A method for preparing a gradient-structured osteochondral regeneration scaffold based on multi-material photocuring printing

Abstract

The present invention discloses a method for preparing a gradient-structured osteochondrocyte regeneration scaffold based on multi-material photocuring printing, which relates to the field of biomedical engineering technology. The preparation method is as follows: different types of materials, cells, and growth factors required for the osteochondrocyte regeneration scaffold are formulated into corresponding inks; the inks used for each layer of the osteochondrocyte regeneration scaffold are integratedly printed using a multi-material photocuring printing device to obtain an osteochondrocyte regeneration scaffold with a stepped or conical structure, wherein the osteochondrocyte regeneration scaffold includes at least one cartilage layer, a transition layer, and a bone layer. This method for preparing a gradient-structured osteochondrocyte regeneration scaffold based on multi-material photocuring printing uses a multi-material photocuring printer to load the materials, cells, and factors required for the cartilage layer, transition layer, and bone layer into different photocuring material troughs, thereby achieving integrated printing of the gradient-structured osteochondrocyte scaffold and providing a technical platform for the manufacture of gradient structures in tissue engineering.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular 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 damage caused by factors such as osteoarthritis and sports injuries is one of the most common types of injuries in clinical practice. Due to the particularity of the gradient structure and cell distribution of osteocartilage, osteochondral defects are difficult to self-repair, and traditional treatment methods have limited effects. The preparation of tissue-engineered osteochondral scaffolds provides a new solution for this type of injury.

[0003] Traditional methods for manufacturing osteochondral scaffolds include mold-based casting and gas foaming. The casting method utilizes porogens to form a porous scaffold in the osteochondral scaffold. The gas foaming method utilizes gas dissolved in a polymer under high pressure, and then rapidly reduces the pressure to allow the gas to escape, forming a porous structure scaffold. However, the porous structures prepared by this traditional method are uneven in size, and the porosity is difficult to control, making it impossible to scale up for mass 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, and cell material assembly technology. Early osteochondral scaffold preparation relied on heating to melt polymers, such as PCL, PVA, and other materials, and then printed layer by layer to form a gradient structure scaffold. Cells or factors can only be loaded after the scaffold is prepared. The development of hydrogels has provided more optional biomaterials for extrusion 3D printing technology. Hydrogels with shear thinning and curing moldability are widely used in cell-laden biological 3D printing, including hydrogels such as gelatin, sodium alginate, and collagen. Extrusion-based biological 3D printing has a high advantage in preparing gradient structures, but its printing accuracy is low. Usually, the diameter of the extruded cell-laden fibers is greater than 100μm, which limits the manufacture of high-precision structures. The introduction of photocuring printing technology provides a reliable solution to the problem of biological 3D printing accuracy. The photosensitive hydrogel is cured by layer-by-layer illumination, which greatly improves the accuracy of the printed structure. Generally speaking, the single layer thickness in photocuring technology can reach 10-30μm, and its printing accuracy is much higher than that of extrusion biological 3D printing. Therefore, photocuring printing technology is widely used in the manufacture of gradient scaffolds with relatively fine structures.

[0004] The osteochondral structure can be divided into three main layers: the outermost layer is the cartilage layer near the joint cavity and without structures such as blood vessels; the middle layer is the transition layer between cartilage and bone; and the innermost layer is the bone layer with structures such as blood vessels and nerves. Different types and morphologies of cells are distributed throughout the three layers. Therefore, the design and manufacture of tissue-engineered osteochondral scaffolds need to consider the gradient structure of the osteochondral material, cells, and blood vessels. A common method currently used in research is to construct osteochondral gradient scaffolds using multi-material extrusion bio-3D printing technology. Printing of gradient structures is achieved by switching the printhead during the printing process. Alternatively, osteochondral scaffolds with material and structural gradients can be constructed in one go using photocuring technology. After printing, different cell types, such as chondrocytes and osteoblasts, can be loaded, or different cell differentiation can be induced in different layers, such as the osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0005] However, this type of manufacturing method is relatively cumbersome in terms of printing steps, has low printing accuracy, and has a limited range of selectable materials. Summary of the Invention

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

[0007] To achieve the above objectives, the present invention provides the following technical solutions: a method for preparing a gradient-structured osteochondral regeneration scaffold based on multi-material photocuring printing, the preparation method of which is as follows: different types of materials, cells and growth factors required for the osteochondral regeneration scaffold are formulated into corresponding inks; the inks used for each layer of the osteochondral regeneration scaffold are integratedly printed using a multi-material photocuring printing device to obtain an osteochondral regeneration scaffold with a stepped or conical structure.

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

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

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

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

[0012] Furthermore, the different types of materials, cells and growth factors required for the osteochondral regeneration scaffold are formulated 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 it is completely dissolved; after the cultured chondrocytes reach a confluence of 90%, they are digested with trypsin and centrifuged, the supernatant is removed, the completely dissolved methacrylamide hyaluronic acid (HAMA) is mixed with the cells, and transforming growth factor-β (TGF-β) is added at a concentration of 1 to 10ng / mL, and the cartilage layer ink is prepared; dissolving methacrylamide hyaluronic acid (HAMA) at 37°C, Glue (GelMA) with a concentration of 10wt% was prepared with ultrapure water. After the material was completely dissolved, it was placed in a water bath at 37°C to 50°C for use; methacrylamide silk fibroin (SilMA) was dissolved at a concentration of 15wt% at 37°C to 50°C and prepared with ultrapure water. After the material was completely dissolved, it was placed in a water bath at 37°C for use; after the confluence of cultured bone marrow mesenchymal stem cells reached 90%, they were digested with trypsin and centrifuged, the supernatant was removed, the completely dissolved methacrylamide silk fibroin (SilMA) was mixed with the cells, and bone morphogenetic protein-2 (BMP-2) was added at a concentration of 10 to 100 ng / mL. At this point, the bone layer ink was prepared.

[0013] Furthermore, the different types of materials, cells and growth factors required for the osteochondral regeneration scaffold are formulated into corresponding inks, which 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 it 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 for use after it is completely dissolved; and 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 it is completely dissolved.

[0014] Furthermore, the method further includes: adding inks of the cartilage layer, transition layer, and bone layer into the three material tanks A, B, and C of the multi-material photocuring printer respectively before using the multi-material photocuring printing device to print the inks used for each layer of the bone cartilage regeneration scaffold in an integrated manner to obtain a bone cartilage regeneration scaffold with a stepped or conical structure; constructing a gradient structure bone cartilage 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; and slicing the model after construction to generate the STL format file required for printing for printing.

[0015] Furthermore, the ink used for each layer of the bone cartilage regeneration scaffold is integratedly printed using a multi-material photocuring printing device to obtain a bone cartilage regeneration scaffold with a stepped or conical structure, specifically including: the print receiving platform first moves to the No. 1 material trough to print the bone layer. After printing is completed, the print receiving platform is lifted and moved to the No. 2 material trough to print the transition layer. After printing is completed, the print receiving platform is lifted and moved to the No. 3 material trough to print the cartilage layer. At this point, the bone cartilage regeneration scaffold with a gradient structure is printed and then placed in a 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-structured osteochondral 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 into different photocuring material troughs respectively, thereby realizing the integrated printing of gradient-structured osteochondral 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 a schematic diagram of the overall structure of a method for preparing a gradient structure bone and cartilage regeneration scaffold based on multi-material photocuring printing of the present invention.

[0019] In the figure: 1. Print receiving platform; 2. Material trough No. 1; 3. Material trough No. 2; 4. Material trough No. 3. DETAILED DESCRIPTION

[0020] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0021] See attached Figure 1 , the preparation method of the gradient structure bone cartilage regeneration scaffold based on multi-material photocuring printing provided by the present invention is now described.

[0022] Example 1

[0023] Preparation of photocurable ink for gradient-structured osteochondral regeneration scaffolds: For the cartilage layer, methacrylamidated hyaluronic acid (HAMA) was dissolved at a concentration of 10 wt% at 37°C and prepared with ultrapure water. After the material was completely dissolved, it was placed in a 37°C water bath for use. After the cultured chondrocytes reached 90% confluence, they were digested with trypsin and centrifuged. The supernatant was removed, and the completely dissolved HAMA was mixed with the cells. Transforming growth factor-β (TGF-β) was added at a concentration of 1-10 ng / mL. The cartilage layer ink was prepared. For the transition layer, methacrylamidated gelatin (GelMA) was dissolved at a concentration of 10 wt% at 37°C and prepared with ultrapure water. After the material was completely dissolved, it was placed in a 37°C water bath for use. For the bone layer, methacrylamide silk fibroin (SilMA) was dissolved at 37°C to a concentration of 15wt% and prepared with ultrapure water. After the material was completely dissolved, it was 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. The supernatant was removed, and the completely dissolved methacrylamide silk fibroin (SilMA) was mixed with the cells, and bone morphogenetic protein-2 (BMP-2) was added at a concentration of 10-100ng / mL. At this point, the bone layer ink was prepared.

[0024] Printing Preparation: Cartilage, transition layer, and bone layer inks were added to the A, B, and C slots of the EFL-BP-8601MIX multi-material stereolithography printer, respectively. A gradient osteochondral model was then constructed using the multi-material stereolithography printer's built-in software, Potato. The cartilage layer was 5 mm thick, the transition layer was 200 μm thick, and the bone layer was 10 mm thick. The fiber spacing in the cartilage layer was 100 μm, the fiber spacing in the transition layer was 20 μm, and the fiber spacing in the bone layer was 200 μm. After model construction, the model was sliced, and the STL file required for printing was generated for printing.

[0025] Printing process: Figure 1 As shown, the print receiving platform first moves to the No. 1 trough to print the bone layer. After printing, the print receiving platform is lifted and moved to the No. 2 trough to print the transition layer. After printing, the print receiving platform is lifted and moved to the No. 3 trough to print the cartilage layer. At this point, the osteochondral regeneration scaffold with a gradient structure is printed and then placed in complete cell culture medium for culture.

[0026] Example 2

[0027] Preparation of the photocurable ink for gradient-structured osteochondral regeneration scaffolds: For the cartilage layer, gelatin methacrylamide (GelMA) was dissolved at a concentration of 8 wt% at 37°C and prepared with ultrapure water. After the material was completely dissolved, it was placed in a 37°C water bath for use. After the cultured chondrocytes reached 90% confluence, they were digested with trypsin and centrifuged. The supernatant was removed, and the completely dissolved gelatin methacrylamide (GelMA) was mixed with the cells. Fibroblast growth factors (FGFs) were added at a concentration of 10-20 ng / mL. This completed the preparation of the cartilage layer ink. For the transition layer, sodium methacrylamide alginate (AlgMA) was dissolved at a concentration of 5 wt% at 37°C and prepared with ultrapure water. After the material was completely dissolved, it was placed in a 37°C water bath for use. For the bone layer, methacrylamide chondroitin sulfate (ChSMA) was dissolved at 50°C to a concentration of 10wt% and prepared with ultrapure water. After the material was completely dissolved, it was 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. The supernatant was removed, and the completely dissolved methacrylamide silk fibroin (SilMA) was mixed with the cells, and bone morphogenetic protein-4 (BMP-4) was added at a concentration of 10-50ng / mL. At this point, the bone layer ink was prepared.

[0028] Printing Preparation: Cartilage, transition layer, and bone layer inks were added to the A, B, and C slots of the EFL-BP-8601MIX multi-material stereolithography printer, respectively. A gradient osteochondral model was then constructed using the multi-material stereolithography printer's built-in software, Potato. The cartilage layer was 5 mm thick, the transition layer was 200 μm thick, and the bone layer was 10 mm thick. The fiber spacing in the cartilage layer was 100 μm, the fiber spacing in the transition layer was 20 μm, and the fiber spacing in the bone layer was 200 μm. After model construction, the model was sliced, and the STL file required for printing was generated for printing.

[0029] Printing process: Figure 1 As shown, the print receiving platform first moves to the No. 1 trough to print the bone layer. After printing, the print receiving platform is lifted and moved to the No. 2 trough to print the transition layer. After printing, the print receiving platform is lifted and moved to the No. 3 trough to print the cartilage layer. At this point, the osteochondral regeneration scaffold with a gradient structure is printed and then placed in complete cell culture medium for culture.

[0030] Example 3

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

[0032] Printing Preparation: Cartilage, transition layer, and bone layer inks were added to the A, B, and C slots of the EFL-BP-8601MIX multi-material stereolithography printer, respectively. A gradient osteochondral model was then constructed using the multi-material stereolithography printer's built-in software, Potato. The cartilage layer was 5 mm thick, the transition layer was 200 μm thick, and the bone layer was 10 mm thick. The fiber spacing in the cartilage layer was 100 μm, the fiber spacing in the transition layer was 20 μm, and the fiber spacing in the bone layer was 200 μm. After model construction, the model was sliced, and the STL file required for printing was generated for printing.

[0033] Printing process: Figure 1 As shown, the print receiving platform first moves to the No. 1 trough to print the bone layer. After printing, the print receiving platform is lifted and moved to the No. 2 trough to print the transition layer. After printing, the print receiving platform is lifted and moved to the No. 3 trough to print the cartilage layer. At this point, the osteochondral regeneration scaffold with a gradient structure is printed and then placed in complete cell culture medium for culture.

[0034] The embodiments of the present invention are presented for purposes of illustration and description and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments are chosen and described in order to better illustrate the principles of the invention and its practical application and to enable those skilled in the art to understand the invention and design various embodiments with various modifications as suited for specific applications.

Claims

1. A method for preparing a gradient structure bone cartilage 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 osteochondrocyte regeneration scaffold are prepared into corresponding inks; the inks used for each layer of the osteochondrocyte regeneration scaffold are integratedly printed using a multi-material light-curing printing device to obtain an osteochondrocyte regeneration scaffold with a stepped or conical structure, wherein the osteochondrocyte regeneration scaffold includes at least one cartilage layer, a transition layer and a bone layer; the different types of materials, cells and growth factors required for the osteochondrocyte regeneration scaffold are prepared into corresponding inks, specifically comprising: dissolving methacrylamidated hyaluronic acid (HAMA) at 37°C, and its concentration is 400mg / L. The ink is prepared by using ultrapure water at a concentration of 10 wt % and placing the material in a water bath at 37°C to 50°C for use after it is completely dissolved. After the confluence of the cultured chondrocytes reaches 90%, the cells are digested with trypsin and centrifuged. The supernatant is removed and the completely dissolved hyaluronic acid (HAMA) is mixed with the cells. Transforming growth factor-β (TGF-β) is added at a concentration of 1 to 10 ng / mL. The cartilage layer ink is thus prepared. Methacrylamide gelatin (GelMA) is dissolved at 37°C at a concentration of 10 wt % and prepared with ultrapure water. After the material is completely dissolved, it is placed in a water bath at 37°C to 50°C for use; SilMA is dissolved at 37°C to 50°C at a concentration of 15wt%, and ultrapure water is used for preparation. After the material is completely dissolved, it is placed in a water bath at 37°C for use; after the confluence of the cultured bone marrow mesenchymal stem cells reaches 90%, they are digested with trypsin and centrifuged, the supernatant is removed, the completely dissolved SilMA is mixed with the cells, and bone morphogenetic protein-2 (BMP-2) is added at a concentration of 10 to 10 0ng / mL, at this point the bone layer ink is prepared, and the multi-material light-curing printing equipment is used to integrate the inks used for each layer of the bone cartilage regeneration scaffold to print, and a bone cartilage regeneration scaffold with a stepped or conical structure is obtained, specifically including: 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 bone cartilage regeneration scaffold with a gradient structure is printed, and then placed in complete cell culture medium for culture.

2. The method for preparing a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 1, characterized in that: The material of the cartilage layer is methacrylamide hyaluronic acid (HAMA) or methacrylamide gelatin (GelMA) or polyethylene glycol diacrylate (PEGDA), the material of the transition layer is methacrylamide gelatin (GelMA) or methacrylamide sodium alginate (AlgMA) or polyether F127 diacrylate (F127-DA), and the material of the bone layer is methacrylamide gelatin (GelMA) or methacrylamide chondroitin sulfate (ChSMA) or methacrylamide silk fibroin (SilMA).

3. The method for preparing a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 2, characterized in that: The cells of the cartilage layer are chondrocytes or bone marrow mesenchymal stem cells or induced pluripotent stem cells, the cells of the transition layer are bone marrow mesenchymal stem cells or induced pluripotent stem cells, and the cells of the bone layer are osteoblasts or bone marrow mesenchymal stem cells or induced pluripotent stem cells.

4. The 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 of the cartilage layer is transforming growth factor-β (TGF-β) or fibroblast growth factor (FGFs) or platelet-derived growth factor (PDGF), the growth factor of the transition layer is transforming growth factor-β (TGF-β) or fibroblast growth factor (FGFs) or platelet-derived growth factor (PDGF), and the growth factor of the bone layer is bone morphogenetic protein-2 (BMP-2) or bone morphogenetic protein-4 (BMP-4) or a combination of dexamethasone, sodium β-glycerophosphate and ascorbic acid.

5. The method for preparing a gradient structure bone cartilage regeneration scaffold based on multi-material photocuring printing according to claim 4, characterized in that: The method of preparing different types of materials, cells and growth factors required for the osteochondral 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 after it is completely dissolved for use; 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 after it is completely dissolved for use; and 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 after it is completely dissolved for use.

6. The method for preparing a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to any one of claim 5, characterized in that: The method uses a multi-material photocuring printing device to integrally print the ink used for each layer of the osteochondral regeneration scaffold to obtain a osteochondral regeneration scaffold with a stepped or conical structure, and further includes: adding ink of the cartilage layer, transition layer, and bone layer to three material tanks A, B, and C of the multi-material photocuring printer, respectively; constructing a gradient structure osteochondral model in the multi-material photocuring printer's built-in software Potato, 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.

7. The method for preparing a gradient structure osteochondral regeneration scaffold based on multi-material photocuring printing according to claim 6, characterized in that: The height of the print receiving platform being raised is 100 μm.

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