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

Abstract

The present application relates to the technical field of artificial dermis, and particularly relates to a preparation method and application of artificial dermis based on biomimetic acellular matrix. The artificial dermis comprises a biomimetic acellular matrix component A and an auxiliary component B, wherein the component A comprises type I collagen, type VI collagen, elastin, fibronectin, laminin, matricellular protein and dextran; the component B comprises antibacterial peptide, antibiotic and 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 steps of respectively preparing A and B component process mother liquor, step-by-step crosslinking to prepare a hydrogel with a double-layer gradient structure, and then freeze-drying and sterilization treatment. The artificial dermis has good biocompatibility and antibacterial performance, can effectively promote wound healing, and is suitable for repairing acute and chronic wounds of limbs, facial parts and trunk parts, and is particularly suitable for repairing complex wounds with exposed bones, tendons, nerves and blood vessels.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of artificial dermis, and particularly relates to a preparation method and application of artificial dermis based on a biomimetic acellular matrix. BACKGROUND

[0002] In the field of skin tissue engineering, dermal regeneration technology is mainly divided into three categories: autologous skin transplantation, allogeneic / xenogeneic acellular dermal matrix (ADM) and synthetic biomimetic scaffold materials. Autologous transplantation is still the clinical gold standard, but its donor site limitation prompts researchers to explore alternative solutions. Animal-derived ADM (such as pig skin, bovine pericardium) retains the extracellular matrix (ECM) structure through decellularization processing, but has the risk of immunogenicity; while synthetic materials (such as collagen-chitosan scaffolds) can be customized, but it is difficult to completely simulate the biological function of natural dermis. In addition, in recent years, some studies have attempted to combine 3D printing or electrospinning technology to prepare biomimetic scaffolds, but there are still many challenges in clinical translation.

[0003] Although autologous skin transplantation has excellent tissue compatibility, its application is limited by the area of the donor site, especially for patients with large-area burns, there is a serious shortage of healthy skin for transplantation. In addition, the skin harvesting surgery can cause hyperplasia, pigmentation or dysfunction of the donor site, and the transplanted skin often appears contracture due to the lack of dermal support, affecting the appearance and function. Although decellularization technology can partially remove xenogeneic antigens, the residual α-Gal epitopes can still trigger a host immune response, leading to accelerated degradation of the graft or fibrous encapsulation. At the same time, the mechanical properties (such as tensile strength, creep) of animal-derived materials are significantly different from human dermis, and the decellularization efficiency of different batches is not uniform, which may affect the repeatability of clinical effects.

[0004] Existing biomimetic scaffolds focus on the structure of the biomimetic (such as mimicking the arrangement of collagen fibers), but ignore the key regulatory role of the dynamic biochemical microenvironment of ECM (such as growth factor gradient, mechanical signal transduction) on cell behavior. For example, electrospinning technology can prepare nanofibers that simulate the topological structure of ECM, but the dense fiber network may hinder the effective infiltration and migration of cells; 3D printing technology promotes the macroscopic pores for vascularization, but it is difficult to accurately match the material degradation rate and the timing requirements of tissue regeneration.

[0005] To solve the above problems, the application provides a kind of bionic acellular matrix, by integrating the biological activity component of natural ECM and the controllable characteristics of synthetic material, innovation is realized in three aspects: (1) immunogenicity control: adopt cross-species conservative ECM protein (such as I / III type collagen) as base material, combined with directional enzymatic technology to completely remove heterologous antigen; (2) dynamic mechanics adaptation: introduce elastic fiber network and gradient crosslinking strategy, so that the material provides mechanical support in the early stage of implantation, and gradually softens to match the mechanical requirements of new tissue as the regeneration process progresses; (3) active regeneration induction: construct a bionic microtubule system in the scaffold to load slow-release cytokines (such as VEGF, FGF-2), to synchronize the regulation of angiogenesis and fibroblast activation. This design is expected to break through the dilemma of "structure bionics and function lag" of existing materials, and provide an integrated regeneration solution for complex skin defects. SUMMARY

[0006] To solve the above problems in the prior art, the application provides a preparation method and application of artificial dermis based on bionic acellular matrix.

[0007] To solve the above technical problems, the technical scheme adopted by the application is:

[0008] The application provides an artificial dermis based on bionic acellular matrix, which includes a bionic acellular matrix component A and an auxiliary component B.

[0009] The bionic acellular matrix component A includes type I collagen, type VI collagen, elastin, fibronectin, laminin, mother cell protein and dextran.

[0010] The auxiliary component B includes antibacterial peptide, antibiotic and high molecular polymer material; the mass ratio of the bionic acellular matrix component A and the auxiliary component B is (4-6):(1-4).

[0011] Preferably, the bionic acellular matrix component A includes the following components with the following mass fractions:

[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] Mother cell protein 1wt%-5wt%;

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

[0019] The biomimetic acellular matrix A component of the present application simulates the acellular dermal matrix in composition, and the type I collagen and type VI collagen jointly construct the fiber framework to provide necessary mechanical support and cell adhesion sites; the elastin endows the material with certain elasticity to buffer the mechanical stress of the wound surface; the fibronectin and the laminin can mediate the adhesion and migration of fibroblasts, keratinocytes and endothelial cells; the matricellular protein and the dextran are beneficial to the enrichment of endogenous growth factors and the regulation of the slow release, so that the biochemical microenvironment similar to the natural dermis is reconstructed without directly using the animal-derived acellular tissue, and the angiogenesis and dermal regeneration are promoted.

[0020] Preferably, the auxiliary material B component comprises the following components in mass fraction:

[0021] Antibacterial peptide 5wt%-15wt%;

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

[0023] High molecular polymer material 70wt%-95wt%.

[0024] The antibacterial peptide and the antibiotic are added in the auxiliary material B component, which can quickly inhibit the proliferation of bacteria and reduce the risk of infection and inflammation; meanwhile, the functionalized carboxymethyl chitosan in the high molecular polymer material is used as the main framework, so that the B component is not only a simple antibacterial carrier, but also a biomimetic polysaccharide substance with multiple functions.

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

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

[0027] The functionalized carboxymethyl chitosan is composed of carboxymethyl chitosan, methacrylated gelatin, dopamine hydrochloride, boric acid and zinc acetate: the carboxymethyl chitosan itself has the characteristics of polysaccharide framework and cation, can have electrostatic interaction with the collagen in the A component, enhance the stability of the whole, and provide basic antibacterial ability through the electrostatic interaction with the bacterial cell membrane; the methacrylated gelatin provides the biological active sequence similar to collagen and the cell adhesion sites, so that the B component forms a continuous transition with the A component in structure, and further improves the cell affinity. The dopamine hydrochloride is partially oxidized under near neutral conditions, and the catechol group thereof on one hand forms a dynamic catechol and boric acid ester crosslinking with the boric acid, endows the gel with good wet adhesion and certain self-repairing ability, ensures that the material can still closely adhere to the wound surface in the environment rich in exudate, and reduces the dead space and exudate accumulation; on the other hand, the catechol group can release Zn 2+Coordination occurs, building metal and catechol crosslinking points, improving the mechanical strength of the gel network, while making Zn 2+ slow and continuous release, play a broad-spectrum antibacterial and promote epithelialization, collagen remodeling and other biological effects. The functional carboxymethyl chitosan shows significant improvement in antibacterial performance, wet adhesion performance, and cell adhesion and infiltration ability, reduces the incidence of infection, and thus obtains faster vascularization process and higher quality dermal regeneration under the same wound conditions.

[0028] Preferably, the preparation method of the functional carboxymethyl chitosan comprises the following steps:

[0029] S1, mixing carboxymethyl chitosan with acetic acid aqueous solution, stirring, standing, to obtain carboxymethyl chitosan solution;

[0030] 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 functional carboxymethyl chitosan.

[0031] Preferably, the mass concentration of the carboxymethyl chitosan solution in step S1 is 3-5%(w / v), and the concentration of the acetic acid aqueous solution is 0.05-0.15%(w / v); the mass concentration of the methacrylated gelatin solution in step S2 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.

[0032] A preparation method of a biomimetic acellular matrix artificial dermis, comprising the following steps:

[0033] (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 the A component mother liquor;

[0034] (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 the B component mother liquor;

[0035] (3) preparing a wound side porous layer blank: mixing part of the A component mother liquor with part of 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%, stirring, then adding EDC aqueous solution and NHS aqueous solution, crosslinking reaction, pouring into a mold, standing, to obtain a wound side porous layer blank;

[0036] (4) Preparing epidermis side layer: mixing part A component mother liquor and part B component mother liquor according to mass ratio 5:3, controlling solid content of mixed solution as 2-4wt%, after stirring, adding EDC aqueous solution and NHS aqueous solution, obtaining second layer crosslinking solution; pouring second layer crosslinking solution on surface of wound side porous layer blank, crosslinking, obtaining double layer hydrogel; freeze-drying, sterilizing, obtaining artificial dermis based on biomimetic acellular matrix.

[0037] Preferably, the artificial dermis based on biomimetic acellular matrix is prepared by the following method:

[0038] (1) Preparing biomimetic acellular matrix A component mother liquor: weighing each substance in biomimetic acellular matrix A component, adding into 800-1400mL physiological saline with concentration of 0.8-1.2wt%, continuously stirring at 2-6℃ and mechanical stirring speed of 600-1000rpm for 12-24h, until each substance is completely dissolved and uniformly dispersed, obtaining A component mother liquor;

[0039] (2) Preparing auxiliary material B component mother liquor: weighing each substance in auxiliary material B component, adding into 80-120mL physiological saline with concentration of 0.8-1.2wt%, continuously stirring at 30-45℃ and mechanical stirring speed of 800rpm for 24-48h, until each substance is completely dissolved and uniformly dispersed, obtaining B component mother liquor;

[0040] (3) Preparing wound side porous layer blank: mixing A component mother liquor prepared in step (1) and B component mother liquor prepared in step (2) according to mass ratio (3-6):(1-3) at room temperature, total solid content is controlled as 0.5-1.5wt%, stirring at 300-600rpm for 8-15min; adding 40-80mL 40-60mM EDC aqueous solution and 20-35mL 20-30mM NHS aqueous solution into above mixed solution, continuously stirring at room temperature for 10-30min, after crosslinking reaction, pouring obtained gel-like mixture into mold with predetermined shape, controlling thickness as 1-3mm, standing for 15-40min, making it preliminarily shaped in mold, obtaining wound side porous layer blank;

[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) The application is not directly using animal-derived acellular dermal matrix, but based on the acellular extracellular matrix material prepared from bovine Achilles tendon as a template, the core functional components are accurately compounded after artificial analysis: including type I collagen, type VI collagen, elastin, fibronectin, laminin, mother cell protein and dextran, which simulates the biological activity of natural extracellular matrix at the molecular level. A material with similar composition to bovine Achilles tendon acellular extracellular matrix is artificially compounded, which can mimic the biological activity of acellular extracellular matrix, and the artificially added composition can effectively avoid the immunogenicity and virus problems of the material after decellularization treatment, so that the use of the finally prepared artificial product is safer and more efficient.

[0046] (2) The application constructs an artificial dermis scaffold with a biomimetic three-dimensional network structure by introducing functional 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 micro-morphology, but more importantly, in order to imitate the three-dimensional structure of collagen in acellular extracellular matrix, the structure of the acellular extracellular matrix is completed, and the composition and structure are combined to realize the gradient change of porosity and mechanical properties, which is more conducive to cell migration, infiltration and vascularization, and realizes the cooperation of composition and structure double simulation.

[0047] (3) The application introduces antibacterial peptides and antibiotics in the auxiliary material B component, and uses the catechol and Zn 2+ coordination system in functional carboxymethyl chitosan to achieve slow release of zinc ions, which together provides strong and persistent broad-spectrum antibacterial ability, can effectively prevent and control post-implantation infection and reduce inflammatory response. At the same time, functional carboxymethyl chitosan itself has good wet-state tissue adhesion and certain self-repairing performance, can closely adhere to the wound surface, reduce the dead space, and provide a stable microenvironment for wound healing.

[0048] (4) The artificial dermis prepared by the application has excellent comprehensive performance and good biocompatibility, can effectively promote the activation and migration of fibroblasts and vascular endothelial cells, and significantly accelerate the wound healing process. Through the material of the application, the limitations of autologous skin transplantation for clinical treatment of dermal regeneration are solved, and the repair of the wound and the regeneration of the dermis have good effects, especially suitable for complex wound repair with bone, tendon and other tissue exposure, which can effectively avoid or reduce secondary skin grafting, shorten the treatment cycle and improve the healing quality. BRIEF DESCRIPTION OF DRAWINGS

[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 be further described in detail below 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] Second step: the cell suspension after ultrasonic treatment is placed at 4℃, centrifuged at a speed of 14000 r / min for 15 min, and the supernatant is collected after centrifugation, freeze-dried, and the mother cell protein is obtained.

[0061] Dextran is purchased from Shanghai McLean Biochemical Technology Co., Ltd., and the item number is D992645.

[0062] Antibacterial peptide, frog skin antibacterial peptide, purchased from Shanghai McLean Biochemical Technology Co., Ltd., item number: D807939.

[0063] Cefuroxime is purchased from Shanghai McLean Biochemical Technology Co., Ltd., and the item number is C922445.

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

[0065] Methacrylated gelatin is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and the item number is G477882-1g.

[0066] Example 1

[0067] An artificial dermis based on a biomimetic acellular matrix, which is composed of a biomimetic acellular matrix A component and an auxiliary material B component;

[0068] The biomimetic acellular matrix A component is composed of the following raw materials, in terms of mass percentage: 70wt% of type I collagen, 10wt% of type VI collagen, 2wt% of elastin, 2wt% of fibronectin, 2wt% of laminin, 2wt% of mother cell protein, and 12wt% of dextran; according to the above proportions, each substance in the biomimetic acellular matrix A component is accurately weighed, and the total mass is 10g;

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

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

[0071] S1, 3.6g of carboxymethyl chitosan is mixed with 90mL of 0.1%(w / v) acetic acid aqueous solution, stirred at room temperature at 600rpm for 2h, and placed at room temperature for 1h to eliminate bubbles, to obtain a uniform transparent carboxymethyl chitosan solution with a mass concentration of 4%(w / v), which is ready for use;

[0072] S2, taking all the carboxymethyl chitosan solution, 10 mL of 10wt% methacrylated gelatin solution was added at 55℃, stirring at 400 rpm for 45 min, cooled to 37℃, then 0.3g dopamine hydrochloride and 0.15g boric acid were added, the solution pH value was adjusted to 7.4 using 1mol / L NaOH aqueous solution, and continued to stir at 37℃ for 8 min to form a gel solution, 15mg zinc acetate was added to the gel solution, stirred at 37℃ for 30 min, and freeze-dried to obtain functionalized carboxymethyl chitosan.

[0073] The mass ratio of the biomimetic acellular matrix A component and the auxiliary B component is 5:2.

[0074] The biomimetic acellular matrix artificial dermis based on the biomimetic acellular matrix is prepared by the following method:

[0075] (1) Preparation of biomimetic acellular matrix A component mother liquor: the weighed biomimetic acellular matrix A component was added to 1000 mL of 0.9wt% physiological saline, and stirred at 800 rpm for 18 h at 4℃ until the substances were completely dissolved and uniformly dispersed, to obtain the A component mother liquor;

[0076] (2) Preparation of auxiliary B component mother liquor: the weighed auxiliary B component was added to 100 mL of 0.9wt% physiological saline, and stirred at 800 rpm for 36 h at 37℃ until the substances were completely dissolved and uniformly dispersed, to obtain the B component mother liquor;

[0077] (3) Preparation of wound side porous layer blank: part of the A component mother liquor prepared in step (1) and part of the B component mother liquor prepared in step (2) were mixed at a mass ratio of 5:2 at room temperature, the total solid content was controlled at 1wt%, and stirred at 500 rpm for 10 min; 50 mL of 50mM EDTA aqueous solution and 25 mL of 25mM NHS aqueous solution were added to the above mixed solution, and stirred at room temperature for 15 min, and the crosslinking reaction was completed; the obtained gel-like mixture was injected into a mold with a predetermined shape, the thickness was controlled at 2.5 mm, and it was placed for 25 min to be initially shaped in the mold, to obtain the wound side porous layer blank;

[0078] (4) At room temperature, the mother liquor of the component A prepared in step (1) and the mother liquor of the component B prepared in step (2) are mixed according to a mass ratio of 5:3, the total solid content is controlled at 3wt%, and stirring is carried out at a rotating speed of 500rpm for 10min; 50mL of 60mM EDTA aqueous solution and 25mL of 35mM NHS aqueous solution are sequentially added into the mixed solution, and the stirring is continuously carried out at room temperature for 15min, so as to obtain a second layer crosslinking solution; when the porous layer blank of the wound side is still in a semi-cured state, the second layer crosslinking solution is slowly poured onto the surface of the porous layer blank, so as to naturally spread and cover, and thus a skin side layer is formed; then, crosslinking is continuously carried out at room temperature for 3h, so that the upper and lower layers are covalently crosslinked and firmly combined at the interface, and thus a double-layer hydrogel with a thickness of 4mm is obtained; the obtained double-layer hydrogel is placed in a refrigerator at-80℃ for 24h, and then freeze-drying and sterilization are carried out, so as to obtain the artificial dermis based on the biomimetic acellular matrix.

[0079] The artificial dermis prepared in the embodiment is shown in the physical object diagram and the SEM diagram of Figure 1 and Figure 2 It can be known from Figure 1 、 Figure 2 that the artificial dermis prepared in the embodiment has a complete appearance and a uniform thickness, and the layers are tightly combined; the SEM microstructure of the artificial dermis is that the skin side is a relatively dense small pore layer, the wound side is a three-dimensional interconnected large pore sponge layer, and an obvious pore size and crosslinking degree gradient is formed along the thickness direction, so that good barrier protection can be provided, and cell infiltration and angiogenesis are also facilitated.

[0080] Embodiment 2

[0081] The embodiment is basically the same as embodiment 1, and the difference lies in that the high molecular polymer material in the auxiliary component B is carboxymethyl chitosan.

[0082] Embodiment 3

[0083] The embodiment is basically the same as embodiment 1, and the difference lies in that the mass ratio of the biomimetic acellular matrix component A and the auxiliary component B is 5:1.

[0084] Embodiment 4

[0085] The embodiment is basically the same as embodiment 1, and the difference lies in that the mass ratio of the biomimetic acellular matrix component A and the auxiliary component B is 5:4.

[0086] Embodiment 5

[0087] The embodiment is basically the same as embodiment 1, and the difference lies in that the biomimetic acellular matrix component A is composed of the following raw materials in terms of mass ratio percentage: 75wt% of type I collagen, 5wt% of type VI collagen, 2wt% of elastin, 2wt% of fibronectin, 2wt% of laminin, 2wt% of mother cell protein and 12wt% of 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] The same as example 1, except that the high molecular polymer material is hyaluronic acid.

[0101] Comparative example 5

[0102] The same as example 1, except that the auxiliary material B component is 100wt% carboxymethyl chitosan.

[0103] Comparative example 6

[0104] The same as example 1, except that dopamine hydrochloride in the preparation process of functional carboxymethyl chitosan is replaced by tannic acid commonly used in the art.

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

[0106] S1, 3.6g carboxymethyl chitosan is mixed with 90mL 0.1%(w / v) acetic acid aqueous solution, stirred at room temperature for 2h at 600rpm, and left at room temperature for 1h to eliminate air bubbles, to obtain a uniform transparent carboxymethyl chitosan solution with a mass concentration of 4%(w / v), which is ready for use;

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

[0108] Test example 1

[0109] Antibacterial performance test: Escherichia coli and Staphylococcus aureus were used as experimental objects, and the antibacterial performance of the artificial dermis of the above examples and comparative examples was tested by plate counting method. Bacterial suspension (concentration 5×10 5 ) was added to the surface of the 4cm×4cm sample, covered with a polyethylene film, and incubated at (37±1)℃, relative humidity >90% for 24h. After incubation, the bacterial solution was eluted with neutralizing solution and serially diluted, and the viable bacterial count (CFU) was counted by pouring method. Sterile high-density polyethylene film was used as negative control. The inhibition rate calculation formula is: R(%)=(B-C) / B×100%, wherein B is the viable bacterial count after 24h of the control sample, and C is the viable bacterial count after 24h of the test sample. Each test was repeated 4 times, and the results were averaged, as shown in Table 1.

[0110] Table 1 antibacterial performance test results

[0111]

[0112] Cytotoxicity test: The extraction solution was prepared according to the method specified in GB / T 16886.12-2023 "Medical devices - Evaluation of biological systems - Part 12: Preparation of samples and reference materials". The complete culture medium (89vol% high-sugar culture medium / 10vol% fetal bovine serum / 1vol% double antibody) was used as the extraction medium, which was first saturated with the extraction medium, and then extracted at a ratio of 0.1 g / mL, and soaked at (37±1) ℃ for (72±2) h. Mouse fibroblasts were inoculated in a 96-well plate at 1×10 4 The cells were cultured for 24 h, the cell culture medium was removed, the extraction solution was used for continuous culture for 24 h, and the cytotoxicity test was performed according to the steps of the CCK-8 kit operation instruction, as shown in Table 2.

[0113] Table 2 Cytotoxicity test results

[0114]

[0115] Test Example 2

[0116] Wound healing rate test: SD rats (body weight 200-250 g) were selected, 10 mice per group, after anesthesia, a full-thickness skin defect wound with a diameter of about 1.5 cm was prepared on the back. Randomly divided into experimental group (implanted with artificial dermis of each embodiment), positive control group (implanted with Integra artificial dermis) and negative control group (vaseline gauze covering). And on D0, D5, D10, D15 and D20, the wound surface was observed and data was collected. The wound surface area of D0, D5, D10, D15 and D20 was measured by software ImageJ, the wound surface area data of each time point of different material groups was obtained, and the wound healing rate was calculated, unit %, and whether the wound surface was infected during the healing process was observed. The negative control group was treated with vaseline gauze covering after wound preparation and traditional pressure bandaging, and the results are shown in Table 3.

[0117] Table 3 Wound healing rate test

[0118]

[0119] From the above results, it can be seen that the artificial dermis prepared by the embodiments of the present application has good antibacterial performance, biocompatibility and healing-promoting ability, which is better than the control group and most of the comparative examples. The bacteriostatic rate of Example 1 on E. coli and S. aureus is close to 100%, Example 2 can still reach more than 80%, Comparative Example 6 is about 90%, and the PBS control group has basically no bacteriostatic effect, which shows that the high molecular polymer material and the antibacterial component in the auxiliary material B component play a key role in resisting infection.

[0120] Examples 2, 3, 4, 5 have some differences in wet adhesion, antibacterial performance and wound healing rate based on Example 1 by introducing functional carboxymethyl chitosan or fine-tuning A / B ratio, collagen composition, etc.: the functional carboxymethyl chitosan coordinates with metals, enhancing wet adhesion and slow-release antibacterial effect, but too high or too low B component ratio will lead to insufficient antibacterial activity or dense pore structure, respectively, resulting in worse but still significantly better than the comparative example; compared with the tannic acid as the polyphenol in the comparative example, the dopamine system can provide stronger mussel-type adhesion and self-repairing ability, so it is more prominent in healing quality and long-term stability.

[0121] In the comparative example, when the high molecular polymer material in the B component is a conventional polysaccharide / protein such as sodium alginate, silk fibroin, hyaluronic acid, etc., the matching degree between the high molecular backbone and collagen and the cationic antibacterial effect are weakened, resulting in a significant decrease in antibacterial rate and healing rate; the formula without antibacterial peptides and antibiotics still has certain physical barrier and scaffold function, but the antibacterial ability is significantly insufficient, and the incidence of infection and delayed healing in animal experiments are most prominent; using one-time mixing and cross-linking to prepare homogeneous sponge instead of the four-step method of the present application to construct a double-layer gradient structure, the cell / vessel infiltration channel on the wound side and the barrier function on the epidermis side are difficult to satisfy simultaneously, and the antibacterial and healing indicators are also lower than the corresponding examples. In summary, through the precise compounding of the seven ECM components in the A component, the multi-dimensional biomimetic function of natural dermal tissue is realized, which is a biochemical microenvironment that existing technologies cannot provide; the high molecular polymer material in the B component is constructed by a five-element composite system of carboxymethyl chitosan, methacrylated gelatin, dopamine hydrochloride, boric acid and zinc acetate, which has a dynamic network structure with wet adhesion, self-repairing and slow-release antibacterial properties, and its antibacterial rate is close to 100%, which is significantly better than the ordinary chitosan or antibiotic scheme.

Claims

1. An artificial dermis based on a biomimetic decellularized matrix, characterized in that, It includes a biomimetic decellularized matrix component A and an excipient component B; The biomimetic decellularized matrix A component includes: type I collagen, type VI collagen, elastin, fibronectin, laminin, blastocyst protein, and dextran; 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). The polymer material is functionalized carboxymethyl chitosan; The preparation method of the functionalized carboxymethyl chitosan includes the following steps: S1. Mix carboxymethyl chitosan with an aqueous acetic acid solution, stir, and let stand to obtain a carboxymethyl chitosan solution; 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. In step S1, the carboxymethyl chitosan solution has a mass concentration of 3-5% (w / v), and the acetic acid aqueous solution has a concentration of 0.05-0.15% (w / v). In step S2, the methacrylamide gelatin solution has a mass concentration of 8-12 wt%, the dopamine hydrochloride is added at 5-10% of the mass of carboxymethyl chitosan, the boric acid is added at 2-5% of the mass of carboxymethyl chitosan, and the zinc acetate is added at 0.5-2% of the mass of carboxymethyl chitosan.

2. The artificial dermis based on biomimetic decellularized matrix according to claim 1, characterized in that, The biomimetic decellularized matrix A component comprises the following components by mass fraction: Type I collagen 55wt%-80wt%; Type VI collagen 5wt%-15wt%; elastin 1wt%-5wt%; Fibronectin 1wt%-5wt%; Laminin 1wt%-5wt%; 1wt%-5wt% of blastocyst protein; Glucan 5wt%-15wt%.

3. The artificial dermis based on biomimetic decellularized matrix according to claim 1, characterized in that, The excipient B component includes the following components by mass fraction: Antimicrobial peptides 5wt%-15wt%; Antibiotics 1wt%-5wt%; Polymer materials 70wt%-95wt%.

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

5. A method for preparing artificial dermis based on a biomimetic decellularized matrix as described in any one of claims 1-4, characterized in that, Includes the following steps: (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; (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; (3) Preparation of porous layer preform on the wound side: Mix the mother liquor of component A and 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. (4) Preparation of the epidermal side layer: Mix part of the mother liquor of component A and part of the mother liquor of component B at a mass ratio of 5:3, control the solid content of the mixed solution to be 2-4wt%, and add EDC aqueous solution and NHS aqueous solution after stirring to obtain the second crosslinking solution; The second cross-linking solution was poured onto the surface of the porous layer preform on the wound side and cross-linked to obtain a double-layer hydrogel. Freeze-drying and sterilization yielded artificial dermis based on a biomimetic decellularized matrix.

6. The method for preparing artificial dermis based on biomimetic decellularized matrix according to claim 5, characterized in that, In step (3), the concentration of the EDC aqueous solution is 40-60 mM and the concentration of the NHS aqueous solution is 20-30 mM; in step (4), the concentration of the EDC aqueous solution is 50-70 mM and the concentration of the NHS aqueous solution is 30-40 mM.

Images