A method for preparing a decellularized matrix containing a bioactive coating

Abstract

This invention discloses a method for preparing a decellularized matrix with a bioactive coating, belonging to the medical aesthetics industry. The key technical points of the method are the following steps: S1, preparation of the decellularized matrix; S2, treatment of the decellularized matrix; S3, rehydrating the decellularized matrix and then sequentially immersing it in an activation solution, a collagen solution, and another activation solution, followed by washing and freeze-drying to obtain a collagen matrix; S4, adding anti-inflammatory microcapsules and growth factor nanocarriers to a hyaluronic acid solution to obtain a mixed solution; S5, immersing the collagen matrix in the activation solution, reacting and rinsing, coating the mixed solution onto the surface of the collagen matrix, then curing, rinsing, and drying to obtain a decellularized matrix with a bioactive coating. This application improves biocompatibility and safety by constructing a covalently linked hyaluronic acid-collagen bilayer structure on the surface of the decellularized matrix, while also solving the problems of unstable structure and poor mechanical strength of traditional decellularized matrices.

Description

Technical Field

[0001] This invention relates to the medical aesthetics industry, and in particular to a method for preparing a decellularized matrix containing a bioactive coating. Background Technology

[0002] Significant progress has been made in the biomedical field with the development of decellularized matrix biomaterials. With the rise of regenerative medicine, the demand for materials with good biocompatibility and the ability to effectively promote tissue regeneration and repair is increasing. As a natural biomaterial, decellularized matrix, due to its preservation of the three-dimensional structure of tissues, has shown great application potential in regenerative medicine fields such as tissue engineering, tissue filling, and wound healing, providing new approaches to addressing tissue damage and treating diseases, and driving the development of regenerative medicine.

[0003] In the application of acellular matrices, to meet diverse clinical needs, acellular matrices with bilayer structures are traditionally prepared. This bilayer design aims to combine different properties to achieve better therapeutic effects. For example, in tissue engineering, bilayer designs with different components or structures may be used to mimic the layered structure of natural tissues, providing a more suitable environment for cell growth and tissue regeneration. In tissue filling, bilayer structures can provide different physical properties, such as elasticity and support, to adapt to the needs of different sites. In the field of wound and repair, bilayer structures may help promote wound healing and tissue repair.

[0004] However, traditional decellularized matrices with a double-layered structure have significant drawbacks. These matrices are prone to disintegration after implantation, and once disintegrated, their physical support capacity decreases significantly. In applications such as tissue engineering and wound repair, physical support capacity is crucial for maintaining tissue morphology and structure. A decrease in physical support capacity affects cell growth and tissue regeneration, thereby impacting treatment outcomes and failing to meet clinical requirements for material stability and effectiveness. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for preparing a decellularized matrix containing a bioactive coating. This application improves biocompatibility and safety by constructing a covalently linked hyaluronic acid-collagen bilayer structure on the surface of the decellularized matrix, while also solving the problems of unstable structure and poor mechanical strength of traditional decellularized matrix.

[0006] The present invention provides a method for preparing a decellularized matrix with a bioactive coating, comprising the following preparation steps:

[0007] S1. Preparation of decellularized matrix;

[0008] S2. Plasma treatment of the decellularized matrix;

[0009] S3. After rehydrating the decellularized matrix obtained in step S2, immerse it in EDC / NHS activation solution and shake it at low temperature. Then transfer it to collagen solution and shake it gently at low temperature for 1 hour. After draining, immerse it in EDC / NHS activation solution again and react at low temperature for 1.5-2 hours. Then react at room temperature for 1-1.5 hours. During the reaction, control the pH of the system at 5.5±0.2. After the reaction, add the collagen matrix to Tris buffer and soak it. Wash it with PBS and ultrapure water and freeze dry to obtain the collagen matrix.

[0010] S4. Add anti-inflammatory microcapsules and growth factor nanocarriers to the hyaluronic acid solution in sequence, vortex mix at low temperature, and then let stand in an ice bath to remove foam and obtain a mixed solution.

[0011] S5. Immerse the collagen matrix obtained in step S3 in EDC / NHS activation solution and react at low temperature for 15-20 min. After rinsing, coat the surface of the activated collagen matrix with the mixture obtained in step S4 and let it stand at low temperature for 50-60 min. Then, spray calcium chloride solution onto the surface of the mixture and cure for 30-35 min. After rinsing and drying, obtain a decellularized matrix with a bioactive coating.

[0012] By adopting the above technical solution, the decellularized matrix is ​​first subjected to plasma treatment to expose more active groups. Then, after rehydration, immersion in EDC / NHS activation solution, and reaction with collagen solution, collagen molecules adhere to the decellularized matrix through physical adsorption and cross-linking with EDC / NHS, as well as through chemical interaction. This enhances the binding force between collagen and the decellularized matrix, thereby constructing a stable framework with a high-strength base coating. This ensures the strong binding of the subsequent sodium hyaluronate solution containing anti-inflammatory microcapsules and growth factor nanocarriers to the decellularized matrix, resulting in a decellularized matrix with good biocompatibility and safety.

[0013] In step S5, after the collagen matrix is ​​immersed in the EDC / NHS activation solution, the surface of the activated collagen matrix carries active groups. Substances in the mixture, such as hyaluronic acid, anti-inflammatory microcapsules, and growth factor nanocarriers, can chemically react with these active groups, such as forming covalent bonds. Hyaluronic acid has good viscosity and moisturizing properties; its combination with the collagen matrix can fill the gaps between collagen fibers, enhancing the density of the structure. Under the action of the activation solution, the anti-inflammatory microcapsules and growth factor nanocarriers, together with the collagen matrix, form a more stable three-dimensional network structure, thereby fixing the anti-inflammatory microcapsules and growth factor carriers within the coating structure. These chemical reactions and structural changes allow the decellularized matrix to better maintain its physical structure after implantation, making it less prone to disintegration and thus improving its mechanical properties. Furthermore, the bioactive coating formed by the anti-inflammatory microcapsules and growth factor nanocarriers combined with the hyaluronic acid solution has anti-inflammatory effects and can also promote tissue growth and repair.

[0014] Preferably, in step S5, after spraying calcium chloride solution onto the surface of the mixture, a polycarbonate porous membrane containing 2mM calcium chloride solution is covered and then cured for 30-35 minutes. The pore size of the polycarbonate porous membrane is 80-100μm.

[0015] By adopting the above technical solution, a small amount of calcium chloride solution is sprayed onto the surface of the mixture to rapidly gel the surface and protect the internal functional components. After covering with a porous polycarbonate membrane, the release rate of calcium ions is limited by the micropores, achieving deep weak cross-linking. This avoids problems such as hardening of hyaluronic acid cross-linking, inactivation of growth factors, and rupture of microcapsules caused by the instantaneous penetration of high concentrations of calcium ions. Moreover, the rigid framework of polycarbonate can improve the flatness of the coating and ensure the feasibility of subsequent surgical operations.

[0016] Preferably, the anti-inflammatory component in the anti-inflammatory microcapsule is composed of dipotassium glycyrrhizate and asiaticoside in a weight ratio of 1:1.

[0017] Preferably, the anti-inflammatory microcapsules are obtained by the following preparation method: chitosan is dissolved in deionized water, sodium alginate is added and stirred, anti-inflammatory components are added and stirred evenly, and then 2-5 wt% calcium chloride solution is added at a dropping rate of 0.5-2 mL / min. After solidification for 10-15 min, the microcapsules are rinsed with deionized water and vacuum dried to obtain anti-inflammatory microcapsules.

[0018] By adopting the above technical solution, the anti-inflammatory components dipotassium glycyrrhizate and asiaticoside are prepared into microcapsules, which not only controls the release rate of the anti-inflammatory components, but also avoids the stimulation or rapid loss that may be caused by direct exposure of the anti-inflammatory components, providing a continuous anti-inflammatory effect, effectively reducing the risk of inflammatory response and immune rejection in the early stage of implantation, and significantly improving biocompatibility.

[0019] In addition, during the preparation of anti-inflammatory microcapsules, if the dropping speed is greater than 2 mL / min, the excessively fast dropping speed results in large microcapsule particle size and wide distribution. When the dropping speed is too low, the efficiency is low and local aggregation is likely to occur. Furthermore, when the concentration of calcium chloride solution is less than 2 wt%, cross-linking is insufficient and the microcapsules are fragile. When the concentration of calcium chloride solution is greater than 5 wt%, the cross-linking is too fast, causing wrinkles on the surface of the microcapsules and the release rate of the anti-inflammatory components is too fast, affecting the efficacy.

[0020] Preferably, the growth factor nanocarrier is obtained using the following preparation method:

[0021] (1) Add growth factor and PCL to solvent and dissolve them under ultrasonication to form oil phase;

[0022] (2) The oil phase was dropped into the PVA aqueous solution and ultrasonically emulsified in an ice-water bath to obtain the primary emulsion;

[0023] (3) The colostrum was dropped into the PVA aqueous solution at a rate of 1 μL / s, the solvent was removed by rotary evaporation under reduced pressure, and the resulting nanoparticle suspension was solidified at -20°C for 10-15 min. Finally, the precipitate was resuspended in PBS solution by centrifugation.

[0024] By adopting the above technical solution, growth factors are protected by nanocarriers, avoiding rapid degradation and inactivation during the initial stages of preparation, storage and implantation. During specific stages of wound healing (such as the late stage of inflammation and the proliferative phase), growth factors are continuously released in response to signals from the wound microenvironment (such as enzyme and pH changes) or as the carrier material degrades, forming a microenvironment conducive to cell behavior locally. This effectively promotes cell proliferation, migration, differentiation, and angiogenesis, thereby accelerating wound healing and stimulating the generation of new tissues.

[0025] Preferably, the growth factors include EGF, FGF and chondroitin sulfate, wherein the contents of EGF, FGF and chondroitin sulfate are 0.1-0.5%, 0.2-1.0% and 5-15% of PCL by weight, respectively.

[0026] Preferably, the plasma treatment conditions in step S2 are 50W for 1-2 minutes.

[0027] Preferably, the hyaluronic acid solution is prepared by dissolving hyaluronic acid in calcium-free HEPES buffer, and then adding 0.1% by weight of trehalose to the hyaluronic acid solution and stirring until homogeneous.

[0028] Preferably, in step S5, the coating thickness of the mixture on the surface of the activated collagen matrix is ​​90-100 μm.

[0029] By adopting the above technical solution, the mixture is coated on the surface of the activated collagen matrix with a thickness of 90-100μm. This ensures that the bioactive coating has a suitable thickness, which can effectively load bioactive ingredients such as anti-inflammatory microcapsules and growth factor nanocarriers to exert anti-inflammatory and tissue repair effects. At the same time, the physical support will not be affected due to excessive coating thickness, or the bioactivity will not be fully exerted due to excessively thin coating. This improves the stability and therapeutic effect of the decellularized matrix containing the bioactive coating.

[0030] In summary, the present invention has the following beneficial effects: By constructing a covalently linked hyaluronic acid-collagen bilayer structure on the surface of the decellularized matrix, this application effectively improves the biocompatibility and safety of the decellularized matrix, and also solves the problems of instability and poor mechanical strength of traditional decellularized matrix structures. Moreover, the anti-inflammatory microcapsules, growth factors, and chondroitin sulfate are encapsulated in nanocarriers and enter the coating network through chemical bonding and physical adsorption, achieving effective fixation. This enables the anti-inflammatory components to achieve a long-lasting anti-inflammatory effect, creating a favorable environment for tissue regeneration. Growth factors and chondroitin sulfate directly promote cell behavior (proliferation, migration, and differentiation). Therefore, the combination of hyaluronic acid and collagen in this application enhances the scaffold for cell adhesion and migration, and regulates water and signal transduction. The synergistic effect of each component significantly enhances the tissue regeneration capacity. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the embodiments. All reagents, unless otherwise specified, are commercially available conventional reagent products.

[0032] Partial solution preparation:

[0033] Preparation of collagen solution: Dissolve type I collagen in 0.01M HCl (pH 3.0) and pre-cool at 4°C to form a collagen solution with a concentration of 5 mg / mL;

[0034] Preparation of hyaluronic acid solution: Dissolve hyaluronic acid in calcium-free HEPES buffer (pH 7.0) at a ratio of 15 mg / mL, add 0.1% trehalose by weight of hyaluronic acid and stir well to obtain hyaluronic acid solution;

[0035] EDC / NHS activation solution preparation: Weigh 0.9762g MES, 0.5755g NHS and 0.9585g EDC in sequence and dissolve them in 100mL of 70% ethanol solution.

[0036] Preparation Example 1.1

[0037] A method for preparing anti-inflammatory microcapsules includes the following steps:

[0038] S1. Dissolve chitosan in deionized water, stir and add sodium alginate while stirring, and continue stirring. The weight ratio of chitosan to sodium alginate is 3:1.

[0039] S2. Add the anti-inflammatory component composed of dipotassium glycyrrhizate and asiaticoside in a weight ratio of 1:1 and stir until homogeneous. The total weight ratio of the anti-inflammatory component to the total weight of sodium alginate and chitosan is 1:2. Then, add it to a 2wt% calcium chloride solution at a dropping rate of 0.5mL / min. After solidification for 15min, rinse with deionized water and vacuum dry to obtain anti-inflammatory microcapsules.

[0040] Preparation Example 1.2

[0041] A method for preparing anti-inflammatory microcapsules includes the following steps:

[0042] S1. Dissolve chitosan in deionized water, stir and add sodium alginate while stirring, and continue stirring. The weight ratio of chitosan to sodium alginate is 3:1.

[0043] S2. Add the anti-inflammatory component composed of dipotassium glycyrrhizate and asiaticoside in a weight ratio of 1:1 and stir until homogeneous. The total weight ratio of the anti-inflammatory component to the total weight of sodium alginate and chitosan is 1:2. Then, add it to a 5wt% calcium chloride solution at a dropping rate of 2mL / min. After solidification for 10min, rinse with deionized water and vacuum dry to obtain anti-inflammatory microcapsules.

[0044] Preparation Example 2.1

[0045] A method for preparing a growth factor nanocarrier includes the following steps:

[0046] (1) Add growth factors and 40 mg PCL (Nw = 20000) to 1 mL of acetone-dichloromethane and dissolve them under ultrasonication to form the oil phase. The growth factors include EGF, FGF and chondroitin sulfate. The contents of EGF, FGF and chondroitin sulfate are 0.1%, 0.2% and 15% of the weight of PCL, respectively.

[0047] (2) The oil phase was dropped into 2 mL of 2 wt% PVA aqueous solution and ultrasonically emulsified in an ice-water bath at 2 °C for 25 min to obtain the primary emulsion;

[0048] (3) The colostrum was dropped into 10 mL of 0.5 wt% PVA aqueous solution at a rate of 1 μL / s. The solvent was removed by rotary evaporation under reduced pressure. The resulting nanoparticle suspension was then solidified at -20 °C for 10 min. Finally, the precipitate was resuspended in PBS solution by centrifugation.

[0049] Preparation Example 2.2

[0050] A method for preparing a growth factor nanocarrier includes the following steps:

[0051] (1) Add growth factors and 40 mg PCL (Nw = 20000) to 1 mL of acetone-dichloromethane and dissolve them under sonication as the oil phase. The growth factors include EGF, FGF and chondroitin sulfate. The contents of EGF, FGF and chondroitin sulfate are 0.5%, 1.0% and 15% of the weight of PCL, respectively.

[0052] (2) The oil phase was dropped into 2 mL of 2 wt% PVA aqueous solution and ultrasonically emulsified in an ice-water bath at 2 °C for 25 min to obtain the primary emulsion;

[0053] (3) The colostrum was dropped into 10 mL of 0.5 wt% PVA aqueous solution at a rate of 1 μL / s. The solvent was removed by rotary evaporation under reduced pressure. The resulting nanoparticle suspension was then solidified at -20 °C for 15 min. Finally, the precipitate was resuspended in PBS solution by centrifugation.

[0054] Example 1

[0055] A method for preparing a decellularized matrix containing a bioactive coating includes the following preparation steps:

[0056] S1. Preparation of decellularized matrix;

[0057] S2. Perform plasma treatment on the decellularized matrix at 50W for 2 minutes.

[0058] S3. The decellularized matrix obtained in step S2 was soaked in PBS (pH 7.4) solution at 4℃ for 1 hour, then immersed in EDC / NHS activation solution and shaken at low temperature. It was then transferred to collagen solution and gently shaken at low temperature for 1 hour. After draining, it was immersed in EDC / NHS activation solution again and reacted at low temperature for 1.5 hours. Then it was reacted at room temperature for 1 hour. During the reaction, the pH of the system was controlled at 5.5±0.2. After the reaction, the collagen matrix was soaked in Tris buffer, washed with PBS and ultrapure water, and freeze-dried to obtain the collagen matrix.

[0059] S4. The anti-inflammatory microcapsules obtained in Preparation Example 1.1 and the growth factor nanocarrier obtained in Preparation Example 2.1 were added sequentially to the hyaluronic acid solution. The mixture was vortexed at 4°C and then allowed to stand in an ice bath to remove bubbles, resulting in a mixed solution. The concentration of the anti-inflammatory microcapsules in the hyaluronic acid solution was 10 mg / mL, and the concentration of the growth factor nanocarrier in the hyaluronic acid solution was 1 mg / mL.

[0060] S5. Immerse the collagen matrix obtained in step S3 in EDC / NHS activation solution and react at low temperature for 15 min. After rinsing, coat the surface of the activated collagen matrix with the mixture obtained in step S4 to a thickness of 90 μm. Let it stand at low temperature for 50 min, and then atomize and spray 0.1M calcium chloride solution (spraying amount of 5 μL / cm) onto the surface of the mixture. 2 After curing for 30 minutes, rinse and dry to obtain a decellularized matrix containing a bioactive coating.

[0061] Example 2

[0062] A method for preparing a decellularized matrix containing a bioactive coating includes the following preparation steps:

[0063] S1. Preparation of decellularized matrix;

[0064] S2. Perform plasma treatment on the decellularized matrix at 50W for 2 minutes.

[0065] S3. The decellularized matrix obtained in step S2 was soaked in PBS (pH 7.4) solution at 4℃ for 1 hour, then immersed in EDC / NHS activation solution and shaken at low temperature. It was then transferred to collagen solution and gently shaken at low temperature for 1 hour. After draining, it was immersed in EDC / NHS activation solution again and reacted at low temperature for 2 hours. Then it was reacted at room temperature for 1.5 hours. During the reaction, the pH of the system was controlled at 5.5±0.2. After the reaction, the collagen matrix was soaked in Tris buffer, washed with PBS and ultrapure water, and freeze-dried to obtain the collagen matrix.

[0066] S4. The anti-inflammatory microcapsules obtained in Preparation Example 1.1 and the growth factor nanocarrier obtained in Preparation Example 2.1 were added sequentially to the hyaluronic acid solution. The mixture was vortexed at 4°C and then allowed to stand in an ice bath to remove bubbles, resulting in a mixed solution. The concentration of the anti-inflammatory microcapsules in the hyaluronic acid solution was 10 mg / mL, and the concentration of the growth factor nanocarrier in the hyaluronic acid solution was 1 mg / mL.

[0067] S5. Immerse the collagen matrix obtained in step S3 in EDC / NHS activation solution and react at low temperature for 20 min. After rinsing, coat the surface of the activated collagen matrix with the mixture obtained in step S4 to a thickness of 100 μm. Let it stand at low temperature for 60 min, and then atomize and spray 0.1 M calcium chloride solution (spraying amount of 5 μL / cm) onto the surface of the mixture. 2 After curing for 35 minutes, the substrate was rinsed and dried to obtain a decellularized matrix containing a bioactive coating.

[0068] Example 3

[0069] A method for preparing a decellularized matrix with a bioactive coating differs from Example 1 in that the anti-inflammatory microcapsules in step S4 are the same as those obtained in Preparation Example 1.2, and the growth factor nanocarriers are the same as those obtained in Preparation Example 2.2.

[0070] Example 4

[0071] A method for preparing a decellularized matrix with a bioactive coating differs from Example 1 in that step S5 is different. Specifically, the collagen matrix obtained in step S3 is immersed in an EDC / NHS activation solution and reacted at low temperature for 15 min. After rinsing, the mixture obtained in step S4 is coated onto the surface of the activated collagen matrix with a coating thickness of 90 μm. The mixture is then allowed to stand at low temperature for 50 min. Finally, a 0.1 M calcium chloride solution (spraying amount of 5 μL / cm) is atomized and sprayed onto the surface of the mixture. 2 Then, a polycarbonate porous membrane containing 2mM calcium chloride solution is applied. The pore size of the polycarbonate porous membrane is 80μm. After curing for 30 minutes, it is rinsed and dried to obtain a decellularized matrix with a bioactive coating. Everything else is the same as in Example 1.

[0072] Example 5

[0073] A method for preparing a decellularized matrix with a bioactive coating differs from Example 1 in that step S5 is different. Specifically, the collagen matrix obtained in step S3 is immersed in an EDC / NHS activation solution and reacted at low temperature for 15 min. After rinsing, the mixture obtained in step S4 is coated onto the surface of the activated collagen matrix with a coating thickness of 90 μm. The mixture is then allowed to stand at low temperature for 50 min. Finally, a 0.1 M calcium chloride solution (spraying amount of 5 μL / cm) is atomized and sprayed onto the surface of the mixture. 2 Then, a polycarbonate porous membrane containing 2mM calcium chloride solution is applied. The pore size of the polycarbonate porous membrane is 100μm. After curing for 30 minutes, it is rinsed and dried to obtain a decellularized matrix with a bioactive coating. Everything else is the same as in Example 1.

[0074] Performance testing

[0075] 1. Mechanical property testing: The decellularized matrix material obtained in the above embodiment was cut into 50mm×4mm rectangles, and the tensile strength and elongation at break of the samples were tested. The tensile rate was 1mm / min, and the test results are shown in Table 1.

[0076] Table 1. Results of Decellularized Matrix Strength Test

[0077] sample Tensile strength / MPa Elongation at break / % Example 1 16.8 78.3 Example 2 16.5 78.5 Example 3 17.1 79.3 Example 4 18.4 79.6 Example 5 18.5 79.5

[0078] As shown in Table 1, the tensile strength of the decellularized matrices obtained in Examples 1-5 is greater than 15 MPa and the elongation at break is greater than 75%, indicating that the decellularized matrices obtained in this application have good mechanical properties, thereby ensuring the structural stability of the double-layer decellularized matrices.

[0079] 2. Biological performance testing: The decellularized matrix obtained in the above examples was evaluated for its biological performance in accordance with the relevant provisions of GB / T16886. The evaluation items included the relative cell proliferation rate after 7 days of encapsulation, sensitization reaction and intradermal stimulation test. The test results are shown in Table 2.

[0080] The cell proliferation experiment was conducted as follows: The decellularized matrix material was prepared into a thin sheet and placed at the bottom of a cell culture plate. Mouse L929 fibroblasts were then seeded onto the surface of the matrix material at a density of 1 × 10⁻⁶ cells / cm². 4 Cells per well. The inoculated cell culture plates were placed in a CO2 cell culture incubator for cell culture. After 1, 3, and 7 days, the cell supernatant was discarded, and 50 μL of MTT solution was added to each well. The plates were incubated at 37°C for 3 hours. The supernatant was then aspirated, and 400 μL of DMSO solution was added to each well. After shaking for 10 minutes, 200 μL of supernatant was aspirated from each well and transferred to a 96-well plate. The optical density (OD) value at 570 nm was measured using a microplate reader. The results are shown in Table 2.

[0081] Table 2. Evaluation results of decellularized matrix biology

[0082]

[0083] As can be seen from the table above, the decellularized matrix obtained in this application has a high relative cell proliferation rate and no sensitization or intradermal irritation reaction, indicating that the decellularized matrix obtained in this application does not have potential toxicity and also has good biocompatibility.

[0084] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a decellularized matrix with a bioactive coating, characterized in that, The preparation steps include the following: S1. Preparation of decellularized matrix; S2. Plasma treatment of the decellularized matrix; S3. After rehydrating the decellularized matrix obtained in step S2, immerse it in EDC / NHS activation solution and shake it at low temperature. Then transfer it to collagen solution and shake it gently at low temperature for 1 hour. After draining, immerse it in EDC / NHS activation solution again and react at low temperature for 1.5-2 hours. Then react at room temperature for 1-1.5 hours. During the reaction, control the pH of the system to 5.5±0.

2. After the reaction, add the decellularized matrix collagen composite material to Tris buffer and soak it. Wash it with PBS and ultrapure water and freeze dry to obtain collagen matrix. S4. Add anti-inflammatory microcapsules and growth factor nanocarriers to the hyaluronic acid solution in sequence, vortex mix at low temperature, and then let stand in an ice bath to remove foam and obtain a mixed solution. S5. Immerse the collagen matrix obtained in step S3 in EDC / NHS activation solution and react at low temperature for 15-20 min. After rinsing, coat the surface of the activated collagen matrix with the mixture obtained in step S4 and let it stand at low temperature for 50-60 min. Then, spray calcium chloride solution onto the surface of the mixture and cover it with a polycarbonate porous membrane containing 2 mM calcium chloride solution. After curing for 30-35 min, rinse and dry to obtain a decellularized matrix with a bioactive coating. The pore size of the polycarbonate porous membrane is 80-100 μm.

2. The method for preparing a decellularized matrix containing a bioactive coating according to claim 1, characterized in that: The anti-inflammatory component in the anti-inflammatory microcapsule is composed of dipotassium glycyrrhizate and asiaticoside in a weight ratio of 1:

1.

3. The method for preparing a decellularized matrix containing a bioactive coating according to claim 2, characterized in that, The anti-inflammatory microcapsules are prepared by the following method: chitosan is dissolved in deionized water, sodium alginate is added and stirred, anti-inflammatory components are added and stirred evenly, and then 2-5 wt% calcium chloride solution is added at a dropping rate of 0.5-2 mL / min. After solidification for 10-15 min, the microcapsules are rinsed with deionized water and vacuum dried to obtain anti-inflammatory microcapsules.

4. The method for preparing a decellularized matrix containing a bioactive coating according to claim 1, characterized in that: The growth factor nanocarrier was obtained using the following preparation method: (1) Add growth factor and PCL to solvent and dissolve under ultrasonication to form oil phase; (2) The oil phase was dropped into the PVA aqueous solution and ultrasonically emulsified in an ice-water bath to obtain the primary emulsion; (3) The colostrum was dropped into the PVA aqueous solution at a rate of 1 μL / s, the solvent was removed by rotary evaporation under reduced pressure, and the resulting nanoparticle suspension was solidified at -20°C for 10-15 min. Finally, the precipitate was resuspended in PBS solution by centrifugation.

5. The method for preparing a decellularized matrix containing a bioactive coating according to claim 4, characterized in that: The growth factors include EGF, FGF and chondroitin sulfate, and the contents of EGF, FGF and chondroitin sulfate are 0.1-0.5%, 0.2-1.0% and 5-15% of PCL by weight, respectively.

6. The method for preparing a decellularized matrix containing a bioactive coating according to claim 1, characterized in that: The plasma treatment conditions in step S2 are 50W for 1-2 minutes.

7. The method for preparing a decellularized matrix containing a bioactive coating according to claim 1, characterized in that: The hyaluronic acid solution is prepared by dissolving hyaluronic acid in calcium-free HEPES buffer, and then adding 0.1% (by weight) of trehalose to the hyaluronic acid solution and stirring until homogeneous.

8. The method for preparing a decellularized matrix containing a bioactive coating according to claim 1, characterized in that: In step S5, the coating thickness of the mixture on the surface of the activated collagen matrix is ​​90-100 μm.

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