM powders at a mass ratio of (2-9):1, dissolve them in an acidic solution containing pepsin, stir continuously until completely digested, add antimicrobial peptides, the amount added is 0.5-5 wt% of the dry weight of the composite matrix, stir thoroughly to form solution 1; (2) Mix dVAM and dAFM powders at a mass ratio of (2-9):1, dissolve them in an acidic solution containing pepsin, stir continuously until completely digested, add gluconic acid in an amount of 0.5-10 wt% of the dry weight of the composite matrix, stir thoroughly to form solution 2; (3) Add EDC / NHS crosslinking agent to solution 1 to make the final concentration of EDC / NHS crosslinking agent 0.05-0.1wt%, mix well and inject into the mold, crosslink at room temperature for 1-2 hours to form a loose porous layer; then, add glutaraldehyde to solution 2 as a crosslinking agent to make the final concentration of glutaraldehyde 0.05-0.25 vol%, mix quickly and inject onto the loose porous layer, crosslink at room temperature for 2-4 hours to form a dense support layer, thus forming a composite double-layer structure; (4) After cross-linking is completed, the residual cross-linking agent is removed by washing with PBS, and the skin repair patch is obtained by sterilization with ultraviolet light or ethylene oxide.

[0025] In a third aspect, the present invention provides the use of the skin repair patch in the preparation of medical dressings for skin injuries.

[0026] In a preferred embodiment of the invention, the skin injury is selected from deep skin trauma, diabetic foot, or burns.

[0027] The technical solution provided by this invention has the following technical advantages: This invention constructs a functionally partitioned, bilayer decellularized matrix skin repair patch. The patch is prepared from decellularized adipose matrix (dAFM), decellularized vascular matrix (dVAM), antimicrobial peptides, and gluconic acid through layered preparation and differentiated cross-linking. In the bilayer structure of the skin repair patch, the layer in contact with the skin has a higher dAFM content and is cross-linked using a mild cross-linking agent to form a loose, porous structure. This soft, porous layer is rich in adipose matrix components and loaded with antimicrobial peptides, providing broad-spectrum antibacterial and anti-inflammatory effects in the early stages of wound healing, while also facilitating early soft tissue reconstruction. The upper composite layer has a higher dVAM content and is cross-linked using a chemical cross-linking agent to form a dense, stable support structure. This dense structure is rich in vascular matrix components and loaded with gluconic acid, serving for anti-scarring regulation and vascular remodeling in the later stages of healing, exhibiting anti-scarring and pro-angiogenic effects. The bilayer structure achieves stable composite formation through a combination of physical embedding and chemical cross-linking, forming an intelligent repair system with spatiotemporally controlled release functionality.

[0028] Furthermore, by adjusting the crosslinking density and component ratio of the two layers, this invention can precisely match the mechanical properties of different layers of skin and achieve the time-sequential release and synergistic effect of drugs. Compared with existing single-function or non-biomimetic decellularized matrix materials, the skin repair patch provided by this invention has excellent biocompatibility, biodegradability, and multi-stage repair functions. It can effectively achieve continuous regulation of "early antibacterial and anti-inflammatory effects—mid-term tissue regeneration—late-stage anti-scarring and angiogenesis promotion," showing significant advantages and broad clinical application prospects in the repair of complex wounds such as deep skin trauma, burns, and diabetic foot. Attached Figure Description

[0029] Figure 1 A diagram or image of a skin repair patch.

[0030] Figure 2 Characterization diagram of the decellularization effect of dAFM or dVAM.

[0031] Figure 3 Polarization of macrophages during growth on skin repair patches, and immunofluorescence staining for polarization markers.

[0032] Figure 4 A plating experiment was conducted after co-culturing skin repair patches with Staphylococcus aureus, and colony counting was performed.

[0033] Figure 5 Images of live / dead cells during fibroblast growth on a skin repair patch.

[0034] Figure 6 The wound healing of a rat model of deep skin wounds was assessed using skin repair patches. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Preparation Example 1: Preparation of decellularized adipose-derived matrix (dAFM) Subcutaneous adipose tissue was collected from healthy SPF-grade adult pigs. After removing surface blood vessels and connective tissue, the adipose tissue was repeatedly rinsed with copious amounts of physiological saline and deionized water until the washing solution became clear, thus removing residual blood and impurities. Subsequently, the washed adipose tissue was cut into tissue blocks of 2-5 mm³ and placed in a decellularization solution for decellularization. The decellularization solution included the following steps: (1) Place the adipose tissue in a buffer solution containing 0.1-1.0% (w / v) Triton X-100 and shake it at 4-25 °C for 12-48 h, changing the water every 4 hours. The buffer solution can be phosphate buffer (PBS) or deionized water, and the ratio of liquid volume to tissue wet weight is 10-30 mL / g. (2) After treatment, rinse repeatedly with PBS 3-5 times, 30 min each time, to remove residual surfactant; (3) The tissue was then transferred to a buffer solution containing 0.1-1.0% (w / v) sodium deoxycholate and shaken at 4-25 °C for 12-48 h. The ratio of liquid volume to tissue wet weight was 10-30 mL / g. (4) After the decellularization process is completed, wash repeatedly with PBS or deionized water 5-10 times until there is no obvious foam in the washing solution and the conductivity is stable.

[0037] After decellularization, the adipose tissue was freeze-fixed at -80 °C and then freeze-dried for 24-72 h. The freeze-dried decellularized adipose matrix was then ground using a low-temperature pulverizing method and sieved through a 40-100 mesh sieve to obtain decellularized adipose matrix powder, which is named dAFM in this invention.

[0038] Preparation Example 2: Preparation of decellularized vascular matrix (dVAM) Harvest the aorta or femoral artery of a healthy SPF-grade adult pig. Remove excess connective tissue from the surrounding vessel tissue and rinse repeatedly with PBS and deionized water to remove residual blood from the lumen. Cut the rinsed vessel tissue into 5–10 mm segments and place them in a decellularization solution for decellularization. The specific steps are as follows: (1) Place the vascular tissue in a buffer solution containing 0.1-1.0% (w / v) Triton X-100 and shake it at 4-25 °C for 12-48 h, changing the water every 4 hours. The buffer solution can be phosphate buffer (PBS) or deionized water, and the ratio of liquid volume to tissue wet weight is 10-30 mL / g. (2) After treatment, rinse repeatedly with PBS 3-5 times, 30 min each time, to remove residual surfactant; (3) The tissue was then transferred to a buffer solution containing 0.1-1.0% (w / v) sodium deoxycholate and shaken at 4-25 °C for 12-48 h. The ratio of liquid volume to tissue wet weight was 10-30 mL / g. (4) After the decellularization process is completed, wash repeatedly with PBS or deionized water 5-10 times until the washing solution is clear.

[0039] The decellularized vascular matrix tissue was frozen at -80 ℃ for 12-24 h and then freeze-dried for 24-72 h. Subsequently, the freeze-dried tissue was ground at low temperature and sieved through a 40-100 mesh sieve to obtain decellularized vascular matrix powder, which is named dVAM in this invention.

[0040] according to Figure 2As shown, the microstructure of decellularized adipose tissue and decellularized vascular tissue was observed using hematoxylin-eosin (HE) staining. No obvious nuclear staining signals (dark purple or blue-purple round / elliptical nuclear staining signals) were observed in the decellularized adipose tissue (left) and decellularized vascular tissue (right), indicating that the cellular components in the adipose and vascular tissues were largely removed after decellularization. The protoplastic region exhibited numerous irregular pore structures, and the overall tissue showed an extracellular matrix structure dominated by eosin staining, with a relatively loose distribution forming a porous network. The decellularized vascular tissue mainly showed continuously distributed eosin-stained areas, a more uniform extracellular matrix structure, relatively smaller pores, and an overall structural density higher than that of the decellularized adipose tissue.

[0041] The HE staining results indicated that the decellularization method effectively removed cellular components from adipose and vascular tissues while preserving the original extracellular matrix structural characteristics of both tissues. Significant differences exist between decellularized adipose and vascular tissues in terms of pore structure and density, providing a structural basis for the subsequent construction of composite skin repair patches with different structural layers.

[0042] Example 1: Preparation method of skin repair patch S1: The dAFM and dVAM powders obtained in the preparation example are mixed at a mass ratio of 2:1, dissolved in an acidic solution containing pepsin, and stirred continuously until completely digested. 1% of the dry weight of the composite matrix of the antimicrobial peptide ε-polylysine is added and stirred thoroughly to form solution 1. S2: Mix the dVAM and dAFM powders obtained in the preparation example at a mass ratio of 2:1, dissolve them in an acidic solution containing pepsin, and continue stirring until completely digested. Add 4% gluconic acid (dry weight of the composite matrix), stir thoroughly to form solution 2. S3: Add EDC / NHS crosslinking agent to solution 1 to make the final concentration of EDC / NHS crosslinking agent 0.1wt%, mix well and inject into the mold. The EDC and NHS are prepared at a mass ratio of 1:1. Crosslink at room temperature for 1 hour to form a loose porous layer. Then, add glutaraldehyde to solution 2 to make the final concentration of glutaraldehyde 0.2 vol%, mix quickly and inject onto the already crosslinked loose porous layer. Crosslink on the loose porous layer at room temperature for 3 hours to form a dense support layer, thus forming a composite double-layer structure. S4: After cross-linking is completed, the residual cross-linking agent is removed by washing with PBS, and the skin repair patch is sterilized by ultraviolet light or ethylene oxide.

[0043] Following the above method, solutions 1 with dAFM to dVAM powder mass ratios of 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1 were prepared, as were solutions 2 with dVAM to dAFM powder mass ratios of 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1. Using a checkerboard method, solutions 1 and 2 were cross-combined to form a total of 64 combinations. Cross-linking and compounding were performed according to step S3 of the above method, followed by cleaning and sterilization to obtain the skin repair patch.

[0044] Example 1: Mechanical Stability Testing Testing method: The maximum stress that patches with different formulations can withstand under pressure is tested using a universal testing machine.

[0045] Patch samples with different formulations prepared in Example 1 were cut into standard sizes (cubes) and then placed on the sample stage of a universal testing machine. Pressure was applied at a standard compression rate until the samples yielded or fractured. The deformation (A) and the maximum force applied (F) of the samples were recorded. max The compressive strength is obtained from the stress-strain formula (compressive strength = F). max / A) The results are shown in Table 1. Gradually increasing the dVAM content resulted in enhanced structural support; a higher dAFM ratio made the patch more flexible and suitable for direct skin contact. When selecting the optimal ratio, a balance needs to be struck between the flexibility of the porous layer and the rigidity of the dense support layer. Based on the practical experience of those skilled in the art, the overall compressive strength of the skin repair patch is preferably controlled within the range of 30-45 kPa. This mechanical range is highly close to the equivalent modulus of human dermis and soft tissue, which helps reduce the mechanical mismatch between the patch and surrounding tissues, thereby reducing stress concentration and promoting stable wound healing. According to the compressive strength test results of different formulations in Table 1, when the mass ratio of dAFM to dVAM in the porous layer of the patch is 2:1-9:1, and the mass ratio of dVAM to dAFM in the dense support layer is 5:1-6:1, the overall compressive strength of the resulting composite bilayer skin repair patch is within the aforementioned preferred range. When the mass ratio of dAFM to dVAM in the loose porous layer of the patch is 2:1-7:1, and the mass ratio of dVAM to dAFM in the dense support layer is 6:1, the overall compressive strength of the resulting composite bilayer skin repair patch is >40 kPa, indicating superior performance.

[0046] Technicians discovered that when the mass ratio of dAFM to dVAM in the porous layer of the patch is 2:1, and the mass ratio of dVAM to dAFM in the dense support layer is 6:1, the compressive strength of the resulting skin repair patch is 42.73 kPa. At this ratio, the patch maintains good flexibility and skin adhesion in the porous layer while providing stable and durable mechanical support through the dense support layer. Its mechanical properties are close to the upper limit of soft tissue but do not exceed the skin's tolerance range. This improves the structural support stability of the wound area without making the patch too stiff, which would affect comfort and the healing process. Therefore, the skin repair patch obtained under these ratios is considered a preferred implementation that balances flexibility and support performance, suitable for most skin wound repair applications.

[0047] Table 1. Compressive strength (kPa) of patches with different formulations In the following efficacy verification experiments, the skin repair patches used all refer to skin repair patches prepared by mixing a loose porous layer with a ratio of dAFM:dVAM=2:1 and a dense support layer with a ratio of dVAM:dAFM=6:1.

[0048] Example 2: Antibacterial effect (1) Skin repair patches have anti-inflammatory effects Mouse-derived macrophage cell lines (such as RAW264.7 macrophages) were selected as the research subjects. Before the experiment, the skin repair patches prepared in this invention were cut into circular pieces matching the size of the cell culture wells. The loose porous layer of the skin repair patch was placed face up on the bottom of the glass culture dish. Pre-wetting and drying were performed to ensure the dried patch adhered fully to the bottom of the culture dish, preventing it from floating. Before cell culture, the cells were sterilized under ultraviolet light for at least 30 minutes, and then macrophages were seeded into each well at a cell density of 1 × 10⁻⁶ cells / well. 5 -5×10 5 Cells were seeded per well. After 6 hours, cells adhered and grew on the patch surface. The culture conditions were 37°C and a 5% CO2 incubator. The control group was cultured under the same conditions, with macrophages seeded onto the surface of a commercially available adhesive bandage.

[0049] After culturing for 24-48 h, cells were fixed with 4% paraformaldehyde and then subjected to permeabilization and blocking treatments. Immunofluorescence staining was then performed on macrophage pro-inflammatory M1 polarization markers using anti-iNOS antibody, while phalloidin was used to label the cytoskeleton and DAPI to label the nuclei. The immunofluorescence results are shown in Figure 3. Compared with the control group, the green fluorescence signal of iNOS in macrophages in the patch group was significantly weakened, and the cell morphology was predominantly round or oval, without obvious spreading or pseudopodia extension; while in the control group, the iNOS expression level was higher, the cell nuclei were significantly larger, and the cells showed a clear activated state.

[0050] The above results indicate that the skin repair patch prepared in this invention can inhibit macrophage polarization towards the pro-inflammatory M1 phenotype under conditions of direct contact with macrophages, and produce a significant anti-inflammatory effect at the site of injury.

[0051] (2) Skin repair patches have antibacterial effects A rat model of deep skin wound infection (Staphylococcus aureus infection) was established. The skin repair patch prepared according to this invention was applied to the wound for 72 hours, and samples were taken for a plaster smear experiment. The control group consisted of ordinary commercially available adhesive bandages applied to the wounds of the rat deep skin wound infection model. The test results are as follows: Figure 4 As shown, the control group had a higher number of colonies, with more than 200 independent colonies; the patch group had 5 colonies. The results indicate that the decellularized matrix patch has a significant antibacterial effect.

[0052] Example 3: Effect of inhibiting scar hyperplasia (1) Skin repair patches have the effect of inhibiting excessive proliferation of fibroblasts. Patch group: Fibroblasts were cultured on the dense support layer of the skin repair patch. Cell viability and mortality were observed after 24 and 72 hours of culture. The control group consisted of ordinary commercially available adhesive bandages. Test results are as follows: Figure 5 As shown, compared with the control group, the fibroblasts in the patch group had less proliferation, but maintained good cell morphology and no dead cells (red) were observed, indicating that the patch effectively inhibited the excessive proliferation of fibroblasts.

[0053] (2) Skin repair patches significantly inhibit scar hyperplasia A rat model of deep skin wound infection was established. The skin repair patch prepared in this invention was applied to the wound. The control group consisted of a commercially available adhesive bandage applied to the wound in the rat deep skin wound infection model. Samples were taken and sectioned for HE staining after 7 days. The results are as follows: Figure 6 As shown, the thickness of newly formed scar connective tissue in the patch group was 2.3 mm, compared with 4.4 mm in the control group, indicating that the patch effectively reduced scar hyperplasia by 47.73%.

[0054] Optimization of crosslinking agent in the experiment The preparation method of the skin repair patch is the same as in Example 1. The mass ratio of dAFM to dVAM powder in solution 1 is 2:1, and the mass ratio of dVAM to dAFM powder in solution 2 is 6:1. Solution 1 and solution 2 are crosslinked according to the types and amounts of crosslinking agents shown in Table 2. The crosslinking time is the same as in Example 1 to form a composite bilayer structure. After cleaning and disinfection, the skin repair patch is obtained, and the composite stability of the skin repair patch is tested.

[0055] Table 2 Compressive Strength (kPa) Considering the comprehensive requirements of skin repair patches in practical applications for flexibility, structural stability, and long-term mechanical support, and based on the compressive strength results of patches with different crosslinking methods shown in Table 2, it can be seen that when Solution 1 uses the EDC / NHS system for crosslinking and Solution 2 uses glutaraldehyde for crosslinking, the constructed composite bilayer structure exhibits the best overall mechanical properties and interlayer stability. Solution 1, as the loose, porous antibacterial layer in contact with the skin, focuses on mimicking the soft mechanical environment of natural adipose tissue. EDC / NHS crosslinks through the formation of stable amide bonds. The loose, porous layer after EDC / NHS crosslinking still maintains low compressive strength and good flexibility, meeting the mechanical requirements of skin patches directly contacting wounds. Solution 2, as the outer dense support and anti-scarring layer, plays a crucial role in providing stable mechanical support to the wound, limiting excessive tissue proliferation, and reducing the risk of scar formation, thus placing higher demands on the structural strength and long-term stability of the material. Glutaraldehyde can undergo multi-point cross-linking with the abundant amino groups in matrix proteins, forming a three-dimensional network structure with high cross-linking density, significantly improving the compressive strength and resistance to deformation of the material. Solution 2 uses glutaraldehyde cross-linking to meet the mechanical stability requirements of the dense support layer.

[0056] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. A skin repair patch, wherein the skin repair patch comprises a two-layer structure consisting of a loose porous layer and a dense support layer, the loose porous layer being in contact with the skin; the loose porous layer is formed by cross-linking decellularized adipose matrix and decellularized vascular matrix at a mass ratio of (2-9):1; the dense support layer is formed by cross-linking decellularized vascular matrix and decellularized adipose matrix at a mass ratio of (2-9):1; the cross-linking method of the loose porous layer is selected from EDC / NHS cross-linking; the cross-linking method of the dense support layer is selected from glutaraldehyde cross-linking; the loose porous layer is loaded with antimicrobial peptides; and the dense support layer is loaded with gluconic acid.

2. The skin repair patch according to claim 1, characterized in that, The loose porous layer is formed by cross-linking decellularized adipose matrix and decellularized vascular matrix in a mass ratio of (2-9):1; the dense support layer is formed by cross-linking decellularized vascular matrix and decellularized adipose matrix in a mass ratio of (5-6):

1.

3. The skin repair patch according to claim 1, characterized in that, The thickness of the loose porous layer is 0.5-5 mm, and the thickness of the dense support layer is 0.1-2 mm.

4. The skin repair patch according to claim 1, characterized in that, The skin repair patch also includes a protective layer that adheres to the porous layer to protect the active ingredients in the porous layer from contamination. When the skin repair patch is used, the protective layer is peeled off. The protective layer is made of polyethylene or polyethylene terephthalate plastic.

5. The skin repair patch according to claim 1, characterized in that, The skin repair patch also includes a backing layer, which is on top of the dense support layer and is used to fix the skin repair patch to the skin surface. The backing layer is made of plastic, non-woven fabric, chemical fiber fabric, pure cotton fabric or silk fabric.

6. A method for preparing the skin repair patch according to claim 1, the method comprising the following steps: (1) Mix the decellularized adipose matrix and decellularized vascular matrix powder at a mass ratio of (2-9):1, dissolve them in an acidic solution containing pepsin, stir continuously until completely digested, add antimicrobial peptides, the amount added is 0.5-5 wt% of the dry weight of the composite matrix, stir thoroughly to form solution 1; (2) Mix the decellularized vascular matrix and decellularized lipid matrix powder at a mass ratio of (2-9):1, dissolve them in an acidic solution containing pepsin, stir continuously until completely digested, add gluconic acid, the amount added is 0.5-10 wt% of the dry weight of the composite matrix, stir thoroughly to form solution 2; (3) Add EDC / NHS crosslinking agent to solution 1 to make the final concentration of EDC / NHS crosslinking agent 0.05-0.1wt%, mix well and inject into the mold, crosslink at room temperature for 1-2 hours to form a loose porous layer; Subsequently, glutaraldehyde was added to solution 2 as a crosslinking agent to make the final concentration of glutaraldehyde 0.05-0.25 vol%. The mixture was quickly mixed and injected onto the loose porous layer. Crosslinking was carried out at room temperature for 2-4 hours to form a dense support layer, thus forming a composite double-layer structure. (4) After cross-linking is completed, the residual cross-linking agent is removed by washing with PBS, and the skin repair patch is obtained by sterilization with ultraviolet light or ethylene oxide.

7. The use of the skin repair patch according to any one of claims 1-5 in the preparation of medical dressings for skin injuries.

8. The application according to claim 7, characterized in that, The skin injury is selected from deep skin trauma, diabetic foot, or burns.

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