3D freeze printing system and printing method of anisotropic scaffold

Abstract

The application relates to a 3D freeze printing system and printing method of an anisotropic support, the system comprising a refrigerant gas transmission device and a freeze printing auxiliary module; the freeze printing auxiliary module comprises a liquid nitrogen refrigeration ring, one or more gas flow channels with a set configuration and arrangement mode are arranged on the liquid nitrogen refrigeration ring, the air inlet of the gas flow channel is connected with the output end of the cold gas transmission device, and the air outlet of the gas flow channel is oriented to the printing platform of an extrusion type 3D printer in a specific direction, so as to guide the formation of an anisotropic microstructure with a specific orientation of a hydrogel support in a printing process. Compared with the prior art, the application can print a large-size anisotropic support with vertical and radial microstructures, strong mechanical properties, guided cell directional migration and endogenous tissue regeneration, and provides a brand-new biomimetic support solution for anisotropic tissue regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomaterial 3D printing equipment technology, and in particular to a liquid nitrogen-assisted anisotropic scaffold 3D cryogenic printing system and printing method. Background Technology

[0002] Natural tissues, such as cartilage, bone, skeletal muscle, tendons, cardiac muscle, nerves, blood vessels, and the cornea, generally exhibit highly ordered anisotropic structures. This directional arrangement endows anisotropic tissues with unique mechanical properties, serving as a key foundation for achieving complex physiological functions. For example, Haversian canals in long bones are aligned with the force line of the bone under mechanical loads, enabling them to withstand compressive loads; highly oriented collagen fibers in the skin help maintain its tensile strength and elasticity. Therefore, this highly ordered anisotropic structure is crucial for maintaining normal tissue function, and the in vitro construction of biomimetic anisotropic scaffolds shows great potential for application in regenerative medicine.

[0003] 3D extrusion printing, as an additive manufacturing technology, constructs three-dimensional scaffolds by continuously extruding and stacking materials layer by layer. Due to its high flexibility and customizability, it is now widely used in the field of tissue regeneration. Hydrogel scaffolds, in particular, have become an important tool in regenerative medicine due to their physicochemical properties highly similar to the natural extracellular matrix (ECM), such as good biocompatibility, adjustable mechanical properties, and abundant cell binding sites. Hydrogel scaffolds not only provide a three-dimensional growth environment for cells but also promote cell adhesion, proliferation, and differentiation, demonstrating enormous clinical translational potential and broad industrial prospects.

[0004] However, as disclosed in Chinese patent application CN201810126051.7, the hydrogel scaffolds prepared by traditional extrusion 3D printing exhibit a disordered microstructure, which differs significantly from the ordered arrangement of normal tissue ECM. Furthermore, as the printing height increases, the scaffold deforms or collapses when the accumulated stress in the lower layer exceeds the material's yield stress, thus limiting the height of models that can be constructed using traditional extrusion 3D printing. This structural mismatch and model size limitation affect its effectiveness in guiding tissue regeneration and clinical translation, especially in the repair of tissues requiring highly ordered arrangement (such as skin and bone).

[0005] Therefore, breaking through the bottlenecks of traditional extrusion printing technology and developing an innovative printing platform capable of integrally constructing complex, large-scale macroscopic geometries and anisotropic microstructures has become an urgent problem to be solved in the field of regenerative medicine. Summary of the Invention

[0006] The purpose of this invention is to provide a 3D cryogenic printing system and method for constructing anisotropic scaffolds.

[0007] The objective of this invention can be achieved through the following technical solutions: As a first aspect of the present invention, a 3D cryo-printing system for anisotropic scaffolds is provided. The 3D cryo-printing system is used to assist an extrusion 3D printer in printing anisotropic hydrogel scaffolds and includes a cooling gas transmission device and a cryo-printing auxiliary module. The cryogenic printing auxiliary module includes a liquid nitrogen cooling ring, on which one or more gas channels with a set configuration and arrangement are provided. The air inlet of the gas channel is connected to the output end of the cold gas transmission device, and the air outlet of the gas channel is oriented towards the printing platform of the extrusion 3D printer in a specific position, so as to guide the hydrogel scaffold to form anisotropic microstructures with a specific orientation during the printing process.

[0008] As a preferred technical solution, the cryogenic printing auxiliary module includes a vertical cryogenic printing module; The vertical cryogenic printing module includes a vertical cooling device and a vertical cooling ring. The vertical cooling device is mounted on the printing platform of the extrusion 3D printer and is used for vertical cryogenic printing below a set height. It includes a thermoelectric cooler and a water-cooling circulation system. The cooling side of the thermoelectric cooler faces the 3D printer nozzle, and the water-cooling circulation system is located on the heat-releasing side of the thermoelectric cooler to remove heat from the heat-releasing side. The vertical cooling ring is installed on the outside of the 3D printer nozzle and is used for cryogenic printing above a set height. The vertical cooling ring is provided with one or more air inlets and an air outlet that faces vertically downward toward the printing platform.

[0009] As a preferred technical solution, the water-cooled circulation system includes a metal heat-conducting block, a water pump, and water pipes. The water pump circulates coolant to the metal heat-conducting block through the water pipes for cooling. The metal heat-conducting block is connected to the heat-dissipating end of the thermoelectric cooler and removes heat from the heat-dissipating side of the thermoelectric cooler.

[0010] As a preferred technical solution, the interior of the vertical cooling ring is divided into an independent cooling air duct and an installation area by a partition; the cooling air duct is provided with one or more air inlets and an air outlet that is vertically downward toward the printing platform; a guide plate is provided in the cooling air duct near the air inlet, and the cross-section of the guide plate is an arc shape with the center of the circle facing the air inlet; the nozzle of the 3D printer vertically penetrates the installation area of ​​the vertical cooling ring and is connected and fixed to the vertical cooling ring.

[0011] As a preferred technical solution, the cryogenic printing auxiliary module includes a radial cryogenic printing module; The radial cryogenic printing module includes a radial cooling ring, which is used to alternately maintain low temperature and freeze the layer-by-layer printed support. The radial cooling ring is arranged on the printing platform of the 3D printer. Multiple radial air inlets are opened on the outer side of the radial cooling ring, and multiple air outlets facing the printing work area are provided on the inner side. The cooling gas transmission device introduces low-temperature gas at a set temperature into the air inlets of the vertical cooling ring and the radial cooling ring, respectively.

[0012] As a preferred technical solution, the cooling gas transmission device includes a gas pump, a gas guide pipe, a liquid nitrogen tank, a liquid nitrogen flow regulating valve, a liquid nitrogen transmission pipe, a liquid nitrogen spray gun, and a multi-turn interface connected in sequence; one end of the multi-turn interface is connected to the liquid nitrogen spray gun; and multiple air outlets at the other end are respectively connected to the air inlet of the liquid nitrogen cooling ring.

[0013] As a preferred technical solution, the 3D cryogenic printing system has a resistance wire wound around the outside of the 3D printer needle. The resistance wire is connected to a power source, and when the resistance wire is energized, it generates heat to heat the needle.

[0014] As a second aspect of the present invention, a 3D cryoprinting method for anisotropic scaffolds is provided. The method is based on the 3D cryoprinting system for anisotropic scaffolds as described above. By setting the configuration and arrangement of the gas flow channels in the liquid nitrogen cooling ring and constructing the corresponding cooling mode, anisotropic hydrogel scaffolds with specific microstructure orientations are printed.

[0015] As a preferred technical solution, the method employs a vertical cryo-printing module to assist a 3D printer in cryo-printing a hydrogel scaffold with a vertical microstructure. The specific steps are as follows: When the printing height is less than the set height threshold, the vertical cooling device in the vertical cryogenic printing module is used to assist printing. The hydrogel scaffold with uniform or gradient internal microstructure is prepared by adjusting the temperature mode of the thermoelectric cooler. When the printing height exceeds the set height threshold, the vertical cooling ring in the vertical cryogenic printing module is used to assist printing, performing layer-by-layer cooling and ultraviolet crosslinking. The specific steps are as follows: After each layer is printed, the printer nozzle is moved so that the air outlet of the vertical cooling ring is aligned with the hydrogel scaffold that has been printed on the printing platform. Liquid nitrogen gas with a temperature lower than the freezing point of the hydrogel is used to cool the hydrogel scaffold, freezing the printed hydrogel and shaping the hydrogel scaffold to form internal vertical micropores. After cooling is completed, the hydrogel scaffold is subjected to ultraviolet crosslinking. When a scaffold with varying internal microstructure pore size is fabricated, the temperature of the thermoelectric cooler is controlled to remain constant. When preparing a scaffold with uniform internal microstructure pore size, the temperature of the thermoelectric cooler is adjusted to decrease with the printing height, and the height of hydrogel freezing during the printing process is controlled to be less than the printing height.

[0016] As a preferred technical solution, the method employs a radial cryo-printing module to assist a 3D printer in cryo-printing a hydrogel scaffold with a radial microstructure. The specific steps are as follows: Based on the designed model, the hydrogel scaffold is printed layer by layer. After each layer is printed, the printer nozzle moves up and then the cooling gas transmission device is activated. The hydrogel scaffold is maintained at low temperature by using liquid nitrogen gas with a temperature between the freezing point and gelation point of the hydrogel through the radial cooling ring. After printing a set number of layers, the hydrogel scaffold is cryogenically molded using liquid nitrogen gas at a temperature lower than the freezing point of the hydrogel through a radial cooling ring, forming internal radial channels; after cryogenic molding, the hydrogel scaffold is cross-linked with ultraviolet light. Repeat the above layer-by-layer printing process to finally obtain an integrated anisotropic scaffold with radial microstructure.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1) The system proposed in this invention can construct a scaffold with complex geometry and anisotropic microstructure in an integrated manner. By integrating 3D extrusion printing and ice template method, it overcomes the limitations of traditional extrusion printing technology in terms of model printing height, complexity and biomimeticity of microstructure.

[0018] 2) The vertical cryo-printing strategy proposed in this invention employs a self-designed vertical liquid nitrogen ring to output liquid nitrogen in a vertical cooling mode, enabling the formation of vertical microstructures within the printed sample. Layer-by-layer cooling with liquid nitrogen rings and ultraviolet crosslinking achieve unrestricted printing height. The resulting vertical microstructure hydrogel scaffold exhibits extremely strong mechanical properties, dozens of times superior to traditional GelMA hydrogel scaffolds. 3) The radial cryo-printing scheme proposed in this invention can achieve different cooling modes in the printing area through liquid nitrogen rings of different designs, thereby constructing customized anisotropic microstructures. For example, radial liquid nitrogen rings are used to alternately maintain the low temperature and freeze-form the layer-by-layer printed scaffold. This enables the printing of hydrogel scaffolds with excellent mechanical properties, large size, and radial microstructures.

[0019] 4) The vertical and radial cryogenic printing schemes proposed in this invention can print large and complex models based on ultra-low viscosity ink (2% GelMA), which greatly expands the range of inks that can be selected for extrusion printing technology.

[0020] 5) The anisotropic scaffold constructed by this invention is not limited to vertical and radial microstructures. By adjusting the design of the liquid nitrogen ring, anisotropic scaffolds with corresponding microstructures can be constructed, which greatly expands its applicability in different biological or engineering scenarios.

[0021] 6) The vertically aligned scaffold (VAS; radially aligned scaffold (RAS) constructed in this invention can guide cells to migrate directionally along the scaffold microstructure in vitro. VAS and RAS scaffolds can guide endogenous bone regeneration processes in vivo. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the 3D vertical cryogenic printing platform of the present invention.

[0023] Figure 2 This is a schematic diagram of the 3D radial cryogenic printing platform of the present invention.

[0024] Figure 3 A schematic diagram of the microstructure of the vertical microstructure hydrogel scaffold prepared for temperature fixation of the cooling chip (-20℃).

[0025] Figure 4 Vertical microstructured hydrogel scaffold microstructures were prepared for reducing the temperature gradient of the cooling chip (-5℃ to -20℃).

[0026] Figure 5 This is a schematic diagram illustrating the mechanical properties of the vertical microstructure support (VAS) printed in this invention.

[0027] Figure 6 This is a schematic diagram of the microstructure of a radial microstructure scaffold printed using 2% and 5% GelMA inks.

[0028] Figure 7 This study compares the printability of vertical / radial cryogenic printing technology with traditional 3D extrusion printing in the construction of large-scale complex models and examines their differences in microstructure.

[0029] Figure 8 This is a schematic diagram of a liquid nitrogen ring that can be sprayed with an angled stream of cooled liquid nitrogen.

[0030] Figure 9 Fluorescence images of anisotropic scaffold microstructures printed using different liquid nitrogen rings.

[0031] Figure 10 This diagram illustrates how vertical / radial microstructure scaffolds guide cells to migrate rapidly along vertical microstructures.

[0032] Figure 11 Comparison of wound healing using traditional hydrogel scaffolds and the anisotropic scaffold of this application for tissue repair.

[0033] Figure 12 Comparison of bone tissue (skull, femur) repair using traditional hydrogel scaffolds and the anisotropic scaffold of this invention.

[0034] As indicated by the numbers in the diagram: 1. Extrusion 3D printer; 2. Vertical cooling device; 21. Thermoelectric cooler; 22. Water cooling device; 3. Vertical cooling ring; 4. Radial cooling ring; 5. Heating needle. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Example 1 This invention proposes a liquid nitrogen-assisted 3D cryoprinting system capable of printing large-scale, high-precision hydrogel scaffolds with anisotropic microstructures (vertical and radial). It overcomes the significant limitations of traditional 3D extrusion printing technology in terms of sample size and internal microstructural disorder. The system specifically includes an extrusion-type 3D printer 1, a cooling gas delivery device, and vertical and radial cryoprinting modules.

[0037] Among them, such as Figure 1 As shown, the vertical cryogenic printing module includes a vertical cooling device 2 and a vertical cooling ring 3. The vertical cooling device 2 is mounted on the printing platform of the extrusion 3D printer 1 and includes a thermoelectric cooler 21 and a water-cooling device 22. The cooling side of the thermoelectric cooler 21 faces the 3D printer nozzle, while the water-cooling device 22 is located on the heat-dissipating side of the thermoelectric cooler 21. The vertical cooling ring 3 is mounted on the outside of the 3D printer nozzle and has one or more air inlets and an air outlet pointing vertically downwards towards the printing platform.

[0038] like Figure 2 As shown, the radial cryogenic printing module includes a radial cooling ring 4, which is arranged on the printing platform of the 3D printer. The radial cooling ring 4 has multiple radial air inlets on its outer side and multiple air outlets facing the printing area on its inner side.

[0039] The air inlets of the vertical cooling ring 3 and the radial cooling ring 4 are respectively supplied with low-temperature gas at a set temperature by a cooling gas transmission device.

[0040] Furthermore, the specific operation process of 3D vertical cryogenic printing using the above-mentioned cryogenic printing system is as follows: When the printing height is less than 5 mm, 3D vertical cryogenic printing uses the lower vertical cooling device 2. By adjusting the temperature mode of the cooling plate, hydrogel scaffolds with uniform or gradient internal microstructures can be fabricated. Figure 3 As shown, when the temperature of the cooling chip is fixed, the fabricated support is a gradient support. Figure 4As shown, the internal aperture of the prepared bracket is uniform as the temperature of the cooling element decreases with the printing height. However, regardless of whether the temperature is fixed or gradient, the vertical cooling device 2 has a limitation on the printing height, that is, it can only maintain a freeze-forming (icing) height of 5 mm at -20℃.

[0041] When the printing height is greater than 5 mm, 3D vertical cryogenic printing uses the upper vertical liquid nitrogen ring 5 for layer-by-layer cooling and ultraviolet cross-linking, and finally prints an anisotropic support with both large size and internal vertical microstructure.

[0042] like Figure 5 As shown, the mechanical properties of the vertical microstructure hydrogel scaffold (VAS) printed using the system and method of this invention are significantly better than those of the traditional GelMA scaffold. This method can overcome the limitations of printing height, and the printed scaffold has an internal microstructure with strong mechanical properties. The pore size and porosity of the scaffold are adjustable.

[0043] Furthermore, the specific operation process of 3D radial cryogenic printing using the above-mentioned cryogenic printing system is as follows: A radial liquid nitrogen ring is attached to the printing platform of an extrusion 3D printer, and then three air inlets are connected to a liquid nitrogen spray gun via liquid nitrogen transfer pipes.

[0044] The cooling mode of this method is divided into low-temperature maintenance (temperature between the freezing point and gelation point of the hydrogel, slightly above 0°C in this embodiment) and freeze-forming (temperature below the freezing point of the hydrogel, -20°C in this embodiment). Specifically: One layer is printed, then the printer nozzle is moved 30mm using a preset G-code. The liquid nitrogen pump is then activated, delivering liquid nitrogen gas at a temperature above 5°C (for 30 seconds; in this embodiment, the hydrogel used is methacrylamide gelatin (GelMA), whose gelation point is around 25°C, so 5°C is sufficient for forming without freezing, above 0°C) into the liquid nitrogen ring. This process is repeated once. When printing the third layer, the printer nozzle is moved 30mm using a preset G-code, and the liquid nitrogen pump is activated, delivering liquid nitrogen gas at -20°C into the liquid nitrogen ring, freezing the first three layers of the sample and simultaneously forming internal radial channels. This is then cross-linked with ultraviolet light for 30 seconds. This process is repeated with three layers as one cycle to print a large-size, radial hydrogel scaffold.

[0045] like Figure 6 The image shows the microstructures of radial microstructure scaffolds printed using GelMA with different concentrations. It is evident that the proposed method can print anisotropic hydrogel models with radial microstructures. Figure 6 As shown in GH, the printed radial microstructure hydrogel scaffold (RAS) exhibits significantly superior pore size, porosity, and mechanical properties compared to traditional non-cryogenic printed hydrogels.

[0046] like Figure 7 As shown, the proposed solution can print large-size complex (circular tube) models, and vertical / radial microstructures are visible within the tube model. The proposed solution far surpasses traditional extrusion printing technology in its ability to print tubular models.

[0047] like Figure 8 , 9 As shown, based on the cryoprinting platform proposed in this invention, different anisotropic microstructures can be constructed by adjusting the design of the liquid nitrogen ring. A unidirectional, angled liquid nitrogen gas flow can form near-vertical or angled microstructure arrangements, with the microstructure pattern determined by the relative relationship between the liquid nitrogen gas flow and the material contact surface. Furthermore, by using liquid nitrogen gas flows in three different directions, anisotropic microstructures corresponding to the direction of the liquid nitrogen gas flow can be constructed in three regions. The cryoprinting platform based on this invention can theoretically print anisotropic scaffolds suitable for different application scenarios.

[0048] like Figure 10-12 As shown, the anisotropic scaffold printed based on the proposed scheme of this invention can guide cells to migrate rapidly and directionally along the microstructure and accelerate the regeneration of various anisotropic tissues, including but not limited to wound healing and bone defect repair. Vertical and radial microstructure scaffolds can guide cells around the damaged area to rapidly infiltrate the scaffold, initiating and accelerating tissue repair. 3D cryo-printing technology can prepare anisotropic biological scaffolds with vertical and radial microstructures. The anisotropic pore design can accurately simulate the structural characteristics of natural tissues and the formation pattern of granulation tissue during tissue regeneration, i.e., the growth patterns in the vertical direction (up and down) and the radial direction (from the periphery to the center). Furthermore, adjustable scaffold parameters (pore size, porosity, mechanical properties) can simulate the structural characteristics of natural tissues and matrix stiffness. Currently used bone graft substitutes in clinical practice suffer from microstructure deficiencies that affect the implant's osteoinduction and osseointegration capabilities. This unique microstructure design helps promote the regeneration of various anisotropic tissues (such as skin, long bones, flat bones, etc.).

[0049] The scaffold of this invention can effectively guide the migration of endogenous cells, accelerate the repair process of tissue damage, and avoid complications such as immune rejection and uncontrolled differentiation that may be caused by the implantation of exogenous cells and growth factors. This anisotropic scaffold can form mature new tissue with a morphology close to that of natural tissue extracellular matrix (ECM), exhibiting good biocompatibility and tissue regeneration effects. This invention has broad application prospects in the biomedical field, can significantly improve the effect of anisotropic tissue regeneration, and provides an innovative technical means for personalized medicine.

[0050] Example 2 As one embodiment of the present invention, this embodiment further provides a specific implementation method for 3D vertical cryogenic printing using the above-mentioned 3D cryogenic printing system, such as... Figure 1 As shown, the 3D vertical cryogenic printing device includes: a vertical cooling unit 2, a liquid nitrogen transfer device, a vertical liquid nitrogen ring, and a heating needle 5. The vertical cooling unit 2 consists of a thermoelectric cooler 21 (TEC) and a water-cooling system. Utilizing the Peltier effect, when direct current passes through the thermoelectric cooler TEC (a couple composed of P-type and N-type semiconductor materials), one end cools while the other end releases heat. The water-cooling system consists of a metal heat-conducting block, a water pump, water pipes, and coolant (a mixture of ice and water), used to remove heat from the heat-releasing side of the TEC. The liquid nitrogen transfer device includes a liquid nitrogen tank, a gas pump, a liquid nitrogen spray gun, a splitter connector, a regulating valve, and a liquid nitrogen transfer pipe. This component is used to precisely control the flow rate (temperature) of liquid nitrogen pumped from the liquid nitrogen tank. One end of the splitter connector connects to the liquid nitrogen spray gun, and the other end has two outlets for connecting to the air inlet of the vertical liquid nitrogen ring 4 and the airflow temperature sensor, respectively. The air inlet of the vertical liquid nitrogen ring 4 transports liquid nitrogen into the ring and forms a vertically downward liquid nitrogen airflow, which is used for the cryogenic molding of hydrogel ink and the formation of microstructures during the printing process. The heated needle 5 is made of a 2 cm long resistance wire wound around a metal needle. When the resistance wire is connected to a power source, it generates heat to raise the temperature of the needle, which is used to prevent the ink from solidifying at low temperatures during the cryogenic printing process and causing the needle to become clogged.

[0051] 1.1) Attach the heat-dissipating end of the cooling chip to the metal block of the water-cooling device, and then attach it to the printing platform of the 3D extrusion printer as a platform for vertical cryogenic printing.

[0052] 1.2) When the height of the printed sample is less than 5 mm, the cryogenic molding of the hydrogel is achieved by the cooling end of the cooling plate. As the printing height increases, the temperature of the cooling end is set to a gradient cooling mode from -5℃ to -20℃ to ensure the uniformity of the pore size of the microstructure inside the cryogenic printing holder.

[0053] Specifically, the power is turned on and the voltage of the thermoelectric cooler is set to 1V, at which point the temperature of the cooling element is -5℃. As the printing height increases, the voltage of the thermoelectric cooler is increased, lowering the temperature of the cooling element and thus increasing the ice-forming height. Simultaneously, during the voltage increase, the ice-forming height is ensured to be slightly less than the printing height to prevent the hydrogel from freezing completely, clogging the printer nozzles, and causing printing failure. Furthermore, this step ensures the continuity of the vertical microstructure of the sample, eliminating printing streaks. When the voltage is adjusted to 6V, the cooling element temperature can be achieved to -20℃, at which point the ice-forming height can reach 5mm.

[0054] 1.3) When the printed sample height is greater than 5 mm, liquid nitrogen ring-assisted printing is used. After each layer is printed, the printer nozzle is moved up 5 mm and to the right via preset code so that the vent is aligned with the printed hydrogel. Then, the liquid nitrogen pump is activated, guiding -20°C liquid nitrogen gas above the hydrogel. This vertical cooling quickly freezes the printed hydrogel (30 seconds), allowing it to solidify and form internal vertical micropores. The printer nozzle is then moved up 30 mm via preset control code to allow space for the UV lamp; subsequently, the printed hydrogel support is cross-linked using ultraviolet light (30 seconds). Repeating these steps increases the printing height.

[0055] like Figure 7 As shown, the hollow cylindrical tube printed using the 3D vertical cryogenic printing module of this invention exhibits uniform pore size and porosity at different printing heights. This demonstrates that the module proposed in this invention can print complex, centimeter-sized samples with uniform vertical microstructures.

[0056] Example 3 As another embodiment of the present invention, this embodiment further provides a specific implementation of 3D radial cryoprinting using the above-mentioned 3D cryoprinting system, such as... Figure 2 As shown, the 3D radial cryogenic printing device includes: a liquid nitrogen transport device, a radial liquid nitrogen ring 4, and a heating needle 5. The liquid nitrogen transport device includes a liquid nitrogen tank, a gas pump, a liquid nitrogen spray gun, a 1-to-4 adapter, a regulating valve, and a liquid nitrogen transport tube. One end of the 1-to-4 adapter connects to the liquid nitrogen spray gun, and the other end has four outlets for connecting to the three inlets of the radial liquid nitrogen ring 6 and the liquid nitrogen gas flow temperature sensor (any one outlet is acceptable). The remaining liquid nitrogen transport device components and the heating needle 5 use the same 3D vertical cryogenic printing module. The radial liquid nitrogen ring 6 transports liquid nitrogen gas into the ring to form a radial liquid nitrogen gas flow for the cryogenic molding of hydrogel ink and the formation of microstructures during the printing process.

[0057] The operation process is as follows: The radial liquid nitrogen ring 6 is attached to the printing platform 2 of a traditional 3D extrusion printer, and then the three air inlets are connected to the liquid nitrogen spray gun through liquid nitrogen transfer pipes. The cooling modes of this method are divided into cryogenic maintenance (slightly above 0°C) and cryogenic forming (-20°C). After the model is designed, the layer-by-layer printing process is started. After printing three layers (the first two layers are cryogenically maintained after printing, and the third layer is cryogenically formed after printing, realizing the cryogenic forming of the three-layer hydrogel scaffold and the formation of internal microstructures), ultraviolet crosslinking is performed once. The above process is repeated to finally print an anisotropic scaffold with both large size and radial microstructure in one piece.

[0058] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A 3D cryogenic printing system for anisotropic scaffolds, characterized in that, The 3D cryogenic printing system is used to assist an extrusion 3D printer (1) in printing anisotropic hydrogel scaffolds, including a cooling gas transmission device and a cryogenic printing auxiliary module; The cryogenic printing auxiliary module includes a liquid nitrogen cooling ring, on which one or more gas channels with a set configuration and arrangement are provided. The air inlet of the gas channel is connected to the output end of the cold gas transmission device, and the air outlet of the gas channel is oriented towards the printing platform of the extrusion 3D printer (1) in a specific direction, so as to guide the hydrogel support to form anisotropic microstructures with a specific orientation during the printing process.

2. The 3D cryogenic printing system for anisotropic scaffolds according to claim 1, characterized in that, The cryogenic printing auxiliary module includes a vertical cryogenic printing module; The vertical cryogenic printing module includes: a vertical cooling device (2) and a vertical cooling ring (3); the vertical cooling device (2) is set on the printing platform of the extrusion 3D printer (1) and is used for vertical cryogenic printing below a set height. It includes a thermoelectric cooler and a water cooling circulation system. The cooling side of the thermoelectric cooler faces the nozzle of the 3D printer. The water cooling circulation system is set on the heat-releasing side of the thermoelectric cooler and is used to remove the heat from the heat-releasing side of the thermoelectric cooler. The vertical cooling ring (3) is installed on the outside of the nozzle of the 3D printer and is used for cryogenic printing above a set height. The vertical cooling ring (3) is provided with one or more air inlets and an air outlet that is vertically downward toward the printing platform.

3. The 3D cryogenic printing system for anisotropic scaffolds according to claim 2, characterized in that, The water-cooled circulation system includes a metal heat-conducting block, a water pump, and water pipes. The water pump circulates coolant to the metal heat-conducting block through the water pipes for cooling. The metal heat-conducting block is connected to the heat-dissipating end of the thermoelectric cooler and carries away the heat from the heat-dissipating side of the thermoelectric cooler.

4. The 3D cryogenic printing system for anisotropic scaffolds according to claim 2, characterized in that, The interior of the vertical cooling ring (3) is divided into an independent cooling air duct and an installation area by a partition; the cooling air duct is provided with one or more air inlets and an air outlet that is vertically downward toward the printing platform; a guide plate is provided in the cooling air duct near the air inlet, and the cross-section of the guide plate is an arc shape with the center of the circle facing the air inlet; the nozzle of the 3D printer vertically penetrates the installation area of ​​the vertical cooling ring (3) and is connected and fixed to the vertical cooling ring (3).

5. The 3D cryogenic printing system for anisotropic scaffolds according to claim 1, characterized in that, The cryogenic printing auxiliary module includes a radial cryogenic printing module; The radial cryogenic printing module includes a radial cooling ring (4) for alternating low-temperature maintenance and cryogenic molding of the layer-by-layer printed support. The radial cooling ring (4) is arranged on the printing platform of the 3D printer. Multiple radial air inlets are provided on the outer side of the radial cooling ring (4), and multiple air outlets facing the printing work area are provided on the inner side. The cooling gas transmission device introduces low-temperature gas at a set temperature into the air inlets of the vertical cooling ring (3) and the radial cooling ring (4), respectively.

6. The 3D cryogenic printing system for anisotropic scaffolds according to claim 1, characterized in that, The cooling gas transmission device includes a gas pump, a gas guide pipe, a liquid nitrogen tank, a liquid nitrogen flow regulating valve, a liquid nitrogen transmission pipe, a liquid nitrogen spray gun, and a multi-turn interface connected in sequence; one end of the multi-turn interface is connected to the liquid nitrogen spray gun; and multiple air outlets at the other end are respectively connected to the air inlet of the liquid nitrogen cooling ring.

7. The 3D cryogenic printing system for anisotropic scaffolds according to claim 1, characterized in that, The 3D cryogenic printing system has a resistance wire wound around the outside of the 3D printer's needle. The resistance wire is connected to a power source, and when energized, it generates heat to heat the needle.

8. A method for 3D cryoprinting an anisotropic scaffold, characterized in that, The method is based on a 3D cryoprinting system for anisotropic scaffolds as described in any one of claims 1-6. By setting the configuration and arrangement of the gas flow channels in a liquid nitrogen cooling ring and constructing a corresponding cooling mode, anisotropic hydrogel scaffolds with specific microstructure orientations are printed.

9. The printing method according to claim 8, characterized in that, The method employs a vertical cryo-printing module to assist a 3D printer in cryo-printing hydrogel scaffolds with vertical microstructures. The specific steps are as follows: When the printing height is less than the set height threshold, the vertical cooling device (2) in the vertical freezing printing module is used to assist printing. The hydrogel scaffold with uniform or gradient internal microstructure is prepared by adjusting the temperature mode of the thermoelectric cooler. When the printing height is greater than the set height threshold, the vertical cooling ring (3) in the vertical freezing printing module is used to assist printing, and the layer-by-layer cooling and ultraviolet crosslinking are carried out. The specific steps are as follows: After each layer is printed, the printer nozzle is controlled to move so that the outlet of the vertical cooling ring (3) is aligned with the hydrogel support printed on the printing platform. Liquid nitrogen gas with a temperature lower than the freezing point of hydrogel is used to cool the hydrogel support, freeze the printed hydrogel and shape the hydrogel support and form the internal vertical micropores. After cooling is completed, the hydrogel scaffold is subjected to ultraviolet crosslinking. When a scaffold with varying internal microstructure pore size is fabricated, the temperature of the thermoelectric cooler is controlled to remain constant. When preparing a scaffold with uniform internal microstructure pore size, the temperature of the thermoelectric cooler is adjusted to decrease with the printing height, and the height of hydrogel freezing during the printing process is controlled to be less than the printing height.

10. The printing method according to claim 8, characterized in that, The method employs a radial cryo-printing module to assist a 3D printer in cryo-printing a hydrogel scaffold with a radial microstructure. The specific steps are as follows: Based on the designed model, the hydrogel scaffold is printed layer by layer. After each layer is printed, the printer nozzle moves up and then the cooling gas transmission device is activated. The hydrogel scaffold is maintained at low temperature by using liquid nitrogen gas with a temperature between the freezing point and gelation point of the hydrogel through the radial cooling ring (4). After printing a set number of layers, the hydrogel scaffold is frozen and molded using liquid nitrogen gas with a temperature lower than the freezing point of the hydrogel through a radial cooling ring (4), and internal radial channels are formed; after the freeze-molding process is completed, the hydrogel scaffold is cross-linked with ultraviolet light. Repeat the above layer-by-layer printing process to finally obtain an integrated anisotropic scaffold with radial microstructure.

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