Preparation method and application of artificial dermis based on bionic acellular matrix

Abstract

The invention relates to the technical field of artificial dermis, in particular to a preparation method and application of artificial dermis based on bionic acellular matrix. The artificial dermis comprises a bionic acellular matrix component A and an auxiliary material component B, wherein the component A comprises type I collagen, type VI collagen, elastin, fibronectin, laminin, mother cell protein and glucan; the component B comprises antibacterial peptide, antibiotics and a high-molecular polymer material; the mass ratio of the component A to the component B is (4-6): (1-4). The preparation method comprises the following steps: respectively preparing procedure mother liquor of the component A and the component B, preparing hydrogel with a double-layer gradient structure through step-by-step crosslinking, and then carrying out freeze drying and sterilization treatment. The artificial dermis disclosed by the invention has good biocompatibility and antibacterial property, can effectively promote wound healing, is suitable for repairing acute and chronic wounds of four limbs, faces and trunk parts, and is particularly suitable for repairing complex wounds with exposed bones, tendons, nerves and blood vessels.

Description

Technical Field

[0001] This invention relates to the field of artificial dermis technology, specifically to a method for preparing and applying artificial dermis based on a biomimetic decellularized matrix. Background Technology

[0002] In the field of skin tissue engineering, dermal regeneration technologies are mainly divided into three categories: autologous skin transplantation, allogeneic / xenogeneic acellular dermal matrix (ADM), and synthetic biomimetic scaffold materials. Autologous transplantation remains the gold standard in clinical practice, but its donor site limitations have prompted researchers to explore alternatives. Animal-derived ADMs (such as porcine skin and bovine pericardium) retain the extracellular matrix (ECM) structure through decellularization, but pose potential immunogenicity risks. While synthetic materials (such as collagen-chitosan scaffolds) can be customized, they are difficult to fully mimic the biological functions of natural dermis. In addition, recent studies have attempted to combine 3D printing or electrospinning techniques to prepare biomimetic scaffolds, but many challenges remain in clinical translation.

[0003] Although autologous skin transplantation boasts excellent tissue compatibility, its application is limited by the donor site area, especially for patients with extensive burns where the availability of healthy skin for transplantation is severely insufficient. Furthermore, the skin harvesting procedure may lead to hypertrophic scarring, pigmentation, or functional impairment at the donor site, and the transplanted skin often experiences contracture due to a lack of dermal support, affecting both aesthetics and function. While decellularization techniques can partially remove xenoantigens, residual α-Gal epitopes may still trigger a host immune response, leading to accelerated graft degradation or fibrous encapsulation. Simultaneously, the mechanical properties (such as tensile strength and creep) of animal-derived materials differ significantly from human dermis, and the decellularization efficiency varies between batches, potentially affecting the reproducibility of clinical outcomes.

[0004] Existing biomimetic scaffolds mostly focus on structural biomimicry (such as mimicking collagen fiber arrangement), but neglect the crucial regulatory role of the dynamic biochemical microenvironment of the ECM (such as growth factor gradients and mechanotransduction) on cell behavior. For example, electrospinning technology can prepare nanofibers that mimic the topology of the ECM, but their dense fiber network may hinder effective cell infiltration and migration; while 3D printing technology promotes macroscopic pores for vascularization, it is difficult to precisely match the material degradation rate with the timing requirements of tissue regeneration.

[0005] To address the aforementioned issues, this application proposes a biomimetic decellularized matrix that achieves innovation at three levels by integrating the bioactive components of natural ECM with the tunable properties of synthetic materials: (1) Immunogenicity control: using cross-species conserved ECM proteins (such as type I / III collagen) as the substrate, combined with targeted enzymatic hydrolysis technology to thoroughly remove xenogeneic antigens; (2) Dynamic mechanical adaptation: introducing an elastic fiber network and a gradient cross-linking strategy to provide mechanical support in the early stages of implantation, and gradually softening as regeneration progresses to match the mechanical needs of newly formed tissue; (3) Active regeneration induction: constructing a biomimetic microtubule system in the scaffold to load and release cytokines (such as VEGF and FGF-2), simultaneously regulating angiogenesis and fibroblast activation. This design is expected to overcome the dilemma of existing materials being "structurally biomimetic but functionally lagging," providing an integrated regeneration solution for complex skin defects. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a method for preparing and applying artificial dermis based on a biomimetic decellularized matrix.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] This invention provides an artificial dermis based on a biomimetic decellularized matrix, comprising a biomimetic decellularized matrix component A and an excipient component B;

[0009] The biomimetic decellularized matrix A component includes: type I collagen, type VI collagen, elastin, fibronectin, laminin, blastocyst protein, and dextran;

[0010] The excipient B component includes: antimicrobial peptides, antibiotics, and high molecular polymer materials; the mass ratio of the biomimetic decellularized matrix component A to the excipient B component is (4-6):(1-4).

[0011] Preferably, the biomimetic decellularized matrix A component comprises the following components by mass fraction:

[0012] Type I collagen 55wt%-80wt%;

[0013] Type VI collagen 5wt%-15wt%;

[0014] elastin 1wt%-5wt%;

[0015] Fibronectin 1wt%-5wt%

[0016] Laminin 1wt%-5wt%;

[0017] 1wt%-5wt% of blastocyst protein;

[0018] Glucan 5wt%-15wt%.

[0019] The biomimetic decellularized matrix component A of this invention mimics the composition of decellularized dermal matrix. Type I and Type VI collagen jointly construct a fibrous skeleton, providing necessary mechanical support and cell adhesion sites. Elastin imparts elasticity to the material, buffering mechanical stress on the wound surface. Fibronectin and laminin can mediate the adhesion and migration of fibroblasts, keratinocytes, and endothelial cells. Steroid proteins and dextran are beneficial for enriching endogenous growth factors and regulating their sustained release. Thus, without directly using animal-derived decellularized tissue, a biochemical microenvironment similar to that of natural dermis can be reconstructed, promoting angiogenesis and dermal regeneration.

[0020] Preferably, the excipient B component comprises the following components by mass fraction:

[0021] Antimicrobial peptides 5wt%-15wt%;

[0022] Antibiotics 1wt%-5wt%;

[0023] Polymer materials 70wt%-95wt%.

[0024] The excipient B component contains antimicrobial peptides and antibiotics, which can rapidly inhibit bacterial proliferation and reduce the risk of infection and inflammation. At the same time, functionalized carboxymethyl chitosan from polymer materials is used as the main framework, making component B not only a simple antimicrobial carrier, but also a biomimetic polysaccharide with multiple functions.

[0025] Preferably, the antibiotic is selected from at least one of cefuroxime, vancomycin, or gentamicin.

[0026] Preferably, the polymer material is selected from at least one of chitosan, carboxymethyl chitosan, and functionalized carboxymethyl chitosan.

[0027] Functionalized carboxymethyl chitosan is composed of carboxymethyl chitosan, methacrylamide gelatin, dopamine hydrochloride, boric acid, and zinc acetate. Carboxymethyl chitosan itself possesses both polysaccharide backbone and cationic properties, enabling electrostatic interactions with collagen in component A, enhancing overall stability, and providing basic antibacterial capabilities through electrostatic interactions with bacterial cell membranes. Methacrylamide gelatin provides collagen-like bioactive sequences and cell adhesion sites, allowing component B to form a continuous structural transition with component A, further improving cell affinity. Dopamine hydrochloride undergoes partial oxidation under near-neutral conditions. Its catechol groups, on the one hand, form dynamic catechol and borate ester crosslinks with boric acid, endowing the gel with good wet adhesion and a certain degree of self-healing ability, ensuring that the material can still adhere tightly to the wound surface in a wound environment rich in exudate, reducing dead space and exudate accumulation; on the other hand, the catechol groups can react with Zn released from zinc acetate. 2+Coordination occurs, forming cross-linking sites between metals and catechols, which improves the mechanical strength of the gel network while enabling Zn to... 2+ It can be slowly and continuously released, exerting broad-spectrum antibacterial effects and promoting epithelialization and collagen remodeling. Functionalized carboxymethyl chitosan shows significant improvements in antibacterial properties, wet adhesion properties, and cell adhesion and infiltration capabilities, reducing the incidence of infection and thus achieving faster vascularization and higher quality dermal regeneration under the same wound conditions.

[0028] Preferably, the preparation method of the functionalized carboxymethyl chitosan includes the following steps:

[0029] S1. Mix carboxymethyl chitosan with an aqueous acetic acid solution, stir, and let stand to obtain a carboxymethyl chitosan solution;

[0030] S2. Add methacrylamide gelatin solution to carboxymethyl chitosan solution, stir, cool and then add dopamine hydrochloride and boric acid to adjust pH to 7.4, stir to form gel solution, add zinc acetate, stir, freeze dry to obtain functionalized carboxymethyl chitosan.

[0031] Preferably, in step S1, the mass concentration of the carboxymethyl chitosan solution is 3-5% (w / v), and the concentration of the acetic acid aqueous solution is 0.05-0.15% (w / v); in step S2, the mass concentration of the methacryloyl gelatin solution is 8-12 wt%, the amount of dopamine hydrochloride added is 5-10% of the mass of carboxymethyl chitosan, the amount of boric acid added is 2-5% of the mass of carboxymethyl chitosan, and the amount of zinc acetate added is 0.5-2% of the mass of carboxymethyl chitosan.

[0032] A method for preparing artificial dermis based on biomimetic decellularized matrix includes the following steps:

[0033] (1) Preparation of the mother liquor of component A of the biomimetic decellularized matrix: Add each substance in component A of the biomimetic decellularized matrix to physiological saline and stir at 4°C for 12-24 h until completely dissolved to obtain the mother liquor of component A;

[0034] (2) Preparation of excipient B component mother liquor: Add each substance in excipient B component to physiological saline and stir at 37°C for 24-48 hours until completely dissolved to obtain excipient B component mother liquor;

[0035] (3) Preparation of porous layer preform on the wound side: Mix part of the mother liquor of component A and part of the mother liquor of component B at a mass ratio of 5:2, control the solid content of the mixed solution to be 0.5-1.5wt%, add EDC aqueous solution and NHS aqueous solution after stirring, crosslinking reaction, inject into mold, let stand, and obtain porous layer preform on the wound side.

[0036] (4) Preparation of the epidermal side layer: Mix part of the A component mother liquor and part of the B component mother liquor at a mass ratio of 5:3, control the solid content of the mixed solution to be 2-4wt%, add EDC aqueous solution and NHS aqueous solution after stirring to obtain the second cross-linking solution; pour the second cross-linking solution onto the surface of the porous layer blank on the wound side, cross-link to obtain a double-layer hydrogel; freeze dry and sterilize to obtain artificial dermis based on biomimetic decellularized matrix.

[0037] Preferably, the biomimetic decellularized matrix-based artificial dermis is prepared using the following method:

[0038] (1) Preparation of biomimetic decellularized matrix A component stock solution: Weigh each substance in the biomimetic decellularized matrix A component and add them sequentially to 800-1400 mL of physiological saline with a concentration of 0.8-1.2 wt%. Stir continuously at a mechanical stirring speed of 600-1000 rpm for 12-24 h at 2-6℃ until each substance is completely dissolved and uniformly dispersed to obtain the A component stock solution;

[0039] (2) Preparation of excipient B component mother liquor: Weigh each substance in the prepared excipient B component and add it to 80-120 mL of physiological saline with a concentration of 0.8-1.2 wt%. Stir continuously at 800 rpm for 24-48 h at 30-45℃ until each substance is completely dissolved and uniformly dispersed to obtain the excipient B component mother liquor.

[0040] (3) Preparation of porous layer preform on the wound side: At room temperature, a portion of the mother liquor of component A prepared in step (1) and a portion of the mother liquor of component B prepared in step (2) are mixed at a mass ratio of (3-6): (1-3), and the total solid content is controlled at 0.5-1.5wt%. The mixture is stirred at a speed of 300-600rpm for 8-15min. 40-80mL of 40-60mMEDC aqueous solution and 20-35mL of 20-30mMNHS aqueous solution are added to the above mixed solution in sequence. The mixture is stirred at room temperature for 10-30min. After the crosslinking reaction is completed, the resulting gel-like mixture is injected into a mold of a predetermined shape, with a thickness of 1-3mm. The mixture is allowed to stand for 15-40min to allow it to initially form in the mold and obtain a porous layer preform on the wound side.

[0041] (4) At room temperature, mix a portion of the mother liquor of component A prepared in step (1) with a portion of the mother liquor of component B prepared in step (2) at a mass ratio of (3-6):(2-4), with the total solid content controlled at 2-4 wt%, and stir at 300-600 rpm for 8-15 min; add 40-70 mL of 50-70 mM EDC aqueous solution and 20-35 mL of 30-40 mM EDC aqueous solution to the above mixed solution in sequence. NHS aqueous solution was stirred at room temperature for 10-30 min to obtain a second cross-linked solution. While the porous layer preform on the wound side was still in a semi-solid state, the second cross-linked solution was slowly poured onto its surface, allowing it to spread and cover naturally to form an epidermal side layer. Cross-linking was then continued at room temperature for 2-4 h to allow the upper and lower layers to covalently cross-link and bond firmly at the interface, resulting in a bilayer hydrogel with a thickness of 2-5 mm. The obtained bilayer hydrogel was stored in a -80℃ freezer for 12-48 h, freeze-dried, and sterilized to obtain an artificial dermis based on a biomimetic decellularized matrix.

[0042] The preparation method of this invention is not a single-step mixing and freeze-drying process, but rather a process that combines the preparation of a large-pore, low-crosslinked layer on the wound side and a small-pore, high-crosslinked layer on the epidermal side, followed by overall freeze-drying. This creates a gradient of pore size and crosslinking degree that varies continuously along the thickness direction within the same artificial dermis. Simultaneously, a dynamic catechol network and metal ion slow-release uniformly embed into the double-layer structure. The resulting biomimetic decellularized matrix artificial dermis has a double-layer gradient structure: the outer layer is a dense structure with a rough surface, which can act as a physical barrier to effectively resist the invasion of external microorganisms and reduce wound moisture evaporation. At the same time, its moderately rough surface morphology is conducive to the adhesion, proliferation, and migration of epidermal cells at the wound edge to the center of the wound, thereby promoting the wound to self-epithelialize and close, reducing or avoiding secondary skin grafting, and shortening the healing time; the lower layer is a three-dimensional porous structure with good biocompatibility and can be gradually degraded in vivo, providing channels for fibroblasts and capillaries to grow rapidly from the wound base and surrounding tissues, shortening the vascularization process of the scaffold. Furthermore, the functionalized carboxymethyl chitosan, antimicrobial peptides, and antibiotics in the biomimetic decellularized matrix component A and excipient component B introduced into the collagen sponge scaffold layer can regulate and enrich endogenous growth factors, synergistically exerting antibacterial / bacteriostatic, anti-inflammatory, and pro-angiogenic effects, and significantly promoting wound healing overall.

[0043] An application of a biomimetic decellularized matrix artificial dermis in the repair of various acute and chronic wounds on the limbs, face and trunk, especially suitable for wound repair of important tissues such as bones, tendons, nerves and blood vessels.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) This invention does not directly use animal-derived decellularized dermal matrix, but uses decellularized extracellular matrix material prepared from bovine Achilles tendon as a template. Through artificial analysis and precise compounding of its core functional components, including type I collagen, type VI collagen, elastin, fibronectin, laminin, blastocysteine, and dextran, it simulates the bioactivity of the natural extracellular matrix at the molecular level. This artificially compounded material, with components similar to those of bovine Achilles tendon decellularized extracellular matrix, can mimic the bioactivity of the decellularized extracellular matrix. Simultaneously, the artificially added components effectively avoid the immunogenicity and viral problems associated with the material after decellularization, resulting in a safer and more efficient artificial product.

[0046] (2) This invention constructs an artificial dermal scaffold with a biomimetic three-dimensional network structure by introducing functionalized carboxymethyl chitosan and other high-molecular polymer materials and using an EDC / NHS crosslinking system. This structure not only simulates the arrangement of collagen fibers in natural dermis in terms of microscopic morphology, but more importantly, in order to imitate the three-dimensional structure of collagen decellularized, it achieves biomimetic decellularized matrix in terms of structure. Combined with the biomimetic interaction of the above-mentioned components, it realizes a gradient change in porosity and mechanical properties, which is more conducive to cell migration, infiltration and vascularization, and achieves synergistic biomimetic effects in both composition and structure.

[0047] (3) In this invention, antimicrobial peptides and antibiotics are introduced into excipient B, and catechol and Zn in functionalized carboxymethyl chitosan are utilized. 2+ The coordination system enables the sustained release of zinc ions, providing a powerful and long-lasting broad-spectrum antibacterial capability that effectively prevents and controls post-implantation infection and reduces inflammatory responses. Simultaneously, the functionalized carboxymethyl chitosan itself possesses excellent wet tissue adhesion and certain self-healing properties, allowing it to closely adhere to the wound surface, reduce dead space, and provide a stable microenvironment for wound healing.

[0048] (4) The artificial dermis prepared by this invention has excellent overall performance and good biocompatibility. It can effectively promote the activation and migration of fibroblasts and vascular endothelial cells, and significantly accelerate the wound healing process. The material of this invention solves the limitation of autologous skin transplantation required for clinical treatment of dermal regeneration. It has good effects on wound repair and dermal regeneration, and is especially suitable for the repair of complex wounds with exposed bone, tendons and other tissues. It can effectively avoid or reduce secondary skin grafting, shorten the treatment cycle and improve the healing quality. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 This is a photograph of the artificial dermis prepared in Example 1;

[0051] Figure 2 The image shows the microstructure of the artificial dermis prepared in Example 1 using SEM. Detailed Implementation

[0052] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0053] The raw materials described in this application are partially described; all other raw materials not described are commercially available.

[0054] Type I collagen was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: H1450900.

[0055] Type VI collagen was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: C390608.

[0056] Elastin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: E754127.

[0057] Fibronectin was purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd., product number lnb-1604.

[0058] The laminin was purchased from Merck Life Sciences, Inc., catalog number: 11243217001.

[0059] The blastocyst protein was prepared in the laboratory from mouse fibroblasts. The preparation method of the blastocyst protein is as follows: Step 1: Take mouse fibroblasts in the logarithmic growth phase, resuspend them in serum-free pH 7.4, 0.01 mol / L PBS buffer, and prepare a cell suspension (1×10⁻⁶). 6 -1×10 7 The cell suspension (cells / mL) was transferred to a pre-cooled 1.5 mL centrifuge tube, placed on ice and allowed to stand for 10 min before being subjected to ultrasonic lysis. The ultrasonic conditions were 25 W power, 5 s each time, 3 s interval, and repeated 4 times to obtain the ultrasonically treated cell suspension.

[0060] Step 2: The cell suspension after sonication was centrifuged at 4°C and 14,000 r / min for 15 min. After centrifugation, the supernatant was collected and freeze-dried to obtain the mother cell protein.

[0061] The dextran was purchased from Shanghai Maclean Biotechnology Co., Ltd., item number: D992645.

[0062] The antimicrobial peptide, made from frog skin, was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number: D807939.

[0063] Cefuroxime was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number: C922445.

[0064] Carboxymethyl chitosan was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number: C304739-25g.

[0065] Methacrylamide gelatin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number: G477882-1g.

[0066] Example 1

[0067] A biomimetic decellularized matrix-based artificial dermis, composed of biomimetic decellularized matrix component A and excipient component B;

[0068] The biomimetic decellularized matrix component A is composed of the following raw materials, in the following mass percentages: Type I collagen 70wt%, Type VI collagen 10wt%, elastin 2wt%, fibronectin 2wt%, laminin 2wt%, blastocyst protein 2wt%, and dextran 12wt%. Each substance in the biomimetic decellularized matrix component A is accurately weighed according to the above proportions, with a total mass of 10g.

[0069] The excipient B component is composed of the following raw materials, by mass percentage: 8 wt% antimicrobial peptide, 2 wt% cefuroxime, and 90 wt% polymer material; each substance in the excipient B component is accurately weighed according to the above proportions, with a total mass of 4 g; the polymer material is functionalized carboxymethyl chitosan.

[0070] The preparation method of the functionalized carboxymethyl chitosan is as follows:

[0071] S1. Mix 3.6g of carboxymethyl chitosan with 90mL of 0.1% (w / v) acetic acid aqueous solution, stir at 600rpm for 2h at room temperature, and let stand at room temperature for 1h to eliminate bubbles, to obtain a homogeneous and transparent carboxymethyl chitosan solution with a mass concentration of 4% (w / v), for later use.

[0072] S2. Take all of the above carboxymethyl chitosan solution, add 10 mL of 10 wt% methacrylamide gelatin solution at 55℃, stir at 400 rpm for 45 min, cool to 37℃, add 0.3 g dopamine hydrochloride and 0.15 g boric acid, adjust the pH of the solution to 7.4 with 1 mol / L NaOH aqueous solution, and continue stirring at 37℃ for 8 min to form a gel solution. Add 15 mg zinc acetate to the gel solution, stir at 37℃ for 30 min, and freeze dry to obtain functionalized carboxymethyl chitosan.

[0073] The mass ratio of the biomimetic decellularized matrix component A to the excipient component B is 5:2.

[0074] The aforementioned biomimetic decellularized matrix-based artificial dermis was prepared using the following method:

[0075] (1) Preparation of biomimetic decellularized matrix A component mother liquor: The weighed substances in biomimetic decellularized matrix A component are added to 1000 mL of physiological saline with a concentration of 0.9 wt% in sequence. The mixture is stirred continuously at 800 rpm for 18 h at 4 °C until all substances are completely dissolved and uniformly dispersed to obtain A component mother liquor.

[0076] (2) Preparation of excipient B component mother liquor: Weigh each substance in the excipient B component and add it to 100 mL of physiological saline with a concentration of 0.9 wt%. Stir continuously at 800 rpm for 36 h at 37 °C until each substance is completely dissolved and evenly dispersed to obtain the excipient B component mother liquor.

[0077] (3) Preparation of porous layer preform on the wound side: At room temperature, a portion of the mother liquor of component A prepared in step (1) and a portion of the mother liquor of component B prepared in step (2) are mixed at a mass ratio of 5:2, and the total solid content is controlled at 1wt%. The mixture is stirred at 500rpm for 10min. 50mL of 50mMEDC aqueous solution and 25mL of 25mMNHS aqueous solution are added to the above mixed solution in sequence. The mixture is stirred at room temperature for 15min. After the crosslinking reaction is completed, the resulting gel-like mixture is injected into a mold of a predetermined shape, with a thickness of 2.5mm. The mixture is left to stand for 25min to allow it to initially form in the mold and obtain a porous layer preform on the wound side.

[0078] (4) At room temperature, a portion of the mother liquor of component A prepared in step (1) and a portion of the mother liquor of component B prepared in step (2) are mixed at a mass ratio of 5:3, and the total solid content is controlled at 3wt%. The mixture is stirred at 500 rpm for 10 min. 50 mL of 60 mMMEDC aqueous solution and 25 mL of 35 mMMNHS aqueous solution are added to the above mixed solution in sequence. Stirring is continued at room temperature for 15 min to obtain the second cross-linking solution. When the porous layer blank on the wound side is still in a semi-cured state, the second cross-linking solution is slowly poured onto its surface to allow it to spread and cover naturally, forming the epidermal side layer. Cross-linking is continued at room temperature for 3 h to allow the upper and lower layers to covalently cross-link and firmly bond at the interface, resulting in a bilayer hydrogel with a thickness of 4 mm. The obtained bilayer hydrogel is stored in a -80℃ refrigerator for 24 h, freeze-dried, and sterilized to obtain an artificial dermis based on a biomimetic decellularized matrix.

[0079] The actual images and SEM images of the prepared artificial dermis are shown below. Figure 1 and Figure 2 ,from Figure 1 , Figure 2 It is known that the artificial dermis prepared by the present invention has a complete appearance, uniform thickness, and tight interlayer bonding; its SEM microstructure shows that the epidermal side is a relatively dense microporous layer and the wound side is a three-dimensionally connected macroporous sponge layer, forming an obvious gradient of pore size and cross-linking degree along the thickness direction, which can provide good barrier protection and is also conducive to cell infiltration and angiogenesis.

[0080] Example 2

[0081] It is basically the same as Example 1, except that the polymer material in component B of the excipient is carboxymethyl chitosan.

[0082] Example 3

[0083] The method is basically the same as in Example 1, except that the mass ratio of the biomimetic decellularized matrix component A to the excipient component B is 5:1.

[0084] Example 4

[0085] The method is basically the same as in Example 1, except that the mass ratio of the biomimetic decellularized matrix component A to the excipient component B is 5:4.

[0086] Example 5

[0087] The composition is basically the same as in Example 1, except that the biomimetic decellularized matrix A component is composed of the following raw materials, in the following percentage by mass: 75wt% type I collagen, 5wt% type VI collagen, 2wt% elastin, 2wt% fibronectin, 2wt% laminin, 2wt% blastocysteine, and 12wt% dextran.

[0088] Example 6

[0089] The process is basically the same as in Example 1, except that the excipient B component is composed of the following raw materials, in the following percentage by mass: 6 wt% antimicrobial peptide, 4 wt% antibiotic, and 90 wt% carboxymethyl chitosan.

[0090] Comparative Example 1

[0091] The process is basically the same as in Example 1, except that the biomimetic decellularized matrix-based artificial dermis is prepared using the following method:

[0092] (1) Preparation of biomimetic decellularized matrix A component mother liquor: The weighed substances in biomimetic decellularized matrix A component are added to 1000 mL of physiological saline with a concentration of 0.9 wt% in sequence. The mixture is stirred continuously at 800 rpm for 18 h at 4 °C until all substances are completely dissolved and uniformly dispersed to obtain A component mother liquor.

[0093] (2) Preparation of excipient B component mother liquor: Weigh each substance in the excipient B component and add it to 100 mL of physiological saline with a concentration of 0.9 wt%. Stir continuously at 800 rpm for 36 h at 37 °C until each substance is completely dissolved and evenly dispersed to obtain the excipient B component mother liquor.

[0094] (3) At room temperature, all the A component mother liquor prepared in step (1) and all the B component mother liquor prepared in step (2) are mixed at a mass ratio of 5:2, and the total solid content is controlled at 1wt%. The mixture is stirred at 500rpm for 10min. 50mL of 50mMEDC aqueous solution and 25mL of 25mMNHS aqueous solution are added to the above mixed solution in sequence. Stirring is continued at room temperature for 15min. After the crosslinking reaction is completed, the resulting gel-like mixture is injected into a mold of a predetermined shape, with a thickness of 2.5mm. The mixture is allowed to stand for 25min. The mold is then placed in a -80℃ refrigerator for 24h, freeze-dried, and sterilized to obtain artificial dermis based on biomimetic decellularized matrix.

[0095] Comparative Example 2

[0096] It is basically the same as Example 1, except that the polymer material is sodium alginate.

[0097] Comparative Example 3

[0098] It is basically the same as Example 1, except that the polymer material is silk fibroin.

[0099] Comparative Example 4

[0100] It is basically the same as Example 1, except that the polymer material is hyaluronic acid.

[0101] Comparative Example 5

[0102] It is basically the same as Example 1, except that: the excipient B component is 100wt% carboxymethyl chitosan.

[0103] Comparative Example 6

[0104] The process is basically the same as in Example 1, except that the dopamine hydrochloride in the preparation of functionalized carboxymethyl chitosan is replaced with tannic acid, which is common in the art.

[0105] The preparation method of the functionalized carboxymethyl chitosan is as follows:

[0106] S1. Mix 3.6g of carboxymethyl chitosan with 90mL of 0.1% (w / v) acetic acid aqueous solution, stir at 600rpm for 2h at room temperature, and let stand at room temperature for 1h to eliminate bubbles, to obtain a homogeneous and transparent carboxymethyl chitosan solution with a mass concentration of 4% (w / v), for later use.

[0107] S2. Take all of the above carboxymethyl chitosan solution, add 10 mL of 10 wt% methacrylamide gelatin solution at 55℃, stir at 400 rpm for 45 min, cool to 37℃, add 0.3 g tannic acid and 0.15 g boric acid, adjust the pH of the solution to 7.4 with 1 mol / L NaOH aqueous solution, and continue stirring at 37℃ for 8 min to form a gel solution. Add 15 mg zinc acetate to the gel solution, stir at 37℃ for 30 min, and freeze dry to obtain functionalized carboxymethyl chitosan.

[0108] Test Example 1

[0109] Antibacterial performance test: Using *Escherichia coli* and *Staphylococcus aureus* as experimental subjects, the antibacterial performance of the artificial dermis from the above examples and comparative examples was tested using the plate count method. Bacterial suspensions (concentration 5 × 10⁻⁶) were used. 5 The sample was dropped onto a 4cm × 4cm sample surface, covered with a polyethylene film, and incubated for 24 h at (37±1)℃ and relative humidity >90%. After incubation, the bacterial suspension was washed with neutralizing solution and serially diluted. The viable colony count (CFU) was counted using the pour method, with a sterile high-density polyethylene film as a negative control. The inhibition rate was calculated using the formula: R(%) = (BC) / B × 100%, where B is the number of viable bacteria in the control sample after 24 h, and C is the number of viable bacteria in the test sample after 24 h. Each test was repeated 4 times, and the average value of the results was taken, as shown in Table 1.

[0110] Table 1. Results of antibacterial performance test

[0111]

[0112] Cytotoxicity test: The extract was prepared according to the method specified in GB / T16886.12-2023 "Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials". Using complete culture medium (89 vol% high glucose medium / 10 vol% fetal bovine serum / 1 vol% penicillin-dextrose antibody) as the extraction medium, the medium was first saturated, and then extraction was performed at an extraction ratio of 0.1 g / mL. The cells were incubated at (37±1)℃ for (72±2) h. Mouse fibroblasts were extracted at 1×10⁻⁶... 4 Cells were seeded per well in 96-well plates and cultured for 24 h. The culture medium was removed, and the cells were cultured for another 24 h using the extraction buffer. Cytotoxicity was then performed according to the CCK-8 kit instructions, as shown in Table 2.

[0113] Table 2 Results of Cytotoxicity Tests

[0114]

[0115] Test Example 2

[0116] Wound healing rate test: SD rats (weighing 200-250g), 10 mice per group, were anesthetized, and a full-thickness skin defect wound with a diameter of approximately 1.5 cm was prepared on the back. They were randomly divided into an experimental group (implanted with artificial dermis from each embodiment), a positive control group (implanted with Integra® artificial dermis), and a negative control group (covered with Vaseline gauze). The wounds were observed and data collected on days 0, 5, 10, 15, and 20 after wound creation. The wound area was measured at D0, D5, D10, D15, and D20 using ImageJ software to obtain the wound area data for different material groups at each time point, and the wound healing rate (%) was calculated. Infection of the wound was observed during the healing process. The negative control group received Vaseline gauze covering and traditional pressure bandaging after wound preparation. The results are shown in Table 3.

[0117] Table 3 Wound healing rate test

[0118]

[0119] The results above show that the artificial dermis prepared in the embodiments of the present invention has good antibacterial properties, biocompatibility, and healing-promoting ability, which are superior to the control group and most comparative examples. Example 1 showed an inhibition rate of nearly 100% against Escherichia coli and Staphylococcus aureus, Example 2 still achieved over 80%, and Comparative Example 6 showed approximately 90%, while the PBS control group showed virtually no antibacterial effect. This indicates that the polymer materials and antibacterial components in excipient B play a crucial role in combating infection.

[0120] Examples 2, 3, 4, and 5, based on Example 1, differed in wet adhesion, antibacterial properties, and wound healing rate by introducing functionalized carboxymethyl chitosan or fine-tuning the A / B ratio and collagen composition. Functionalized carboxymethyl chitosan, coordinated with metals, enhanced wet adhesion and sustained-release antibacterial effects. However, excessively high or low proportions of component B could lead to insufficient antibacterial activity or a denser pore structure, resulting in effects that were worse than Example 1 but still significantly better than the comparative example. Compared to the comparative example using tannic acid as the polyphenol, the dopamine system provided stronger mussel-like adhesion and self-repair capabilities, thus exhibiting superior healing quality and long-term stability.

[0121] In the comparative example, when the polymer materials in component B were conventional polysaccharides / proteins such as sodium alginate, silk fibroin, and hyaluronic acid, the matching degree between the polymer backbone and collagen and the cationic antibacterial effect were weakened, resulting in a significant decrease in both antibacterial rate and healing rate. Although the formulation that removed antimicrobial peptides and antibiotics still had a certain physical barrier and scaffold function, its antibacterial ability was significantly insufficient, and the infection rate and delayed healing were the most prominent in animal experiments. When a homogeneous sponge was prepared by one-time mixing and cross-linking instead of constructing a double-layer gradient structure using the four-step method of this invention, it was difficult to simultaneously satisfy the cell / vascular infiltration channels on the wound side and the barrier function on the epidermal side, which also resulted in a comprehensive decrease in antibacterial and healing indicators compared to the corresponding examples. In summary, the precise combination of seven ECM components in component A achieves multi-dimensional biomimetic functions of natural dermal tissue, a biochemical microenvironment that existing technologies cannot provide. The polymer materials in component B construct a dynamic network structure with wet adhesion, self-repair, and sustained-release antibacterial properties through a five-element composite system of carboxymethyl chitosan, methacrylamide gelatin, dopamine hydrochloride, boric acid, and zinc acetate. Its antibacterial rate is close to 100%, which is significantly better than ordinary chitosan or antibiotic solutions.

Claims

1. A biomimetic acellular matrix-based artificial dermis, characterized by, The biomimetic acellular matrix A component and the auxiliary B component are included; The biomimetic acellular matrix A component includes type I collagen, type VI collagen, elastin, fibronectin, laminin, matricellular protein and dextran; The auxiliary B component includes antibacterial peptide, antibiotic and high molecular polymer material; the mass ratio of the biomimetic acellular matrix A component and the auxiliary B component is (4-6):(1-4).

2. The biomimetic acellular matrix-based artificial dermis according to claim 1, characterized by, The biomimetic acellular matrix A component includes the following components with the following mass fractions: Type I collagen 55wt%-80wt%; Type VI collagen 5wt%-15wt%; Elastin 1wt%-5wt%; Fibronectin 1wt%-5wt%; Laminin 1wt%-5wt%; Matricellular protein 1wt%-5wt%; Dextran 5wt%-15wt%.

3. The biomimetic acellular matrix-based artificial dermis according to claim 1, characterized by, The auxiliary B component includes the following components with the following mass fractions: Antibacterial peptide 5wt%-15wt%; Antibiotic 1wt%-5wt%; High molecular polymer material 70wt%-95wt%.

4. The biomimetic acellular matrix-based artificial dermis according to claim 1 or 3, characterized in that, The antibiotic is at least one selected from cefuroxime, vancomycin or gentamicin.

5. The biomimetic acellular matrix-based artificial dermis according to claim 1 or 3, characterized in that, The high molecular polymer material is at least one selected from chitosan, carboxymethyl chitosan and functionalized carboxymethyl chitosan.

6. The biomimetic acellular matrix-based artificial dermis according to claim 5, characterized in that, The preparation method of the functionalized carboxymethyl chitosan includes the following steps: S1, mixing carboxymethyl chitosan with acetic acid aqueous solution, stirring, standing, to obtain a carboxymethyl chitosan solution; S2, adding methacrylated gelatin solution to the carboxymethyl chitosan solution, stirring, adding dopamine hydrochloride and boric acid after cooling, adjusting pH to 7.4, stirring to form a gel solution, adding zinc acetate, stirring, freeze-drying to obtain functionalized carboxymethyl chitosan.

7. The biomimetic acellular matrix-based artificial dermis according to claim 6, characterized in that, In the step S1, the mass concentration of the carboxymethyl chitosan solution is 3-5%(w / v), and the concentration of the acetic acid aqueous solution is 0.05-0.15%(w / v); in the step S2, the mass concentration of the methacrylated gelatin solution is 8-12wt%, the addition amount of dopamine hydrochloride is 5-10% of the mass of carboxymethyl chitosan, the addition amount of boric acid is 2-5% of the mass of carboxymethyl chitosan, and the addition amount of zinc acetate is 0.5-2% of the mass of carboxymethyl chitosan.

8. A method for the preparation of a biomimetic acellular matrix-based artificial dermis according to any one of claims 1 to 7, characterized in that, The method includes the following steps: (1) preparing a biomimetic acellular matrix A component mother liquor: adding each substance in the biomimetic acellular matrix A component to physiological saline, stirring at 4℃ for 12-24h until completely dissolved, to obtain an A component mother liquor; (2) preparing an auxiliary B component mother liquor: adding each substance in the auxiliary B component to physiological saline, stirring at 37℃ for 24-48h until completely dissolved, to obtain a B component mother liquor; (3) preparing a wound side porous layer blank: mixing the A component mother liquor and the B component mother liquor according to a mass ratio of 5:2, controlling the solid content of the mixed solution to be 0.5-1.5wt%, adding EDC aqueous solution and NHS aqueous solution after stirring, crosslinking, pouring into a mold, standing, to obtain a wound side porous layer blank; (4) Preparation of epidermal side layer: mix part A component mother liquor and part B component mother liquor according to mass ratio 5:3, control solid content of mixed solution to be 2-4wt%, after stirring, add EDC aqueous solution and NHS aqueous solution, to obtain second layer crosslinking solution; Pour the second layer crosslinking solution on the surface of the wound side porous layer blank, crosslink, to obtain double-layer hydrogel; Freeze-drying, sterilization, to obtain biomimetic acellular matrix-based artificial dermis.

9. The method for preparing a biomimetic acellular matrix-based artificial dermis according to claim 8, characterized by, The concentration of the EDC aqueous solution in the step (3) is 40-60mM, and the concentration of the NHS aqueous solution is 20-30mM; the concentration of the EDC aqueous solution in the step (4) is 50-70mM, and the concentration of the NHS aqueous solution is 30-40mM.

10. Application of the biomimetic acellular matrix-based artificial dermis according to any one of claims 1-7 in the process of repairing various types of acute and chronic wounds of limbs, facial parts and trunk parts, suitable for wound repair of important tissue exposure of skeleton, tendon, nerve and blood vessel.

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