Collagen-based multilayer biological material and preparation method thereof

Abstract

The invention discloses a collagen-based multilayer biological material and a preparation method thereof. The preparation method comprises the following steps: 1) dividing a collagen solution, a collagen Pickering emulsion and a water-soluble polymer solution into n parts of feed liquid; the n parts of feed liquid at least comprise 1 part of collagen solution or collagen Pickering emulsion; (2) a directional freezing device is arranged, a freezing feed liquid mold in the directional freezing device is divided into n independent channels through n-1 baffles, and n parts of feed liquid are poured into the channels respectively; (3) liquid nitrogen is added to the bottom of the device for pre-cooling, all the baffles are slowly lifted upwards from the bottom of the mold, and the lifting speed of the baffles should be larger than the maximum advancing speed of the ice peak; and 4) after the baffle plate is completely pulled out of the mold, carrying out freeze drying to obtain the collagen-based multilayer biological material. According to the method, the problems that the collagen-based multi-layer biological material is high in preparation difficulty, poor in layer interface stability and the like are solved, and a novel universal technology is provided for manufacturing the multi-layer biological material which is free of adhesion and firm in interlayer interface.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a collagen-based multilayer biomaterial and a preparation method thereof. Background Art

[0002] Collagen is the most abundant structural protein in animals and is widely present in animal tissues such as skin, bones, tendons and cartilage. It has many advantages such as low antigenicity, biodegradability and biocompatibility. It has great application value and development potential in the field of biomedical materials.

[0003] In recent years, among the booming biomedical materials, composite materials composed of multilayer materials with a layered structure and no less than two layers have become increasingly popular in the market. The outstanding advantages of multilayer biomedical materials are: (1) by selecting different materials for multilayer combination, multilayer materials can exhibit good biological properties such as biocompatibility and anti-adhesion, thereby reducing the possibility of rejection and tissue adhesion; (2) by using multilayer structures to introduce gradient structural characteristics, it can better promote tissue repair, such as controlling the release cycle of active substances, drugs, etc. In short, by adjusting the number of layers, or adjusting the structural and functional characteristics of each layer, each sublayer of the multilayer material can perform its own functions, thereby meeting the specific clinical needs of users and achieving precision medicine.

[0004] In the prior art, collagen-based multilayer biomaterials are usually prepared by conventional physical pressing, dipping or spraying methods, which makes it difficult to form a stable and lasting interface bond between layers; and in the preparation process, most of them use bonding to achieve interlayer interface connection. The biomaterials obtained in this way are very likely to experience interface detachment when exposed to water. At the same time, the use of adhesives may also have a negative impact on the biocompatibility of the material. Summary of the invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a technical method with good process controllability and adjustability and wide applicability of raw materials for preparing collagen-based biomedical materials. Without using any adhesive components, the manufactured collagen-based multilayer biomedical materials can have strong interface stability.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] A method for preparing a collagen-based multilayer biomaterial, comprising:

[0008] 1) dividing the collagen solution, collagen Pickering emulsion, and water-soluble polymer solution into n portions of liquid feeds; wherein the n portions of liquid feeds include at least one portion of the collagen solution or collagen Pickering emulsion;

[0009] 2) a directional freezing device is provided, wherein the freezing liquid mold in the directional freezing device is divided into n independent channels by n-1 baffles, and n portions of liquid are respectively poured into the n independent separated channels;

[0010] 3) After adding liquid nitrogen to the bottom of the device for precooling, each baffle is slowly lifted upward from the bottom of the mold, wherein the lifting rate of the baffle should be greater than the maximum forward speed of the ice peak;

[0011] 4) After the baffles are completely pulled out of the mold, freeze drying is performed to obtain the collagen-based multilayer biomaterial.

[0012] Furthermore, the concentration of the collagen solution is 1-15 mg / mL; the preparation method of the collagen solution is as follows:

[0013] At 10°C, the purified collagen was dissolved in a 0.1-0.5 mol / L acetic acid solution and stirred to dissolve evenly to obtain a collagen solution.

[0014] Furthermore, the preparation method of the collagen Pickering emulsion comprises the following steps:

[0015] A) dissolving the purified collagen in a 0.1-0.5 mol / L acetic acid solution at 10° C. and stirring to dissolve uniformly to obtain a collagen solution; the concentration of the collagen solution is 1-15 mg / mL;

[0016] B) Adding the oil phase to the collagen solution and emulsifying it into a collagen Pickering emulsion by high-speed stirring and shearing.

[0017] Furthermore, in step A), the volume ratio of the collagen solution to the oil phase is 1-9:1; the oil phase includes but is not limited to dodecane; in step B), the high-speed stirring speed is 5000-20000 r / min, and the stirring time is 1-5 min.

[0018] Furthermore, the preparation method of the refined collagen is: using the acid-enzyme method to extract and purify collagen from animal connective tissue, and obtaining a refined collagen sponge after drying, which is used as a refined collagen raw material for standby use;

[0019] The animal connective tissue includes but is not limited to animal skin and Achilles tendon.

[0020] Furthermore, the concentration of the water-soluble polymer solution is 1-40 mg / mL, and the preparation method of the water-soluble polymer solution is:

[0021] Adding a non-toxic water-soluble polymer raw material suitable for use in the field of biomedical materials into an aqueous solution and stirring and dissolving the mixture uniformly to obtain a water-soluble polymer solution;

[0022] The non-toxic water-soluble polymer raw materials suitable for use in the field of biomedical materials include, but are not limited to, water-soluble carboxymethyl chitosan, various cellulose derivatives, sodium alginate, hyaluronic acid, chondroitin sulfate, polyvinyl alcohol, polyethylene glycol, preferably carboxymethyl chitosan, cellulose derivatives, polyethylene glycol.

[0023] Furthermore, the directional freezing device includes a liquid nitrogen tank, a brass cylindrical conductor, a freezing liquid mold, a baffle and a traction device;

[0024] The brass cylindrical conductor is placed in a liquid nitrogen tank, the frozen liquid mold is placed on the top of the brass cylindrical conductor, one or more baffles are longitudinally inserted into the frozen liquid mold to separate it into multiple independent channels, and the traction device is arranged above the baffle to traction the baffle to lift it.

[0025] Furthermore, the freezing liquid mold is made of polytetrafluoroethylene, the baffle is made of stainless steel, and a PDMS wedge is built into the bottom of the freezing liquid mold;

[0026] The traction device comprises a traction rope and a lifting device. The traction rope is fixed to the upper end of each baffle, and the lifting device lifts the baffle upwards through the traction rope.

[0027] Furthermore, the precooling time in step 3) is 5-15 minutes; the freeze-drying temperature in step 4) is -50°C, and the freeze-drying time is 48-72 hours.

[0028] The present invention also provides a collagen-based multilayer biomaterial, which is prepared by the preparation method of the collagen-based multilayer biomaterial.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1) The present invention can make the manufactured collagen-based multilayer biomedical material have strong interface stability without using any adhesive components; by controlling the ice peak freezing rate and the baffle lifting rate, the slurry between different channels can be frozen and fixed at low temperature when it diffuses with each other but not excessively diffuses, so as to easily realize one-time molding of multilayer materials with stable interfaces.

[0031] 2) The present invention sets up multiple channels separated by baffles, and different channels can be loaded with different slurries. The lifting rate of the baffles is controlled to achieve simple manufacturing of multi-layer biomaterials with firm interlayer interfaces. This not only breaks through the traditional mindset of using adhesives to achieve interlayer adhesion, but also provides a disruptive technology to innovate traditional directional freezing technology. In addition, this method is not only applicable to collagen systems, but also to other water-soluble polymer systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the appearance picture of the multilayer collagen sponge material and the SEM morphology picture at the layer interface;

[0033] Figure 2 is the interfacial peel strength of multilayer collagen sponge material;

[0034] Figure 3 is a schematic diagram of a dual-channel directional freezing device;

[0035] Figure 4 This is a physical picture of the PDMS wedge. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The present invention provides a method for preparing a collagen-based multilayer biomaterial, comprising:

[0038] 1) dividing the collagen solution, collagen Pickering emulsion, and water-soluble polymer solution into n portions of liquid (n≥2); wherein the n portions of liquid include at least one portion of collagen solution or collagen Pickering emulsion;

[0039] 2) A directional freezing device is provided, wherein the frozen liquid mold in the directional freezing device is divided into n independent channels by n-1 baffles, and n portions of liquid are poured into the n independent separated channels respectively; the volume of each portion of liquid does not exceed the inner volume of the corresponding compartment, and the inner volume size of each compartment is flexibly adjusted according to the structure of the target product.

[0040] The dual-channel directional freezing device of the present invention comprises a liquid nitrogen tank 1, a brass cylindrical conductor 2, a freezing liquid mold 3 (the freezing liquid mold 3 is made of polytetrafluoroethylene material for easy demoulding), a baffle 4 (the baffle 4 is a stainless steel baffle) and a traction device;

[0041] The brass cylindrical conductor 2 is placed in the liquid nitrogen tank 1, and the freezing liquid mold 3 is placed on the top of the brass cylindrical conductor 2. One or more baffles 4 are longitudinally inserted into the freezing liquid mold 3 to separate it into multiple independent channels. The traction device is arranged above the baffle 4 to pull the baffle 4 to lift; the traction device includes a traction rope 5 and a lifting device 6. The traction rope 5 is fixed to the upper end of each baffle 4, and the lifting device 6 lifts the baffle 4 upward through the traction rope 5.

[0042] Furthermore, an opening is provided in the middle of the freezing liquid mold 3. For heat conduction, a heat conduction block can be further provided at the bottom of the freezing liquid mold 3. The heat conduction block can contact the liquid material inside the opening of the freezing liquid mold 3 for heat conduction. Specifically, when a PDMS wedge is built into the bottom of the freezing liquid mold, it is a bidirectional directional freezing mode (when the temperature is conducted through this PDMS wedge, the heat transfer gap caused by its angle will produce one more dimension of heat transfer than the unidirectional one); otherwise, it is a unidirectional directional freezing mode.

[0043] 3) After adding liquid nitrogen to the bottom of the device for pre-cooling for 2-10 minutes, each baffle is slowly lifted upward from the bottom of the mold. In the bottom area without the baffle, the liquid in the compartments on both sides immediately come into contact, and interface interaction and diffusion occur. At the same time, the liquid including the diffusion interface begins to be frozen and formed from the bottom. To prevent the baffle from freezing, the lifting rate of the baffle should be greater than the maximum forward rate of the ice peak (for example: when brass is heat conductive, liquid nitrogen is the cold source, and a PDMS wedge with a 10° inclination angle is installed at the bottom of the mold, the maximum pre-ice rate is measured to be 0.6mm / min; then the baffle can be slowly lifted upward from the bottom at a fixed rate greater than 0.6mm / min).

[0044] 4) When the baffles are completely pulled out of the mold, n portions of the liquid (including n-1 contact interfaces) are also immediately frozen and formed. After freeze drying at -50°C for 48-72 hours, a multi-layer collagen-based sponge scaffold material with strong interface bonding (i.e., collagen-based multi-layer biomaterial) is obtained.

[0045] The concentration of the above collagen solution is 1-15 mg / mL; the preparation method of the collagen solution is: at 10°C, dissolve the purified collagen in 0.1-0.5 mol / L acetic acid solution and stir to dissolve evenly to obtain the collagen solution.

[0046] The preparation method of collagen Pickering emulsion comprises the following steps:

[0047] A) dissolving the purified collagen in a 0.1-0.5 mol / L acetic acid solution at 10° C. and stirring to dissolve uniformly to obtain a collagen solution; the concentration of the collagen solution is 1-15 mg / mL;

[0048] B) Add the oil phase to the collagen solution at an oil-water ratio (1-9:1) and emulsify it into a collagen Pickering emulsion by high-speed stirring and shearing (5000-20000 r / min, 1-5 min).

[0049] The preparation method of the above-mentioned refined collagen is: using the acid-enzyme method to extract and purify collagen from animal connective tissue, and obtaining a refined collagen sponge after drying, which is used as a refined collagen raw material; wherein the animal connective tissue includes but is not limited to animal skin and Achilles tendon.

[0050] The concentration of the water-soluble polymer solution is 1-40 mg / mL. The preparation method of the water-soluble polymer solution is: add non-toxic water-soluble polymer raw materials suitable for use in the field of biomedical materials (raw materials include but are not limited to water-soluble carboxymethyl chitosan, various cellulose derivatives, sodium alginate, hyaluronic acid, chondroitin sulfate, polyvinyl alcohol, polyethylene glycol, preferably carboxymethyl chitosan, cellulose derivatives, polyethylene glycol) into an aqueous solution and stir to dissolve evenly to obtain a water-soluble polymer solution.

[0051] Example 1

[0052] Weigh 0.1g, 0.2g, and 0.3g of collagen respectively, add an equal volume of 20mL of 0.1mol / L acetic acid solution, and prepare them into collagen solutions with mass volume concentrations of 5mg / mL, 10mg / mL, and 15mg / mL respectively. Further, according to the internal dimensions of the mold made of polytetrafluoroethylene (top view: 60mm long × 20mm wide), a stainless steel baffle (0.5mm thick) is selected, and both sides of the baffle are wrapped with waterproof glue. This customized baffle is placed in the middle of the mold (such as Figure 3 As shown in the figure, a mold with two independent channels is formed. The above-mentioned 5mg / mL, 10mg / mL, and 15mg / mL collagen solutions are combined in three concentration gradients: (10 / 5) mg / mL, (15 / 5) mg / mL, and (15 / 10) mg / mL. Then they are poured into the independent channels on both sides respectively. The brass bottom is cooled with liquid nitrogen. After precooling for 5 minutes, the baffle is lifted by the lifting device (at a speed of 0.6mm / min). After the sample is completely frozen, it is freeze-dried at -50°C for 48 hours to obtain a collagen composite double-layer sponge (pure collagen double layer).

[0053] Example 2

[0054] On the basis of Example 1, 5 mg / mL, 10 mg / mL, and 15 mg / mL collagen solutions were prepared in the same manner. Dodecane was then added to the collagen solution at a fixed oil-water ratio (oil: water = 3:7), and emulsified by high-speed shear (4°C, 0.1 MPa, 10000 rpm) for 3 min. 5 mg / mL, 10 mg / mL, and 15 mg / mL collagen Pickering emulsions were obtained respectively. Subsequently, they were poured into independent channels on both sides of the mold according to the three concentration gradient combinations in Example 1. The brass bottom was cooled with liquid nitrogen. After precooling for 10 minutes, the baffle was lifted by a lifting device (at a speed of 0.6 mm / min). After the sample was completely frozen, it was freeze-dried at -50°C for 72 hours to obtain a composite double-layer sponge composed of pure collagen emulsion.

[0055] Example 3

[0056] On the basis of Example 1, two stainless steel baffles are cut according to the internal size of the beaker or the mold, and the two sides of the cardboard are wrapped with waterproof glue. Then the customized baffles are placed in the mold at the positions of 1 / 3 and 2 / 3 to form three independent channel spaces (the number of spaces depends on the size of the mold). The 5mg / mL, 10mg / mL, and 15mg / mL collagen solutions prepared in Example 1 are poured into the three channels of the mold from large to small. The brass bottom is cooled with liquid nitrogen. After precooling for 9 minutes, the baffle is lifted by a lifting device (at a speed of 0.7mm / min). A composite three-layer gradient sponge made of pure collagen solution can be obtained.

[0057] Example 4

[0058] On the basis of Example 2, two stainless steel baffles are cut according to the internal dimensions of the beaker or the mold, and the baffles are wrapped with waterproof glue on both sides. Then the customized baffles are placed in the mold at the positions of 1 / 3 and 2 / 3 to form three independent channel spaces (the number of spaces depends on the size of the mold). The 5mg / mL, 10mg / mL, and 15mg / mL collagen Pickering emulsions prepared in Example 2 are poured into the three channels respectively, and the brass bottom is cooled with liquid nitrogen. After precooling for 10 minutes, the baffle is lifted by a lifting device (at a speed of 0.8mm / min). A three-layer gradient structure sponge made of collagen emulsion can be obtained.

[0059] Example 5

[0060] On the basis of Examples 1 and 2, the collagen solution prepared in Example 1 was combined with the collagen Pickering emulsion prepared in Example 2: solution 15 mg / mL / emulsion 15 mg / mL, solution 15 mg / mL / emulsion 10 mg / mL, solution 15 mg / mL / emulsion 5 mg / mL; solution 10 mg / mL / emulsion 15 mg / mL, solution 10 mg / mL / emulsion 10 mg / mL, solution 10 mg / mL / emulsion 5 mg / mL. The two molds with independent channels in Example 1 were used for preparation respectively. The brass bottom was cooled with liquid nitrogen. After precooling for 5 minutes, the baffle was lifted by a lifting device (speed 0.6 mm / min). A composite double-layer sponge (solution layer / emulsion layer) can be obtained.

[0061] Example 6

[0062] Weigh 0.3g collagen, add 20mL of 0.5mol / L acetic acid solvent, and stir for 30min at 0.1MPa and 4℃. Centrifuge at 10000rpm to make it into a uniform glue stock solution with a concentration of 15mg / mL. Similarly, weigh powdered PEG (molecular weight 100000g / mol) and dissolve it in four portions of deionized water (20mL each), prepare it according to the concentration gradient of 5mg / mL, 10mg / mL, 20mg / mL, and 30mg / mL, and stir it at room temperature until it is completely dissolved to obtain a PEG solution. Then, add 15mg / mL glue stock solution and four portions of PEG solutions of different concentrations (collagen solution 15mg / mL / PEG solution 5mg / mL, collagen solution 15mg / mL / PEG solution 10mg / mL, collagen solution 15mg / mL / PEG solution 15mg / mL) into the double-channel mold respectively, and place the mold on the brass bottom. Cool the brass bottom with liquid nitrogen. After precooling for 9 minutes, the baffle was lifted by a lifting device (speed was 0.7 mm / min). After the sample was completely frozen, it was freeze-dried at -50°C for 60 hours to obtain a series of collagen layer / PEG composite double-layer sponges.

[0063] Example 7

[0064] Weigh 0.3g collagen, add 20mL of 0.25mol / L acetic acid solvent, and stir for 30min at 0.1MPa and 4℃. Centrifuge at 10000rpm to make it into a 15mg / mL uniform concentration of collagen stock solution. Similarly, weigh powdered CMCS (substitution degree ≥80%) and dissolve it in four portions of deionized water (20mL each), prepare according to the mass concentration gradient of 5mg / mL, 10mg / mL, 15mg / mL, and 20mg / mL, stir at room temperature until completely dissolved, and obtain CMCS solution. Then, adjust the solution systems of COL and CMCS to the same pH (pH=6-7), respectively, match the collagen and four portions of CMCS of different concentrations in a double-channel mold, and place the mold on the brass bottom. Cool the brass bottom with liquid nitrogen. After precooling for 10min, lift the baffle by the lifting device (speed is 0.6mm / min). After the sample was completely frozen, it was freeze-dried at -50°C for 48 h to obtain a series of collagen layer / CMCS composite double-layer sponges.

[0065] Example 8

[0066] Weigh 0.3g collagen, add 20mL of 0.1mol / L acetic acid solvent, and stir for 30min at 0.1MPa and 4℃. Centrifuge at 10000rpm to make it into a 15mg / mL uniform concentration of collagen raw material liquid. Similarly, weigh powdered CMCS (substitution degree ≥80%) and dissolve it in four portions of deionized water (each with a concentration of 20mL), with a mass concentration gradient of 5, 10, 15, and 20mg / mL in turn. Adjust the pH to 6-7 at room temperature and stir until completely dissolved. Then, 10mg / mL collagen solution and four portions of CMCS solutions of different concentrations are respectively mixed and modified (by volume 1:1) to obtain 4 portions of blended solutions containing collagen and CMCS. Subsequently, the blended solution of collagen and CMCS and the pure 15mg / mL collagen solution are respectively matched in a double-channel mold, and the mold is placed on a brass bottom. The brass bottom is cooled with liquid nitrogen. After precooling for 8 minutes, the baffle was lifted by a lifting device (speed was 0.8 mm / min). After the sample was completely frozen, it was freeze-dried at -50°C for 72 hours to obtain a series of collagen layers / collagen and CMCS blended double-layer sponges.

[0067] Characterization and testing

[0068] From the attached Figure 1 It can be seen that the double layer ( Figure 1 A) or three layers ( Figure 1 C) The scaffold has obvious stratification characteristics due to the different phases of collagen solution and emulsion. The interface between the layers is tight and seamless. The fusion between the interfaces can also be verified at the microscopic level with the help of SEM. Figure 1 B clearly shows the microscopic diffusion bonding phenomenon of different components at the interface of the double-layer material (see arrows).

[0069] From the attached Figure 2 It can be seen that the test results of Examples 1 (pure collagen solution group) and 2 (pure collagen emulsion group) show that the peeling strength increases with the increase in concentration, and the peeling strength of the double-layer sample interface is not less than the strength of the matrix (i.e., the sample obtained by a single channel) itself; at the same time, the greater the concentration difference between the two layers, the greater the interface strength. The test results of Example 5 (collagen solution / collagen emulsion group) show that the double-layer material obtained by the collagen solution / collagen emulsion group also has a high interface peeling strength. The test results of Examples 6 to 8 (collagen solution / other water-soluble polymer solution groups) show that the interface peeling strength of heterogeneous double-layer materials is generally higher than the strength of samples obtained from pure collagen solutions, further proving that collagen can be paired with different water-soluble polymers, and a two-channel or even multi-channel bidirectional freezing device can be used to prepare multilayer biomaterials with a strong interface.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a collagen-based multilayer biomaterial, characterized in that: include: 1) dividing the collagen solution, collagen Pickering emulsion, and water-soluble polymer solution into n portions of liquid feeds; wherein the n portions of liquid feeds include at least one portion of the collagen solution or collagen Pickering emulsion; 2) a directional freezing device is provided, wherein the freezing liquid mold in the directional freezing device is divided into n independent channels by n-1 baffles, and n portions of liquid are respectively poured into the n independent separated channels; 3) After adding liquid nitrogen to the bottom of the device for precooling, each baffle is slowly lifted upward from the bottom of the mold, wherein the lifting rate of the baffle should be greater than the maximum forward speed of the ice peak; 4) After the baffles are completely pulled out of the mold, freeze drying is performed to obtain the collagen-based multilayer biomaterial.

2. The method for preparing a collagen-based multilayer biomaterial according to claim 1, characterized in that: The concentration of the collagen solution is 1-15 mg / mL; the preparation method of the collagen solution is as follows: At 10°C, the purified collagen was dissolved in a 0.1-0.5 mol / L acetic acid solution and stirred to dissolve evenly to obtain a collagen solution.

3. The method for preparing a collagen-based multilayer biomaterial according to claim 2, characterized in that: The preparation method of the collagen Pickering emulsion comprises the following steps: A) dissolving the purified collagen in a 0.1-0.5 mol / L acetic acid solution at 10° C. and stirring to dissolve uniformly to obtain a collagen solution; the concentration of the collagen solution is 1-15 mg / mL; B) Adding the oil phase to the collagen solution and emulsifying it into a collagen Pickering emulsion by high-speed stirring and shearing.

4. The method for preparing a collagen-based multilayer biomaterial according to claim 3, characterized in that: In step A), the volume ratio of the collagen solution to the oil phase is 1-9:1; the oil phase includes but is not limited to dodecane; in step B), the high-speed stirring speed is 5000-20000 r / min, and the stirring time is 1-5 min.

5. The method for preparing a collagen-based multilayer biomaterial according to claim 3, characterized in that: The preparation method of the refined collagen is as follows: using the acid-enzyme method to extract and purify collagen from animal connective tissue, and obtaining a refined collagen sponge after drying, which is used as a refined collagen raw material for standby use; The animal connective tissue includes but is not limited to animal skin and Achilles tendon.

6. The method for preparing a collagen-based multilayer biomaterial according to claim 1, characterized in that: The concentration of the water-soluble polymer solution is 1-40 mg / mL, and the preparation method of the water-soluble polymer solution is: Adding a non-toxic water-soluble polymer raw material suitable for use in the field of biomedical materials into an aqueous solution and stirring and dissolving the mixture uniformly to obtain a water-soluble polymer solution; The non-toxic water-soluble polymer raw materials suitable for use in the field of biomedical materials include, but are not limited to, water-soluble carboxymethyl chitosan, various cellulose derivatives, sodium alginate, hyaluronic acid, chondroitin sulfate, polyvinyl alcohol, polyethylene glycol, preferably carboxymethyl chitosan, cellulose derivatives, polyethylene glycol.

7. The method for preparing a collagen-based multilayer biomaterial according to claim 1, characterized in that: The directional freezing device comprises a liquid nitrogen tank (1), a brass cylindrical conductor (2), a freezing liquid mold (3), a baffle (4) and a traction device; The brass cylindrical conductor (2) is placed in a liquid nitrogen tank (1), the freezing liquid mold (3) is placed on top of the brass cylindrical conductor (2), one or more baffles (4) are longitudinally inserted into the freezing liquid mold (3) for dividing it into a plurality of independent channels, and the traction device is arranged above the baffle (4) for traction and lifting the baffle (4).

8. The method for preparing a collagen-based multilayer biomaterial according to claim 7, characterized in that: The freezing liquid mold (3) is made of polytetrafluoroethylene, the baffle (4) is made of stainless steel, and a PDMS wedge is built into the bottom of the freezing liquid mold; The traction device comprises a traction rope (5) and a lifting device (6); the traction rope (5) is fixed to the upper end of each baffle (4); and the lifting device (6) lifts the baffle (4) upwards via the traction rope (5).

9. The method for preparing a collagen-based multilayer biomaterial according to claim 1, characterized in that: The precooling time in step 3) is 5-15 minutes; the freeze-drying temperature in step 4) is -50°C, and the freeze-drying time is 48-72 hours.

10. A collagen-based multilayer biomaterial, characterized in that: The collagen-based multilayer biomaterial is prepared by the preparation method of any one of claims 1 to 9.

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