Preparation method of gradient-structure osteochondral regeneration scaffold based on multi-material photocuring printing

Abstract

The invention discloses a preparation method of a gradient-structure osteochondral regeneration scaffold based on multi-material photocuring printing, and relates to the technical field of biomedical engineering.The preparation method comprises the following steps that different kinds of materials, cells and growth factors needed by the osteochondral regeneration scaffold are prepared into corresponding ink; the multi-material photocuring printing equipment is used for conducting integrated printing on the ink used by all the layers of osteochondral regeneration scaffolds, the osteochondral regeneration scaffold of a step-shaped or conical structure is obtained, and the osteochondral regeneration scaffold comprises at least one cartilage layer, a transition layer and a bone layer. According to the preparation method of the gradient-structure osteochondral regeneration scaffold based on multi-material photocuring printing, materials, cells and factors required by a cartilage layer, a transition layer and a bone layer are respectively loaded in different photocuring material troughs through a multi-material photocuring printer, so that integrated printing of the gradient-structure osteochondral regeneration scaffold is realized; and a technical platform is provided for manufacturing of a tissue engineering gradient structure.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and specifically to a method for preparing a gradient-structured osteochondral regeneration scaffold based on multi-material photocuring printing. Background Art

[0002] With the development of tissue engineering technology, researchers have begun to explore the use of biomaterials, cells and growth factors to repair or replace damaged tissues or organs. Bone and cartilage injuries caused by factors such as osteoarthritis and sports injuries are one of the common clinical injury types. Due to the special nature of the osteochondral gradient structure and cell distribution, osteochondral defects are difficult to self-repair, and the effects of traditional treatment methods are limited. The preparation of tissue-engineered osteochondral scaffolds provides a new solution for such injuries.

[0003] Traditional methods for manufacturing osteochondral scaffolds include mold-based casting methods and gas foaming methods. The casting method uses porogens to form a porous scaffold in the osteochondral scaffold. The gas foaming method uses gas to dissolve in the polymer under high pressure and rapidly depressurizes to allow the gas to escape, forming a porous structure scaffold. However, the porous structures prepared by such traditional methods have uneven sizes, and the porosity is difficult to control. It is impossible to form a large-scale batch production, and organic solvents may remain. With the development of additive manufacturing technology, 3D printing-based osteochondral manufacturing methods have been widely used, including extrusion-based 3D printing technology, electrospinning technology, cell material assembly technology, etc. In the early stage, the preparation of osteochondral scaffolds relied on heating to melt polymers such as PCL and PVA, and a gradient structure scaffold was formed after layer-by-layer printing. Cells or factors could be loaded only after the scaffold was prepared. The development of hydrogels has provided more optional biomaterials for extrusion-based 3D printing technology. Hydrogels with shear thinning and curing formability have been widely used in cell-laden bio-3D printing, including gelatin, sodium alginate, collagen and other hydrogels. Extrusion-based bio-3D printing has high advantages in preparing gradient structures, but its printing accuracy is low. Usually, the diameter of the cell-laden fibers extruded is greater than 100 μm, which limits the manufacture of high-precision structures. The introduction of photocuring printing technology provides a reliable solution to solve the problem of bio-3D printing accuracy. By curing photosensitive hydrogels layer by layer through light irradiation, the accuracy of the printed structure is greatly improved. Generally speaking, the single-layer thickness in photocuring technology can reach 10-30 μm, and its printing accuracy is much higher than that of extrusion-based bio-3D printing. Therefore, photocuring printing technology has been widely used in the manufacture of gradient scaffolds with relatively fine structures.

[0004] The osteochondral structure can be mainly divided into three layers. The outermost layer is the cartilage layer close to the joint cavity without structures such as blood vessels. The middle layer is the transitional layer between cartilage and bone. The innermost layer is the bone layer with structures such as blood vessels and nerves. Moreover, different types and morphologies of cells are distributed in the three-layer structure. Therefore, the design and manufacture of tissue-engineered osteochondral scaffolds need to consider gradient structures including the materials, cells, and vascular factors of osteochondral. The commonly used method in current research is to construct osteochondral gradient scaffolds through multi-material extrusion-based bioprinting technology, switching the printing nozzles during the printing process to achieve the printing of gradient structures. Additionally, osteochondral scaffolds with material and structural gradients can be constructed in one step through photocuring technology. After printing, different types of cells, such as chondrocytes and osteoblasts, are loaded, or different cell inductions and differentiations are carried out in different layers, such as the osteogenic and chondrogenic differentiations of bone marrow mesenchymal stem cells.

[0005] However, such manufacturing methods are rather cumbersome in the printing steps, have relatively low printing accuracy, and the range of selectable materials is limited. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method for a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing, so as to solve the problems of cumbersome printing steps, relatively low printing accuracy, and limited range of selectable materials in the manufacturing method mentioned in the above background technology.

[0007] To achieve the above purpose, the present invention provides the following technical solution: A preparation method for a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing, and the preparation method is as follows: Prepare inks corresponding to different types of materials, cells, and growth factors required for the osteochondral regeneration scaffold; Use a multi-material photocuring printing device to integrally print the inks used for each layer of the osteochondral regeneration scaffold to obtain an osteochondral regeneration scaffold with a stepped or conical structure.

[0008] Further, the osteochondral regeneration scaffold includes at least one cartilage layer, a transitional layer, and a bone layer.

[0009] Further, the material of the cartilage layer is hyaluronic acid methacrylamide (HAMA) or gelatin methacrylamide (GelMA) or polyethylene glycol diacrylate (PEGDA), the material of the transitional layer is gelatin methacrylamide (GelMA) or sodium alginate methacrylamide (AlgMA) or polyether F127 diacrylate (F127-DA), and the material of the bone layer is gelatin methacrylamide (GelMA) or chondroitin sulfate methacrylamide (ChSMA) or silk fibroin methacrylamide (SilMA).

[0010] Furthermore, the cells in the cartilage layer are chondrocytes, bone marrow mesenchymal stem cells or induced pluripotent stem cells; the cells in the transition layer are bone marrow mesenchymal stem cells or induced pluripotent stem cells; and the cells in the bone layer are osteoblasts, bone marrow mesenchymal stem cells or induced pluripotent stem cells.

[0011] Furthermore, the growth factors in the cartilage layer are transforming growth factor-β (TGF-β), fibroblast growth factors (FGFs) or platelet-derived growth factor (PDGF); the growth factors in the transition layer are transforming growth factor-β (TGF-β), fibroblast growth factors (FGFs) or platelet-derived growth factor (PDGF); and the growth factors in the bone layer are bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4) or a combination of dexamethasone, β-glycerophosphate and ascorbic acid.

[0012] Furthermore, different types of materials, cells and growth factors required for the osteochondral regeneration scaffold are formulated into corresponding inks, specifically including: dissolving methacrylated hyaluronic acid (HAMA) at 37 °C with a concentration of 10 wt%, formulated with ultrapure water, and placing it in a water bath at 37 °C - 50 °C for use after complete dissolution; after the confluence of cultured chondrocytes reaches 90%, digesting with trypsin, centrifuging, removing the supernatant, mixing the completely dissolved methacrylated hyaluronic acid (HAMA) with the cells, and adding transforming growth factor-β (TGF-β) with a concentration of 1 - 10 ng / mL, thus completing the formulation of the cartilage layer ink; dissolving methacrylated gelatin (GelMA) at 37 °C with a concentration of 10 wt%, formulated with ultrapure water, and placing it in a water bath at 37 °C - 50 °C for use after complete dissolution; dissolving methacrylated silk fibroin (SilMA) at 37 °C - 50 °C with a concentration of 15 wt%, formulated with ultrapure water, and placing it in a water bath at 37 °C for use after complete dissolution; after the confluence of cultured bone marrow mesenchymal stem cells reaches 90%, digesting with trypsin, centrifuging, removing the supernatant, mixing the completely dissolved methacrylated silk fibroin (SilMA) with the cells, and adding bone morphogenetic protein-2 (BMP-2) with a concentration of 10 - 100 ng / mL, thus completing the formulation of the bone layer ink.

[0013] Furthermore, the method of preparing the different types of materials, cells and growth factors required for the osteochondrocyte regeneration scaffold into corresponding inks also includes: dissolving methacrylamide gelatin (GelMA) at a concentration of 10wt% at 37°C, using ultrapure water for preparation, and placing the material in a water bath at 37°C to 50°C for use after the material is completely dissolved; dissolving methacrylamide chondroitin sulfate (ChSMA) at a concentration of 15wt% at 37°C to 50°C, using ultrapure water for preparation, and placing the material in a water bath at 37°C to 37°C for use after the material is completely dissolved; dissolving methacrylamide sodium alginate (AlgMA) at a concentration of 10wt% at 37°C, using ultrapure water for preparation, and placing the material in a water bath at 37°C to 50°C for use after the material is completely dissolved.

[0014] Furthermore, the method of using a multi-material photocuring printing device to integrally print the ink used for each layer of the osteochondrocyte regeneration scaffold to obtain a osteochondrocyte regeneration scaffold with a stepped or conical structure also includes: adding cartilage layer, transition layer, and bone layer inks to the three material tanks A, B, and C of the multi-material photocuring printer respectively; constructing a gradient structure osteochondrocyte model in the software Potato provided by the multi-material photocuring printer, wherein the thickness of the cartilage layer is 5 mm, the thickness of the transition layer is 200 μm, the thickness of the bone layer is 10 mm, the fiber spacing of the cartilage layer is 100 μm, the fiber spacing of the transition layer is 20 μm, and the fiber spacing of the bone layer is 200 μm; after the model is constructed, slicing is performed to generate the STL format file required for printing, and printing is performed.

[0015] Furthermore, the ink used for each layer of the bone cartilage regeneration scaffold is integratedly printed using a multi-material light-curing printing device to obtain a bone cartilage regeneration scaffold with a stepped or conical structure, specifically including: the print receiving platform is first moved to material trough No. 1 to print the bone layer. After printing, the print receiving platform is lifted and moved to material trough No. 2 to print the transition layer. After printing, the print receiving platform is lifted and moved to material trough No. 3 to print the cartilage layer. At this point, the bone cartilage regeneration scaffold with a gradient structure is printed and then placed in complete cell culture medium for culture.

[0016] Furthermore, the height of the print receiving platform being lifted is 100 μm.

[0017] The present invention provides a method for preparing a gradient structure bone cartilage regeneration scaffold based on multi-material photocuring printing, which has the following beneficial effects: the present invention uses a multi-material photocuring printer to load the materials, cells and factors required for the cartilage layer, transition layer and bone layer in different photocuring material troughs respectively, thereby realizing the integrated printing of gradient structure bone cartilage scaffolds and providing a technical platform for the manufacture of gradient structures in tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the overall structural schematic diagram of a preparation method for a gradient - structured osteochondral regeneration scaffold based on multi - material photocuring printing according to the present invention.

[0019] In the figure: 1. Printing receiving platform; 2. No. 1 material tank; 3. No. 2 material tank; 4. No. 3 material tank. Specific implementation manners

[0020] The following further describes the implementation manners of the present invention in detail with reference to the drawings and embodiments. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0021] Refer to the attached Figure 1 , and now a preparation method for a gradient - structured osteochondral regeneration scaffold based on multi - material photocuring printing provided by the present invention will be described.

[0022] Embodiment 1

[0023] Preparation of photocuring printing ink for the gradient - structured osteochondral regeneration scaffold: For the cartilage layer, dissolve methacryloylated hyaluronic acid (HAMA) at 37 °C, with a concentration of 10 wt%, and prepare it with ultrapure water. After the material is completely dissolved, place it in a 37 °C water bath for use. After the confluence of the cultured chondrocytes reaches 90%, digest and centrifuge them using trypsin, remove the supernatant, mix the completely dissolved methacryloylated hyaluronic acid (HAMA) with the cells, and add transforming growth factor - β (TGF - β) with a concentration of 1 - 10 ng / mL. Thus, the cartilage - layer ink is prepared. For the transition layer, dissolve methacryloylated gelatin (GelMA) at 37 °C, with a concentration of 10 wt%, and prepare it with ultrapure water. After the material is completely dissolved, place it in a 37 °C water bath for use. For the bone layer, dissolve methacryloylated silk fibroin (SilMA) at 37 °C, with a concentration of 15 wt%, and prepare it with ultrapure water. After the material is completely dissolved, place it in a 37 °C water bath for use. After the confluence of the cultured bone marrow mesenchymal stem cells reaches 90%, digest and centrifuge them using trypsin, remove the supernatant, mix the completely dissolved methacryloylated silk fibroin (SilMA) with the cells, and add bone morphogenetic protein - 2 (BMP - 2) with a concentration of 10 - 100 ng / mL. Thus, the bone - layer ink is prepared.

[0024] Preparation before printing: Add cartilage layer ink, transition layer ink, and bone layer ink into the three material tanks A, B, and C of the multi-material stereolithography printer with the model number EFL-BP-8601MIX, respectively. Subsequently, construct a gradient-structured osteochondral model in the built-in software Potato of the multi-material stereolithography printer. Among them, the thickness of the cartilage layer is 5 mm, the thickness of the transition layer is 200 μm, and the thickness of the bone layer is 10 mm. The fiber spacing of the cartilage layer fiber printing is 100 μm, the fiber spacing of the transition layer printing is 20 μm, and the fiber spacing of the bone layer printing is 200 μm. After the model is constructed, slice it to generate the STL format file required for printing and then perform printing.

[0025] Printing process: As Figure 1 shown, the printing receiving platform first moves to the No. 1 material tank to print the bone layer. After printing is completed, the printing receiving platform rises and moves to the No. 2 material tank to print the transition layer. After printing is completed, the printing receiving platform rises and moves to the No. 3 material tank to print the cartilage layer. Thus, the osteochondral regeneration scaffold with a gradient structure is printed. Subsequently, it is placed in a complete cell culture medium for cultivation.

[0026] Example 2

[0027] Preparation of the stereolithography printing ink for the gradient-structured osteochondral regeneration scaffold: For the cartilage layer, dissolve methacrylated gelatin (GelMA) at 37 °C, with a concentration of 8 wt%, and prepare it with ultrapure water. After the material is completely dissolved, place it in a 37 °C water bath for use. After the confluence of the cultured chondrocytes reaches 90%, digest and centrifuge them with trypsin, remove the supernatant, mix the completely dissolved methacrylated gelatin (GelMA) with the cells, and add fibroblast growth factor (FGFs) with a concentration of 10 - 20 ng / mL. Thus, the cartilage layer ink is prepared. For the transition layer, dissolve methacrylated sodium alginate (AlgMA) at 37 °C, with a concentration of 5 wt%, and prepare it with ultrapure water. After the material is completely dissolved, place it in a 37 °C water bath for use. For the bone layer, dissolve methacrylated chondroitin sulfate (ChSMA) at 50 °C, with a concentration of 10 wt%, and prepare it with ultrapure water. After the material is completely dissolved, place it in a 37 °C water bath for use. After the confluence of the cultured bone marrow mesenchymal stem cells reaches 90%, digest and centrifuge them with trypsin, remove the supernatant, mix the completely dissolved methacrylated silk fibroin (SilMA) with the cells, and add bone morphogenetic protein-4 (BMP-4) with a concentration of 10 - 50 ng / mL. Thus, the bone layer ink is prepared.

[0028] Preparation before printing: Add cartilage layer ink, transition layer ink, and bone layer ink into the three material tanks A, B, and C of the multi-material stereolithography printer with the model number EFL-BP-8601MIX respectively. Subsequently, construct a gradient structure osteochondral model in the built-in software Potato of the multi-material stereolithography printer. Among them, the thickness of the cartilage layer is 5 mm, the thickness of the transition layer is 200 μm, and the thickness of the bone layer is 10 mm. The fiber spacing of the cartilage layer fiber printing is 100 μm, the fiber spacing of the transition layer printing is 20 μm, and the fiber spacing of the bone layer printing is 200 μm. After the model is constructed, slice it to generate the STL format file required for printing and then perform printing.

[0029] Printing process: As Figure 1 shown, the printing receiving platform first moves to the No. 1 material tank to print the bone layer. After printing, the printing receiving platform lifts and moves to the No. 2 material tank to print the transition layer. After printing, the printing receiving platform lifts and moves to the No. 3 material tank to print the cartilage layer. Thus, the osteochondral regeneration scaffold with a gradient structure is printed. Subsequently, it is placed in a complete cell culture medium for cultivation.

[0030] Example 3

[0031] Preparation of photocurable printing ink for gradient-structured osteochondral regeneration scaffold: For the cartilage layer, methacryloylated hyaluronic acid (HAMA) and polyethylene glycol diacrylate (PEGDA) were dissolved at 37 °C with concentrations of 5 wt% and 2 wt% respectively, and were prepared with ultrapure water. After the materials were completely dissolved, they were placed in a 37 °C water bath for use. After the confluence of the cultured chondrocytes reached 90%, they were digested with trypsin and centrifuged to remove the supernatant. The completely dissolved methacryloylated hyaluronic acid (HAMA) and polyethylene glycol diacrylate (PEGDA) were mixed with the cells, and transforming growth factor-β (TGF-β) with a concentration of 1-10 ng / mL was added. Thus, the cartilage layer ink was prepared. For the transition layer, methacryloylated gelatin (GelMA) and polyether F127 diacrylate (F127-DA) were dissolved at 37 °C with concentrations of 8 wt% and 10 wt% respectively, and were prepared with ultrapure water. After the materials were completely dissolved, they were placed in a 37 °C water bath for use. For the bone layer, methacryloylated silk fibroin (SilMA) and methacryloylated chondroitin sulfate (ChSMA) were dissolved at 37 °C with concentrations of 10 wt% and 5 wt% respectively, and were prepared with ultrapure water. After the materials were completely dissolved, they were placed in a 37 °C water bath for use. After the confluence of the cultured bone marrow mesenchymal stem cells reached 90%, they were digested with trypsin and centrifuged to remove the supernatant. The completely dissolved methacryloylated silk fibroin (SilMA) was mixed with the cells, and bone morphogenetic protein-2 (BMP-2) with a concentration of 10-100 ng / mL was added. Thus, the bone layer ink was prepared.

[0032] Preparation before printing: The cartilage layer, transition layer, and bone layer inks were respectively added to three material slots A, B, and C of a multi-material photocurable printer with the model number EFL-BP-8601MIX. Subsequently, a gradient-structured osteochondral model was constructed in the built-in software Potato of the multi-material photocurable printer. Among them, the thickness of the cartilage layer was 5 mm, the thickness of the transition layer was 200 μm, and the thickness of the bone layer was 10 mm. The fiber printing spacing of the cartilage layer was 100 μm, the fiber printing spacing of the transition layer was 20 μm, and the fiber printing spacing of the bone layer was 200 μm. After the model was constructed, it was sliced to generate an STL format file required for printing and then printed.

[0033] Printing process: As Figure 1 shown, the printing receiving platform first moves to the No. 1 material slot to print the bone layer. After printing, the printing receiving platform is lifted and moves to the No. 2 material slot to print the transition layer. After printing, the printing receiving platform is lifted and moves to the No. 3 material slot to print the cartilage layer. Thus, the gradient-structured osteochondral regeneration scaffold is printed. Subsequently, it is placed in a complete cell culture medium for cultivation.

[0034] The embodiments of the present invention are given for purposes of illustration and description, and are not exhaustive or limit the invention to the disclosed forms. Many modifications and variations are obvious to those of ordinary skill in the art. The embodiments are chosen and described in order to best explain the principles of the invention and its practical application, and to enable those of ordinary skill in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing, characterized in that, The preparation method is as follows: Different types of materials, cells, and growth factors required for the osteochondral regeneration scaffold are formulated into corresponding inks; an integrated printing of the inks used for each layer of the osteochondral regeneration scaffold is performed using a multi-material photocuring printing device to obtain an osteochondral regeneration scaffold with a stepped or conical structure.

2. The preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 1, characterized in that The osteochondral regeneration scaffold includes at least one cartilage layer, a transition layer, and a bone layer.

3. A preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 2, characterized in that, The material of the cartilage layer is hyaluronic acid methacrylamide (HAMA) or gelatin methacrylamide (GelMA) or polyethylene glycol diacrylate (PEGDA), the material of the transition layer is gelatin methacrylamide (GelMA) or sodium alginate methacrylamide (AlgMA) or polyether F127 diacrylate (F127-DA), and the material of the bone layer is gelatin methacrylamide (GelMA) or chondroitin sulfate methacrylamide (ChSMA) or silk fibroin methacrylamide (SilMA).

4. The preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 3, wherein, The cells in the cartilage layer are chondrocytes or bone marrow mesenchymal stem cells or induced pluripotent stem cells, the cells in the transition layer are bone marrow mesenchymal stem cells or induced pluripotent stem cells, and the cells in the bone layer are osteoblasts or bone marrow mesenchymal stem cells or induced pluripotent stem cells.

5. A method for preparing a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 3, characterized in that, The growth factor in the cartilage layer is transforming growth factor-β (TGF-β) or fibroblast growth factors (FGFs) or platelet-derived growth factor (PDGF), the growth factor in the transition layer is transforming growth factor-β (TGF-β) or fibroblast growth factors (FGFs) or platelet-derived growth factor (PDGF), and the growth factor in the bone layer is bone morphogenetic protein-2 (BMP-2) or bone morphogenetic protein-4 (BMP-4) or a combination of dexamethasone, β-glycerophosphate, and ascorbic acid.

6. A preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to any one of claims 1-5, characterized in that, The different types of materials, cells and growth factors required for the osteochondrocyte regeneration scaffold are prepared into corresponding inks, specifically including: dissolving methacrylamide hyaluronic acid (HAMA) at a concentration of 10wt% at 37°C, using ultrapure water for preparation, and placing the material in a 37°C to 50°C water bath for use after the material is completely dissolved; after the confluence of the cultured chondrocytes reaches 90%, digesting and centrifuging with trypsin, removing the supernatant, mixing the completely dissolved methacrylamide hyaluronic acid (HAMA) with the cells, and adding transforming growth factor-β (TGF-β) at a concentration of 1 to 10ng / mL, so that the cartilage layer ink is prepared; dissolving methacrylamide gelatin ( The method comprises the following steps: preparing a 10wt% gel matrix and placing the SilMA in a water bath at 37°C to 50°C for use; dissolving methacrylamide silk fibroin (SilMA) at a concentration of 15wt% and using ultrapure water for preparation at 37°C to 50°C for use; digesting the cultured bone marrow mesenchymal stem cells with a confluence of 90% and centrifuging them, removing the supernatant, mixing the completely dissolved methacrylamide silk fibroin (SilMA) with the cells, and adding bone morphogenetic protein-2 (BMP-2) at a concentration of 10 to 100 ng / mL, thereby completing the preparation of the bone layer ink.

7. A preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 6, characterized in that, The method of preparing the corresponding inks with different types of materials, cells and growth factors required for the osteochondral regeneration scaffold also includes: dissolving methacrylamide gelatin (GelMA) at a concentration of 10wt% at 37°C, using ultrapure water for preparation, and placing the material in a water bath at 37°C to 50°C for use after the material is completely dissolved; dissolving methacrylamide chondroitin sulfate (ChSMA) at a concentration of 15wt% at 37°C to 50°C, using ultrapure water for preparation, and placing the material in a water bath at 37°C to 37°C for use after the material is completely dissolved; dissolving methacrylamide sodium alginate (AlgMA) at a concentration of 10wt% at 37°C, using ultrapure water for preparation, and placing the material in a water bath at 37°C to 50°C for use after the material is completely dissolved.

8. A preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to any one of claims 1-5, characterized in that, The method uses a multi-material photocuring printing device to integrally print the ink used for each layer of the osteochondrocyte regeneration scaffold to obtain a osteochondrocyte regeneration scaffold with a stepped or conical structure, and also includes: adding cartilage layer, transition layer, and bone layer inks to three material tanks A, B, and C of the multi-material photocuring printer respectively; constructing a gradient structure osteochondrocyte model in the software Potato provided by the multi-material photocuring printer, wherein the thickness of the cartilage layer is 5 mm, the thickness of the transition layer is 200 μm, the thickness of the bone layer is 10 mm, the fiber spacing of the cartilage layer is 100 μm, the fiber spacing of the transition layer is 20 μm, and the fiber spacing of the bone layer is 200 μm; after the model is constructed, slicing is performed to generate an STL format file required for printing, and printing is performed.

9. The preparation method of a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 8, characterized in that, The multi-material light-curing printing device is used to perform integrated printing on the ink used for each layer of the osteochondrium regeneration scaffold to obtain a osteochondrium regeneration scaffold with a stepped or conical structure, specifically comprising: the print receiving platform is first moved to the No. 1 material tank to print the bone layer, after printing is completed, the print receiving platform is lifted and moved to the No. 2 material tank to print the transition layer, after printing is completed, the print receiving platform is lifted and moved to the No. 3 material tank to print the cartilage layer, at this point the osteochondrium regeneration scaffold with a gradient structure is printed, and then placed in a complete cell culture medium for culture.

10. A method for preparing a gradient-structured osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 9, characterized in that The height of the print receiving platform being lifted is 100 μm.

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