CN110201225A - 3D printing fibroin/gelatin bracket and preparation method thereof for repair of cartilage - Google Patents

Abstract

The invention discloses a 3D printed silk fibroin/gelatin scaffold for cartilage repair and a preparation method thereof. The porous hydrogel scaffold was constructed by 3D printing technology, and the enzymatic cross-linking was achieved by soaking in hydrogen peroxide, and the mechanical properties were further enhanced by alcohol solution treatment. The scaffold of the invention combines the excellent properties of gelatin and silk fibroin, has good biocompatibility, mechanical properties and compression fatigue resistance, and can be applied to cartilage tissue regeneration and repair.

Description

3D printed silk fibroin/gelatin scaffold for cartilage repair and its preparation method

technical field

The invention relates to the technical field of biomedical materials, in particular to a 3D printing silk fibroin/gelatin scaffold for cartilage repair and a preparation method thereof.

Background technique

Once the cartilage tissue is damaged, it is difficult to achieve self-repair. Although many techniques have been applied to the treatment of cartilage defects clinically, there are deficiencies in various therapies. The research and development of tissue engineered cartilage has brought new ideas for the repair of cartilage defects. Cartilage scaffold materials need to have good biocompatibility, degradation rate and mechanical properties that match cartilage tissue regeneration; there are many ways to construct cartilage tissue engineering scaffolds. Compared with some traditional scaffold molding methods, 3D printing can More precise control over the structure of the scaffold. Hydrogel has a structure and water content similar to human soft tissue. In recent years, the research on 3D printing hydrogel has also attracted much attention and many achievements have been made.

3D printing hydrogels have certain requirements for materials, and using gelatin temperature sensitivity to achieve 3D printing can obtain a three-dimensional structure with better fidelity. The advantage of using gelatin as a cartilage repair material is that the molecular chain is known to allow cells to recognize and adhere to the site, which has good biocompatibility, but gelatin also has defects such as poor mechanical properties and short degradation time in vivo. On the contrary, silk fibroin, as a natural polymer extracted from silk, has good mechanical properties and a relatively controllable degradation rate, but there is no cell adhesion site on the natural silk fibroin molecular chain, which is not conducive to cell adhesion. Migration, adhesion and growth thereon. Combining the two materials to build a 3D printing scaffold can realize the complementary advantages of the two materials.

Horseradish peroxidase-catalyzed cross-linking is a fast and gentle way of cross-linking. Gelatin can achieve enzymatic cross-linking by modifying tyramine groups on the molecular chain; while the natural silk fibroin molecular chain itself has tyrosine residues for enzyme-catalyzed cross-linking. The two can be covalently cross-linked by enzymatic cross-linking reaction to realize the complementary advantages of the two. In addition, the mechanical properties of the silk fibroin hydrogel after enzymatic crossover can be significantly enhanced after being treated with alcohol solution to induce conformational transformation.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and propose a 3D printing silk fibroin/gelatin scaffold for cartilage repair and its preparation method. The scaffold combines the excellent properties of gelatin and silk fibroin, and has good biological Compatibility, mechanical properties, and compression fatigue resistance can be applied to cartilage tissue regeneration and repair.

In order to achieve the above object, the technical solution provided by the present invention is: a 3D printing silk fibroin/gelatin scaffold for cartilage repair, the scaffold is made of silk fibroin, tyramide grafted modified gelatin, and horseradish peroxidase Slurry, using 3D printing technology to construct porous hydrogel scaffolds, realize enzyme cross-linking by soaking in hydrogen peroxide, and further use alcohol solution treatment to enhance mechanical properties.

The preparation method of the described 3D printing silk fibroin/gelatin support for cartilage repair comprises the following steps:

1) Printing material preparation

1.1) Preparation of tyramide-grafted modified gelatin: prepare 500ml of 50mM morpholine ethanesulfonic acid buffer solution, add 10g of gelatin powder, stir at 50°C, fully dissolve, add 5g of tyramine hydrochloride, stir, After the solution is fully dissolved and cooled to room temperature, add carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to activate the gelatin molecular chain Carboxyl group, react at room temperature for 12 hours; put the reaction product into a dialysis bag with a molecular weight cut-off of 10,000-12,000, dialyze in deionized water for 4 days, and finally use a freeze dryer to remove water to obtain a white spongy modified product, moisture-proof Store in the cabinet, spare;

1.2) Preparation of silk fibroin solution: add 4g of degummed silk to a beaker, then add 20ml of 9.3mol/l lithium bromide solution; place the beaker in a water bath at 60°C and heat to dissolve the degummed silk for 4 hours; after fully dissolving, transfer the solution to In a dialysis bag with a molecular weight cut-off of 3500, dialyze in deionized water for 3 days, and change the water 2-3 times a day; after the dialysis, centrifuge the solution in the dialysis bag twice in a centrifuge to remove insoluble impurities; Finally, a clear silk fibroin solution is obtained, the concentration of the silk fibroin solution is obtained by the drying specific gravity method, and it is stored in a refrigerator at 4°C for use within three weeks;

2) Configuration of printing paste

Add the modified gelatin to the silk fibroin solution diluted to the specified concentration, dissolve it at 50°C for about 2 hours, and mix well; add horseradish peroxidase after the gelatin is dissolved; transfer it to the barrel after mixing, and set aside; Among them, the concentration of gelatin is 15wt%, the concentration of silk fibroin is 0-5w/v%, and the concentration of horseradish peroxidase is 30-240 Units/ml;

3) Printing and post-processing of hydrogel scaffolds

Transfer the cylinder containing the printing paste in step 2) to the 3D printer, after setting the temperature of the cylinder and the temperature of the printing deposition platform, let the cylinder keep warm; after setting the parameters of the internal and external structures of the bracket , start printing after adjusting the extrusion pressure and drawing speed; after printing, soak the printed structure in hydrogen peroxide solution to trigger the enzyme-catalyzed cross-linking between silk fibroin and modified gelatin; finally use alcohol solution to treat the enzyme cross-link The combined hydrogel scaffold can promote the conformational transformation of silk fibroin to enhance the mechanical properties of the scaffold; after the treatment, wash it with deionized water three times to remove the residual alcohol solution, and obtain a 3D printing silk fibroin/gelatin water Gel stand.

In step 1.1), the dosages of the carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are respectively: 0.37g, 0.11 g.

In step 3), the temperature of the barrel is set at 28-30°C, and the platform temperature is set at 4-8°C.

In step 3), the shape and size of the external structure of the stent can be designed according to actual needs; the internal structure of the stent is fiber filaments with a spacing of 0.4 to 1 mm, and the angle between each two layers of fiber filaments is 90°. The thickness is 180 μm to 400 μm.

In step 3), the extrusion pressure is 1.5-4 bar; the drawing speed is 10-20 mm/s.

In step 3), the hydrogen peroxide concentration is 5-10 mM, and the soaking time is 30-60 min.

In step 3), the alcohol solution is 70-90w/v% methanol or ethanol aqueous solution, and the soaking time is 6-10 hours.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The materials used in the present invention are two natural renewable polymers, gelatin and silk fibroin, which have a wide range of sources and are easy to obtain, and have good biocompatibility.

2. The 3D printing silk fibroin/gelatin scaffold prepared by the present invention has an internally connected pore structure, which is conducive to the growth and proliferation of cells inside the scaffold.

3. The 3D printed silk fibroin/gelatin scaffold prepared by the present invention has high compression modulus and compression fatigue resistance, and can adapt to the stress of natural cartilage tissue.

4. The preparation process proposed by the present invention is simple and easy, and can efficiently and stably construct a three-dimensional porous scaffold for cartilage tissue engineering repair.

Description of drawings

Figure 1 is a schematic diagram of the scaffold 3D printing preparation process.

Fig. 2 is a scanning electron microscope image of the freeze-dried scaffolds prepared in Examples 1-5.

Fig. 3 is a histogram of the cell proliferation status characterized by the CCK-8 method after the scaffolds prepared in Examples 1-5 are inoculated with human adipose-derived mesenchymal stem cells.

FIG. 4 is a histogram of the compression modulus of the scaffolds prepared in Examples 1-5.

Fig. 5 is the stress-strain curves of the stents prepared in Examples 1-4 in the linear cyclic loading and unloading compression test.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto.

Example 1

1) Printing material preparation

1.1) Preparation of tyramide-grafted modified gelatin: prepare 500ml of 50mM morpholineethanesulfonic acid buffer, add 10g of gelatin powder, stir at 50°C, and fully dissolve. Add 5g of tyramine hydrochloride, stir and dissolve fully; after the solution is cooled to room temperature, add carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride 0.37g/0.11g, to activate the carboxyl group on the gelatin molecular chain, react at room temperature for 12h; put the reaction product into a dialysis bag with a molecular weight cut-off of 10000-12000, and dialyze for 4 days under a deionized water environment. Finally, the water was removed by a freeze dryer to obtain a white spongy modified product, which was stored in a moisture-proof cabinet for future use.

1.2) Preparation of silk fibroin solution: add 4g of degummed silk to a beaker, then add 20ml of 9.3mol/l lithium bromide solution; place the beaker in a water bath at 60°C and heat to dissolve the degummed silk for 4 hours; after fully dissolving, transfer the solution to In a dialysis bag with a molecular weight cut-off of 3500, dialyze with ionized water for 2 days, and change the water 2-3 times a day; after the dialysis, centrifuge the solution in the dialysis bag twice in a centrifuge to remove insoluble impurities; to obtain a clear silk fibroin solution, the concentration of the silk fibroin solution is obtained by drying the specific gravity method, and the silk fibroin solution is diluted to 2.5w/v% with deionized water.

2) Configuration of printing paste

Add modified gelatin to the silk fibroin solution with a concentration of 2.5w/v% to a concentration of 15w/v%, dissolve it at 50°C for about 2 hours, and mix well; add horseradish peroxidation after the gelatin is dissolved Phytase, so that the concentration is 60Units/ml; transfer to the barrel after mixing.

3) Printing and post-processing of hydrogel scaffolds

Transfer the barrel containing the printing paste in step 2) to the 3D printer, set the temperature of the barrel to 29°C, set the temperature of the printing deposition platform to 4°C, and keep the barrel warm for 2 hours; set the external shape of the bracket The appearance is a cuboid of 10*10*4mm, and the internal structure is: the distance between the fiber filaments is 0.6mm, and the angle between each two layers of fiber filaments is 90°; after setting the extrusion pressure to 2bar and the drafting speed to 15mm/s Start printing; after printing, soak the printed structure in hydrogen peroxide solution for 30 minutes to trigger enzyme-catalyzed cross-linking between silk fibroin and modified gelatin, wash with deionized water three times, and obtain 3D printed SF2.5GT15 hydrogel stand.

Example 2

1) Printing material preparation

1.1) Preparation of tyramide-grafted modified gelatin: prepare 500ml of 50mM morpholineethanesulfonic acid buffer, add 10g of gelatin powder, stir at 50°C, and fully dissolve. Add 5g of tyramine hydrochloride, stir and dissolve fully; after the solution is cooled to room temperature, add carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride 0.37g/0.11g, to activate the carboxyl group on the gelatin molecular chain, react at room temperature for 12h; put the reaction product into a dialysis bag with a molecular weight cut-off of 10000-12000, and dialyze for 4 days under a deionized water environment. Finally, the water was removed by a freeze dryer to obtain a white spongy modified product, which was stored in a moisture-proof cabinet for future use.

1.2) Preparation of silk fibroin solution: add 4g of degummed silk to a beaker, then add 20ml of 9.3mol/l lithium bromide solution; place the beaker in a water bath at 60°C and heat to dissolve the degummed silk for 4 hours; after fully dissolving, transfer the solution to In a dialysis bag with a molecular weight cut-off of 3500, dialyze with ionized water for 2 days, and change the water 2-3 times a day; after the dialysis, centrifuge the solution in the dialysis bag twice in a centrifuge to remove insoluble impurities; after the centrifugation, A clear silk fibroin solution is obtained, the concentration of the silk fibroin solution is obtained by drying the specific gravity method, and the silk fibroin solution is diluted to 2.5 w/v% with deionized water.

2) Configuration of printing paste

Add modified gelatin to the silk fibroin solution with a concentration of 2.5w/v% to a concentration of 15w/v%, dissolve it at 50°C for about 2 hours, and mix well; after the gelatin is dissolved, add horseradish peroxidase to make Its concentration is 60Units/ml; transfer to the cylinder after mixing.

3) Printing and post-processing of hydrogel scaffolds

Transfer the barrel containing the printing paste in step 2) to the 3D printer, set the temperature of the barrel to 29°C, set the temperature of the printing deposition platform to 4°C, and keep the barrel warm for 2 hours; set the external shape of the bracket The appearance is a cuboid of 10*10*4mm, the internal structure is: the distance between fiber filaments is 0.6mm, the angle between each two layers of fiber filaments is 90°, the extrusion pressure is set at 2.5bar, and the drafting speed is 15mm/s Then start printing; after printing, soak the printed structure in hydrogen peroxide solution for 30 minutes to trigger the enzyme-catalyzed cross-linking between silk fibroin and modified gelatin, and finally soak the enzyme-crosslinked hydrogel in 75wt% methanol solution The scaffold was 10h; after the treatment, it was washed three times with deionized water to obtain a 3D printed SF2.5GT15/alcohol-treated hydrogel scaffold.

Example 3

1) Printing material preparation

1.1) Preparation of tyramide-grafted modified gelatin: prepare 500ml of 50mM morpholineethanesulfonic acid buffer, add 10g of gelatin powder, stir at 50°C, and fully dissolve. Add 5g of tyramine hydrochloride, stir and dissolve fully; after the solution is cooled to room temperature, add carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride 0.37g/0.11g, to activate the carboxyl group on the gelatin molecular chain, react at room temperature for 12h; put the reaction product into a dialysis bag with a molecular weight cut-off of 10000-12000, and dialyze for 4 days under a deionized water environment. Finally, the water was removed by a freeze dryer to obtain a white spongy modified product, which was stored in a moisture-proof cabinet for future use.

1.2) Preparation of silk fibroin solution: add 4g of degummed silk to a beaker, then add 20ml of 9.3mol/l lithium bromide solution; place the beaker in a water bath at 60°C and heat to dissolve the degummed silk for 4 hours; after fully dissolving, transfer the solution to In a dialysis bag with a molecular weight cut-off of 3500, dialyze in deionized water for 2 days, and change the water 2-3 times a day; after the dialysis, centrifuge the solution in the dialysis bag twice in a centrifuge to remove insoluble impurities; Finally, a clear silk fibroin solution is obtained, the concentration of the silk fibroin solution is obtained by drying the specific gravity method, and the silk fibroin solution is diluted to 5 w/v% with deionized water.

2) Configuration of printing paste:

Add modified gelatin to the silk fibroin solution with a concentration of 5w/v% to a concentration of 15w/v%, dissolve it at 50°C for about 2 hours, and mix well; after the gelatin is dissolved, add horseradish peroxidase to make it The concentration is 60U/ml; transfer to the cylinder after mixing.

3) Printing and post-processing of hydrogel scaffolds

Transfer the barrel containing the printing paste in step 2) to the 3D printer, set the temperature of the barrel to 30°C, set the temperature of the printing deposition platform to 4°C, and keep the barrel warm for 2 hours; set the external shape of the bracket Appearance: 10*10*4mm cuboid, internal structure: the distance between fiber filaments is 0.6mm, the angle between each two layers of fiber filaments is 90°, the extrusion pressure is set to 3bar, and the drafting speed is 15mm/s Then start printing; after printing, soak the printed structure in hydrogen peroxide solution for 30 minutes to trigger enzyme-catalyzed cross-linking between silk fibroin and modified gelatin, and wash it three times with deionized water to obtain a 3D printed SF5GT15 hydrogel scaffold .

Example 4

1) Printing material preparation

1.1) Preparation of tyramide-grafted modified gelatin: prepare 500ml of 50mM morpholineethanesulfonic acid buffer, add 10g of gelatin powder, stir at 50°C, and fully dissolve. Add 5g of tyramine hydrochloride, stir and dissolve fully; after the solution is cooled to room temperature, add carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride 0.37g/0.11g, to activate the carboxyl group on the gelatin molecular chain, react at room temperature for 12h; put the reaction product into a dialysis bag with a molecular weight cut-off of 10000-12000, and dialyze for 4 days under a deionized water environment. Finally, the water was removed by a freeze dryer to obtain a white spongy modified product, which was stored in a moisture-proof cabinet for future use.

1.2) Preparation of silk fibroin solution: add 4g of degummed silk to a beaker, then add 20ml of 9.3mol/l lithium bromide solution; place the beaker in a water bath at 60°C and heat to dissolve the degummed silk for 4 hours; after fully dissolving, transfer the solution to In a dialysis bag with a molecular weight cut-off of 3500, dialyze in deionized water for 3 days, and change the water 2-3 times a day; after the dialysis, centrifuge the solution in the dialysis bag twice in a centrifuge to remove insoluble impurities; Finally, a clear silk fibroin solution is obtained, the concentration of the silk fibroin solution is obtained by drying the specific gravity method, and the silk fibroin solution is diluted to 5 w/v% with deionized water.

2) Configuration of printing paste

Add modified gelatin to the silk fibroin solution with a concentration of 5w/v% to a concentration of 15w/v%, dissolve it at 50°C for about 2 hours, and mix well; after the gelatin is dissolved, add horseradish peroxidase to make it The concentration is 60Units/ml; transfer to the cylinder after mixing

3) Printing and post-processing of hydrogel scaffolds

Transfer the barrel containing the printing paste in step 2) to the 3D printer, set the temperature of the barrel to 30°C, set the temperature of the printing deposition platform to 4°C, and keep the barrel warm for 2 hours; set the external shape of the bracket Appearance: 10*10*4mm cuboid, the internal structure is: the distance between fiber filaments is 0.6mm, the angle between each two layers of fiber filaments is 90°, after setting the extrusion pressure to 3bar and the drafting speed to 15mm/s Start printing; after printing, soak the printed structure in hydrogen peroxide solution for 30 minutes to trigger enzyme-catalyzed cross-linking between silk fibroin and modified gelatin; finally soak the enzyme-crosslinked hydrogel scaffold in 75wt% methanol solution 10h; after the treatment, wash three times with deionized water to remove the residual alcohol solution, and obtain the 3D printed SF5GT15/alcohol-treated hydrogel scaffold.

Embodiment 5 (comparative example)

1) Printing material preparation

1.1) Preparation of tyramide-grafted modified gelatin: prepare 500ml of 50mM morpholineethanesulfonic acid buffer, add 10g of gelatin powder, stir at 50°C, and fully dissolve. Add 5g of tyramine hydrochloride, stir and dissolve fully; after the solution is cooled to room temperature, add carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride 0.37g/0.11g, to activate the carboxyl group on the gelatin molecular chain, react at room temperature for 12h; put the reaction product into a dialysis bag with a molecular weight cut-off of 10000-12000, and dialyze for 4 days under a deionized water environment. Finally, the water was removed by a freeze dryer to obtain a white spongy modified product, which was stored in a moisture-proof cabinet for future use.

2) Configuration of printing paste

Add modified gelatin to deionized water to make the concentration 15w/v%, dissolve at 50°C for about 2 hours, and mix well; after the gelatin is dissolved, add horseradish peroxidase to make the concentration 60U/ml; Mix and transfer to a barrel.

3) Printing and post-processing of hydrogel scaffolds

Transfer the barrel containing the printing paste in step 2) to the 3D printer, set the temperature of the barrel to 28°C, set the temperature of the printing deposition platform to 4°C, and keep the barrel warm for 2 hours; set the external shape of the bracket Appearance: 10*10*4mm rectangular parallelepiped bracket, internal structure: the distance between fiber filaments is 0.6mm, and the angle between each two layers of fiber filaments is 90°. After adjusting the extrusion pressure and drafting speed, start printing; print ends Finally, soak the printed structure in hydrogen peroxide solution for 30 minutes to trigger enzyme-catalyzed cross-linking between silk fibroin and modified gelatin; finally soak the enzyme-crosslinked hydrogel scaffold in 75wt% methanol solution for 10 hours; after the treatment , washed three times with deionized water to remove residual alcohol solution, and obtained 3D printed pure gelatin hydrogel scaffold.

In summary, the present invention mainly uses horseradish peroxidase, modified gelatin, and silk fibroin as printing pastes to construct porous hydrogel scaffolds through temperature-controlled 3D printing, soaking in hydrogen peroxide to initiate crosslinking of the scaffolds, and then further Treatment with alcohol solution triggers the conformational transformation of silk fibroin, improves the physical and chemical properties of the material, and obtains a scaffold suitable for cartilage tissue regeneration. Refer to Figures 1 to 5, which are schematic diagrams of the scaffold 3D printing preparation process; scanning electron micrographs of the freeze-dried scaffolds prepared in Examples 1 to 5; After mesenchymal stem cells, use the CCK-8 method to characterize the histogram of cell proliferation; the histogram of the compression modulus of the scaffolds prepared in Examples 1-5; the scaffolds prepared in Examples 1-4 in the linear cyclic loading and unloading compression test the stress-strain curve.

However, the implementation of the present invention is not limited by the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention are all equivalent replacement methods, including Within the protection scope of the present invention.

Claims (8)

1. 3D printing silk fibroin/gelatin scaffold for cartilage repair, characterized in that: the scaffold uses silk fibroin, tyramide grafted modified gelatin, and horseradish peroxidase as slurry, and is constructed by 3D printing technology The porous hydrogel scaffold is enzymatically cross-linked by soaking in hydrogen peroxide, and the mechanical properties are further enhanced by alcohol solution treatment. 2. the preparation method of the 3D printing silk fibroin/gelatin support that is used for cartilage repair as claimed in claim 1, is characterized in that, comprises the following steps: 1) Printing material preparation 1.1) Preparation of tyramide-grafted modified gelatin: prepare 500ml of 50mM morpholine ethanesulfonic acid buffer solution, add 10g of gelatin powder, stir at 50°C, fully dissolve, add 5g of tyramine hydrochloride, stir, After the solution is fully dissolved and cooled to room temperature, add carboxyl activator N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to activate the gelatin molecular chain Carboxyl group, react at room temperature for 12 hours; put the reaction product into a dialysis bag with a molecular weight cut-off of 10,000-12,000, dialyze in deionized water for 4 days, and finally use a freeze dryer to remove water to obtain a white spongy modified product, moisture-proof Store in the cabinet, spare; 1.2) Preparation of silk fibroin solution: add 4g of degummed silk to a beaker, then add 20ml of 9.3mol/l lithium bromide solution; place the beaker in a water bath at 60°C and heat to dissolve the degummed silk for 4 hours; after fully dissolving, transfer the solution to In a dialysis bag with a molecular weight cut-off of 3500, dialyze in deionized water for 3 days, and change the water 2-3 times a day; after the dialysis, centrifuge the solution in the dialysis bag twice in a centrifuge to remove insoluble impurities; Finally, a clear silk fibroin solution is obtained, the concentration of the silk fibroin solution is obtained by the drying specific gravity method, and it is stored in a refrigerator at 4°C for use within three weeks; 2) Configuration of printing paste Add the modified gelatin to the silk fibroin solution diluted to the specified concentration, dissolve it at 50°C for about 2 hours, and mix well; add horseradish peroxidase after the gelatin is dissolved; transfer it to the barrel after mixing, and set aside; Among them, the concentration of gelatin is 15wt%, the concentration of silk fibroin is 0-5w/v%, and the concentration of horseradish peroxidase is 30-240 Units/ml; 3) Printing and post-processing of hydrogel scaffolds Transfer the cylinder containing the printing paste in step 2) to the 3D printer, after setting the temperature of the cylinder and the temperature of the printing deposition platform, let the cylinder keep warm; after setting the parameters of the internal and external structures of the bracket , start printing after adjusting the extrusion pressure and drawing speed; after printing, soak the printed structure in hydrogen peroxide solution to trigger the enzyme-catalyzed cross-linking between silk fibroin and modified gelatin; finally use alcohol solution to treat the enzyme cross-link The combined hydrogel scaffold can promote the conformational transformation of silk fibroin to enhance the mechanical properties of the scaffold; after the treatment, wash it with deionized water three times to remove the residual alcohol solution, and obtain a 3D printing silk fibroin/gelatin water Gel stand. 3. the preparation method of the 3D printing silk fibroin/gelatin support that is used for cartilage repair according to claim 2, is characterized in that: in step 1.1), described carboxyl activator N-hydroxyl succinimide and 1- The dosages of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are: 0.37g and 0.11g respectively. 4. The method for preparing a 3D printed silk fibroin/gelatin scaffold for cartilage repair according to claim 2, characterized in that: in step 3), the temperature of the barrel is set at 28-30°C, and the platform temperature It is 4 ~ 8 ℃. 5. The preparation method of the 3D printing silk fibroin/gelatin support for cartilage repair according to claim 2, characterized in that: in step 3), the shape and size of the external structure of the support can be designed according to actual needs ; The internal structure of the stent is fiber filaments with a spacing of 0.4-1 mm, the angle between each two layers of fiber filaments is 90°, and the layer thickness is 180 μm-400 μm. 6. The preparation method of 3D printing silk fibroin/gelatin scaffold for cartilage repair according to claim 2, characterized in that: in step 3), the extrusion pressure is 1.5-4bar; the drafting speed It is 10-20mm/s. 7. The preparation method of 3D printing silk fibroin/gelatin scaffold for cartilage repair according to claim 2, characterized in that: in step 3), the hydrogen peroxide concentration is 5-10 mM, and the soaking time is 30-60 min . 8. The preparation method of 3D printing silk fibroin/gelatin scaffold for cartilage repair according to claim 2, characterized in that: in step 3), the alcohol solution is 70-90w/v% methanol or ethanol aqueous solution , soaking time is 6 ~ 10h.