A 3D printing preparation method for meniscus scaffold

Abstract

The present invention discloses a 3D printing preparation method for a meniscus scaffold, based on a 3D printing device and a meniscus scaffold biomaterial having the following structures: the 3D printing device comprises a constant temperature molding chamber (1), wherein the constant temperature molding chamber (1) is provided with a printing platform (2), a constant temperature extrusion nozzle (3) and a cross-linking agent nozzle (4); the meniscus scaffold biomaterial comprises poly (N-isopropyl acrylamide) and type I collagen; the present invention improves 3D printing conditions and combines specific 3D printing extrusion materials, thereby not only enabling rapid 3D printing molding, but also avoiding the introduction of relatively complex components, so that the prepared meniscus scaffold has good biosafety and bioactivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering material preparation, and particularly relates to a 3D printing preparation method for a meniscus scaffold. Background Art

[0002] Meniscus injury is one of the most common orthopedic diseases. Located between the femur and tibia, the meniscus is responsible for lubricating the joint, transferring weight, and stabilizing the joint. Injury can cause joint dysfunction, induce knee osteoarthritis, and seriously affect the patient's daily life. After a meniscus injury, due to the lack of blood supply to the inner 2 / 3 of the meniscus, it is often difficult to repair itself. After surgical repairs such as meniscus repair and meniscus plasty, the healing failure rate is also high due to the same reason. Therefore, how to effectively treat meniscus injuries has always been a hot and difficult problem that needs to be solved in the medical community.

[0003] In the application of meniscus repair, there is an urgent need to develop a 3D printing preparation method for meniscus scaffolds, so that the meniscus scaffolds can not only be quickly 3D printed, but also the prepared meniscus scaffolds have good biosafety and bioactivity, have suitable mechanical properties, contribute mechanical functions to the local meniscus, can load stem cells or stem cell secretions, and transport them to the site of meniscus damage to play a repair role. Summary of the Invention

[0004] The present invention aims to at least partially overcome the above-mentioned and / or other potential problems existing in the prior art: to provide a 3D printing preparation method for a meniscus scaffold, and to achieve a meniscus scaffold with high biocompatibility and strong mechanical properties.

[0005] The technical solution of the present invention is as follows: A 3D printing method for preparing a meniscus scaffold is based on a 3D printing device and a meniscus scaffold biomaterial having the following structure: the 3D printing device includes a constant temperature molding chamber, wherein the constant temperature molding chamber is provided with a printing platform, a constant temperature extrusion nozzle, and a crosslinker nozzle; the meniscus scaffold biomaterial includes poly (N-isopropylacrylamide) and type I collagen;

[0006] The specific preparation method comprises the following steps:

[0007] 1) Slicing the three-dimensional model of the meniscus scaffold using slicing software, importing the sliced data into the above-mentioned 3D printing device to start printing, wherein the extrusion material in the extrusion nozzle of the 3D printing device includes the following components in parts by weight: 10-20 parts of poly (N-isopropylacrylamide); 100 parts of water; and 0.1-1 parts of type I collagen;

[0008] 2) During the printing process, the temperature of the extruder nozzle is controlled at 10-25°C, the temperature of the constant temperature molding chamber is controlled at 30-45°C, and the temperature of the printing platform is controlled at 30-45°C;

[0009] 3) After the 3D printing device prints and forms a layer of material on the printing platform, a cross-linking agent is evenly sprayed on the surface of the layer through a cross-linking agent nozzle, and the extrusion nozzle starts to print the next layer; finally, the meniscus scaffold is printed layer by layer.

[0010] As an optimization, the type I collagen is water-soluble collagen.

[0011] In step 3), the mass concentration of the cross-linking agent is 2-10 wt% glutaraldehyde solution or 1 wt%-5 wt% EDC / NHS cross-linking agent.

[0012] In step 3), the temperature of the crosslinking agent is 30-45°C and the spraying amount is 0.5-1 mL / cm 2 .

[0013] The temperature of the extrusion cavity of the extrusion nozzle is controlled by semiconductor cooling sheets and temperature sensors arranged around the extrusion cavity.

[0014] The temperature in the constant temperature molding cavity is controlled by introducing hot air at 30-45°C.

[0015] A heating wire and a temperature sensor connected to the main controller are provided at the bottom of the printing platform.

[0016] A heating wire and a temperature sensor connected to the main controller are provided at the bottom of the printing platform.

[0017] The beneficial effects of the present invention include: Poly (N-isopropylacrylamide) (PNIPAM) is a thermosensitive polymer material that is liquid at room temperature and has excellent rheological properties, making it easy to extrude. It transforms into a solid state above 30°C, exhibiting mechanical properties similar to those of soft tissue and high biosafety. Type I collagen (COLLAGEN-I), a major component of the meniscus, has high bioactivity and can promote the differentiation of stem cells into chondrocytes. During the preparation process, water-soluble COLLAGEN-I can dissolve with PNIPAM in aqueous solutions below 30°C to form a cross-linked network, resulting in a low-viscosity, easily extrudable printing solution. The meniscus scaffold prepared by the present invention can load stem cells or materials secreted by stem cells and deliver them to the site of meniscus damage to exert a repair effect. It has both biosafety and bioactivity to promote the effectiveness of stem cells; it also has suitable mechanical properties to ensure the retention of stem cells and contribute mechanical functions to the local meniscus. The extrusion material of the present invention has a simple and uncomplicated composition, is water-soluble, does not introduce organic solvents, and can effectively reduce side effects such as inflammatory reactions. The present invention improves 3D printing conditions and combines specific 3D printing extrusion materials, which not only enables rapid 3D printing and molding, but also does not introduce more complex components, so that the prepared meniscus scaffold has good biosafety and bioactivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the structure of the 3D printing device in the embodiment.

[0019] Figure 2 The microscope photos and graphs of Example 2 are shown.

[0020] Figure 3 Phase transition temperature curves of extruded materials with different compositions.

[0021] Figure 1 :1. Constant temperature molding chamber; 2. Printing platform; 3. Constant temperature extrusion nozzle; 4. Cross-linking agent nozzle; 5. Semiconductor refrigeration chip.

[0022] Figure 2 : A is an optical microscope photograph of rat chondrocytes seeded on a meniscus scaffold containing 0.5% type I collagen; B is a cell proliferation curve of rat chondrocytes seeded on meniscus scaffolds with different compositions for 24-96 hours. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to specific examples, but the present invention is not limited to the following specific examples.

[0024] Example 1

[0025] This embodiment provides a 3D printing method for preparing a meniscus scaffold, such as Figure 1As shown, this embodiment is based on a 3D printing device with the following structure, wherein the 3D printing device includes a constant temperature molding chamber 1, in which a printing platform 2, a constant temperature extrusion nozzle 3 and a cross-linking agent nozzle 4 are provided; the other parts of the 3D printing device are the same as the 3D printing device based on the FDM (Fused Deposition Modeling) process in the prior art.

[0026] The preparation method comprises the following steps:

[0027] 1) After slicing the three-dimensional model of the meniscus scaffold using slicing software, the sliced data is imported into the above-mentioned 3D printing device to start printing. The extrusion material in the extrusion nozzle 3 of the 3D printing device includes the following components in parts by weight: 10 parts of poly (N-isopropylacrylamide); 100 parts of water; and 0.5 parts of type I collagen;

[0028] 2) Control the temperature of the extrusion nozzle 3 to 20°C, the temperature in the constant temperature molding chamber 1 to 37°C; and the temperature of the printing platform 2 to 37°C;

[0029] 3) After the 3D printing device prints a layer of material on the printing platform 2, a crosslinking agent is evenly sprayed on the surface of the layer through the crosslinking agent nozzle 4. The crosslinking agent is a 10 wt% glutaraldehyde solution at a temperature of 37°C and a spraying volume of 0.5 mL / cm 2 ; Then the nozzle 3 is extruded and the next layer is printed; finally, the meniscus bracket is printed layer by layer.

[0030] The type I collagen is water-soluble collagen.

[0031] In step 1), a three-dimensional model of the meniscus scaffold can be constructed by importing the knee joint MRI data into the medical reverse engineering software Mimics; the constructed three-dimensional model of the meniscus scaffold is sliced using a slicing software, and the sliced data is imported into the above-mentioned 3D printing device to start printing;

[0032] The temperature of the extrusion cavity of the extrusion nozzle 3 is controlled to be 20° C. In this embodiment, the temperature control is achieved by means of a semiconductor cooling plate 5 and a temperature sensor arranged around the extrusion cavity.

[0033] The temperature in the constant temperature molding cavity 1 is 37° C.; this embodiment is specifically achieved by introducing 37° C. hot air into the constant temperature molding cavity 1 .

[0034] The printing platform 2 has a temperature control function. Specifically, a heating wire and a temperature sensor connected to the main controller are provided at the bottom thereof. In step 2), the temperature of the printing platform 2 is heated and controlled at 37°C.

[0035] To ensure that the printed meniscus scaffold can still maintain its solid state after being taken out to room temperature, each layer of material needs to be cross-linked and shaped after printing. In step 3), after the 3D printing equipment prints and shapes a layer of material on the printing platform 2, the cross-linking agent is pumped into the cross-linking agent nozzle 4, and a layer of cross-linking agent is evenly sprayed on the surface of the layer through the cross-linking agent nozzle 4. After the extrusion nozzle 3 rises one layer height, it starts to print the next layer; thus, the meniscus scaffold is finally printed layer by layer.

[0036] The suspended gap between the three-dimensional model of the meniscus scaffold and the printing platform 2 can be filled by a support structure model; this is a common choice in the 3D printing process and will not be described in detail here.

[0037] Example 2

[0038] Figure 2 A is an optical microscope photograph of mouse chondrocytes seeded on a meniscus scaffold containing 0.5% type I collagen. Figure 2 B shows the results of a cell proliferation experiment in which mouse chondrocytes were inoculated on meniscus scaffolds with different compositions (0.5 parts collagen, 0.2 parts collagen, 0.1 parts collagen, and a control group without collagen, with the remaining compositions and printing conditions being the same as in Example 1) for 24-96 hours. It can be seen that all experimental groups containing collagen have a stronger ability to promote cell proliferation than the control group, with the 0.5 part collagen group having the highest ability.

[0039] Figure 3 Phase transition temperature curves of extruded materials with different compositions.

[0040]

[0041]

[0042] The table above shows the viscoelastic mechanical test data for extrusion materials of different compositions and the meniscus scaffolds constructed from them. As can be seen from the table, the extrusion materials exhibit good extrusion properties before printing (i.e., at room temperature and without cross-linking), facilitating printing. The resulting meniscus scaffolds (at 37°C, after cross-linking and setting) exhibit suitable mechanical strength, facilitating the mechanical function of the meniscus and providing a mechanical environment for meniscus tissue regeneration.

[0043] The above are only examples of the features of the present invention and do not constitute any limitation on the scope of protection of the present invention. Any technical solution formed by equivalent exchange or equivalent replacement falls within the scope of protection of the present invention.

Claims

1. A 3D printing preparation method for a meniscus scaffold, characterized in that: Based on a 3D printing device and a meniscus scaffold biomaterial having the following structure, the 3D printing device comprises a constant temperature molding chamber (1), wherein the constant temperature molding chamber (1) is provided with a printing platform (2), a constant temperature extrusion nozzle (3), and a cross-linking agent nozzle (4); the meniscus scaffold biomaterial comprises poly (N-isopropylacrylamide) and type I collagen; The specific preparation method comprises the following steps: 1) After slicing the three-dimensional model of the meniscus scaffold using slicing software, the slice data is imported into the above-mentioned 3D printing device to start printing, wherein the extrusion material in the extrusion nozzle (3) of the 3D printing device includes the following components in parts by weight: 10-20 parts of poly (N-isopropyl acrylamide); 100 parts of water; and 0.1-1 parts of type I collagen; 2) During the printing process, the temperature of the extrusion nozzle (3) is controlled to be 10-25°C, the temperature in the constant temperature molding chamber (1) is controlled to be 30-45°C; and the temperature of the printing platform (2) is controlled to be 30-45°C; 3) After the 3D printing device prints and forms a layer of material on the printing platform (2), a layer of cross-linking agent is evenly sprayed on the surface of the layer through the cross-linking agent nozzle (4), and the next layer is printed through the extrusion nozzle (3); finally, a meniscus scaffold is printed layer by layer.

2. The 3D printing preparation method of the meniscus scaffold according to claim 1, characterized in that: The type I collagen is water-soluble collagen.

3. The 3D printing preparation method of the meniscus scaffold according to claim 2, characterized in that: In step 3), the mass concentration of the cross-linking agent is 2-10 wt % glutaraldehyde solution or 1 wt % -5 wt % EDC / NHS cross-linking agent.

4. The 3D printing preparation method of the meniscus scaffold according to claim 3, characterized in that: In step 3), the temperature of the crosslinking agent is 30-45°C and the spraying amount is 0.5-1 mL / cm 2 .

5. The 3D printing preparation method of the meniscus scaffold according to claim 4, characterized in that: The temperature of the extrusion cavity of the extrusion nozzle (3) is controlled by a semiconductor cooling plate (5) and a temperature sensor arranged around the extrusion cavity.

6. The 3D printing preparation method of the meniscus scaffold according to claim 5, characterized in that: The temperature in the constant temperature molding cavity (1) is controlled by introducing hot air at 30-45°C.

7. The 3D printing preparation method of the meniscus scaffold according to claim 6, characterized in that: A heating wire and a temperature sensor connected to a main controller are provided at the bottom of the printing platform (2).

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