A biomimetic collagen double-layered scaffold and a preparation method thereof

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to a kind of biomimetic collagen double-layered scaffold and a preparation method thereof, with low-cost pig peritoneum as raw material, the same thickness and dense structure of human skin epidermis layer are prepared by defatting and decellularization process Decellularized matrix, and further utilize the decellularized matrix cell matrix to prepare the hydrogel layer with loose porous, can provide suitable microenvironment for cell adhesion and growth proliferation, the double-layered scaffold formed in this way is more close to human tissue in structure, provides microenvironment conducive to cell adhesion, proliferation and differentiation, while maintaining appropriate porosity and mechanical properties, solve the technical problem that the existing double-layered scaffold is prone to layer separation in actual use.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a kind of biomimetic collagen double-layer scaffold and preparation method thereof. BACKGROUND

[0002] In the fields of tissue repair, wound management and cartilage repair, the use of scaffold materials to assist biological tissue regeneration and repair has become a hot research field. Scaffold materials often need to have similar biological properties to human tissues in order to be accepted by the body tissue and reduce rejection. To meet this demand, biomimetic material technology has gradually become a research hotspot in the field of biomedicine. It can simulate the structure and function of human tissues and organs to prepare new materials with similar biological materials. These new materials have good biocompatibility and biological activity and can play an important role in the field of biomedicine.

[0003] Collagen has been widely used in medicine and beauty due to its biocompatibility, low immunogenicity and biodegradability. However, the application of traditional single-layer collagen scaffolds in tissue repair and regeneration has been greatly limited. Single-layer scaffolds can only meet one of the conditions of structural density or porous structure, and cannot meet the dual needs of the microenvironment required for cell adhesion, proliferation and differentiation and the protection of the material for the inner cell tissue. This poses a great challenge to tissue repair and regeneration.

[0004] Currently, the double-layer interface combination of double-layer scaffolds generally uses a solution pouring combination with freeze-drying method. The interface combination relies only on the physical bonding of polymer solution penetrating into the dense layer tissue under the action of gravity. However, human skin itself has a dense stratum corneum and few surface groups, resulting in slow polymer penetration speed and low interface adhesion capacity. The double-layer scaffold is prone to layer separation during actual use. SUMMARY

[0005] The purpose of the present application is to provide a kind of biomimetic collagen double-layer scaffold and preparation method thereof, which is more similar in structure to human tissue, provides a microenvironment conducive to cell adhesion, proliferation and differentiation, and can maintain appropriate porosity and mechanical properties.

[0006] To achieve the above purpose, the present application provides a kind of biomimetic collagen double-layer scaffold and preparation method thereof, comprising the following steps:

[0007] Step 1: preparation of dense layer decellularized matrix using pig peritoneum;

[0008] Step 2: preparation of loose layer collagen solution;

[0009] Step 3: ultrasonic treatment of dense layer decellularized matrix;

[0010] Step 4: Crosslinking to obtain a double-layer scaffold.

[0011] The process of preparing a dense, decellularized matrix using porcine peritoneum includes the following steps:

[0012] Defatting of porcine peritoneum;

[0013] Decellularization of porcine peritoneum.

[0014] The process of defatting the pig peritoneum involves manually removing the fat attached to the pig peritoneum tissue and repeatedly washing it with physiological saline, 3 to 8 times. After washing, the pig peritoneum is soaked in an organic reagent at room temperature for further defatting, and then the defatted pig peritoneum is repeatedly soaked and washed in pure water 3 to 8 times.

[0015] The process of decellularizing porcine peritoneum involves immersing the defatted porcine peritoneum in an alkaline solution to remove cells, followed by repeated washing in pure water until the solution becomes neutral, resulting in a dense, decellularized matrix.

[0016] The preparation process of the loose layer collagen solution includes decellularized matrix pulverization, enzymatic hydrolysis, and dissolution. Specifically, the dense layer decellularized matrix is ​​freeze-dried and then cut into fragments, which are then pulverized into powder using a pulverizer. The powder is mixed with a pepsin solution and then filtered through a filter cloth to obtain a collagen suspension. An acetic acid solution with a concentration of 0.1% to 1% v / v is added to the collagen suspension to dissolve the suspended particles. Finally, the mixed solution is dialyzed in pure water to remove excess impurities, thus obtaining the loose layer collagen solution.

[0017] The freeze-drying process involved pre-freezing at -30℃ for 12 hours, followed by drying at -20 to 10℃ and 0 to 10 Pa for 12 to 24 hours. The ratio of powder to pepsin solution was 1:50 to 1:100 w / w, with pepsin activity ranging from 100 to 500 U / mL.

[0018] In the process of ultrasonic treatment of the dense layer decellularized matrix, the loose layer collagen solution with a concentration of 5-20 mg / mL is spread on the dense layer decellularized matrix, and then the loose layer collagen solution is exposed to a cell disruptor for cyclic ultrasonic treatment, so that the loose layer collagen solution penetrates into the dense layer decellularized matrix.

[0019] In the cyclic ultrasound treatment process, the ultrasonic emission power is 1200W, the intensity is 50-200W / cm2, the distance between the amplitude rod of the cell disruptor and the decellularized matrix of the dense layer is 1-2cm, the treatment time is 0.5-3min per treatment, the interval time is 1-5min, and the number of treatments is 5-20.

[0020] The process of obtaining a bilayer scaffold through cross-linking involves pouring a mixture of loose collagen and chemical cross-linking agent onto the dense, decellularized matrix treated in step 3. The mixture is then placed at a constant temperature of 5–37°C for 1–24 hours for cross-linking. After the loose collagen solution gels, the obtained bilayer scaffold is repeatedly rinsed in pure water to remove any residual chemical cross-linking agent.

[0021] The present invention also proposes a biomimetic collagen bilayer scaffold, which is prepared by the aforementioned biomimetic collagen bilayer scaffold preparation method and includes a dense layer of decellularized matrix and a loose layer of collagen hydrogel. The lower layer of the biomimetic collagen bilayer scaffold is a dense layer of decellularized matrix, and the upper layer is a loose layer of collagen hydrogel.

[0022] This invention provides a biomimetic collagen bilayer scaffold and its preparation method. Using inexpensive porcine peritoneum as raw material, a decellularized matrix with the same thickness and dense structure as the epidermis of human skin is prepared through a degreasing and decellularization process. Furthermore, a loose and porous hydrogel layer is prepared using this decellularized matrix, which can provide a suitable microenvironment for cell adhesion, growth and proliferation. The bilayer scaffold constructed in this way is structurally closer to human tissue, providing a microenvironment conducive to cell adhesion, proliferation and differentiation, while maintaining appropriate porosity and mechanical properties. This solves the technical problem of layer separation that easily occurs in existing bilayer scaffolds during actual use. Attached Figure Description

[0023] 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.

[0024] Figure 1 This is a schematic flowchart of a method for preparing a biomimetic collagen bilayer scaffold according to the present invention.

[0025] Figure 2 This is a schematic diagram of the biomimetic collagen bilayer scaffold prepared in Specific Embodiment 1 of the present invention placed in a medium water cup.

[0026] Figure 3 This is a schematic diagram of the biomimetic collagen bilayer scaffold prepared in specific embodiment 2 of the present invention and after cold drying treatment.

[0027] Figure 4 This is a multi-view schematic diagram of the dried biomimetic collagen bilayer scaffold prepared in specific embodiment 3 of the present invention.

[0028] Figure 5This is a scanning electron microscope image of the hydrogel layer of the dried biomimetic collagen bilayer scaffold prepared in specific embodiment 3 of the present invention.

[0029] Figure 6 This is an optical microscope image of the decellularized matrix layer of the dried biomimetic collagen bilayer scaffold prepared in specific embodiment 3 of the present invention.

[0030] Figure 7 This is a stress-strain curve of the dried biomimetic collagen bilayer scaffold prepared in specific embodiment 3 of the present invention. Detailed Implementation

[0031] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0032] Please see Figure 1 This invention provides a method for preparing a biomimetic collagen bilayer scaffold, comprising the following steps:

[0033] S1: Preparation of dense decellularized matrix using porcine peritoneum;

[0034] S2: Preparation of collagen solution for loose layer;

[0035] S3: Ultrasonic treatment of the decellularized matrix in the dense layer;

[0036] S4: Crosslinking to obtain a double-layer scaffold.

[0037] The process of preparing a dense, decellularized matrix using porcine peritoneum includes the following steps:

[0038] Defatting of porcine peritoneum;

[0039] Decellularization of porcine peritoneum.

[0040] The process of defatting the pig peritoneum involves manually removing the fat attached to the peritoneal tissue and repeatedly washing it with physiological saline, 3 to 8 times. After washing, the pig peritoneum is soaked in an organic reagent at room temperature for further defatting, and then the defatted pig peritoneum is repeatedly soaked and washed in pure water 3 to 8 times.

[0041] Specifically, the organic reagent is any one of acetone, isopropanol, diethyl ether, methanol, chloroform, ethyl acetate, and n-hexane.

[0042] Furthermore, the ratio of the porcine peritoneum to the organic reagent is 1:6 to 1:14 (w / v).

[0043] The process of decellularizing porcine peritoneum involves immersing the defatted porcine peritoneum in an alkaline solution to remove cells, followed by repeated washing in pure water until the solution becomes neutral, resulting in a dense, decellularized matrix.

[0044] Specifically, the alkaline solution is a mixture of sodium hydroxide and sodium chloride, with a sodium hydroxide concentration of 0.1% to 2% (w / w) and a sodium chloride concentration of 0.5% to 2% (w / w).

[0045] Furthermore, the ratio of defatted porcine peritoneum to alkaline solution is 1:5 to 1:10 (w / v).

[0046] Furthermore, the number of times the pure water is rinsed is 3 to 8.

[0047] The preparation process of the loose layer collagen solution includes decellularized matrix pulverization, enzymatic hydrolysis, and dissolution. Specifically, the dense layer decellularized matrix is ​​freeze-dried and then cut into fragments, which are then pulverized into powder using a pulverizer. The powder is mixed with a pepsin solution and then filtered through a filter cloth to obtain a collagen suspension. An acetic acid solution with a concentration of 0.1% to 1% v / v is added to the collagen suspension to dissolve the suspended particles. Finally, the mixed solution is dialyzed in pure water to remove excess impurities, thus obtaining the loose layer collagen solution.

[0048] The freeze-drying conditions were pre-freezing at -30℃ for 12 hours, followed by drying at -20~10℃ and 0~10Pa for 12~24 hours; the ratio of powder to pepsin solution was 1:50~1:100 w / w, and the pepsin activity was 100~500U / mL.

[0049] During the ultrasonic treatment of the dense layer decellularized matrix, the loose layer collagen solution with a concentration of 5-20 mg / mL is spread on the dense layer decellularized matrix. Then, the loose layer collagen solution is exposed to a cell disruptor for cyclic ultrasonic treatment, so that the loose layer collagen solution penetrates into the dense layer decellularized matrix.

[0050] The ultrasonic emission power during the cyclic ultrasonic treatment is 1200W, the intensity is 50-200W / cm2, the distance between the amplitude rod of the cell disruptor and the decellularized matrix of the dense layer is 1-2cm, the treatment time is 0.5-3min per treatment, the interval is 1-5min, and the number of treatments is 5-20.

[0051] The process of obtaining a bilayer scaffold through cross-linking involves pouring a mixture of loose collagen and chemical cross-linking agent onto the dense, decellularized matrix layer after step S3. The mixture is then placed at a constant temperature of 5–37°C for 1–24 hours for cross-linking. After the loose collagen solution gels, the obtained bilayer scaffold is repeatedly rinsed in pure water to remove any residual chemical cross-linking agent.

[0052] The chemical cross-linking reagents include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / 2-(N-morpholino)ethanesulfonic acid, genipin, 1,4-butanediol diglycidyl ether, glutaraldehyde, divalent metal ions, riboflavin, proanthocyanidins, tannic acid, etc.

[0053] The number of rinsing cycles is 3 to 8.

[0054] Furthermore, the present invention also proposes a biomimetic collagen bilayer scaffold, which is prepared using the aforementioned biomimetic collagen bilayer scaffold preparation method, comprising a dense layer of decellularized matrix and a loose layer of collagen hydrogel. The lower layer of the biomimetic collagen bilayer scaffold is a dense layer of decellularized matrix, and the upper layer is a loose layer of collagen hydrogel.

[0055] For details, please refer to Figures 2 to 7 The present invention will be further illustrated through three specific embodiments:

[0056] Example 1:

[0057] 1. Preparation of dense layer decellularized matrix:

[0058] (1) Degreasing treatment of pig peritoneum: Most of the fat attached to the pig peritoneum tissue was removed manually and repeatedly washed with physiological saline. After washing, the pig peritoneum was soaked in an organic reagent at room temperature for a certain period of time for degreasing. After degreasing, the pig peritoneum was repeatedly soaked and washed in pure water to replace the remaining organic solvent in the pig peritoneum.

[0059] The saline solution was used for 5 washes.

[0060] Furthermore, the organic reagent is isopropanol.

[0061] Furthermore, the ratio of the porcine peritoneum to the organic reagent is 1:6 (w / v).

[0062] Furthermore, the pure water rinsing is performed 5 times.

[0063] (2) Decellularization of porcine peritoneum: After the above defatting process, the porcine peritoneum is soaked in an alkaline solution to remove cells, and then repeatedly washed in pure water until the solution is neutral to obtain a dense decellularized matrix.

[0064] The alkaline solution is a mixture of sodium hydroxide and sodium chloride, with a sodium hydroxide concentration of 0.1% (w / w) and a sodium chloride concentration of 0.5% (w / w).

[0065] Furthermore, the ratio of the defatted porcine peritoneum to the alkali solution is 1:10 (w / v).

[0066] Furthermore, the pure water rinsing is performed 5 times.

[0067] 2. Preparation of collagen solution for loose layer:

[0068] Decellularized matrix pulverization, enzymatic hydrolysis, and dissolution: The above-mentioned decellularized matrix was freeze-dried, cut into fragments, and then pulverized into powder of a certain particle size using a pulverizer. The powder was mixed with pepsin solution in a certain proportion for a certain period of time. The larger particles were then filtered through a filter cloth to obtain a collagen suspension. A certain mass fraction of acetic acid solution was added to the collagen suspension to dissolve the suspended particles. Finally, the solution was dialyzed in pure water to remove excess impurities, yielding the final collagen solution.

[0069] The freeze-drying process involves pre-freezing at -30°C for 12 hours, followed by drying at 5°C and 10Pa for 24 hours.

[0070] Furthermore, the ratio of the powder to pepsin is 1:50 (w / w), and the pepsin activity is 500 U / mL.

[0071] Furthermore, the acetic acid concentration is 0.1% (v / v).

[0072] 3. Ultrasonic treatment of the dense decellularized matrix obtained above: Spread the 5 mg / mL collagen solution prepared above on the decellularized matrix, and then expose the collagen solution to a cell disruptor (ultrasonic power of 1200W, ultrasonic intensity of 120W / cm2, the amplitude rod of the cell disruptor is kept 1cm away from the dense decellularized matrix, and the interval is 5min) for 5 cycles of ultrasonic treatment, so that the collagen solution penetrates into the dense decellularized matrix.

[0073] 4. Crosslinking of the bilayer scaffold: A mixture of collagen and a chemical crosslinking agent (5 mg / mL collagen solution, 0.05% glutaraldehyde) was poured onto the sonicated decellularized matrix and placed at 25°C for 24 hours for crosslinking. After the collagen solution gelled, the bilayer scaffold was rinsed five times in pure water to remove residual chemical crosslinking agent. This yielded a bilayer scaffold with a dense decellularized matrix layer at the bottom and a loose collagen hydrogel layer on top.

[0074] Figure 2 This is a schematic diagram showing the biomimetic collagen bilayer scaffold prepared in Example 1 placed in a water cup. As can be seen from the diagram, the dense layer of decellularized matrix and the loose layer of collagen hydrogel have excellent interfacial bonding, and layer-to-layer separation does not occur even when immersed in water.

[0075] Example 2

[0076] Following the same ultrasonic treatment steps for the decellularized matrix of the dense layer as in Example 1, the concentration of the glutaraldehyde crosslinking agent was changed to 0.025% by mass. The specific steps are as follows:

[0077] A mixture of collagen and chemical cross-linking agent (5 mg / mL collagen solution, 0.025% glutaraldehyde) was poured onto the sonicated decellularized matrix and then placed at 25°C for 24 hours for cross-linking. After the collagen solution gelled, the bilayer scaffold was rinsed five times in pure water to remove residual chemical cross-linking agent. This yielded a bilayer scaffold with a dense decellularized matrix layer and a loose collagen hydrogel layer.

[0078] Figure 3 A is the biomimetic collagen bilayer scaffold prepared in Example 2. Because the concentration of glutaraldehyde used is relatively high, the resulting bilayer collagen scaffold appears pale yellow, which is due to the high degree of collagen cross-linking.

[0079] Figure 3 B is the bilayer scaffold of biomimetic collagen prepared in Example 2 after freeze-drying. Due to the high degree of cross-linking, the decellularized matrix warped, but the collagen hydrogel in the upper layer still adhered to the dense decellularized matrix after freeze-drying, which further indicates that there is good interfacial bonding between the two layers.

[0080] Example 3

[0081] The ultrasonic intensity was changed to 250 W / cm² and the crosslinking agent concentration was changed to 0.01%, while the other steps remained the same. The specific implementation steps are as follows:

[0082] 3.3. Ultrasonic treatment of the dense decellularized matrix obtained above: The 5 mg / mL collagen solution prepared above is spread on the decellularized matrix, and then the collagen solution is exposed to a cell disruptor (ultrasonic power of 1200W, ultrasonic intensity of 250W / cm2, the amplitude rod of the cell disruptor is kept 1cm away from the dense decellularized matrix, and the interval is 5min) for 5 cycles of ultrasonic treatment, so that the collagen solution penetrates into the dense decellularized matrix.

[0083] 3.4 Crosslinking of the bilayer scaffold: A mixture of collagen and chemical crosslinking agent (5 mg / mL collagen solution, 0.01% glutaraldehyde) was poured onto the sonicated decellularized matrix and placed at 25°C for 24 hours for crosslinking. After the collagen solution gelled, the bilayer scaffold was rinsed five times in pure water to remove residual chemical crosslinking agent. This yielded a bilayer scaffold with a dense decellularized matrix layer and a loose collagen hydrogel layer. The bilayer scaffold was then freeze-dried to obtain a dried bilayer scaffold.

[0084] Figure 4 A is a top view of the dried biomimetic collagen bilayer scaffold prepared in Example 3. Figure 4 B is a side view of the dried biomimetic collagen bilayer scaffold prepared in Example 3. It can be seen that the dense layer of decellularized matrix and the loose layer of collagen hydrogel still have good interfacial bonding after freeze-drying. Even though the lower layer of decellularized matrix warped after drying, no separation between layers occurred.

[0085] Figure 5 The image shows a scanning electron microscope (SEM) image of the hydrogel layer of the dried biomimetic collagen bilayer scaffold prepared in Example 3. It can be seen that the pore diameter of the hydrogel is about 100-500 micrometers, and this porous structure is conducive to cell adhesion, growth and proliferation.

[0086] Figure 6 This is an optical microscope image of the decellularized matrix layer of the dried biomimetic collagen bilayer scaffold prepared in Example 3. The image shows that the decellularized matrix layer still retains a dense network structure formed by collagen fiber entanglement, which has a protective effect on the inner cell tissue when used for tissue repair, wound management, cartilage repair, etc.

[0087] Figure 7 The figure shows the stress-strain curve of the dried biomimetic collagen bilayer scaffold prepared in Example 3. The tensile strength of the cross-linked bilayer scaffold is 14.8 MPa, indicating that the material has excellent mechanical properties.

[0088] In summary, compared with the prior art, the present invention has the following advantages:

[0089] 1. This invention uses inexpensive porcine peritoneum as raw material and prepares a decellularized matrix with the same thickness (0.2 mm) and dense structure as the human skin epidermis through a defatting and decellularization process. This layer has excellent mechanical properties (tensile strength 14.8 MPa) as a double-layer scaffold. Furthermore, this decellularized matrix is ​​used to prepare a loosely porous hydrogel layer (pore size approximately 100–500 μm), which can provide a suitable microenvironment for cell adhesion, growth, and proliferation.

[0090] 2. This invention utilizes ultrasonic cavitation to infiltrate collagen macromolecules into the dense, decellularized matrix, and further enhances the interfacial bonding of the bilayer scaffold through the cross-linking effect of a chemical cross-linking agent. This prevents delamination of the bilayer scaffold during practical applications such as tissue repair, wound management, and cartilage repair. The method of altering the interfacial adhesion of biological tissues using ultrasound combined with chemical cross-linking can serve as a simple and universally applicable method for constructing other similar bilayer scaffold structures.

[0091] 3. The biomimetic collagen bilayer scaffold prepared by the present invention can control the mechanical properties, pore structure and degradation time of the bilayer scaffold by adjusting the type of crosslinking agent, the action time of the crosslinking agent and the crosslinking temperature, so as to meet the performance requirements of the material for related application scenarios such as tissue repair, wound management and cartilage repair.

[0092] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a biomimetic collagen bilayer scaffold, characterized in that, Includes the following steps: Step 1: Preparation of dense decellularized matrix using porcine peritoneum; The process of preparing a dense, decellularized matrix using porcine peritoneum includes the following steps: Defatting of porcine peritoneum; Decellularization of porcine peritoneum; The process of defatting the pig peritoneum involves manually removing the fat attached to the pig peritoneum tissue and repeatedly washing it with physiological saline, 3 to 8 times. After washing, the pig peritoneum is soaked in an organic reagent at room temperature for further defatting, and then the defatted pig peritoneum is repeatedly soaked and washed in pure water 3 to 8 times. The process of decellularizing porcine peritoneum involves immersing the defatted porcine peritoneum in an alkaline solution to remove cells, followed by repeated washing in pure water until the solution becomes neutral, resulting in a dense, decellularized matrix. The alkaline solution is a mixture of sodium hydroxide and sodium chloride, with a sodium hydroxide concentration of 0.1%~2% w / w and a sodium chloride concentration of 0.5%~2% w / w; the ratio of defatted porcine peritoneum to alkaline solution is 1:10 w / w. Step 2: Preparation of collagen solution for the loose layer; Step 3: Ultrasonic treatment of the dense layer decellularized matrix; During the ultrasonic treatment of the dense layer decellularized matrix, the loose layer collagen solution with a concentration of 5~20 mg / mL is spread on the dense layer decellularized matrix, and then the loose layer collagen solution is exposed to a cell disruptor for cyclic ultrasonic treatment, so that the loose layer collagen solution penetrates into the dense layer decellularized matrix. The ultrasonic emission power during the cyclic ultrasonic treatment process is 1200W, the intensity is 50~200W / cm2, the distance between the amplitude rod of the cell disruptor and the decellularized matrix of the dense layer is 1~2cm, the treatment time is 0.5~3min, the interval is 1~5min, and the number of treatments is 5~20. Step 4: Crosslinking to obtain a double-layer scaffold.

2. The method for preparing a biomimetic collagen bilayer scaffold as described in claim 1, characterized in that, The preparation process of the loose layer collagen solution includes decellularized matrix pulverization, enzymatic hydrolysis, and dissolution. Specifically, the dense layer decellularized matrix is ​​freeze-dried and then cut into fragments, which are then pulverized into powder using a pulverizer. The powder is mixed with a pepsin solution and then filtered through a filter cloth to obtain a collagen suspension. An acetic acid solution with a concentration of 0.1% to 1% v / v is added to the collagen suspension to dissolve the suspended particles. Finally, the mixed solution is dialyzed in pure water to remove excess impurities, thus obtaining the loose layer collagen solution.

3. The method for preparing a biomimetic collagen bilayer scaffold as described in claim 2, characterized in that, The freeze-drying conditions are: pre-freezing at -30℃ for 12 hours, followed by drying at -20~10℃ and 0~10Pa for 12~24 hours; the ratio of powder to pepsin solution is 1:50~1:100 w / w, and the pepsin activity is 100~500U / mL.

4. The method for preparing a biomimetic collagen bilayer scaffold as described in claim 3, characterized in that, The process of obtaining a bilayer scaffold through cross-linking involves pouring a mixture of loose collagen and chemical cross-linking agent onto the dense, decellularized matrix layer after step 3. The mixture is then placed at a constant temperature of 5-37°C for 1-24 hours for cross-linking. After the loose collagen solution gels, the obtained bilayer scaffold is repeatedly rinsed in pure water to remove any residual chemical cross-linking agent.

5. A biomimetic collagen bilayer scaffold, prepared using the biomimetic collagen bilayer scaffold preparation method as described in claim 1, characterized in that, It includes a dense layer of decellularized matrix and a loose layer of collagen hydrogel. The lower layer of the biomimetic collagen bilayer scaffold is a dense layer of decellularized matrix, and the upper layer is a loose layer of collagen hydrogel.

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