CN107007883B

CN107007883B - Cartilage repair support and preparation method thereof

Info

Publication number: CN107007883B
Application number: CN201710084882.8A
Authority: CN
Inventors: 敖英芳; 史尉利; 孙牧旸; 陈海峰; 胡晓青
Original assignee: Peking University Third Hospital
Current assignee: Peking University Third Hospital
Priority date: 2017-02-16
Filing date: 2017-02-16
Publication date: 2020-02-07
Anticipated expiration: 2037-02-16
Also published as: CN107007883A

Abstract

The invention discloses a preparation method of a cartilage repair scaffold. The method comprises the following steps: printing a mold of the support by using a 3D printing technology; the bottom surface of the mould is provided with a plurality of first convex ridges which are parallel to each other; a plurality of second ribs parallel to each other and connected to the upper surface of the first rib; and a plurality of third ribs parallel to each other connected to the upper surface of the second rib; the width of the three is 100-500 μm; the first rib and the second rib are crossed to form a preset angle; the second rib and the third rib are crossed to form a preset angle; preparing a mixed solution of fibroin and gelatin; pouring the mixed solution into a mould, so that the liquid level of the mixed solution is not higher than that of the third rib; after the mixed solution is solidified into gel, removing the mould to obtain a material main body of the cartilage repair scaffold; subjecting to protein denaturation and chemical crosslinking, and freeze drying. The scaffold prepared by the method provides more attachment sites and a suitable growth microenvironment for the enrichment of BMSC, and promotes the repair of damaged cartilage.

Description

Cartilage repair support and preparation method thereof

Technical Field

The invention relates to the technical field of cartilage repair scaffolds, in particular to a cartilage repair scaffold and a preparation method thereof.

Background

Articular cartilage damage is more common with increasing levels of exercise and participation in physical activity. Articular cartilage has poor self-repairing ability after injury due to its special anatomical and histological characteristics. Such injuries, if not effectively treated in a timely manner, can progress to osteoarthritis, placing a heavy burden on the patient and society.

Currently, the commonly used methods for repairing articular cartilage damage mainly include bone marrow stimulation techniques (drilling and microfracture), osteochondral transplantation techniques, chondrocyte transplantation techniques, and the like.

Bone marrow stimulation techniques (drilling and microfracture) utilize mesenchymal stem cells contained in a blood clot formed from released bone marrow blood to differentiate to form new cartilage and repair the defect area. The technology has a certain effect on cartilage injury, but the differentiation of bone marrow mesenchymal stem cells generates fibrocartilage, the biological performance and the mechanical performance of the fibrocartilage are lower than those of normal cartilage, and meanwhile, released blood cannot be stored locally and rapidly to influence the repair effect, so that the repair is more difficult especially for larger defects.

Osteochondral transplantation techniques include autologous cartilage transplantation and allogeneic cartilage transplantation. The technology can relieve pain of patients and improve joint function to a certain extent, however, researches show that autologous osteochondral transplantation may cause cell death of a supply area to cause corresponding symptoms of the supply area, and meanwhile, the application of the technology is limited due to the problems of poor compatibility with normal cartilage, reduced cell activity during storage, limited source of donor grafts, risk of disease transmission and the like.

The chondrocyte transplantation technology has disadvantages in that it is still inevitable to face problems of dedifferentiation during chondrocyte culture, secondary surgical trauma and high cost, and it does not have significant clinical therapeutic advantages compared to microfracture surgery.

Therefore, the research on cartilage damage repair remains a difficult point and a hot spot in clinical research.

In recent years, tissue engineering technology is rapidly developed, and a new idea is brought to repair of articular cartilage damage. The tissue engineering technology mainly generates new tissues through the interaction and regulation of a bracket, seed cells and biological factors, and achieves the purposes of repairing tissue damage caused by trauma, diseases and aging and recovering physiological functions. Tissue engineering techniques have made articular cartilage considered one of the most promising tissues and organs for successful regeneration by tissue engineering techniques.

The requirements of the cartilage repair scaffold in the tissue engineering technology on design are as follows: 1. the biodegradation does not generate toxic substances; 2. good mechanical support is provided for the new tissue; 3. the degradation speed is matched with the tissue regeneration speed; 4. can have a porous property, allowing diffusion of nutrients and metabolites; 5. matching the compression characteristics of the scaffold and normal cartilage.

The common scaffold for cartilage tissue engineering at present is divided into the following materials according to the manufacturing materials: scaffolds made of synthetic materials and scaffolds made of biological natural materials. The scaffold made of the artificial material has certain advantages in material control, but is poor in biocompatibility and degradability. The scaffold made of natural materials, especially made by biological methods, can promote the generation and remodeling of normal extracellular matrix, and the scaffold made of natural materials has incomparable advantages in biocompatibility, biodegradation and chondrogenic differentiation.

Biological natural materials are classified into proteins such as fibroin, fibrin, collagen, gelatin, and the like, and saccharides such as agarose, alginate, hyaluronic acid, chitosan, and the like. The fibroin has the advantages of rich source, good biocompatibility, no toxic or side effect of degradation products, good mechanical property, low price and the like, so that the fibroin becomes an excellent scaffold material for cartilage repair; but the degradation speed is too slow, which influences the regeneration and repair of cartilage to a certain extent. Collagen is the major component of the cartilage matrix, which adheres well and supports cell growth, but collagen of animal origin is often immunogenic. Gelatin is used as a substitute material of collagen, has the characteristic of low immunogenicity, and has good effect in the field of tissue engineering, but the gelatin has poor mechanical property and is limited to be used as a scaffold material independently due to too high degradation speed.

In the research, the spatial structure of the scaffold, particularly the three-dimensional structure and the pore size of the scaffold, have important significance for cartilage regeneration and repair besides the biological characteristics of the scaffold material. Research has shown that scaffolds with three-dimensional structures are more favorable for chondrocyte differentiation and proliferation than pure planar structures.

The microfracture technology utilizes the multidirectional differentiation potential of seed cells, combines a support made of biological natural materials, and is supplemented with cytokines to repair cartilage defects, and has wide clinical prospects due to simplicity, practicability and low cost. The microfracture technology can make bone marrow mesenchymal stem cells (a seed cell) escape, and the bone marrow mesenchymal stem cells are stimulated by mechanics and biological factors in a bracket and are differentiated towards cartilage cells to achieve the purpose of repairing cartilage defects. However, after microfracture, the effective bone marrow mesenchymal stem cells have low content, are easy to flow out of a cartilage defect area and are not easy to remain in a local area, so that the mechanics of the new cartilage tissue is weak, the new cartilage tissue cannot bear the movement of joints, and the cartilage repair effect is influenced.

In the process of implementing the invention, the inventor finds that the prior art has at least the following problems: the traditional method for directly freeze-drying the biological natural material cannot realize fine design, the prepared scaffold is of a planar structure, does not have a three-dimensional structure of fine pores, has poor mechanical strength, and is not beneficial to enriching mesenchymal stem cells on the scaffold, retaining the mesenchymal stem cells in a defect area and differentiating the mesenchymal stem cells into cartilage cells to form new cartilage.

Disclosure of Invention

In order to solve the technical problems, the invention provides a cartilage repair scaffold and a preparation method thereof. The technical scheme is as follows:

in a first aspect, the present invention provides a method for preparing a cartilage repair scaffold, comprising:

printing a mould of the cartilage repair scaffold by using a 3D printing technology; the bottom surface of the mould is provided with a plurality of first convex ridges which are parallel to each other; a plurality of second ribs parallel to each other and connected to the upper surface of the first rib; and a plurality of third ribs parallel to each other connected to the upper surface of the second rib; the width of the first rib is 100-500 μm; the width of the second protruding edge is 100-500 μm; the width of the third rib is 100-500 mu m; the first rib and the second rib are crossed to form a preset angle; the second rib and the third rib are crossed to form a preset angle;

preparing a mixed solution of fibroin and gelatin;

pouring the mixed solution into a mold, so that the liquid level of the mixed solution does not exceed the third rib;

after the mixed solution is solidified into gel, removing the mould to obtain a material main body of the cartilage repair scaffold;

performing protein denaturation treatment on the material main body of the cartilage repair scaffold;

and carrying out chemical crosslinking treatment on the material main body of the cartilage repair scaffold subjected to protein denaturation treatment, and then carrying out freeze drying to obtain the cartilage repair scaffold.

Preferably, the first rib is perpendicular to the second rib; the second rib is perpendicular to the third rib.

Preferably, the width of the first rib is 300-500 μm; the width of the second protruding edge is 300-500 μm; the width of the third rib is 300-.

More preferably, the first rib, the second rib and the third rib each have a width of 400 μm.

Specifically, the gap between two adjacent first ribs is 0.4-0.7 mm; the gap between every two adjacent second convex edges is 0.4-0.7 mm; the gap between every two adjacent third convex edges is 0.4-0.7 mm.

Specifically, the height of the liquid surface of the mixed solution is 0.1-0.3mm higher than the sum of the heights of the first rib, the second rib and the third rib.

Preferably, the mass fraction of the fibroin in the mixed solution is 2.3% -4.6%; the mass fraction of the gelatin in the mixed solution is 2.3% -4.6%.

More preferably, the sum of the mass fractions of the fibroin and the gelatin in the mixed solution is 6.9%.

Specifically, the protein denaturation treatment is to place the repair scaffold precursor in an ethanol solution with a volume fraction of 95% overnight.

Specifically, the chemical crosslinking treatment is carried out on the material main body of the cartilage repair scaffold subjected to the protein denaturation treatment, namely the material main body of the cartilage repair scaffold subjected to the protein denaturation treatment is placed in 0.3-1% genipin solution by mass percentage for overnight at room temperature.

Specifically, the preparation method further comprises the following steps: and (4) sterilizing the cartilage repair support.

More preferably, the preparing further comprises: coupling a mesenchymal stem cell affinity polypeptide to the surface of the cartilage repair scaffold; the amino acid sequence of the bone marrow mesenchymal stem cell affinity polypeptide is shown as SEQ ID NO: 1 is shown.

Specifically, the operation steps of coupling the mesenchymal stem cell affinity polypeptide to the surface of the cartilage repair scaffold are as follows:

firstly, carrying out amination treatment on the cartilage repair scaffold;

soaking the aminated cartilage repairing scaffold in a solution containing a bifunctional cross-linking agent under the condition of keeping out of the sun;

then placing the cartilage repair scaffold containing the double-heterofunctional-group cross-linking agent in a solution containing the bone marrow mesenchymal stem cell affinity polypeptide for incubation;

and finally, carrying out freeze drying on the cartilage repair scaffold modified with the bone marrow mesenchymal stem cell affinity polypeptide on the surface to prepare the cartilage repair scaffold coupled with the bone marrow mesenchymal stem cell affinity polypeptide.

More specifically, the amination treatment of the cartilage repair scaffold is to soak the cartilage repair scaffold in an ethylenediamine isopropanol solution with the volume fraction of 5% -15%, and keep the cartilage repair scaffold for more than 1 hour at the temperature of 37 ℃.

More specifically, the solution containing the heterobifunctional crosslinking agent is a dimethyl sulfoxide solution of sodium 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimide ester with the mass volume fraction of 5-15%; the immersion time of the aminated cartilage repair scaffold in the solution of the heterobifunctional cross-linking agent is more than 1 hour.

More specifically, the solution containing the mesenchymal stem cell affinity polypeptide is a phosphate buffer solution with the concentration of the mesenchymal stem cell affinity polypeptide of 0.05-0.15 mg/mL; the incubation time is 1 hour or more.

In a second aspect, the present invention provides a cartilage repair scaffold prepared by any one of the above-described preparation methods.

The technical scheme provided by the embodiment of the invention has the beneficial effects that:

1. according to the preparation method of the cartilage repair support, the mould of the cartilage repair support with the three-layer fine convex edge structure is printed by using a 3D printing technology, so that the cartilage repair support with the fine three-dimensional net structure is prepared, and the cartilage repair support has better mechanical strength.

2. Through the design to the width of the bead of mould, the size of the pore of the three-dimensional network structure of cartilage repair support is controlled, and the liquid level of mixed solution does not pass through the third bead, make cartilage repair support have a base, form compact membrane structure, prevent mesenchymal stem cells that marrow released from flowing into the joint chamber effectively, avoid the scouring of joint fluid to forming blood clot simultaneously, for mesenchymal stem cells (BMSC) enrichment provides more attachment sites and suitable growth microenvironment on the support, make more mesenchymal stem cells remain in the defective area and differentiate into chondrocyte, form new cartilage, promote the repair of damaged cartilage.

3. The cartilage repair scaffold used by the preparation method provided by the invention is prepared from a mixed solution of fibroin and gelatin, has good biocompatibility and degradability, and does not generate toxic substances after degradation.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

Fig. 1 is a perspective view of a mold for a cartilage repair scaffold according to an exemplary embodiment of the present invention;

fig. 2 is a sectional view of a mold for a cartilage repair scaffold according to an exemplary embodiment of the present invention;

fig. 3 is a top view of a mold for a cartilage repair scaffold according to an exemplary embodiment of the present invention;

FIG. 4 is a bottom view of a cartilage repair scaffold shown after cutting in accordance with an exemplary embodiment of the present invention;

FIG. 5 is an electron micrograph of FIG. 4;

FIG. 6 is a top view of a cartilage repair scaffold shown after cutting in accordance with an exemplary embodiment of the present invention;

FIG. 7 is an electron micrograph of FIG. 6;

FIG. 8 is a graph showing the results of mechanical strength measurement of the cartilage repair scaffold provided in example 3 of the present invention;

FIG. 9 is a graph showing the results of an in vitro degradation assay performed on a cartilage repair scaffold provided in example 3 of the present invention;

FIG. 10 is a graph showing the results of CCK-8 assay of cartilage repair scaffolds provided in examples 3 and 5 of the present invention;

FIG. 11 is a graph showing the results of DNA assay of cartilage repair scaffolds provided in examples 3 and 5 of the present invention;

FIG. 12 is a graph showing the results of glycosaminoglycan (GAG) assay performed on the cartilage repair scaffolds provided in examples 3 and 5 of the present invention;

FIG. 13 is a diagram showing the observation result of a confocal laser scanning microscope according to embodiment 7 of the present invention;

FIG. 14 is a graph showing the results of cartilage repair in an animal experiment provided in example 8 of the present invention.

Detailed Description

In order to make the technical solutions and advantages of the present invention clearer, the following will describe embodiments of the present invention in further detail with reference to the accompanying drawings.

printing a mould of the cartilage repair scaffold by using a 3D printing technology; as shown in fig. 1 to 3, the mold is a groove, and the bottom surface of the groove is provided with a plurality of first ribs 1 which are parallel to each other; a plurality of second ribs 2 parallel to each other connected to the upper surface of the first rib 1; and a plurality of third ribs 3 parallel to each other connected to the upper surface of the second rib 2; the width of the first rib 1 may be 100-500 μm; the width of the second rib 2 may be 100-500 μm; the width of the third rib 3 may be 100-500 μm; the first rib 1 and the second rib 2 are crossed to form a preset angle; the second rib 2 and the third rib 3 are crossed to form a preset angle;

preparing a mixed solution of fibroin and gelatin;

pouring the mixed solution into a mould, so that the liquid level of the mixed solution is not higher than that of the third rib;

carrying out protein denaturation treatment on the material main body of the cartilage repair scaffold;

The preparation method of the cartilage repair scaffold provided by the invention has the beneficial effects that:

2. Through the design of the width of the convex edge of the die, the size of the pore of the three-dimensional reticular structure of the cartilage repair support is controlled, and the liquid level of the mixed solution does not exceed the third convex edge, so that the cartilage repair support is provided with a base (compact membrane structure), mesenchymal stem cells released by bone marrow are effectively prevented from flowing into a joint cavity, meanwhile, the joint fluid is prevented from scouring blood clots formed, more attachment sites and suitable growth microenvironments are provided for the enrichment of the mesenchymal stem cells (BMSC) on the support, more mesenchymal stem cells are kept in a defect area and are differentiated into cartilage cells, new cartilage is formed, and the repair of damaged cartilage is promoted.

The skilled person can use 3D mapping technique to map the shape of the mould of the cartilage repair scaffold, and then use 3D printing technique to print out the mould of the cartilage repair scaffold. The mold may be a slot as shown in fig. 1. The bottom surface of the groove is provided with a plurality of parallel first ribs 1 with the width of 100-. Because the mould of the cartilage repair scaffold has the stereo-crossing reticular structure, the prepared cartilage repair scaffold has the stereo-crossing reticular structure. The mixed solution of fibroin and gelatin is poured into a mold, and the liquid surface of the mixed solution does not pass through the third rib, so that a three-dimensional net structure with a base (a compact membrane structure) and a plurality of pores of 100-. The cartilage repair scaffold with the three-dimensional mesh structure with the pores has high mechanical strength, can effectively prevent mesenchymal stem cells released by bone marrow from flowing into a joint cavity, avoids scouring of joint fluid on formed blood clots, provides more attachment sites and a proper growth microenvironment for the enrichment of the mesenchymal stem cells (BMSC) on the scaffold, enables more mesenchymal stem cells to be reserved in a defect area and differentiated into chondrocytes, forms new cartilage, and promotes the repair of damaged cartilage.

The first rib and the second rib are crossed to form a preset angle, 0 degrees < the preset angle is less than or equal to 90 degrees, preferably 45 degrees to 90 degrees, more preferably 60 degrees to 90 degrees, even more preferably 75 degrees to 90 degrees, and most preferably 90 degrees for convenience in processing and the like. The second rib and the third rib are crossed to form a preset angle, 0 degrees < the preset angle is less than or equal to 90 degrees, preferably 45 degrees to 90 degrees, more preferably 60 degrees to 90 degrees, even more preferably 75 degrees to 90 degrees, and most preferably 90 degrees for convenience of processing and the like.

As shown in fig. 1 to 3, the first rib 1 and the second rib 2 may be perpendicular to each other to form an included angle of 90 °; the second rib 2 and the third rib 3 can be vertical to each other in different planes to form an included angle of 90 degrees; the first rib 1 is parallel to the third rib 3; as shown in fig. 3, the third rib 3 is not located right above the first rib 1, i.e. the plane of the center lines of the two is not perpendicular to the horizontal plane. Of course, it is also conceivable for the person skilled in the art that the included angle formed by the first rib and the second rib is smaller than 90 °, the included angle formed by the second rib and the third rib is smaller than 90 °, and the two included angles formed may be the same or different; the third rib may be located directly above the first rib, i.e. the plane of the centre lines of the first and second ribs is perpendicular to the horizontal plane, although the first and third ribs may not be parallel. The position relationship of the first rib, the second rib and the third rib can form a fine three-dimensional crossing net structure required by the invention, and the cartilage repair scaffold prepared according to the mould has a base (a compact membrane structure) and a plurality of pores of 500 μm × 100-.

It should be noted that, the steps of printing the mold of the cartilage repair scaffold by using the 3D printing technology and preparing the mixed solution of fibroin and gelatin according to the present invention have no obvious logical order, and those skilled in the art may first perform any of the two steps.

The printing material used in the 3D printing technique is common knowledge in the art. In order to obtain the cartilage repair scaffold of the present invention without damage, the material of the mold is preferably high-strength polystyrene or polystyrene propylene, and the polystyrene or polystyrene propylene can be dissolved in D-limonene to remove the D-limonene, and the D-limonene does not damage the structure of the cartilage repair scaffold and does not cause environmental pollution. It will be appreciated by those skilled in the art that the removal of the mold is performed at a temperature below the melting point of gelatin to prevent the gelatin from melting into a liquid, destroying the structure of the cartilage repair scaffold, and degrading the mechanical properties of the cartilage repair scaffold.

In practical cases, after the mold is removed, the material body of the obtained cartilage repair scaffold can be repeatedly washed by using an ethanol solution with a volume fraction of 95%, and D-limonene adhered to the material body can be removed by washing for 3 to 5 times.

The pores of the cartilage repair scaffold of the present invention are very important for the enrichment and growth of bone marrow mesenchymal stem cells on the cartilage repair scaffold. In order to obtain pores which are more beneficial to enrichment and growth of bone marrow mesenchymal stem cells, the invention carries out a series of optimization on the convex edge of the mould of the cartilage repair scaffold:

in one specific embodiment of the mold for cartilage repair scaffold, as shown in fig. 1 and 2, the first rib 1 is perpendicular to the second rib 2; the second rib 2 is perpendicular to the third rib 3. The third rib 3 is positioned right above the first rib 1, that is, the center line of the first rib and the center line of the third rib are positioned on the same plane.

In a preferred embodiment of the mold for cartilage repair scaffold, the width of the first rib may be 300-500 μm; the width of the second rib can be 300-500 μm; the width of the third rib may be 300-500 μm. It will be appreciated by those skilled in the art that the widths of the first, second and third ribs may be the same or different.

The length, width and height referred to herein are those understood in the general sense of the present invention. Taking the first rib as an example, as shown in fig. 2, the length of the first rib 1 is the distance from the front end to the rear end of the first rib 1, the width of the first rib 1 is the distance from the left side to the right side of the first rib, and the height of the first rib 1 is the distance from the upper end to the lower end of the first rib 1.

The gap between two adjacent first ribs of the mould for the cartilage repair scaffold can be 0.4-0.7 mm; the gap between two adjacent second convex edges can be 0.4-0.7 mm; the gap between two adjacent third convex edges can be 0.4-0.7mm, and the three convex edges can be the same or different. The gap between two adjacent first ribs referred to herein means a distance from the rightmost edge of the first rib 1 located on the left side to the leftmost edge of the first rib 1 located on the right side of the two adjacent first ribs 1, as shown in fig. 2. The definition of the gap between two adjacent third ribs and the definition of the gap between two adjacent second ribs are the same as the definition of the gap between two adjacent first ribs, please refer to the definition of the gap between two adjacent first ribs.

In one embodiment of the mold for the cartilage repair scaffold, the mold may have a groove with a height of 0.5-1cm, and the first, second, and third ribs may each have a width of 400 μm; the gap between two adjacent first ribs can be 0.6 mm; the gap between two adjacent second convex edges can be 0.6 mm; the gap between every two adjacent third ribs can be 0.6 mm; the side walls of the mould may be 1mm thick and the base 1mm thick.

The steps involved in the method for preparing the cartilage repair scaffold disclosed in the present invention will be described in detail below.

The preparation method comprises the following steps of preparing a mixed solution of fibroin and gelatin, wherein the mixed solution is prepared by a common technical means in the field, namely dissolving the fibroin and the gelatin in water respectively to form respective aqueous solutions, and proportioning according to the required concentration.

In practical application, the fibroin has good mechanical property but slow degradation rate, while the gelatin has good immunogenicity, but poor mechanical property and fast degradation rate, so that the mass fraction of the fibroin in the mixed solution is 2.3% -4.6%, preferably 2.3% in order to ensure that the cartilage repair scaffold has good mechanical property and the degradation rate of the cartilage repair scaffold is matched with the regeneration rate of cartilage; the mass fraction of the gelatin in the mixed solution is 2.3% -4.6%, preferably 4.6%, and further, the sum of the mass fractions of the fibroin and the gelatin in the mixed solution is 6.9%.

The skilled person can determine from practical experience when the mixed solution solidifies to form a gel. The mixed solution is usually kept still in the mould for more than 5 minutes, so that the mixed solution can be solidified into gel, namely the material body of the cartilage repair scaffold is formed.

The fibroin can be dissolved in water and must be subjected to denaturation treatment, and the gelatin is melted at high temperature, so that the protein is not easy to denature at high temperature, and the toxicity problem is also considered, therefore, the material main body of the repair scaffold is placed in an ethanol solution with the volume fraction of 95% overnight, the overnight time can be usually 8-12 hours, the fibroin is denatured, after the protein denaturation treatment, the material main body of the cartilage repair scaffold has certain strength, and the fibroin cannot be dissolved in the subsequent treatment process. By fully playing respective advantages of fibroin and gelatin, the optimization of the mechanical and degradation performances of the cartilage repair scaffold is realized.

In practical application, before the chemical crosslinking treatment is carried out on the material body of the cartilage repair scaffold, ethanol adhered to the material body of the cartilage repair scaffold is washed away by water. The water used in the biological field is usually distilled water, double distilled water, deionized water, ultrapure water or the like, unless otherwise specified.

The chemical crosslinking treatment of the material body of the cartilage repair scaffold after the protein denaturation treatment can generally use 0.01 to 0.3 mass percent of carbodiimide solution, 0.3 to 1 mass percent of genipin solution or 0.1 to 2 mass percent of glutaraldehyde and the like, for example, the material body of the cartilage repair scaffold after the protein denaturation treatment can be placed in 0.5 mass percent of genipin solution at room temperature overnight. The overnight period is generally understood to be 8-12 hours. Room temperature is generally understood to be 18-25 ℃.

The material main body of the cartilage repair scaffold after the chemical crosslinking treatment is subjected to freeze drying, and specifically, a freeze dryer can be used for vacuum freeze drying treatment, which is a conventional technical means in the field and can be mastered by a person skilled in the art according to actual conditions, and the details are not repeated herein.

Before the cartilage repair scaffold is used for repairing damaged cartilage, the cartilage repair scaffold is sterilized or is sterilized when the preparation is finished, and the cartilage repair scaffold is packaged to be made into a finished product.

In a preferred embodiment of the present invention, the preparation method further comprises: coupling the bone marrow mesenchymal stem cell affinity polypeptide to the surface of the cartilage repair scaffold; the amino acid sequence of the bone marrow mesenchymal stem cell affinity polypeptide is shown as SEQ ID NO: 1, EPLQLKM, having 7 amino acids, abbreviated as E7. The affinity polypeptide of the mesenchymal stem cells can effectively recruit BMSCs and has no species specificity. The bone marrow mesenchymal stem cell affinity polypeptide has stable property, can effectively recruit bone marrow mesenchymal stem cells released by microfracture operation from the aspect of function, and enables the cells to be retained in the defect area, thereby providing more repairing cells for the cartilage defect area. The properties of the affinity polypeptide for mesenchymal stem cells have been reported in detail in patent publication No. CN102229646A, and the present invention is not repeated herein.

The bone marrow mesenchymal stem cell affinity polypeptide is an artificially synthesized sequence and is synthesized by Beijing Zhongke Sudoku Biotech Co. Synthesizing the bone marrow mesenchymal stem cell affinity polypeptide on polyethylene glycol (PEG-PS) resin by a solid phase method, wherein the synthesis comprises the following steps:

a. activating 3-maleimido propionic acid by dicyclohexyl carbodiimide under a nitrogen environment;

b. reacting the activated 3-maleimide propionic acid with an amino acid residue of a polypeptide molecule connected to a resin, so that maleimide is coupled to the tail end of the polypeptide; the polypeptide molecule activated by maleimide and trifluoroacetic acid are cut off from PEG-PS resin, freeze-dried and packaged.

The operation steps of coupling the bone marrow mesenchymal stem cell affinity polypeptide to the surface of the cartilage repair scaffold are as follows:

firstly, carrying out amination treatment on a cartilage repair scaffold;

then placing the cartilage repair scaffold containing the double-heterofunctional-group cross-linking agent in a solution containing bone marrow mesenchymal stem cell affinity polypeptide for incubation;

The following will describe in detail the procedure of coupling the mesenchymal stem cell affinity polypeptide to the surface of the cartilage repair scaffold.

The amination treatment of the cartilage repair scaffold can be carried out by soaking the cartilage repair scaffold in an ethylenediamine isopropanol solution with the volume fraction of 5% -15% and keeping the solution at the temperature of 37 ℃ for more than 1 hour.

The solution containing the heterobifunctional cross-linking agent can be dimethyl sulfoxide solution of 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium with the mass volume fraction of 5-15%; the immersion time of the aminated cartilage repair scaffold in the solution of the bifunctional cross-linking agent is more than 1 hour.

The solution containing the bone marrow mesenchymal stem cell affinity polypeptide is a phosphate buffer solution (0.01M, pH value of 7.2) with the concentration of the bone marrow mesenchymal stem cell affinity polypeptide of 0.05-0.15mg/mL, preferably the concentration of the bone marrow mesenchymal stem cell affinity polypeptide of 0.1 mg/mL; the incubation time is 1 hour or more.

In a second aspect, the present invention also provides a cartilage repair scaffold prepared using any one of the above-described preparation methods.

As can be seen from the above description of the mold for cartilage repair scaffold, the present invention determines the structure of the prepared cartilage repair scaffold by defining the structure of the mold for cartilage repair scaffold. The cartilage repair scaffold has a base (dense membrane structure) by controlling the liquid level of the mixed solution to be higher than the sum of the heights of the first rib, the second rib and the third rib over the third rib, namely the liquid level of the mixed solution is higher than the sum of the heights of the first rib, the second rib and the third rib. The structure of the mould of the cartilage repair support is opposite to that of the cartilage repair support, namely the convex edges of the mould correspond to the grooves of the cartilage repair support.

In one embodiment of the cartilage repair scaffold, the cartilage repair scaffold has a base (as shown in fig. 4) and a plurality of grooves formed on the base, the grooves are intersected to form pores (as shown in fig. 6), and the base is a dense membrane structure formed by the liquid level of the mixed solution passing through the third convex ribs; the shape of the groove corresponds to the shape of the rib of the die. As shown in fig. 6, the upper end surface of the cartilage repair scaffold was fitted to the bottom surface of the mold after the mixed solution was poured into the mold. The cartilage repair scaffold with the structure can effectively prevent bone marrow mesenchymal stem cells released by bone marrow from flowing into a joint cavity, simultaneously avoid blood clots formed by washing joint fluid, provide more attachment sites and a proper growth microenvironment for the enrichment of the bone marrow mesenchymal stem cells (BMSCs) on the scaffold, enable more bone marrow mesenchymal stem cells to be reserved in a defect area and to be differentiated into chondrocytes, form new cartilage and promote the repair of damaged cartilage.

The electron microscope images of the cartilage repair scaffold are shown in fig. 5 and 7, the cartilage repair scaffold has a three-dimensional reticular space structure of a base, the three-dimensional reticular space structure is formed by 3D printing design and consists of edges and a middle pore structure, and a more appropriate living microenvironment can be provided for bone marrow mesenchymal stem cells in articular cartilage injury repair; the base is connected with the three-dimensional reticular structure, the structure of the base is a layer of compact fibroin-gelatin, bone marrow blood released by microfracture can be effectively prevented from entering a joint cavity when articular cartilage damage is repaired, and bone marrow mesenchymal stem cells required by cartilage repair are effectively retained in the three-dimensional reticular structure and the top surface membrane structure, so that repair of damaged cartilage is more effectively promoted.

It can be seen that the thickness of the base of the cartilage repair scaffold according to the present invention is about 0.1 to 0.3mm, based on the fact that the height of the liquid surface of the mixed solution is 0.1 to 0.3mm higher than the sum of the heights of the first rib, the second rib and the third rib.

In practical applications, the height of the cartilage repair scaffold of the present invention can be designed according to the thickness of cartilage that needs to be repaired.

The cartilage repair scaffold is prepared from a mixed solution of fibroin and gelatin, and can be referred to as an SFG scaffold for short. Both fibroin and gelatin are natural biomaterials with good biocompatibility, degradability and nontoxicity. Preferably, the mass fraction of the fibroin in the mixed solution is 2.3% -4.6%; the mass fraction of the gelatin in the mixed solution is 2.3-4.6%. The degradation rate of the cartilage repair scaffold is obtained to match the rate of cartilage regeneration within this mass component range. More preferably, the sum of the mass fractions of the fibroin and the gelatin in the mixed solution is 6.9%.

In a preferred embodiment of the cartilage repair scaffold, the surface of the cartilage repair scaffold is coupled with a mesenchymal stem cell affinity polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, EPLQLKM, abbreviated as E7 polypeptide. The cartilage repair scaffold with the E7 polypeptide can specifically recruit bone marrow mesenchymal stem cells released by microfracture surgery, and retain the cells in the defect area, providing more repair cells for the cartilage defect area.

Example 1 extraction of fibroin

A beaker was charged with 2L of ultrapure water, boiled, and weighed to take 4.24g of Na₂CO₃Slowly pouring the mixture into the container; shearing 5g of silkworm cocoon (Guangdong silkworm base) with scissors, pouring into boiling water, soaking for 30min, and stirring to dissolve sericin in silkworm cocoon in water completely to remove sericin in silkworm cocoon;

boiling for 30min, taking out flocculent silk fibroin, cooling with ultrapure water, extruding out excessive water, placing into 1L beaker, adding ultrapure water, stirring for 30min, extruding out water, and repeating for 2-3 times;

taking out pure silk fibroin which is silk fibroin, spreading the silk fibroin on an aluminum foil, and airing the silk fibroin in a ventilated place overnight;

weighing LiBr with corresponding mass according to the weight ratio of fibroin to LiBr (lithium bromide) being 1:4, and preparing 9.3M LiBr aqueous solution by using water. Pouring LiBr aqueous solution onto the fibroin to completely cover the fibroin;

the mixed solution was heated to 60 ℃ and maintained for 4 hours to completely dissolve it, giving a transparent amber color. Subsequently dialyzed with ultrapure water for 3 days, and the dialyzed solution was centrifuged at a low temperature for 20min (4 ℃, 9000 rpm);

the supernatant was removed and centrifuged again, and the resulting fibroin was stored in a sealed condition at 4 ℃.

The invention is not related to the point of obtaining fibroin, and the invention provides only a method for obtaining fibroin with higher purity, which can be obtained by other conventional means or purchased from the market by those skilled in the art.

EXAMPLE 23D mold design and preparation

And drawing a mould of the cartilage repair scaffold by using AutoCAD software. Wherein the height of the mould is 1 cm; the thickness of the side wall and the bottom surface of the mold is 1 mm. The bottom surface of the mould is provided with 28 mutually parallel first convex ridges; 28 second ribs which are vertical to the different surfaces of the first ribs and are connected with the upper surfaces of the first ribs and are parallel to each other; and 28 mutually parallel third ribs which are vertical to the different surfaces of the second ribs and are connected with the upper surfaces of the second ribs; 3 layers of convex edges are formed, the width and height of the first convex edge, the second convex edge and the third convex edge are all 0.4mm, and the length is 3 cm;

the mold of the cartilage repair scaffold was then printed out using a desktop 3D printer (three-dimensional, china first) with the selected wire being HIPS (high strength polystyrene, 1.75mm) (flash casting technology, china).

Example 3 preparation of cartilage repair scaffolds

Mixing 0% and 6.9% (first group), 2.3% and 4.6% (second group), 4.6% and 2.3% (third group) of fibroin and gelatin in the mixed solution, fully stirring, pouring the mixture into a mold before gelatin gelling (the mixed solution should not have a third rib to ensure a compact top surface membrane structure), placing in a vacuum drying oven at 60 ℃ for 10min to completely fill the mold with the composite solution, and taking out;

after the mixed solution is solidified into gel at room temperature (5min), placing the mold into D-limonene (Jiuzhong Kogyo, China) for 3 days, and removing the mold;

the temperature is required to be kept lower than the melting temperature of the gelatin in the process of removing the mould, and the solution is changed at regular time. After the mold is removed, the resulting porous scaffold is lyophilized and placed again in limonene to remove the remaining mold.

Washing off D-limonene by using 95% ethanol, putting the scaffold into 95% ethanol overnight after cleaning, and denaturing and curing the fibroin;

after the treatment was completed, the scaffold was placed in 0.5 wt% genipin (tianfeng biotechnology, china) aqueous solution and left overnight at room temperature to complete the crosslinking of gelatin and genipin. Taking out the crosslinked stent, cleaning with ultrapure water, drilling the massive crosslinked stent into a cylindrical stent by using a trephine (phi 4.00mm), freeze-drying to obtain the cylindrical cartilage repair stent, and performing cobalt 60 irradiation disinfection on the cylindrical cartilage repair stent before use.

Since the cartilage repair scaffold is made of fibroin and gelatin, the cartilage repair scaffold of the present invention may also be referred to as a fibroin-gelatin scaffold (SFG scaffold).

Example 4 determination of mechanical Strength and in vitro degradation of the fibroin-gelatin scaffolds prepared in example 3

Taking the three fibroin-gelatin scaffolds prepared in example 3, measuring compression deformation and strength by using a mechanical compression instrument, and calculating the mechanical strength of the three fibroin-gelatin scaffolds; as shown in fig. 8, the mechanical strength of the fibroin-gelatin scaffold increased with increasing fibroin concentration.

The three fibroin-gelatin scaffolds described above were weighed, immersed in PBS (0.01M, pH 7.2), SFG was taken out on

days

7, 14, and 21, freeze-dried and weighed, and the in vitro degradation rate was calculated. As shown in fig. 9, the cartilage repair scaffold prepared by using gelatin alone was rapidly degraded, while the degradation rate was significantly slowed down when the mass fraction of fibroin in the mixed solution was increased to 4.6%, and to match the regeneration rate of cartilage, the second group was selected as the final fibroin-gelatin scaffold concentration ratio for the subsequent experiments.

Wherein, the first group, the second group and the third group respectively represent that the mass fractions of the fibroin and the gelatin in the mixed solution are respectively 0% and 6.9%, 2.3% and 4.6%, 4.6% and 2.3%.

Example 5 coupling of E7 Polypeptides to fibroin-gelatin scaffolds with concentration ratios of the second group

Preparing 10% ethylenediamine solution with isopropanol, soaking fibroin and gelatin scaffold with concentration ratio of the second group in the solution (37 deg.C, 1 hr), and performing amination treatment.

Washing with double distilled water for 3 times; 10mg of a heterobifunctional crosslinking reagent (sodium 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimide ester, Sulfo-smcc) was dissolved in 100. mu.l of a Dimethyl sulfoxide (DMSO) solution and prepared. And soaking the aminated fibroin-gelatin scaffold for 1 hour in a dark environment.

Phosphate Buffered Saline (PBS) (0.01M, PH 7.2) of 0.1M ethylenediaminetetraacetic acid (EDTA) was prepared, and the fibroin-gelatin scaffold was washed 3 times with the buffer; dissolving bone marrow mesenchymal stem cell affinity polypeptide (E7 polypeptide) in PBS (the concentration is 0.1mg/ml), adding into the scaffold, incubating at room temperature for 2 hours to fully couple the E7 polypeptide to the scaffold, realizing the surface modification of the fibroin-gelatin scaffold by the E7 polypeptide, and freeze-drying for 24 hours for later use.

Prior to use, the fibroin-gelatin scaffolds coupled with E7 polypeptide were cobalt 60 radiation sterilized.

Example 6 CCK-8 assay and DNA and glycosaminoglycan (GAG) assay

Mixing 1.5X 10⁵Bone marrow mesenchymal stem cells per ml are respectively inoculated on a fibroin-gelatin bracket (SFG bracket) and a fibroin-gelatin bracket (SFG-E7 bracket) coupled with E7 polypeptide, complete culture medium is added for culture, the culture medium is respectively discarded on

days

1, 3, 5 and 7, PBS buffer solution is washed for 2 times, and then 200 microliters of new culture medium containing 10 microliters of CCK-8 working solution is added. 37 ℃ and 5% CO₂Incubated under conditions for 2 hours. 100 microliters of the above solution was added to a 96-well plate, and the OD value was measured, and the results are shown in fig. 10, and both the fibroin-gelatin scaffold (SFG scaffold) and the fibroin-gelatin scaffold coupled with E7 polypeptide (SFG-E7 scaffold) could well support the proliferation of bone marrow mesenchymal stem cells, and the fibroin-gelatin scaffold coupled with E7 polypeptide was superior to the fibroin-gelatin scaffold.

Mixing 1.5X 10⁵Inoculating bone marrow mesenchymal stem cells/ml onto SFG and SFG-E7, respectively, adding complete culture medium, culturing on 7 th, 14 th and 21 th days, discarding culture medium, washing with PBS for 2 times, adding 200 μ l papain at 60 deg.COvernight, the scaffold and cells were completely lysed;

after 20. mu.l of the above-mentioned solution was dropped into a 96-well plate, 200. mu.l of Hoechst33258 solution or 200. mu.l of DMMB solution was added thereto, and incubated at 37 ℃ for 30 minutes to determine the OD value. DNA determination conditions: excitation light 360nm, emission light 460 nm. Determination of GAG conditions: OD was measured at 520 nm. The results are shown in fig. 11 and 12, respectively, the two scaffolds can well support the chondrogenic differentiation of the in vitro mesenchymal stem cells, and the fibroin-gelatin scaffold coupled with the E7 polypeptide has a better effect on promoting the chondrogenic differentiation of the in vitro mesenchymal stem cells.

Example 7 confocal laser confocal microscopy for observing growth and distribution of mesenchymal stem cells on cartilage repair scaffold

The density is 1.5 multiplied by 10⁵Dropping the culture medium of bone marrow mesenchymal stem cells per ml on SFG and SFG-E7, and incubating for 1 hour at 37 ℃;

adding 1ml of MEM- α medium (containing no nucleotide and deoxynucleotide) into a 6-well plate, and culturing at 37 ℃ in an incubator;

after 3 days of culture, the scaffolds were removed and washed 2 times with PBS;

fixing by using 4% of paraformaldehyde, and staining a cytoskeleton for 4 hours in a dark place by using rhodamine-phalloidin (160nM, 2 ml);

PBS washing for 2 times, and re-staining cell nucleus for 1 hour by Hoechst33258(1:800, 2 ml);

and (5) washing with PBS for 2 times, and observing the distribution condition of the mesenchymal stem cells by using a laser confocal microscope.

FIG. 13 is a diagram showing the results of confocal laser scanning microscopy. Fig. 13 shows the growth and distribution of bone marrow mesenchymal stem cells on two scaffolds. Both the two scaffolds can well support the adhesion and growth of stem cells, and the fibroin-gelatin scaffold coupled with the E7 polypeptide can adhere more mesenchymal stem cells, which shows that the fibroin-gelatin scaffold coupled with the E7 polypeptide is more beneficial to the enrichment of mesenchymal stem cells.

Example 8 animal experiments-repair of cartilage defects in rabbits

Adult male New Zealand white rabbits are taken, and the weight of the adult male New Zealand white rabbits is 2.9 plus or minus 0.3 kg. Randomized into 3 groups: MF group (simple microfracture), SFG group (SFG scaffold + microfracture), SFG-E7 group (SFG scaffold coupled with E7 polypeptide + microfracture).

3 groups of New Zealand white rabbits are anesthetized by 3% pentobarbital through ear marginal intravenous injection, and the dosage is 1 mL/kg. After the rabbits are successfully anesthetized, the supine position is taken, the head and the four limbs are fixed, the right rear knee joint is exposed, the skin of the operation area is prepared, the iodophor is disinfected conventionally, the alcohol is deiodinated, and a sterile operation sheet is laid; the knee joint is flexed, the skin is cut through the anterior median approach of the knee, and the joint cavity is opened continuously through the medial peripatellar approach; extension of the knee-valgus-flexion of the knee expose the femoral trochlear. A4 mm diameter corneal ring is vertically drilled from the surface of the femoral trochlear (the depth is 1mm), and the cylindrical cartilage bolt drilled is taken out to prepare the full-thickness cartilage defect area of the femoral trochlear. The operation needs to be careful and avoid damaging tissues such as synovium, muscle, meniscus and the like.

MF group: the cartilage defect area is only treated by microfracture (the aperture is 0.5mm, the hole interval is 1mm, the depth is based on the exudation of blood and lipid drops), and no stent is filled.

SFG group: firstly, performing micro-fracture operation on a cartilage defect area, then implanting an SFG bracket into the cartilage defect part of a femoral pulley, filling the defect, and suturing the joint cavity layer by layer.

SFG-E7 group: firstly, performing micro-fracture operation on a cartilage defect area, then implanting an SFG-E7 stent into the cartilage defect of a femoral pulley, filling the defect, and suturing the joint cavity layer by layer.

Knee joints of all groups of rabbits do not carry out any external fixation treatment after the operation; 80 million U/penicillin is injected into the muscle for 3 days continuously; feeding with standard animal feed, keeping at 15-20 deg.C, drinking water, raising in cages, and moving freely.

The healing of cartilage damage was examined 6 months after microarthrosis in the 3 groups of rabbits. The results are shown in FIG. 14:

microfracture group (MF): gross observation shows that after 6 months of microfracture operation, the cartilage defect area can not be filled, and hyperosteogeny is shown around the defect; the nuclear Magnetic Resonance Imaging (MRI) and hematoxylin-eosin (HE) staining show that the cartilage defect is not filled, toluidine blue staining shows that the content of proteoglycan in the repair tissue of the cartilage defect area is low, the repair tissue is used for repairing fibrocartilage, and type II collagen immunohistochemical staining shows that the content of type II collagen in the tissue is low.

Fibroin-gelatin Scaffold (SFG) group: in general, defects are filled, but not complete; MRI can see the discontinuity of cartilage and the cystic surface of subchondral bone; HE and toluidine blue staining showed filling of the defect, with slightly lower GAG and type ii collagen content.

Group of fibroin-gelatin scaffolds (SFG-E7) coupled with E7 polypeptide: the general form is similar to that of normal cartilage, the defect is completely filled, and the cartilage is glossy; the MRI result shows that the cartilage is continuous, and the subchondral bone has no cystoid change and other abnormalities; HE. Toluidine blue and immunohistochemistry showed that the defect was well filled, tightly bound to surrounding tissues, and GAG and type ii collagen content was close to that of normal cartilage.

The rabbit in-vivo cartilage defect repair experiment shows that the cartilage repair scaffold coupled with the mesenchymal stem cell affinity polypeptide and the cartilage repair scaffold not coupled with the mesenchymal stem cell affinity polypeptide can promote cartilage repair, wherein the cartilage repair scaffold coupled with the mesenchymal stem cell affinity polypeptide has a better effect of promoting cartilage repair.

The above description is only for facilitating the understanding of the technical solutions of the present invention by those skilled in the art, and is not intended to limit the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> third Hospital of Beijing university

<120> cartilage repair scaffold and preparation method thereof

<160>1

<170>PatentIn version 3.3

<210>1

<211>7

<212>PRT

<213>Artificial

<220>

<223> artificially synthesized amino acid sequence

<400>1

Glu Pro Leu Gln Leu Lys Met

1 5

Claims

1. A preparation method of a cartilage repair scaffold is characterized by comprising the following steps:

preparing a mixed solution of fibroin and gelatin;

2. The method of manufacturing of claim 1, wherein the first rib is perpendicular to the second rib; the second rib is perpendicular to the third rib.

3. The method as claimed in claim 1, wherein the width of the first rib is 300-500 μm; the width of the second protruding edge is 300-500 μm; the width of the third rib is 300-.

4. The production method according to claim 3, wherein the first ridge, the second ridge, and the third ridge each have a width of 400 μm.

5. The manufacturing method according to claim 1, wherein a gap between adjacent two first ribs is 0.4 to 0.7 mm; the gap between every two adjacent second convex edges is 0.4-0.7 mm; the gap between every two adjacent third convex edges is 0.4-0.7 mm.

6. The manufacturing method according to claim 1, wherein the liquid level height of the mixed solution is 0.1 to 0.3mm higher than the sum of the heights of the first rib, the second rib, and the third rib.

7. The preparation method according to claim 1, characterized in that the mass fraction of the fibroin in the mixed solution is 2.3% -4.6%; the mass fraction of the gelatin in the mixed solution is 2.3% -4.6%.

8. The method according to claim 7, wherein the sum of the mass fractions of the fibroin and the gelatin in the mixed solution is 6.9%.

9. The method for preparing a scaffold material according to claim 1, wherein the protein denaturation treatment is carried out by placing the material body of the scaffold in an ethanol solution with a volume fraction of 95% overnight.

10. The preparation method according to claim 1, characterized in that the chemical crosslinking treatment is carried out on the material main body of the cartilage repair scaffold subjected to the protein denaturation treatment, and the material main body of the cartilage repair scaffold subjected to the protein denaturation treatment is placed in 0.3-1% by mass of genipin solution at room temperature overnight.

11. The method of claim 1, further comprising: and (4) sterilizing the cartilage repair support.

12. The production method according to any one of claims 1 to 11, characterized by further comprising: coupling a mesenchymal stem cell affinity polypeptide to the surface of the cartilage repair scaffold; the amino acid sequence of the bone marrow mesenchymal stem cell affinity polypeptide is shown as SEQ ID NO: 1 is shown.

13. The method of claim 12, wherein the step of coupling the mesenchymal stem cell affinity polypeptide to the surface of the cartilage repair scaffold comprises:

firstly, carrying out amination treatment on the cartilage repair scaffold;

14. The method for preparing the cartilage repair scaffold according to claim 13, wherein the amination treatment of the cartilage repair scaffold comprises soaking the cartilage repair scaffold in an ethylenediamine isopropanol solution with a volume fraction of 5% -15%, and keeping the solution at 37 ℃ for more than 1 hour.

15. The production method according to claim 13, characterized in that the solution containing the heterobifunctional crosslinking agent is a dimethyl sulfoxide solution of sodium sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate in a mass volume fraction of 5% to 15%; the immersion time of the aminated cartilage repair scaffold in the solution of the heterobifunctional cross-linking agent is more than 1 hour.

16. The preparation method according to claim 13, wherein the solution containing the mesenchymal stem cell affinity polypeptide is a phosphate buffer solution having a concentration of the mesenchymal stem cell affinity polypeptide of 0.05 to 0.15 mg/mL; the incubation time is 1 hour or more.

17. A cartilage repair scaffold prepared by the preparation method according to any one of claims 1 to 16.