Collagen scaffolds with controllable removal of telopeptides, methods of making and uses thereof

Abstract

The application aims to realize self-adhesive fixation of cartilage repair scaffolds, and provides a collagen scaffold preparation method with controllable removal of terminal peptides, comprising the following steps: S1, cleaning and shearing collagen raw materials to obtain granular collagen materials; S2, dehydrating, centrifuging and dispersing the granular collagen materials to obtain dispersed collagen materials; S3, performing enzymatic treatment on the dispersed collagen materials to obtain a collagen solution; and S4, preparing a collagen scaffold by using the collagen solution, wherein the removal rate of terminal peptides in the collagen solution is controlled at 60%-80% in step S3, which comprises: adding water, glacial acetic acid and pepsin into the dispersed collagen materials according to the mass ratio of dispersed collagen materials:water:glacial acetic acid:pepsin=1:(5-25):(0.3-1.0):(0.05-1.5); keeping at a temperature of 4-8 DEG C for 48 h-120 h, and stirring several times during the keeping period; and performing low-temperature gradient dialysis under a temperature condition of 4-8 DEG C to remove residual enzyme solution, thereby obtaining the collagen solution. The application further provides the collagen scaffold prepared by the method and application thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a collagen scaffold with controllable removal of telopeptide as well as a preparation method and application thereof. BACKGROUND

[0002] Articular cartilage is a highly differentiated hyaline cartilage tissue, and once damaged, its self-repairing ability is very limited. With the development of biomaterials, tissue engineering and regenerative medicine, cartilage repair treatment technology has made remarkable progress in recent years. Common clinical treatment methods include joint cleaning, microfracture, autologous chondrocyte implantation, autologous cartilage transplantation and matrix-induced autologous chondrocyte technology.

[0003] At present, microfracture is one of the most commonly used cartilage repair treatment technologies in clinic, which fills the defect site by promoting bone marrow stem cells to fill the defect site through microfracture in subchondral bone, thereby promoting the self-repair of articular cartilage and subchondral bone. However, the cartilage repaired by this method is fibrocartilage rather than hyaline cartilage, which has a large difference in structure and biomechanical properties from normal cartilage. Matrix-induced autologous chondrocyte technology is a new cartilage repair technology based on microfracture, which mainly uses absorbable biomaterials on the basis of microfracture. This material can temporarily replace the damaged cartilage tissue and provide a good environment for cell growth.

[0004] Matrix-induced autologous chondrocyte technology has a broad application prospect due to its small damage and good repair effect, and the appearance of a suitable and easy-to-operate scaffold material in the development of this technology has a milestone significance. For example, Chondro-Gide double-layer scaffold for cartilage repair produced by Swiss Geistlich Pharma AG is a collagen scaffold product that can effectively repair cartilage damage, but this product needs to be sutured and fixed under arthroscopy, and the surgical process is complicated, which can cause damage to the surrounding cartilage and requires a long operation time. For another example, CN101066475A and CN105381504A both disclose multi-layer collagen scaffolds with good cartilage repair capacity, but these scaffolds cannot realize self-adhesion fixation, and often need to be assisted by additional fixation support means when repairing larger cartilage defects, which limits their application.

[0005] There are some biological materials that can be achieved in the prior art, for example, CN211327153U discloses a biological membrane product which can be sutured and attached, which utilizes the advantages of the porous collagen sponge layer that can absorb and coagulate blood, solving the problem of dense connective tissue that needs to be sutured and fixed during use, but the strength and adhesion performance of such sponge material is limited, and cannot meet the requirements of cartilage repair scaffolds for high mechanical strength and complex mechanical action. Therefore, there is an urgent need for cartilage repair scaffolds with high strength and high adhesion performance in arthroscopic minimally invasive cartilage repair. SUMMARY

[0006] In view of the above problems in the prior art, the inventors of the present application have conducted extensive research on collagen materials of different sources and their characteristics in the preparation process of the scaffold, and have found that:

[0007] When the scaffold material is used as a cell carrier, the control of the collagen molecular structure in the three-dimensional direction is very critical. Among various collagen materials of different sources, animal tendons are mainly composed of type I collagen, which has a triple helix structure formed by three alpha chains winding around each other. The N-terminal and C-terminal of the collagen molecule each have a non-triple helix terminal peptide region, and the terminal peptide in this region can be removed during collagen enzymatic treatment. Since the terminal peptide is one of the important factors causing immunogenicity, it is traditionally believed that the terminal peptide should be removed as much as possible, i.e., the higher the removal rate, the better. However, the inventors have found that when the removal rate of the terminal peptide in this region is too high, the interaction and cross-linking ability between collagen molecules will be significantly reduced, and the fibrous ability of collagen will be significantly weakened, especially the adhesion between the collagen scaffold and the cartilage tissue will be significantly reduced. Therefore, by controlling the enzymatic process, the removal rate of the terminal peptide can be controlled, and on this basis, by cross-linking process, the controllable cross-linking of the unremoved terminal peptide can also be realized, so that more free triple helix molecules can be retained in the final collagen scaffold material, which can realize molecular mutual winding with the collagen of the cartilage or bone injury site, thereby improving the adhesion strength between the scaffold and the tissue, and realizing the self-adhesion fixation of the cartilage repair scaffold.

[0008] Based on the above findings, the present application provides a collagen scaffold preparation method with controllable removal of terminal peptides, comprising the following steps:

[0009] Step S1, cleaning and shearing the collagen raw material to obtain a granular collagen material;

[0010] Step S2, dehydrating the granular collagen material with a dehydrating agent, removing the dehydrating agent by centrifugation and performing dispersion treatment to obtain a dispersed collagen material;

[0011] Step S3, enzymatically treating the dispersed collagen material to obtain a collagen solution;

[0012] Step S4, preparing a collagen scaffold by cross-linking with the collagen solution,

[0013] In the step S3, the removal rate of the telopeptide in the collagen solution is controlled at 60%-80% by using an enzymatic method of controllable telopeptide removal, including:

[0014] Step S3-1, water, glacial acetic acid and pepsin are added to the dispersed collagen material according to the mass ratio of dispersed collagen material : water : glacial acetic acid : pepsin = 1 : (5-25) : (0.3-1.0) : (0.05-1.5);

[0015] Step S3-2, keeping at 4-8℃ for 48 h-120 h, and stirring several times during the keeping to completely dissolve and sufficiently react the dispersed collagen material;

[0016] Step S3-3, removing the residual enzyme solution by low-temperature gradient dialysis under the condition of 4-8℃ to obtain the collagen solution.

[0017] In the collagen scaffold preparation method of controllable telopeptide removal provided by the present application, the collagen raw material of step S1 can be Achilles tendon, and the size of the particulate collagen material can be 3-10 mm.

[0018] In addition, in the collagen scaffold preparation method of controllable telopeptide removal provided by the present application, step S4 can further include:

[0019] Step S4-1, the collagen solution is laid flat in a mold for low-temperature freezing molding to form a frozen collagen;

[0020] Step S4-2, the frozen collagen is soaked in a prepared salt solution for a predetermined time to form a first layer of collagen;

[0021] Step S4-3, the collagen solution is laid again on the first layer of collagen, and after standing until the surface is flat, freeze-drying is performed to form a double-layer collagen;

[0022] Step S4-4, the double-layer collagen is immersed in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution for overall cross-linking to form a double-layer scaffold;

[0023] Step S4-5, the double-layer scaffold is washed with water and then subjected to secondary freeze-drying to obtain the collagen scaffold.

[0024] In the collagen scaffold preparation method of controllable telopeptide removal provided by the present application, the salt solution in step S4-2 can be a phosphate buffer solution (PBS solution) containing 5%-40% NaCl (w / v).

[0025] In addition, in the method for preparing the collagen scaffold with controllable removal of terminal peptides, the concentration of the EDC solution in step S4-4 can be 5 mg / ml-30 mg / ml, and the crosslinking time can be 0.5-6 hours.

[0026] In the method for preparing the collagen scaffold with controllable removal of terminal peptides, the freezing temperature in step S4-1 can be-4℃--80℃, and the freezing time can be 8-24 hours.

[0027] In addition, the dehydrating agent in step S2 can be ethanol.

[0028] The application further provides a collagen scaffold, which is prepared by using the method for preparing the collagen scaffold with controllable removal of terminal peptides according to any one of the above.

[0029] The application further provides the use of the collagen scaffold in the preparation of a material for repairing cartilage defects.

[0030] Effects of the application

[0031] According to the collagen scaffold with controllable removal of terminal peptides and the preparation method thereof, since the removal rate of the terminal peptides is controlled to be 60%-80% in the enzymatic treatment of the collagen, the collagen molecules with the removed terminal peptides can be physically entangled with the surrounding cartilage and subchondral bone tissue through the triple helix structure after the collagen solution obtained by the enzymatic treatment is crosslinked to prepare the collagen scaffold, so that the adhesion performance between the collagen scaffold and the bone defect site is greatly improved, and the adhesion strength can reach 1 N or more, so that the bone defect site can be repaired, and especially, a larger bone defect site can be repaired without additional support means.

[0032] In addition, the collagen raw material of the application is Achilles tendon, which is mainly type I collagen and mainly has a triple helix molecular structure. The Achilles tendon is cut into 3mm-10mm granules after washing, and the size is suitable for retaining the triple helix structure. The Achilles tendon is also subjected to dehydration and dispersion treatment after cutting, and then subjected to enzymatic treatment, so that the triple helix structure in the collagen molecules can be effectively retained. Therefore, the proportion of the triple helix structure in the collagen scaffold can be further improved, and the realization of the adhesion performance is further ensured. DETAILED DESCRIPTION

[0033] The collagen scaffold with controllable removal of terminal peptides and the preparation method thereof will be described below in combination with embodiments.

[0034] The collagen raw material used in the following examples and comparative examples is Achilles tendon, which is obtained by peeling from commercially available quarantine qualified pig trotters. In addition, the reagent materials not specified in the examples and comparative examples are commonly commercially available, and the operation method not specified in the specific process refers to the conventional operation method of the prior art.

[0035] Example 1

[0036] The present example provides a collagen scaffold with controllable removal of telopeptide and a preparation method thereof.

[0037] The preparation method of the present example comprises the following steps:

[0038] Step S1, cleaning and shearing the collagen raw material to obtain a particulate collagen material, specifically including:

[0039] Step S1-1, removing the surface fat and fascia of the peeled Achilles tendon;

[0040] Step S1-2, shearing the peeled Achilles tendon obtained in step S1-1 into particles of about 3 mm in size, i.e. particulate collagen material.

[0041] Step S2, dehydrating the particulate collagen material with a dehydrating agent, centrifuging to remove the dehydrating agent and performing dispersion treatment to obtain a dispersed collagen material. The dehydrating agent is ethanol, and the volume of ethanol used for dehydrating is more than 3 times the volume of the particulate collagen material; the dispersion treatment is to separate and loosen the solid material after centrifugation to form loose particulate or fibrous solid material.

[0042] Step S3, enzymatic treatment of the dispersed collagen material obtained in step S2 to obtain a collagen solution, specifically including:

[0043] Step S3-1, adding water, glacial acetic acid and pepsin to the dispersed collagen material according to the mass ratio of dispersed collagen material : water : glacial acetic acid : pepsin = 1 : 10 : 0.3 : 0.05;

[0044] Step S3-2, maintaining at a temperature of 4-8℃ for 72h, and stirring several times during the maintaining period to completely dissolve and fully react the dispersed collagen material;

[0045] Step S3-3, after the reaction is completed, low-temperature gradient dialysis is performed at a temperature of 4-8℃ to remove the residual enzyme solution, and the obtained solution is the collagen solution.

[0046] Step S4, preparing a collagen scaffold using the collagen solution, specifically including:

[0047] Step S4-1, the collagen solution is laid in the mold and low-temperature frozen to form a frozen collagen, wherein the low-temperature freezing temperature is -4℃ to -80℃, and the freezing time is 8 to 24 hours;

[0048] Step S4-2, the frozen collagen is soaked in the prepared salt solution for a predetermined time to form a first layer of collagen, wherein the salt solution is a PBS solution containing 5% to 40% NaCl (w / v), and in this embodiment, the salt solution specifically contains NaCl 32 g / L, Na2HPO4 2.436 g / L, and KH2PO4 0.8 g / L, and the soaking time is 24 hours;

[0049] Step S4-3, collagen solution is laid again on the first layer of collagen, and freeze-drying is performed after standing until the surface is flat to form a double-layer collagen;

[0050] Step S4-4, the double-layer collagen is immersed in an EDC solution for overall crosslinking to form a double-layer scaffold, wherein the concentration of the EDC solution is 20 mg / ml, and the crosslinking time is 3 hours;

[0051] Step S4-5, the double-layer scaffold is washed with water and then subjected to secondary freeze-drying to obtain a collagen scaffold. The collagen scaffold can be cut into a desired size and stored at room temperature after irradiation sterilization.

[0052] Example 2

[0053] This embodiment provides another collagen scaffold with controllable removal of terminal peptides and a preparation method thereof.

[0054] The preparation method steps of this embodiment are the same as those of Example 1, but the specific conditions are different, as follows:

[0055] In step S1-2, the Achilles tendon is cut into particles with a size of about 4 mm;

[0056] In step S3-1, the mass ratio of the dispersed collagen material : water : glacial acetic acid : pepsin is 1 : 12.5 : 0.4 : 0.15;

[0057] In step S3-2, the holding time at a temperature of 4-8℃ is 80 h.

[0058] Example 3

[0059] This embodiment provides yet another collagen scaffold with controllable removal of terminal peptides and a preparation method thereof.

[0060] The preparation method steps of this embodiment are the same as those of Example 1, but the specific conditions are different, as follows:

[0061] In step S1-2, the Achilles tendon is cut into particles with a size of about 6 mm;

[0062] In step S3-1, the mass ratio of the dispersed collagen material : water : ice acetic acid : pepsin is 1 : 15 : 0.5 : 0.40;

[0063] In step S3-2, the holding time at a temperature of 4-8℃ is 48 h.

[0064] Example 4

[0065] This example provides yet another collagen scaffold with controllable removal of telopeptides and a preparation method thereof.

[0066] The preparation method steps of this example are the same as those of Example 1, but the specific conditions are different, as follows:

[0067] In step S1-2, the Achilles tendon is cut into particles with a size of about 8 mm;

[0068] In step S3-1, the mass ratio of the dispersed collagen material : water : ice acetic acid : pepsin is 1 : 20 : 0.7 : 0.22;

[0069] In step S3-2, the holding time at a temperature of 4-8℃ is 96 h.

[0070] Example 5

[0071] This example provides yet another collagen scaffold with controllable removal of telopeptides and a preparation method thereof.

[0072] The preparation method steps of this example are the same as those of Example 1, but the specific conditions are different, as follows:

[0073] In step S1-2, the Achilles tendon is cut into particles with a size of about 10 mm;

[0074] In step S3-1, the mass ratio of the dispersed collagen material : water : ice acetic acid : pepsin is 1 : 25 : 1.0 : 0.5;

[0075] In step S3-2, the holding time at a temperature of 4-8℃ is 120 h.

[0076] Comparative Example 1

[0077] This comparative example provides a collagen scaffold for comparison and a preparation method thereof.

[0078] The preparation method steps of this comparative example are the same as those of Example 1, but the specific conditions are different, as follows:

[0079] In step S1-2, the Achilles tendon is cut into particles with a size of about 7 mm;

[0080] In step S3-1, the mass ratio of the dispersed collagen material : water : ice acetic acid : pepsin was 1 : 16 : 2.0 : 0.0, i.e. no pepsin was added;

[0081] In step S3-2, the holding time at a temperature of 4-8°C was 100 h.

[0082] Comparative Example 2

[0083] This comparative example provides another comparative collagen scaffold and its preparation method.

[0084] The preparation method steps of this comparative example are the same as Example 1, but the specific conditions are different, as follows:

[0085] In step S1-2, the Achilles tendon was cut into particles of about 1 mm in size;

[0086] In step S3-1, the mass ratio of the dispersed collagen material : water : ice acetic acid : pepsin was 1 : 16 : 2.0 : 0.03;

[0087] In step S3-2, the holding time at a temperature of 4-8°C was 24 h.

[0088] Comparative Example 3

[0089] This comparative example provides another comparative collagen scaffold and its preparation method.

[0090] The preparation method steps of this comparative example are the same as Example 1, but the specific conditions are different, as follows:

[0091] In step S1-2, the Achilles tendon was cut into particles of about 15 mm in size;

[0092] In step S3-1, the mass ratio of the dispersed collagen material : water : ice acetic acid : pepsin was 1 : 20 : 1.2 : 1.2;

[0093] In step S3-2, the holding time at a temperature of 4-8°C was 150 h.

[0094] The Achilles tendon cutting size in step S1-2, the mass ratio of the dispersed collagen material : water : ice acetic acid : pepsin in step S3-1, and the holding time in step S3-2 in each of the examples and comparative examples are summarized as shown in Table 1 below:

[0095]

[0096] Test Example 1: End Peptide Removal Rate Test

[0097] The test example is the end peptide removal rate test of the collagen solution in each embodiment and the comparative example. Specifically, a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) is used to quantitatively detect the end peptide contained in each collagen solution.

[0098] The specific operation is as follows:

[0099] The sample, standard, and HRP-labeled detection antibody are sequentially added to the coated microwell coated with pig type I procollagen C-terminal propeptide (PICP) / pig type I procollagen N-terminal propeptide (PINP) capture antibody, and then incubated and thoroughly washed. Color development is performed using the substrate TMB, which is converted into blue under the catalysis of hydrogen peroxidase and into final yellow under the action of acid. The color depth is positively correlated with the content of pig type I collagen C-terminal propeptide / pig type I collagen N-terminal propeptide in the sample. The absorbance value (OD value) is measured at a wavelength of 450 nm using an enzyme-labeled instrument, and the sample concentration is calculated.

[0100] The standard curve graph is drawn according to the experimental results, and the content of pig type I collagen N-terminal propeptide / pig type I collagen C-terminal propeptide in the control is calculated according to the linear relationship.

[0101] The end peptide removal rate is calculated according to the following formula:

[0102] The end peptide removal rate of the experimental sample is calculated according to the following formula:

[0103] In the above process, the collagen solution of each embodiment and comparative examples 2-3 is used as the experimental sample; the collagen of comparative example 1 is only subjected to acid hydrolysis treatment without enzyme hydrolysis treatment, and the end peptide of the collagen molecules in the obtained collagen solution is not removed, so the collagen solution of comparative example 1 is used as the control sample, and the end peptide removal rate is 0.

[0104] Test Example 2: Adhesion test of collagen scaffold in cartilage repair

[0105] The test example is an adhesion test of different collagen scaffolds prepared in each embodiment and the comparative example in the application of cartilage repair. A pig is used as an animal model of cartilage defect for testing.

[0106] The specific operation is as follows:

[0107] After the pig is anesthetized, a circular cartilage defect (diameter 8.0 mm, depth 2.5 mm) is made at the femoral condyle and trochlear groove, and then a collagen scaffold with the same size as the defect is implanted. The sample is taken within 7 days after the operation (the implanted collagen scaffold and surrounding cartilage tissue are taken together), and the adhesion of the scaffold material in the cartilage tissue is tested using a universal material testing machine.

[0108] The test process is as follows: the obtained cartilage tissue is fixed at the lower clamp of the testing machine, so that the scaffold implantation site is in the middle; the upper clamp is a cylindrical indenter with a diameter of 7.8 mm (the top of the indenter is glued), the indenter is slowly lowered and contacted with the implanted scaffold, and then pressed at a loading rate of 5 mm / min to test the adhesive strength, and the force strength data during the whole process of pressing the scaffold to fall off are recorded, and the maximum force value in the process is recorded as the adhesive strength.

[0109] The end peptide removal rate and adhesive strength of each example and comparative example measured in the above Test Example 1 and Test Example 2 are shown in Table 2 below:

[0110] Table 2 End peptide removal rate and adhesive strength of examples and comparative examples

[0111]

[0112] As can be seen from Table 2 above, the C-terminal end peptide removal rate of Examples 1-5 is between 62% and 81%, and the N-terminal end peptide removal rate is between 60% and 80%. Compared with these examples, the end peptide of Comparative Example 1 is not removed, and the C-terminal and N-terminal end peptide removal rates of Comparative Example 2 are both about 30%, which is lower than Examples 1-5; the C-terminal and N-terminal end peptide removal rates of Comparative Example 3 are both about 90%, which is higher than Examples 1-5.

[0113] In addition, in Test Example 2, the collagen scaffolds of Examples 1-5 did not fall off during implantation; tests show that the adhesive strength of Examples 1-5 is relatively high, significantly higher than that of Comparative Examples 1-3, indicating that the adhesive strength of the scaffold has a strong correlation with the end peptide removal rate, and low end peptide removal rate (Comparative Example 1, Comparative Example 2) or high end peptide removal rate (Comparative Example 3) will result in a decrease in adhesive strength.

[0114] The above results show that by controlled removal of end peptides, the adhesive strength of collagen scaffold materials during cartilage repair can be controlled, and the reason may be that after partial removal of end peptides, the collagen molecules retaining end peptides can crosslink through end peptides, while the collagen molecules removing end peptides cannot form crosslinking through end peptides, but can form physical entanglement with the surrounding cartilage and subchondral bone tissue through its triple helix structure, which greatly improves the adhesion performance between the collagen scaffold and the bone defect site.

[0115] Especially, when the removal rate of C-terminal end peptide and N-terminal end peptide is controlled between about 60% and 80%, the adhesive strength of the collagen scaffold can reach more than 1N, which is 3-5 times higher than that of the collagen scaffold outside the removal rate range, and can greatly improve the self-adhesion performance of the collagen scaffold, repair the bone defect site, especially the large size bone defect site without additional support means. Therefore, only the size needs to be trimmed appropriately without the need to prepare additional support materials, the collagen scaffold with controllable removal of end peptides can be used as a repair material for cartilage defects, especially for large size cartilage defects.

Claims

1. A method for preparing a controllably end-peptide-removed collagen scaffold, characterized by, The collagen scaffold is a cartilage repair scaffold for self-fixing, and the preparation method comprises the following steps: Step S1: cleaning and shearing treatment is performed on collagen raw materials to obtain granular collagen materials, and the size of the granular collagen materials is 3-10 mm; Step S2: a dehydrating agent is used to perform dehydration treatment on the granular collagen materials, the dehydrating agent is removed by centrifugation, and dispersion treatment is performed to obtain dispersed collagen materials; Step S3: the dispersed collagen materials are subjected to enzymatic hydrolysis treatment to obtain a collagen solution; Step S4: the collagen solution is used to prepare a collagen scaffold by cross-linking, In the step S3, a controllable end peptide removal enzymatic hydrolysis method is used to control the end peptide removal rate of the collagen solution to be 60%-80%, and the step S3 comprises the following steps: Step S3-1: water, glacial acetic acid and pepsin are added to the dispersed collagen materials according to the mass ratio of dispersed collagen materials: water: glacial acetic acid: pepsin = 1: (5-25): (0.3-1.0): (0.05-1.5); Step S3-2: the temperature is kept at 4-8 ℃ for 48-120 h, and the dispersed collagen materials are completely dissolved and fully reacted by stirring several times during the keeping period; Step S3-3: low-temperature gradient dialysis is performed under the condition of 4-8 ℃ to remove the residual enzyme solution to obtain the collagen solution.

2. The collagen scaffold preparation method according to claim 1, wherein: wherein, The collagen raw materials in the step S1 are Achilles tendons.

3. The collagen scaffold preparation method according to claim 1, wherein: wherein The end peptide removal rate comprises a C-terminal end peptide removal rate and an N-terminal end peptide removal rate, The C-terminal end peptide removal rate and the N-terminal end peptide removal rate are both controlled to be 60%-80%.

4. The method of claim 1, wherein the collagen scaffold is prepared by a controllable removal of telopeptides. In the step S4, the step S4 comprises the following steps: Step S4-1: the collagen solution is laid flat in a mold to be low-temperature frozen to form a frozen collagen; Step S4-2: the frozen collagen is soaked in a prepared salt solution for a predetermined time to form a first layer of collagen; Step S4-3: the collagen solution is laid again on the first layer of collagen, and freeze-drying is performed after the surface is flat to form a double-layer collagen; Step S4-4: the double-layer collagen is immersed in an EDC solution for overall cross-linking to form a double-layer scaffold; Step S4-5: the double-layer scaffold is washed with water, and secondary freeze-drying is performed to obtain the collagen scaffold.

5. The collagen scaffold preparation method according to claim 4, wherein: The salt solution in the step S4-2 is a phosphate buffer solution containing 5%-40% NaCl (w / v). wherein 6. The collagen scaffold preparation method according to claim 4, wherein: The concentration of the EDC solution in the step S4-4 is 5-30 mg / ml, and the cross-linking time is 0.5-6 hours. wherein 7. The collagen scaffold preparation method according to claim 4, wherein: The freezing temperature in the step S4-1 is -4 ℃ to -80 ℃, and the freezing time is 8-24 hours. wherein, ​ 8. The method of claim 1, wherein the collagen scaffold is prepared by the method of claim 1. wherein The dehydrating agent of step S2 is ethanol.

9. A cartilage repair scaffold for self-fixation, comprising: The collagen scaffold is prepared by the method of any one of claims 1-8.

10. Use of the collagen scaffold of claim 9 in the preparation of a cartilage defect repair material.

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