CN114848909A

CN114848909A - Collagen aggregate scaffold material and preparation method thereof

Info

Publication number: CN114848909A
Application number: CN202210430561.XA
Authority: CN
Inventors: 但卫华; 何晓棠; 朱美峰; 但年华
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2022-08-05
Anticipated expiration: 2042-04-22
Also published as: CN114848909B

Abstract

The invention provides a collagen aggregate scaffold material and a preparation method thereof, and relates to the technical field of biomedical materials. The method comprises the following steps: (A) soaking collagen aggregate extracted from Achilles tendon of cattle in the medium; (B) transferring the fully soaked collagen aggregate and the medium to a vacuum homogenizer to prepare a collagen aggregate dispersion liquid; (C) transferring the collagen aggregate dispersion liquid into a screen or a receiving container, shaking up, and naturally drying; (D) soaking the naturally dried transparent material into purified water for water absorption and swelling, then pre-freezing at a certain freezing temperature and freeze-drying to obtain a material with a certain interlayer spacing or pore structure; (E) preparing a bracket material with certain tensile strength and flexibility by adopting a cross-linking agent under certain cross-linking conditions; (F) and fully cleaning the scaffold material, then pre-freezing at a certain freezing temperature and freeze-drying to obtain the collagen aggregate scaffold material.

Description

Collagen aggregate scaffold material and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a collagen aggregate scaffold material and a preparation method thereof.

Background

Collagen is the structural protein with the largest content and the widest distribution in animals. The collagen enables the extracellular matrix to have good mechanical property and structural integrity, has the functions of supporting organs and protecting organisms, and plays a key role in maintaining the structure and physiological functions of the organisms. Collagen has the advantages of good biocompatibility, weak antigenicity, biodegradability and the like as a natural biological material. However, pure collagen has poor mechanical strength and flexibility, and it is due to its lack of mechanical strength and flexibility that limits its wide application in tissue engineering and regenerative medicine.

In order to overcome the characteristic of poor mechanical property of pure collagen, the following methods are commonly adopted in the prior art to improve the mechanical property of collagen: first, crosslinking is the most common method of improving its mechanical strength and stability. The crosslinking comprises chemical crosslinking, physical crosslinking and biological crosslinking, wherein the chemical crosslinking (glutaraldehyde (GTA), EDC-NHS) is the most widely and effectively applied method at present due to the advantages of high crosslinking uniformity, strong crosslinking capability and the like. While physical crosslinking (ultraviolet radiation (UV), dehydrothermal treatment (DHT)) is generally used as a secondary crosslinking means. Although the biological cross-linking agent (genipin, riboflavin and transglutaminase 2(TG2)) has low cytotoxicity and is not easy to cause collagen denaturation, the wide application of the biological cross-linking agent is limited due to the high price and high cost. Thus, although crosslinked collagen materials are widely used in biomedicine, there is currently no standard crosslinking protocol that can achieve a perfect balance between collagen stability and functional remodeling. Second, adding inorganic or organic materials, adding inorganic (metal ions such as Ag) ⁺ 、Ca ²⁺ 、Fe ³⁺ ) Or organic materials (cellulose nanofibers, cellulose nanocrystals, keratin, casein and gelatin) to some extent with collagen addedMechanical strength. However, the material prepared by the method increases the mechanical property of the material, increases the brittleness of the material, has a compact internal structure, and is rarely researched as a material for repairing tissues. Third, mechanical properties of pure gum materials are improved by techniques such as microfluidics, which typically handle small volumes of fluid in channels with dimensions of tens to hundreds of microns, and do not allow scaffold materials to be formed with a certain scale and size. Plastic compression, which is a method that substantially increases the mechanical properties of a material by increasing the density of collagen, is dense, has no pore structure, and requires appropriate production equipment for mass production, and is not suitable for mass production at present. The raw materials used in the method mainly extract the lowest level collagen molecules, the materials prepared from the raw materials have compact structures, have no pore structures beneficial to the permeation of cells and nutrient substances, lack certain flexibility and are easy to break during operation.

In conclusion, the pure collagen scaffold material prepared by the current preparation method has low mechanical strength and is not suitable for mechanical integration of tissues, and the material is compact and is not beneficial to infiltration of cells, so that a new strategy is urgently needed to improve the mechanical strength and flexibility of collagen, so that the pure collagen scaffold material can be used for tissue repair of force bearing parts, such as hernia, tendons, blood vessels, dura mater, skin and other parts, in addition, a proper pore structure is beneficial to infiltration and vascularization of cells, so that the tissue repair is promoted, and therefore, how to construct a collagen scaffold material with high mechanical strength, flexibility and a proper pore structure is still a problem to be solved at present.

Disclosure of Invention

The invention aims to provide a porous collagen aggregate scaffold material with high strength, flexibility and tissue regeneration promotion and a preparation method thereof, and aims to solve the technical problems of low mechanical strength, no proper pore structure and flexibility of a pure collagen scaffold in the prior art. The technical effects that can be produced by the preferred technical scheme in the technical schemes provided by the invention are described in detail in the following.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a preparation method of a collagen aggregate scaffold material, which comprises the following steps:

(A) soaking: soaking collagen aggregates extracted from bovine achilles tendon in a medium, wherein the medium comprises purified water, a PBS aqueous solution, or an acid solvent;

(B) homogenizing in vacuum: transferring the fully soaked collagen aggregate and the medium into a vacuum homogenizer to prepare a collagen aggregate dispersion liquid, and performing vacuumizing and bubble removal treatment;

(C) and (3) naturally drying: transferring the collagen aggregate dispersion liquid obtained in the step (B) into a screen or a receiving container, shaking uniformly, and naturally drying for 24-96 hours;

(D) soaking treatment: immersing the transparent material obtained after natural air drying into purified water for water absorption and swelling, then pre-freezing the material subjected to water absorption and swelling at a certain freezing temperature, and then performing freeze-drying treatment to obtain a material with a certain interlayer spacing or pore structure;

(E) and (3) crosslinking: preparing the material with the interlayer spacing or the pore structure obtained in the step (D) by using a cross-linking agent under a certain cross-linking condition to obtain a bracket material with certain tensile strength and flexibility;

(F) cleaning and freeze-drying treatment: and fully cleaning the scaffold material, then pre-freezing at a certain freezing temperature, and then performing freeze-drying treatment to obtain the collagen aggregate scaffold material.

According to a preferred embodiment, in step (a), the collagen aggregates comprise collagen fibrils, collagen fibers or collagen fiber bundles;

the pH value of the medium is 5-7.5, and the acid solution comprises acetic acid, hydrochloric acid or sulfuric acid.

The raw material used in the preparation method of the application is bovine achilles tendon, and the species of the cattle include Bracteal bulls, Haford cattle, Angus cattle, Chinese buffalo, yellow cattle and Simmental cattle, but other species of cattle are not excluded.

According to a preferred embodiment, in step (a), the soaking process further comprises: fully soaking the collagen aggregate in the medium for 4-24 hours at the temperature of 2-8 ℃.

According to a preferred embodiment, in the step (B), the method for preparing the collagen aggregate dispersion further comprises: and transferring the fully soaked collagen aggregate and the medium into a vacuum homogenizer at 10000-40000 r/min and 3-6 grades for 10-30S, and repeating for 1-5 times to obtain the collagen aggregate dispersion liquid with the mass fraction concentration of 0.2-5%.

According to a preferred embodiment, in the step (C), the diameter of the screen is 5-30 cm, and the receiving container is a 304 stainless steel container.

According to a preferred embodiment, in the step (D), the transparent material obtained after natural air drying is immersed in purified water for 0-3 hours for water absorption and swelling, and then the material after water absorption and swelling is pre-frozen by adopting liquid nitrogen at-20 ℃, at-80 ℃ and then is freeze-dried, so as to prepare the materials with different interlayer spacing or pore structures.

According to a preferred embodiment, in the step (E), the crosslinking treatment further comprises: preparing a bracket material with various different tensile strengths and flexibilities by using various different cross-linking agents under different cross-linking conditions;

the adopted crosslinking agent comprises EDC/NHS, aldehydes or genipin, the mass fraction concentration of the adopted crosslinking agent is 0.05-1%, and the solvent adopted for crosslinking is 0-100% of ethanol solution.

According to a preferred embodiment, in the step (F), the crosslinked stent material is cleaned by using an automatic water changer, wherein the water changing frequency of the automatic water changer is 1-2 hours/time, and the cleaning time is 48-96 hours.

The invention also provides a collagen aggregate scaffold material which is prepared by the preparation method of any one of the claims 1 to 8, has the tensile strength of 9-22 MPa and the surface roughness of 40-90, and shows the periodic transverse striation D belt peculiar to collagen fibrils or collagen fibers;

under the dry condition, the collagen aggregate scaffold material cannot be broken after being folded for 50-100 times, and under the wet condition, when the collagen aggregate scaffold material deforms for 5-25%, the collagen aggregate scaffold material is circularly loaded for 10-20 times, and the scaffold still keeps complete;

the collagen aggregate scaffold material has a layered pore structure, and the interlayer spacing is 5-80 mu m;

the collagen aggregate scaffold material has a cytotoxicity rating of 0.

According to a preferred embodiment, the collagen aggregate scaffold material has the appearance of a white, yellowish or brown scaffold material without macroscopic impurities.

Based on the technical scheme, the collagen aggregate scaffold material and the preparation method thereof have the following technical effects:

(1) compared with the traditional preparation method in which collagen molecules are used as raw materials, the collagen aggregate scaffold material provided by the invention has a better natural bionic three-dimensional structure, better mechanical properties and more cell action binding sites.

(2) The preparation method can select different soaking time and different freezing temperatures to control the pore structure of the material, and compared with the existing preparation method, the prepared material has controllable pore structure and mechanical strength and can adapt to the repair and regeneration of different tissues.

(3) The preparation method of the invention can select different cross-linking agents and different cross-linking conditions for cross-linking, and compared with the existing preparation method, the different cross-linking conditions can lead the material to form different mechanical strength and degradation time, so that the proper stent material can be selected according to the length of the repair part and the implantation time of the implant.

(4) Compared with other existing preparation methods, the preparation method provided by the invention adopts a vacuum homogenization and natural air drying treatment method, is simple to operate, and is more beneficial to large-scale production.

(5) Compared with the existing material, the scaffold material prepared by the preparation method has a pore structure infiltrated by cells, high tensile strength and suture strength, is favorable for integration with host tissues and promotion of macrophage polarization, and further promotes tissue repair or regeneration.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is the maximum tensile strength of a collagen aggregate scaffold material prepared in example 1 of the present invention;

FIG. 2 is a scanning electron microscope image of a longitudinal section of a collagen aggregate scaffold material prepared in example 1 of the present invention;

FIGS. 3 and 4 are roughness of the collagen aggregate scaffold prepared in example 1 of the present invention and the scaffold prepared from the existing collagen molecules;

FIG. 5 is a schematic representation of the wet flexibility of a collagen aggregate scaffold material prepared according to an embodiment of the present invention;

FIGS. 6 and 7 are graphs showing the effect of a conventional scaffold material L-col made of collagen molecules and a collagen aggregate scaffold material H-col prepared in examples 1 to 4 on pore structures in a preparation process of different soaking times and freezing temperatures;

FIG. 8 shows the results of collagenase degradation tests of a scaffold material L-col made of conventional collagen molecules and a collagen aggregate scaffold material H-col prepared in example 1 of the present application;

FIG. 9 shows the effect of different soaking times and freezing temperatures on the mechanical strength of a scaffold material L-col made of conventional collagen molecular material and a collagen aggregate scaffold material H-col prepared in examples 1 to 4 of the present application;

FIG. 10 is the effect of different cross-linking concentrations on the mechanical strength of the scaffold material in example 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1:

this example provides a preferred method of making an aggregate scaffold material:

soaking collagen aggregates, specifically collagen fibrils, extracted from bovine achilles tendon in purified water, wherein the pH of the purified water is 6-7, the mass fraction concentration of the purified water is 4%, and storing the purified water in a refrigerator at 2-8 ℃ for 4 hours.

Then, the fully soaked 4% collagen aggregate solution was transferred to a vacuum homogenizer at 20000 rpm, 3 steps, 10S/time, for a total of 3 times. And vacuumizing to remove bubbles to obtain uniformly dispersed collagen fibril dispersion liquid.

Then, the collagen fibril dispersion was slowly poured into a 20 cm-diameter sieve having 80 mesh nylon net laid therein, the solution was slowly shaken up, and the 80 mesh nylon net was further laid thereon. The mixture was left at room temperature and air-dried for 48 hours. Subsequently, the obtained transparent film was air-dried and soaked in purified water for 1 hour. Prefreezing at-20 deg.C, quickly transferring to a lyophilizer, and lyophilizing for 48 hours.

Subsequently, the lyophilized white-like scaffold material was purified with 80% ethanol solution, EDC/NHS ═ 4: 1, EDC concentration of 0.3% by mass was crosslinked for 6 hours.

Then, the reaction mixture was washed with purified water for 48 hours, and the water was changed every 2 hours. And then pre-freezing at-20 ℃, and freeze-drying for 48 hours to obtain the porous collagen aggregate scaffold material with high strength, flexibility and tissue regeneration promotion.

As shown in fig. 1, fig. 1 shows the tensile strength of the collagen aggregate scaffold material prepared in this example, and it can be seen from fig. 1 that the maximum tensile strength of the collagen aggregate scaffold material prepared in this example can reach 20Mpa and the maximum elongation is 35% as the tensile strain of the scaffold material increases. FIG. 2 shows a longitudinal sectional scanning electron microscope image of the collagen aggregate scaffold material prepared in the present example; as can be seen from figure 2, the scaffold material has a layered pore structure, and the interlayer spacing is 5-80 um. The surface roughness of the collagen aggregate scaffold material prepared by the invention is 40-90, as shown in fig. 4, and fig. 3 and 4 show the roughness comparison of the collagen aggregate scaffold material H-col prepared in example 1 and the scaffold material L-col prepared by the existing collagen molecules. As shown in fig. 3, the surface of the collagen aggregate scaffold material of the present invention shows periodic striation D bands peculiar to collagen fibrils or collagen fibers. As shown in fig. 5, fig. 5 shows the wet flexibility of the collagen aggregate scaffold material prepared in this example, and as can be seen from fig. 5, the collagen aggregate scaffold material has a white appearance without macroscopic impurities. In the wet state, the stent remains intact after deformation.

Example 2:

soaking collagen aggregates, specifically collagen fibers, extracted from bovine achilles tendon in a PBS (phosphate buffer solution) reagent, wherein the pH value of the PBS reagent is 7.2-7.4, the mass fraction concentration is 2%, and storing the PBS reagent in a refrigerator at 2-8 ℃ for 12 hours.

Then, the fully soaked 2% collagen aggregate solution was transferred to a vacuum homogenizer at 40000 rpm, 5 steps, 30S/time for 1 total. And vacuumizing to remove bubbles to obtain uniformly dispersed collagen fiber dispersion liquid.

Then, slowly pouring the collagen fiber dispersion liquid into a screen with the diameter of 10cm, paving a 80-mesh nylon net in the screen, slowly shaking up the solution, and paving the 80-mesh nylon net on the solution. The mixture was left at room temperature and air-dried for 24 hours. Subsequently, the obtained transparent film was air-dried and soaked in purified water for 3 hours. Prefreezing at-20 deg.C, quickly transferring to a lyophilizer, and lyophilizing for 24 hours.

Subsequently, the lyophilized white-like scaffold material was purified by dissolving in 50% ethanol, EDC/NHS 4: 1, EDC at 0.15% mass concentration for crosslinking for 4 hours.

Then, the reaction mixture was washed with purified water for 24 hours, and the water was changed every 2 hours. And then pre-freezing at-20 ℃, and freeze-drying for 24 hours to obtain the porous collagen aggregate scaffold material with high strength, flexibility and tissue regeneration promotion.

Example 3

Soaking a collagen aggregate, specifically a collagen fiber bundle, extracted from the bovine achilles tendon in purified water, wherein the pH of the purified water is 6-7, the mass fraction concentration of the purified water is 5%, and the purified water is stored in a refrigerator at the temperature of 2-8 ℃ for 24 hours.

Then, the fully soaked 5% collagen aggregate solution was transferred to a vacuum homogenizer at 40000 rpm, 6 steps, 20S/time, for a total of 4 times. Vacuumizing to remove air bubbles, and obtaining the uniformly dispersed collagen fiber bundle dispersion liquid.

Then, slowly pouring the collagen fiber bundle dispersion liquid into a screen with the diameter of 10cm, paving a 80-mesh nylon net in the screen, slowly shaking up the solution, and paving the 80-mesh nylon net on the solution. The mixture was left at room temperature and air-dried for 24 hours. Subsequently, the obtained transparent film was air-dried and soaked in purified water for 1 hour. Pre-freezing at-80 deg.C, quickly transferring to a freeze dryer, and freeze-drying for 48 hr.

Subsequently, the lyophilized white-like scaffold material was purified by dissolving in 50% ethanol, EDC/NHS 4: 1, EDC mass concentration of 0.15% for crosslinking for 4 hours.

Then, the reaction mixture was washed with purified water for 24 hours, and the water was changed every 2 hours. And then pre-freezing at-80 ℃, and freeze-drying for 48 hours to obtain the porous collagen aggregate scaffold material with high strength, flexibility and tissue regeneration promotion.

Example 4

Then, slowly pouring the collagen fiber dispersion liquid into a screen with the diameter of 10cm, paving a 80-mesh nylon net in the screen, slowly shaking up the solution, and paving the 80-mesh nylon net on the solution. The mixture was left at room temperature and air-dried for 24 hours. Subsequently, the obtained transparent film was air-dried and soaked in purified water for 3 hours. Pre-freezing at-80 deg.C, quickly transferring to a freeze dryer, and freeze-drying for 24 hr.

Then, the reaction mixture was washed with purified water for 24 hours, and the water was changed every 2 hours. And then pre-freezing at-80 ℃, and freeze-drying for 24 hours to obtain the porous collagen aggregate scaffold material with high strength, flexibility and tissue regeneration promotion.

Fig. 6 and 7 show the effect of the conventional scaffold material L-col made of collagen molecules and the collagen aggregate scaffold material H-col prepared in examples 1 to 4 of the present application on the pore structure under the preparation processes of different soaking times and different freezing temperatures. As can be seen from fig. 6, the collagen aggregate scaffold material prepared by the present application has a different layered pore structure from the collagen molecular scaffold material, and different soaking times and freezing temperatures cause the layered pore structure of the collagen aggregate scaffold material to have different sizes, so that the pore structure size of the material can be selectively controlled by using different soaking times and different freezing temperatures according to the application requirements.

Fig. 8 shows the results of the collagenase degradation test of the scaffold material L-col prepared from conventional collagen molecules and the collagen aggregate scaffold material H-col prepared in example 1 of the present application, and it can be seen from fig. 8 that the collagen aggregate scaffold material H-col prepared by the preparation method of the present application is less likely to be degraded than the scaffold material prepared from conventional collagen molecules as the degradation time is prolonged.

Fig. 9 shows the influence of different soaking times and freezing temperatures on the mechanical strength of the L-col scaffold prepared from the conventional common collagen molecular material and the H-col scaffold prepared from the collagen aggregate in examples 1 to 4 of the present application, and it can be seen from fig. 9 that the collagen aggregate scaffold prepared by the preparation method of the present invention has higher mechanical strength.

Example 5:

soaking a collagen aggregate mixture extracted from the bovine achilles tendon in 0.01mol/L acetic acid solution, wherein the pH is 5-5.5, the mass fraction concentration is 4%, and storing the mixture in a refrigerator at the temperature of 2-8 ℃ for 1 hour.

Then, the fully soaked 4% collagen aggregate solution was transferred to a vacuum homogenizer at 20000 rpm, 2 steps, 10S/time, for 2 times in total. And vacuumizing to remove bubbles to obtain the uniformly dispersed collagen aggregate dispersion liquid.

Then, the collagen aggregate dispersion was slowly poured into a stainless steel pan having a diameter of 10cm, a 80-mesh nylon net was laid in the pan, the solution was slowly shaken up, and a 80-mesh nylon net was further laid on the pan. The mixture was left at room temperature and air-dried for 24 hours. Subsequently, the obtained transparent film was air-dried and immersed in purified water for 10 minutes. Prefreezing at-20 deg.C, quickly transferring to a lyophilizer, and lyophilizing for 48 hours.

Subsequently, the lyophilized white-like scaffold material was crosslinked with EDC/NHS at concentrations of 0.2%, 0.3%, 0.4%, 0.5%, 0.6% and 1% by mass for 6 hours, respectively. Then, the reaction mixture was washed with purified water for 48 hours, and the water was changed every 2 hours. Then prefreezing at-20 deg.C, and freeze-drying for 48 hr to obtain 6 porous collagen aggregate scaffold materials with high strength and flexibility for promoting tissue regeneration and prepared from different cross-linking agent concentrations.

As shown in fig. 10, fig. 10 shows the effect of different crosslinking concentrations on the mechanical strength of the scaffold material in example 5, and as can be seen from fig. 10, the mechanical strength increases and then decreases with increasing crosslinking concentration, and the mechanical strength is optimal at a crosslinking concentration of 0.3%.

The scaffold material for promoting tissue regeneration, which is prepared by the invention, has high strength, flexibility and porous collagen aggregate has the tensile strength of 9-22 Mpa and the surface roughness of 40-90, and shows collagen fibrils or specific periodic transverse striation D bands of the collagen fibers. Under the dry state, the support can be folded for 50-100 times without breaking, and under the wet state, when the deformation is 5-25%, the support can be circularly loaded for 10-20 times, and still keeps complete. The support material has a lamellar pore structure, and the interlayer spacing is 5-80 um. The cytotoxicity rating of the collagen aggregate scaffold material was 0. The porous collagen aggregate scaffold material with high strength, flexibility and capability of promoting tissue regeneration, which is prepared by the invention, can be used for promoting repair or regeneration of rectus abdominis or blood vessels.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A preparation method of a collagen aggregate scaffold material is characterized by comprising the following steps:

2. The method for preparing a collagen aggregate scaffold material according to claim 1, wherein in step (a), said collagen aggregate comprises collagen fibrils, collagen fibers or collagen fiber bundles;

3. The method for preparing a collagen aggregate scaffold material according to claim 1, wherein in the step (A), the soaking process further comprises: fully soaking the collagen aggregate in the medium for 4-24 hours at the temperature of 2-8 ℃.

4. The method for producing a collagen aggregate scaffold material according to claim 1, wherein in the step (B), the method for producing a collagen aggregate dispersion further comprises: and transferring the fully soaked collagen aggregate and the medium into a vacuum homogenizer at 10000-40000 r/min and 3-6 grades for 10-30S, and repeating for 1-5 times to obtain the collagen aggregate dispersion liquid with the mass fraction concentration of 0.2-5%.

5. The method for preparing a collagen aggregate scaffold material according to claim 1, wherein in the step (C), the mesh has a diameter of 5 to 30cm, and the receiving vessel is a 304 stainless steel vessel.

6. The method for preparing a collagen aggregate scaffold material according to claim 1, wherein in the step (D), the transparent material obtained by natural air drying is immersed in purified water for 0 to 3 hours to undergo water-absorbing swelling, and then the water-absorbing swollen material is pre-frozen and then freeze-dried at-20 ℃ to-80 ℃ and liquid nitrogen, respectively, to prepare materials having different interlamellar spacings or pore structures.

7. The method for preparing a collagen aggregate scaffold material according to claim 1, wherein in the step (E), said crosslinking treatment further comprises: preparing a bracket material with various different tensile strengths and flexibilities by using various different cross-linking agents under different cross-linking conditions;

8. The method for preparing a collagen aggregate scaffold material according to claim 1, wherein in step (F), the crosslinked scaffold material is washed with an automatic water exchanger, wherein the water exchange frequency of the automatic water exchanger is 1-2 hours/time, and the washing time is 48-96 hours.

9. A collagen aggregate scaffold material, which is produced by the production method according to any one of claims 1 to 8, has a tensile strength of 9 to 22Mpa and a surface roughness of 40 to 90, and exhibits a periodic cross-grain D band peculiar to collagen fibrils or collagen fibers;

the collagen aggregate scaffold material has a cytotoxicity rating of 0.

10. The collagen aggregate scaffold material according to claim 9, wherein said collagen aggregate scaffold material has the appearance of a white, yellowish or brownish scaffold material without macroscopic impurities.