Collagen scaffold capable of controllably removing terminal peptide as well as preparation method and application of collagen scaffold

Abstract

The invention aims to realize self-adhesion fixation of a cartilage repair scaffold, and provides a collagen scaffold preparation method capable of controllably removing terminal peptide, which comprises the following steps: S1, cleaning and shearing a collagen raw material to obtain a granular collagen raw material; s2, dehydrating, centrifuging and dispersing the granular collagen material to obtain a dispersed collagen material; s3, performing enzymolysis treatment on the dispersed collagen material to obtain a collagen solution; step S4, preparing a collagen scaffold by adopting a collagen solution, in the step S3, the removal rate of terminal peptides in the collagen solution is controlled to be 60%-80%, and the step S3 comprises the following steps: adding water, glacial acetic acid and pepsase into the dispersed collagen material according to the mass ratio of the dispersed collagen material to the water to the glacial acetic acid to the pepsase being 1: (5-25): (0.3-1.0): (0.05-1.5); keeping the temperature at 4-8 DEG C for 48-120 hours, and stirring for multiple times during the keeping period; and performing low-temperature gradient dialysis at the temperature of 4-8 DEG C to remove enzyme solution residues, thereby obtaining the collagen solution. The invention also provides the collagen scaffold prepared by the method and application of the collagen scaffold.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a collagen scaffold with controllable removal of terminal peptides, and a preparation method and application thereof. Background Art

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

[0003] Currently, microfracture is one of the most commonly used cartilage repair treatment techniques in clinical practice. It creates microfractures in the subchondral bone to encourage bone marrow stem cells to fill the defect site, thereby promoting the self-repair of articular cartilage and subchondral bone. However, the cartilage repaired by this method forms fibrocartilage rather than hyaline cartilage, which is quite different from normal cartilage in structure and biomechanical properties. Matrix-induced autologous chondrocyte technology is a new cartilage repair technology based on microfracture. It mainly uses absorbable biomaterials superimposed on microfracture. This material can temporarily replace damaged cartilage tissue and provide a good environment for cell growth.

[0004] The matrix-induced autologous chondrocyte technology has little damage and good repair effect, and has broad application prospects. The emergence of scaffold materials with suitable performance and easy operation is of milestone significance in the development of this technology. For example, the Chondro-Gide double-layer scaffold for cartilage repair produced by Geistlich Pharma AG in Switzerland is a collagen scaffold product that can effectively repair cartilage damage, but this product is implanted under arthroscopy and requires suture fixation. The surgical process is cumbersome, will cause damage to the surrounding cartilage, and the operation time is long. For another example, CN101066475A and CN105381504A both disclose multi-layer collagen scaffolds with good cartilage repair capabilities, but these scaffolds cannot achieve self-adhesive fixation, and often require additional fixing and support means when repairing larger cartilage defects, which limits their application.

[0005] There are some biomaterials that can be realized in the prior art. For example, CN211327153U discloses a biofilm product that can be sutured or attached. It uses the advantage of the porous collagen sponge layer to absorb and coagulate blood, solving the problem that dense connective tissue needs to be sutured and fixed during use. However, the strength and adhesion of this sponge material are limited, and it cannot meet the requirements of stent fixation in cartilage, which has high mechanical strength and complex mechanical effects. Therefore, there is an urgent need for a cartilage repair stent with high strength and high adhesion in arthroscopic minimally invasive cartilage repair. Summary of the invention

[0006] In view of the above problems in the prior art, the inventors of the present invention have conducted in-depth and extensive research on collagen materials from different sources and their characteristics in the scaffold preparation process, and found that: When used as a scaffold material for cell carriers, it is critical to control the structure of collagen molecules in three dimensions. Among the collagen materials from various sources, animal Achilles tendons are mainly type I collagen, which has a triple helix structure formed by three α chains entangled with each other. The N-terminus and C-terminus of the collagen molecule each have a non-triple helix telopeptide region, and the telopeptides in this region can be removed during the collagenase hydrolysis process. Since the telopeptide is one of the important factors causing immunogenicity, it is traditionally believed that the telopeptide should be removed as much as possible, that is, the higher the removal rate, the better. However, the inventors found that when the telopeptide removal rate of the telopeptide region is too high, the interaction and cross-linking ability between the collagen molecules will be significantly reduced, and the fibrogenic ability of the collagen will be significantly weakened, especially the adhesion between the collagen scaffold and the cartilage tissue will be significantly reduced. Therefore, the removal rate of the telopeptide can be controlled by controlling the enzymatic hydrolysis process. On this basis, the controllable cross-linking of the telopeptide that has not been removed can be achieved through the cross-linking process, so that more free triple-helical molecules can be retained in the final collagen scaffold material, which can achieve molecular entanglement with the collagen in the cartilage or bone damage site, thereby improving the bonding strength between the scaffold and the tissue and achieving self-adhesive fixation of the cartilage repair scaffold.

[0007] Based on the above findings, the present invention provides a method for preparing a collagen scaffold with controllable removal of telopeptides, comprising the following steps: Step S1, cleaning and shearing the collagen raw material to obtain a granular collagen material; Step S2, dehydrating the granular collagen material with a dehydrating agent, removing the dehydrating agent by centrifugation and dispersing the material to obtain a dispersed collagen material; Step S3, enzymatically hydrolyzing the dispersed collagen material to obtain a collagen solution; Step S4, preparing a collagen scaffold by cross-linking the collagen solution, Wherein, step S3 adopts a controllable enzymatic hydrolysis method for removing telopeptides to control the telopeptide removal rate in the collagen solution to 60%-80%, including: 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: (5-25): (0.3-1.0): (0.05-1.5); Step S3-2, maintaining the temperature at 4-8°C for 48 h to 120 h, stirring several times during the maintaining period to allow the dispersed collagen material to be completely dissolved and fully reacted; Step S3-3, performing low-temperature gradient dialysis at a temperature of 4-8°C to remove the residual enzyme solution to obtain a collagen solution.

[0008] In the method for preparing the collagen scaffold with controllable removal of telopeptides provided by the present invention, the collagen raw material in step S1 can be Achilles tendon, and the size of the granular collagen material can be 3mm-10mm.

[0009] In addition, in the method for preparing the collagen scaffold with controllable removal of telopeptides provided by the present invention, step S4 may further include: Step S4-1, spreading the collagen solution in a mold and freezing it at low temperature to form frozen collagen; Step S4-2, soaking the frozen collagen in a prepared saline solution for a predetermined time to form a first layer of collagen; Step S4-3, spreading collagen solution on the first collagen layer again, letting it stand until the surface is flat, and then freeze-drying to form a double collagen layer; Step S4-4, immersing the double-layer collagen in a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution for overall cross-linking to form a double-layer scaffold; Step S4-5, washing the double-layer scaffold with water, and then freeze-drying it for a second time to obtain a collagen scaffold.

[0010] In the method for preparing the collagen scaffold with controllable removal of telopeptides provided by the present invention, the saline solution in step S4-2 can be a phosphate buffered saline solution (PBS solution) containing 5%-40% NaCl (w / v).

[0011] In addition, in the method for preparing the above-mentioned collagen scaffold with controllable removal of telopeptides provided by the present invention, the concentration of the EDC solution in step S4-4 can be 5 mg / ml-30 mg / ml, and the cross-linking time can be 0.5-6 hours.

[0012] In the method for preparing the collagen scaffold with controllable removal of telopeptides provided by the present invention, the freezing temperature of step S4-1 can be -4°C to -80°C, and the freezing time can be 8-24 hours.

[0013] In addition, the dehydrating agent in step S2 may be ethanol.

[0014] The present invention also provides a collagen scaffold, characterized in that it is prepared by using any of the above methods for preparing a collagen scaffold with controllable removal of telopeptides.

[0015] The present invention also provides the use of the above collagen scaffold in preparing cartilage defect repairing materials.

[0016] Function and Effect of the Invention

[0017] According to the collagen scaffold with controllable removal of telopeptides and the preparation method thereof provided by the present invention, since the removal rate of telopeptides is controlled between 60% and 80% in the enzymatic hydrolysis of collagen, after the collagen solution obtained by such enzymatic hydrolysis is cross-linked to prepare the collagen scaffold, the collagen molecules with the telopeptides removed can be physically entangled with the surrounding cartilage and subchondral bone tissue through their triple helix structure, greatly improving the adhesion performance between the collagen scaffold and the bone defect site, and the bonding strength can reach more than 1N, achieving the beneficial effect of repairing the bone defect site, especially repairing the larger bone defect site without the need for additional support means.

[0018] In addition, the collagen raw material of the present invention uses Achilles tendon, which is mainly type I collagen, mainly a triple helix molecular structure; the Achilles tendon is sheared into 3mm-10mm particles after cleaning, and the size is suitable for retaining the triple helix structure; the Achilles tendon is also dehydrated and dispersed after shearing, and then enzymatically hydrolyzed, and such treatment can also effectively retain the triple helix structure in the collagen molecule. Therefore, the proportion of the triple helix structure in the collagen scaffold can be further increased, and the above-mentioned adhesion performance can be further guaranteed. DETAILED DESCRIPTION

[0019] The collagen scaffold with controllable removal of telopeptides and the preparation method thereof of the present invention are described below with reference to the examples.

[0020] The collagen raw materials used in the following examples and comparative examples are all Achilles tendons, which are obtained by peeling from commercially available quarantined pig trotters. In addition, the reagent materials whose sources are not specified in the examples and comparative examples are all conventionally available on the market, and the operating methods without specific processes are referred to the conventional operating methods of the prior art.

[0021] Example 1

[0022] This embodiment provides a collagen scaffold with controllable removal of telopeptides and a preparation method thereof.

[0023] The preparation method of this embodiment comprises the following steps: Step S1, cleaning and shearing the collagen raw material to obtain a granular collagen material, specifically comprising: Step S1-1, removing the fat and fascia on the surface of the Achilles tendon obtained by peeling; Step S1-2, cutting the Achilles tendon obtained after peeling in step S1-1 into pieces to form particles of about 3 mm in size, namely, the granular collagen material.

[0024] Step S2, dehydrating the granular collagen material with a dehydrating agent, removing the dehydrating agent by centrifugation and performing a dispersion treatment to obtain a dispersed collagen material. The dehydrating agent is ethanol, and the volume of ethanol used for the dehydration treatment is about 3 times or more of the volume of the granular collagen material; the dispersion treatment is to separate and loosen the solid matter after centrifugation to form loose granular or fibrous solid matter.

[0025] Step S3, enzymatically hydrolyzing the dispersed collagen material obtained in step S2 to obtain a collagen solution, specifically comprising: 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; Step S3-2, maintaining the mixture at 4-8°C for 72 hours, stirring the mixture several times during the maintaining period to allow the dispersed collagen material to be completely dissolved and reacted sufficiently; Step S3-3, after the reaction is completed, low-temperature gradient dialysis is performed at a temperature of 4-8°C to remove the residual enzyme solution, and the obtained solution is the collagen solution.

[0026] Step S4, using the collagen solution to prepare a collagen scaffold, specifically comprising: Step S4-1, spreading the collagen solution in a mold, freezing it at low temperature to form frozen collagen, wherein the freezing temperature is -4°C to -80°C, and the freezing time is 8-24 hours; Step S4-2, soaking the frozen collagen in a prepared saline solution for a predetermined time to form a first layer of collagen, wherein the saline solution is a PBS solution containing 5%-40% NaCl (w / v), and in this embodiment, the saline solution specifically contains 32 g / L NaCl, 2.436 g / L Na2HPO4, and 0.8 g / L KH2PO4, and the soaking time is 24 hours; Step S4-3, spreading collagen solution on the first collagen layer again, letting it stand until the surface is flat, and then freeze-drying to form a double collagen layer; Step S4-4, immersing the double-layer collagen in an EDC solution for overall cross-linking to form a double-layer scaffold, wherein the concentration of the EDC solution is 20 mg / ml and the cross-linking time is 3 hours; Step S4-5, washing the double-layer scaffold with water, and then freeze-drying it twice to obtain a collagen scaffold. The collagen scaffold can be cut into a desired size, sterilized by irradiation, and stored at room temperature.

[0027] Example 2

[0028] This embodiment provides another collagen scaffold with controllable removal of telopeptides and a preparation method thereof.

[0029] The preparation method steps of this embodiment are the same as those of embodiment 1, but the specific conditions are different, as follows: In step S1-2, the Achilles tendon is cut into pieces to form particles of about 4 mm in size; In step S3-1, the mass ratio of dispersed collagen material: water: glacial acetic acid: pepsin is 1: 12.5: 0.4: 0.15; In step S3-2, the holding time at 4-8°C is 80 h.

[0030] Example 3

[0031] This embodiment provides another collagen scaffold with controllable removal of telopeptides and a preparation method thereof.

[0032] The preparation method steps of this embodiment are the same as those of embodiment 1, but the specific conditions are different, as follows: In step S1-2, the Achilles tendon is cut into pieces to form particles of about 6 mm in size; In step S3-1, the mass ratio of dispersed collagen material: water: glacial acetic acid: pepsin is 1:15:0.5:0.40; In step S3-2, the holding time at 4-8°C is 48 h.

[0033] Example 4

[0034] This embodiment provides another collagen scaffold with controllable removal of telopeptides and a preparation method thereof.

[0035] The preparation method steps of this embodiment are the same as those of embodiment 1, but the specific conditions are different, as follows: In step S1-2, the Achilles tendon is cut into pieces to form particles of about 8 mm in size; In step S3-1, the mass ratio of dispersed collagen material: water: glacial acetic acid: pepsin is 1:20:0.7:0.22; In step S3-2, the holding time at 4-8°C is 96 h.

[0036] Example 5

[0037] This embodiment provides another collagen scaffold with controllable removal of telopeptides and a preparation method thereof.

[0038] The preparation method steps of this embodiment are the same as those of embodiment 1, but the specific conditions are different, as follows: In step S1-2, the Achilles tendon is cut into pieces to form particles of about 10 mm in size; In step S3-1, the mass ratio of dispersed collagen material: water: glacial acetic acid: pepsin is 1:25:1.0:0.5; In step S3-2, the holding time at 4-8°C is 120 h.

[0039] Comparative Example 1

[0040] This comparative example provides a collagen scaffold and a preparation method thereof for comparison.

[0041] The preparation method steps of this comparative example are the same as those of Example 1, but the specific conditions are different, as follows: In step S1-2, the Achilles tendon is cut into pieces to form particles of about 7 mm in size; In step S3-1, the mass ratio of dispersed collagen material: water: glacial acetic acid: pepsin is 1:16:2.0:0.0, i.e., pepsin is not added; In step S3-2, the holding time at 4-8°C is 100 h.

[0042] Comparative Example 2

[0043] This comparative example provides another collagen scaffold and its preparation method for comparison.

[0044] The preparation method steps of this comparative example are the same as those of Example 1, but the specific conditions are different, as follows: In step S1-2, the Achilles tendon is cut into pieces to form particles of about 1 mm in size; In step S3-1, the mass ratio of dispersed collagen material: water: glacial acetic acid: pepsin is 1:16:2.0:0.03; In step S3-2, the holding time at 4-8°C is 24 h.

[0045] Comparative Example 3

[0046] This comparative example provides another collagen scaffold and its preparation method for comparison.

[0047] The preparation method steps of this comparative example are the same as those of Example 1, but the specific conditions are different, as follows: In step S1-2, the Achilles tendon is cut into pieces to form particles of about 15 mm in size; In step S3-1, the mass ratio of dispersed collagen material: water: glacial acetic acid: pepsin is 1:20:1.2:1.2; In step S3-2, the holding time at 4-8°C is 150 h.

[0048] The size of the Achilles tendon cut into pieces in step S1-2 in each embodiment and comparative example, the mass ratio of the dispersed collagen material: water: glacial acetic acid: pepsin in step S3-1, and the holding time in step S3-2 are summarized in Table 1 below:

[0049] Test Example 1: Telopeptide Removal Rate Test This test example is a test of the telopeptide removal rate of the collagen solutions in the embodiments and comparative examples. Specifically, a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) is used to quantitatively detect the telopeptides contained in each collagen solution.

[0050] The specific operations are as follows: Samples, standards, and HRP-labeled detection antibodies were added to the coated microwells pre-coated with porcine type I procollagen C-terminal propeptide (PICP) / porcine type I procollagen N-terminal propeptide (PINP) capture antibodies, followed by warm bath and thorough washing. The substrate TMB was used for color development. TMB was converted into blue under the catalysis of catalase and into the final yellow under the action of acid. The depth of the color was positively correlated with the content of porcine type I collagen C-terminal propeptide / porcine type I collagen N-terminal propeptide in the sample. The absorbance value (OD value) was measured at a wavelength of 450 nm using an enzyme reader to calculate the sample concentration.

[0051] A standard curve was drawn according to the experimental results, and the content of porcine type I collagen N-terminal propeptide / porcine type I collagen C-terminal propeptide in the control was calculated according to the relevant linear relationship.

[0052] The telopeptide removal rate was calculated according to the following formula: The removal rate of the telopeptide of the experimental sample = (the telopeptide content of the control sample - the telopeptide content of the experimental sample) / the telopeptide content of the control sample × 100%.

[0053] In the above process, the collagen solutions of each embodiment and comparative examples 2-3 were used as experimental samples; the collagen of comparative example 1 was only subjected to acid hydrolysis treatment but not enzymatic hydrolysis treatment, and the telopeptides of the collagen molecules in the obtained collagen solution were not removed. Therefore, the collagen solution of comparative example 1 was used as a control sample, and its telopeptide removal rate was recorded as 0.

[0054] Test Example 2: Adhesion test of collagen scaffold in cartilage repair

[0055] This test example is a test of the adhesion of different collagen scaffolds prepared in various embodiments and comparative examples in cartilage repair applications, and pigs are used as cartilage defect animal models for testing.

[0056] The specific operations are as follows: After the pigs were anesthetized, circular (8.0 mm in diameter, 2.5 mm in depth) cartilage defects were made in the femoral condyle and trochlear groove, respectively. Collagen scaffolds of the same size as the defects were implanted, and the samples were removed within 7 days after the operation (the implanted collagen scaffolds and the surrounding cartilage tissues were removed together). The adhesion of the scaffold material in the cartilage tissue was tested using a universal material testing machine.

[0057] The test process is as follows: fix the obtained cartilage tissue on the lower fixture of the testing machine so that the stent implantation site is in the middle; the upper fixture is a cylindrical indenter with a diameter of 7.8 mm (with glue on the top of the indenter), slowly lower the indenter and place it on the implanted stent, making complete contact and close contact, then press down at a loading rate of 5 mm / min to test the bonding strength, record the force strength data of the stent from pressing down to stent falling off, and record the maximum force in the process as the bonding strength.

[0058] The telopeptide removal rates and bonding strengths of the various embodiments and comparative examples measured in the above-mentioned Test Example 1 and Test Example 2 are shown in Table 2 below: Table 2 The removal rate of terminal peptides and the bonding strength of the examples and comparative examples

[0059] As can be seen from Table 2 above, the C-terminal telopeptide removal rates of Examples 1-5 are between 62% and 81%, and the N-terminal telopeptide removal rates are between 60% and 80%. Compared with these examples, the telopeptide of Comparative Example 1 was not removed, and the C-terminal and N-terminal telopeptide removal rates of Comparative Example 2 were both about 30%, which was lower than that of Examples 1-5; the C-terminal and N-terminal telopeptide removal rates of Comparative Example 3 were both about 90%, which was higher than that of Examples 1-5.

[0060] In addition, in Test Example 2, the collagen scaffolds of Examples 1-5 did not fall off during implantation; the test showed that the bonding strengths of Examples 1-5 were relatively high, significantly higher than those of Comparative Examples 1-3, indicating that the bonding strength of the scaffolds was strongly correlated with the telopeptide removal rate, and that a low telopeptide removal rate (Comparative Example 1, Comparative Example 2) or a high telopeptide removal rate (Comparative Example 3) would result in reduced bonding strength.

[0061] The above results indicate that the controllable removal of telopeptides can achieve control over the adhesion strength of collagen scaffold materials during cartilage repair. The reason may be that after the telopeptides are partially removed, the collagen molecules that retain the telopeptides can be cross-linked through the telopeptides. Although the collagen molecules from which the telopeptides have been removed cannot form cross-links through the telopeptides, they can be physically entangled with the surrounding cartilage and subchondral bone tissue through their triple helix structures. This physical entanglement greatly enhances the adhesion performance between the collagen scaffold and the bone defect site.

[0062] In particular, when the removal rate of the C-terminal telopeptide and the N-terminal telopeptide 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 the adhesive strength of the collagen scaffold outside this removal rate range, and can greatly improve the self-adhesion performance of the collagen scaffold, so as to achieve the beneficial effect of repairing bone defects, especially repairing large bone defects without additional support means. Therefore, this collagen scaffold with controllable removal of telopeptides can be used as a repair material for cartilage defects, and is particularly suitable for use as a repair material for large cartilage defects, only by trimming the size appropriately without preparing additional support materials.

Claims

1. A method for preparing a collagen scaffold with controllable removal of telopeptides, characterized in that: The steps include: Step S1, cleaning and shearing the collagen raw material to obtain a granular collagen material; Step S2, dehydrating the granular collagen material with a dehydrating agent, removing the dehydrating agent by centrifugation and dispersing the granular collagen material to obtain a dispersed collagen material; Step S3, enzymatically hydrolyzing the dispersed collagen material to obtain a collagen solution; Step S4, preparing a collagen scaffold by cross-linking the collagen solution, Wherein, the step S3 adopts an enzymatic hydrolysis method for controllable removal of telopeptides to control the telopeptide removal rate in the collagen solution to 60%-80%, comprising: 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: (5-25): (0.3-1.0): (0.05-1.5); Step S3-2, maintaining the mixture at a temperature of 4-8° C. for 48 h to 120 h, stirring the mixture several times during the maintaining period to allow the dispersed collagen material to be completely dissolved and reacted sufficiently; Step S3-3, performing low-temperature gradient dialysis at a temperature of 4-8°C to remove the residual enzyme solution to obtain the collagen solution.

2. The method for preparing a collagen scaffold with controllable removal of telopeptides according to claim 1, characterized in that: in, The collagen raw material in step S1 is Achilles tendon, and the size of the granular collagen material is 3 mm-10 mm.

3. The method for preparing a collagen scaffold with controllable removal of telopeptides according to claim 1, characterized in that: in, The telopeptide removal rate includes the C-terminal telopeptide removal rate and the N-terminal telopeptide removal rate. The C-terminal telopeptide removal rate and the N-terminal telopeptide removal rate are both controlled at 60%-80%.

4. The method for preparing a collagen scaffold with controllable removal of telopeptides according to claim 1, Features: Wherein, the step S4 comprises: Step S4-1, spreading the collagen solution in a mold and freezing it at low temperature to form frozen collagen; Step S4-2, soaking the frozen collagen in a prepared saline solution for a predetermined time to form a first layer of collagen; Step S4-3, spreading collagen solution on the first collagen layer again, letting it stand until the surface is flat, and then freeze-drying to form a double-layer collagen; Step S4-4, immersing the double-layer collagen in an EDC solution for overall cross-linking to form a double-layer scaffold; Step S4-5, washing the double-layer scaffold with water, and then freeze-drying it for a second time to obtain the collagen scaffold.

5. The method for preparing a collagen scaffold with controllable removal of telopeptides according to claim 4, characterized in that: in, The saline solution in step S4-2 is a phosphate buffer solution containing 5%-40% NaCl (w / v).

6. The method for preparing a collagen scaffold with controllable removal of telopeptides according to claim 4, characterized in that: in, The concentration of the EDC solution in step S4-4 is 5 mg / ml-30 mg / ml, and the cross-linking time is 0.5-6 hours.

7. The method for preparing a collagen scaffold with controllable removal of telopeptides according to claim 4, characterized in that: in, The freezing temperature of step S4-1 is -4°C to -80°C, and the freezing time is 8 to 24 hours.

8. The method for preparing a collagen scaffold with controllable removal of telopeptides according to claim 1, characterized in that: in, The dehydrating agent in step S2 is ethanol.

9. A collagen scaffold, characterized in that: The collagen scaffold is prepared by the method for preparing a collagen scaffold with controllable removal of telopeptides as described in any one of claims 1 to 8.

10. Use of the collagen scaffold according to claim 9 in preparing cartilage defect repair materials.

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