A frozen field biological 3D printing forming device and application thereof

Abstract

This invention belongs to the field of additive manufacturing technology, and particularly relates to a cryogenic bio-3D printing molding device and its application. The molding device includes an extrusion module, a freezing module, and a molding platform. The extrusion module is positioned above the molding platform, and the freezing module provides a freezing environment for the molding platform. The freezing module includes a freezing zone, which comprises a freezing chamber and a freezing component. The freezing component provides a low-temperature environment for the freezing chamber. The freezing chamber includes a supporting bottom and a freezing outer wall, with the outer wall surrounding the supporting bottom. The height of the freezing outer wall is greater than 1 cm. The molding platform is positioned on the supporting bottom. The ink extruded by the extrusion module is deposited, cooled, and solidified on the molding platform. This invention is applicable to slurries of different concentrations, including low-concentration slurries, and can print products with relatively high heights.

Description

Technical Field

[0001] This invention belongs to the field of additive manufacturing technology, and particularly relates to a cryogenic biological 3D printing molding device and its application, specifically to a cryogenic biological 3D printing molding device and a method for printing inorganic / organic composite porous biological scaffolds using the device. Background Technology

[0002] Tissue and organ defects and failures caused by diseases, traumas, etc. seriously affect human life and health. The human body has limited self-repair capabilities. When the damage reaches a certain level, in-situ treatment can no longer meet the actual needs, and transplantation is required. As a result, tissue engineering for tissue and organ replacement has become a research focus in the fields of medicine, biology, and materials science.

[0003] Based on their origin, grafts can be divided into two main categories: biological grafts and artificial grafts. Biological grafts include four types: autologous grafts, allogeneic grafts, allogeneic grafts, and xenografts. They have disadvantages such as easy damage to the donor site, donor shortage, and immune rejection, resulting in a low success rate. Therefore, the development of artificial transplant tissues / organs holds great promise.

[0004] The term tissue engineering was established in the 1980s, referring to the emerging discipline that combines cell biology and materials science to construct tissues or organs in vitro or in vivo to restore damaged tissues or organs. The three essential elements of tissue engineering are cells, scaffolds, and the culture environment. Cells refer to stem cells, which can develop into any type of tissue and organ; they are highly plastic and are also known as "pluripotent cells." The culture environment refers to the signaling molecules required for cell growth, namely proteinaceous substances that have biological effects such as inducing and stimulating cell proliferation and maintaining cell survival. Scaffolds are supporting materials used to support cell growth and proliferation, and come in several categories, including polymers, metals, bioceramics, and their composites. To improve cell adhesion, promote cell proliferation and differentiation, and facilitate cell ingrowth into the scaffold, the supporting material is often a three-dimensional porous structure with interconnected structures.

[0005] Methods for preparing three-dimensional porous materials include freeze-drying, template methods, etching, high internal phase emulsion polymerization, and 3D printing. Among these, 3D printing, also known as additive manufacturing, is a technology that uses digital model files as a basis to create designed structures through layer-by-layer printing. It offers advantages such as saving raw materials, high precision, and customization. Therefore, bio-3D printing technology has received increasing attention and is widely used in tissue-engineered heart valves, tissue-engineered blood vessels, tissue-engineered trachea, tissue-engineered intestines, tissue-engineered lungs, tissue-engineered kidneys, and tissue-engineered bone. To maintain the good structure of three-dimensional porous materials, the printing ink used in 3D printing must be able to solidify in a timely manner. Solidification methods include solvent evaporation, ionic crosslinking, and freezing. Solvent evaporation requires solvents with low boiling points and easy volatility, and they are mostly organic solvents that are difficult to completely remove through post-processing. Ionic crosslinking methods, such as sodium alginate-calcium ion systems, although the crosslinking process is mild, can still damage cells if the cells are immersed in high-osmotic-pressure, high-ion-concentration solutions for a long time. Freezing field methods not only achieve rapid molding but also have the advantages of being non-toxic and easy to operate, making them an ideal strategy for solidifying bio-3D printing materials.

[0006] There are currently three main types of freezing methods: The first involves printing the material at room temperature and then transferring it to a freeze dryer for freeze-setting and drying. However, this method is not suitable for slurries with low concentrations, and the three-dimensional structure of the material is prone to collapse during the printing or transfer process, making it difficult to achieve good shape control. The second method involves pouring the slurry into a model, placing it in a low-temperature environment, and directly molding and freezing it. This method is difficult to achieve personalized customization or porous structures. The third method involves printing the material directly in a low-temperature environment. However, this low-temperature environment is only provided by a low-temperature plane. The temperature of the printing environment gradually increases at a certain distance from the low-temperature plane. For structures larger than 1 cm, the low-temperature plane cannot provide the necessary freezing and setting conditions, which greatly limits the molding height and makes it difficult to print products with a height greater than 1 cm. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a cryogenic biological 3D printing molding apparatus and its application. This apparatus can improve at least one of the following problems: it is suitable for slurries of different concentrations, including slurries with lower concentrations, and it can print products with higher heights, such as greater than 1 cm.

[0008] This invention provides the following technical solution:

[0009] In a first aspect, a cryogenic biological 3D printing molding device includes an extrusion module, a freezing module, and a molding platform. The extrusion module is disposed above the molding platform, and the freezing module is used to provide a freezing environment for the molding platform.

[0010] The freezing module includes a freezing field, which includes a freezing chamber and a freezing component. The freezing component is used to provide a low-temperature environment for the freezing chamber. The freezing chamber includes a supporting bottom and a freezing outer wall. The outer wall is wrapped around the supporting bottom and the height of the freezing outer wall is greater than 1 cm. The forming platform is set on the supporting bottom. The ink extruded by the extrusion module is deposited, cooled and shaped on the forming platform.

[0011] According to an embodiment of the present invention, the height of the frozen outer wall is greater than or equal to 3cm, preferably greater than or equal to 4cm, for example 4.5cm. When the height of the frozen outer wall is 4.5cm, it is possible to print products with a height of less than or equal to 3cm.

[0012] According to an embodiment of the present invention, the refrigeration assembly includes a refrigeration unit and a refrigeration circulation group. The refrigeration unit is used to provide a refrigeration medium to the refrigeration chamber, and the refrigeration medium can circulate between the refrigeration chamber and the refrigeration unit. The refrigeration circulation group surrounds the refrigeration chamber and provides a refrigeration environment for the refrigeration chamber.

[0013] According to an embodiment of the present invention, the freezing field includes a freezing chamber and a freezing circulation group surrounding the freezing chamber, wherein the top of the cooling circulation group is a groove-type open structure, and the groove is the freezing chamber.

[0014] According to an embodiment of the present invention, the molding apparatus further includes a temperature measurement module, which includes a plurality of temperature detectors for detecting the temperature at different locations, such as the temperature inside the refrigeration unit or the freezing chamber.

[0015] According to an embodiment of the present invention, the molding apparatus further includes an external control system, which includes a computer or the like.

[0016] According to an embodiment of the present invention, the extrusion module is used to store and extrude bio-ink, the extrusion module includes a barrel and a heating cylinder, the barrel being placed in the heating cylinder, and the heating cylinder being insertable into a freezing chamber.

[0017] According to an embodiment of the present invention, the heating cylinder is connected to a position adjustment mechanism, the position adjustment mechanism including a Y-axis linear motion mechanism, a double Z-axis linear motion mechanism and an X-axis linear motion mechanism.

[0018] According to an embodiment of the present invention, the position adjustment mechanism further includes a base frame, a Y-axis linear motion mechanism is disposed at the bottom of the base frame, and a freezing cycle assembly is disposed at the top of the Y-axis linear motion mechanism. The Y-axis linear motion mechanism includes a sliding track, and the freezing cycle assembly is capable of sliding on the sliding track.

[0019] According to an embodiment of the present invention, a double Z-axis linear motion mechanism is provided on opposite sides of the base frame, and an X-axis linear motion mechanism is provided on the upper part of the double Z-axis linear motion mechanism, and the heating cylinder is connected to the X-axis linear motion mechanism.

[0020] According to an embodiment of the present invention, when the X-axis linear motion mechanism moves up and down on the double Z-axis linear motion mechanism, the distance between the heating cylinder and the forming platform can be changed; when the double Z-axis linear motion mechanism moves on the base frame, the distance between the heating cylinder and the inner wall of the freezing chamber can be changed; when the freezing cycle group slides on the Y-axis linear motion mechanism, the distance between the heating cylinder and the inner wall of the freezing chamber can be changed.

[0021] According to an embodiment of the present invention, the Y-axis linear motion mechanism, the double Z-axis linear motion mechanism, and the X-axis linear motion mechanism are driven by cylinders to perform corresponding actions.

[0022] According to an embodiment of the present invention, limit sensors are provided at the ends of the Y-axis linear motion mechanism, the dual Z-axis linear motion mechanism and the X-axis linear motion mechanism to limit the movement range of the print head and ensure the safe operation of the motion mechanism.

[0023] According to an embodiment of the present invention, the adjustment mechanism may further include a rotary motion mechanism for changing the position of the heating cylinder to meet the printing requirements of more complex parts.

[0024] According to an embodiment of the present invention, an air pressure adjustment mechanism is further provided outside the heating cylinder. The air pressure adjustment mechanism is connected to the material cylinder inside the heating cylinder and is used to adjust the air pressure inside the material cylinder to squeeze out the ink.

[0025] According to an embodiment of the present invention, the air pressure adjustment mechanism includes an air compressor, and the air compressor is connected to the heating cylinder via a connecting pipe.

[0026] According to an embodiment of the present invention, a temperature and pressure control system is provided between the air compressor and the heating cylinder, and the temperature and pressure control system is provided with a temperature adjustment knob and a pressure adjustment knob for adjusting the pressure and temperature of the gas entering the heating cylinder.

[0027] According to an embodiment of the present invention, the cooling circulation group includes a circulation cavity, which is a space for containing coolant. The outer wall of the circulation cavity is provided with a coolant circulation outlet and a coolant circulation inlet. The upper surface of the circulation cavity has a grooved open structure, which can better provide a low-temperature freezing field.

[0028] According to an embodiment of the present invention, a heat insulation layer is provided on the outer wall of the circulation cavity. The heat insulation layer can be made of any material capable of heat insulation, such as foam.

[0029] According to an embodiment of the present invention, the circulation chamber is connected to the refrigeration unit via a low-temperature insulated pipe.

[0030] According to an embodiment of the present invention, the heating cylinder includes a heating cylinder body, the interior of the heating cylinder body is used to accommodate a material cylinder, a printing needle is provided at the bottom of the material cylinder, the material cylinder is used to contain bio-ink, and a material cylinder fixing buckle is provided at the top of the heating cylinder body to fix the material cylinder. The material cylinder fixing buckle is provided with a gas interface.

[0031] According to an embodiment of the present invention, the gas provided by the air compressor is connected to the heating cylinder via an interface. After the bio-ink is loaded into the cartridge, it is placed in the heating cylinder for heating, ensuring that the ink does not freeze prematurely at the needle tip or inside the printing syringe and thus cannot be extruded.

[0032] According to an embodiment of the present invention, the connecting pipe and the low-temperature insulated pipe may be equipped with flow control valves for controlling the flow rate of the corresponding liquid or gas.

[0033] According to an embodiment of the present invention, the temperature detection module, flow control valve, and cylinder are connected to an external control system, such as a wired or wireless connection, for controlling and collecting relevant position, temperature, and other information.

[0034] According to an embodiment of the present invention, the refrigeration module can be directly refrigerated with liquid nitrogen, purged with liquid nitrogen converted into cold nitrogen gas, or refrigerated with coolant. Optionally, when refrigeration with coolant is used, in order to achieve temperature adjustment from 30 to -120°C to meet the printing needs of different inks, the refrigeration unit contains two compressors, namely a main compressor and an auxiliary compressor.

[0035] According to an embodiment of the present invention, an illumination unit, such as an LED light source, is provided inside or outside the circulation cavity, and the illumination unit can shine into the freezing cavity to perform curing printing.

[0036] According to an embodiment of the present invention, the outer wall of the circulation cavity is made of a metal with high thermal conductivity, such as copper.

[0037] According to an embodiment of the present invention, the connecting channel between the refrigeration unit and the circulation chamber is a low-temperature silicone tube, and to maintain the temperature of the cooling medium, it is wrapped with a layer of antifreeze and heat-insulating foam.

[0038] According to an embodiment of the present invention, the number of printheads is at least one, for example, one, two, three, four or more; optionally, when two or more printheads are provided, it can be coaxial printing or simple multi-head printing.

[0039] According to an embodiment of the present invention, the extrusion module can be a pneumatic extrusion module, a screw extrusion module, or a mechanical extrusion module; optionally, when a pneumatic extrusion module is used, a gas inlet is provided, and the gas is supplied by an air compressor, thereby realizing the smooth extrusion and precise stopping of the bio-ink.

[0040] According to an embodiment of the present invention, the heating cylinder has a temperature adjustment function, and its temperature setting range is 10 to 100°C.

[0041] According to an embodiment of the present invention, the heating cylinder is made of a metal with good thermal conductivity, such as copper, and the exterior is made of polytetrafluoroethylene, which is temperature-controlled and prevents burns.

[0042] According to an embodiment of the present invention, the diameter of the print head can be 0.16 to 3 mm, for example, the inner diameter can be 0.16 mm, 0.21 mm, 0.26 mm, 0.34 mm, 0.41 mm, 0.51 mm, 0.60 mm, 0.84 mm, 1.20 mm, 1.36 mm, 1.55 mm, 2 mm, 2.5 mm or 3 mm.

[0043] According to an embodiment of the present invention, the molding platform is made of a metal with good thermal conductivity, such as copper.

[0044] According to an embodiment of the present invention, the size of the molding platform is slightly smaller than the size of the freezing chamber.

[0045] According to an embodiment of the present invention, the molding platform is provided with a connector, such as a hook, to facilitate the removal of the molding platform.

[0046] According to an embodiment of the present invention, the temperature of the freezing chamber can be controlled by the refrigeration unit and monitored in real time by the digital display screen contained therein. Since the cooling medium circulates outside the refrigeration unit, the actual temperature of the circulation chamber will be slightly lower than the set temperature.

[0047] According to an embodiment of the present invention, the actual temperature of the freezing area can be monitored by an external temperature measurement module, such as a near-infrared thermal imager, an infrared thermometer, or a low-temperature recorder with a temperature-sensing probe.

[0048] Secondly, the present invention provides an application of the above-described molding apparatus in 3D printing.

[0049] Thirdly, the present invention also provides a method for 3D printing using the above-mentioned molding apparatus, comprising the following steps:

[0050] Add bio-ink to the cartridge, insert the insulation cylinder, adjust the position of the insulation cylinder in the freezing chamber, and run the 3D printer with the pre-set slicing program to obtain a solidified support under the action of the freezing zone.

[0051] According to an embodiment of the present invention, after obtaining the cured scaffold, the following step is further included: freeze-drying the cured scaffold.

[0052] According to an embodiment of the present invention, the bio-ink is selected from existing bio-inks.

[0053] According to an embodiment of the present invention, the bio-ink comprises an inorganic phase solid powder, an organic phase solid powder, and a solvent.

[0054] According to an embodiment of the present invention, the inorganic phase solid powder and the organic phase solid powder are solutes, and the mass ratio of the solute to the solvent is 5-30%, preferably 10-20%.

[0055] According to an embodiment of the present invention, the mass ratio of the inorganic phase solid powder to the organic phase solid powder is (4-7):(3-6).

[0056] According to an embodiment of the present invention, when a pneumatic extrusion module is used, its pressure can be 0.1 to 0.8 MPa, for example, 0.2 MPa, 0.3 MPa, 0.4 MPa, or 0.6 MPa.

[0057] According to an embodiment of the present invention, the heating temperature of the heating cylinder can be 10 to 100°C, for example, 30°C, 40°C, 50°C, 60°C, or 80°C.

[0058] According to an embodiment of the present invention, the inorganic phase is bioceramic, bioglass, or a mixture thereof, such as one or more of hydroxyapatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, calcium hydrogen phosphate, calcium sulfate, calcium silicate, etc.

[0059] According to an embodiment of the present invention, the inorganic phase may be a natural polymer, an artificial polymer, or a mixture thereof and its modified form, such as sodium alginate, gelatin, collagen, chitosan, cellulose, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyurethane, polydioxanone, polyetheretherketone, polyetherketoneketone, etc.

[0060] According to an embodiment of the present invention, the ratio of inorganic components to organic components in the inorganic-organic composite scaffold can be 1:(0.3 to 1.5), for example 4:6, 5:5, 6:4, 7:3, etc.

[0061] According to an embodiment of the present invention, the bio-ink further comprises at least one of, for example, cells, drugs, growth factors, peptides, etc.

[0062] According to an embodiment of the present invention, the shape of the inorganic-organic composite scaffold can be customized into regular or irregular shapes, such as a column or cylinder; the filling density can be 40-90%, such as 50%, 60%, 70%, 80%, etc.

[0063] According to an embodiment of the present invention, the post-processing method of the inorganic-organic composite scaffold is freeze-drying, so as to better preserve the original macroscopic morphology of the scaffold. The freeze dryer can be a commercially available freeze dryer, such as a temperature-controlled one or a non-temperature-controlled one. The freeze-drying temperature is -30 to -80°C, for example, -35°C, 45°C, or 65°C; the freeze-drying time is 24 to 72 hours, for example, 30 hours, 45 hours, or 60 hours.

[0064] Beneficial effects

[0065] 1. The device of the present invention integrates the 3D printing and freeze-drying process of bio-ink, which significantly shortens the post-processing time of the scaffold and improves efficiency. The efficiency is higher than that of the existing methods of first 3D printing and then freeze-drying or simply molding and freezing.

[0066] 2. The freezing chamber of the device of the present invention has a semi-enclosed structure. Except for the open structure at the top, its side walls and bottom are closed, and the side walls have a certain height, which can better provide a low-temperature freezing field. The bottom and inner wall of the freezing chamber are in contact with the freezing medium, thus forming an enveloping freezing field. Compared with the freezing field of the prior art that only includes a low-temperature bottom surface, the present invention can print ink with a lower concentration (e.g., concentration less than or equal to 20%), and the minimum printing concentration can reach 10%. It can also print products with a higher height, such as 3 cm or higher, without collapsing.

[0067] 3. The device of the present invention includes a heating cylinder with a temperature regulation function, which ensures that the ink will not freeze prematurely at the needle tip or inside the printing syringe in a freezing environment and thus cannot be extruded; and an insulation layer is provided outside the heating cylinder to prevent the temperature of the heating cylinder from spreading to the freezing area and affecting the low temperature environment in the freezing area.

[0068] 4. The forming platform of the device of the present invention is placed inside the freezing chamber. When printing, the ink is deposited on the forming platform from bottom to top in a set manner. Under the action of the freezing field, it is quickly frozen and solidified. Then the sample stage is taken out and the sample is freeze-dried together with the sample for subsequent post-processing. This can avoid the frozen sample from becoming brittle or damaged during the transfer process, which is more convenient and faster, and does not affect subsequent printing tasks.

[0069] 5. The device of the present invention broadens the limitations of 3D printing bio-inks. Bio-inks with lower concentration and lower viscosity are beneficial to cell survival. However, the resulting low energy modulus makes it impossible for them to be well formed in ordinary 3D printing environments. Under the action of the freezing field, the ink can achieve rapid freezing and forming. Attached Figure Description

[0070] Figure 1 This is a schematic diagram of the structure of the cryogenic biological 3D printing molding device in Example 1; wherein, 1-1 is the refrigeration unit, 1-2 is the refrigeration cycle group, 1-3 is the heating cylinder, 1-4 is the Y-axis linear motion mechanism, 1-5 is the double Z-axis linear motion mechanism, 1-6 is the X-axis linear motion mechanism, 1-7 is the air pressure and temperature controller, 1-8 is the temperature adjustment knob, 1-9 is the air pressure adjustment knob, and 1-10 is the air compressor.

[0071] Figure 2 This is a schematic diagram of the refrigeration circulation unit; where 2-1 is the coolant circulation outlet and 2-2 is the coolant circulation inlet.

[0072] Figure 3 This is a cross-sectional view of the refrigeration cycle unit; where 3-1 is the forming platform, 3-2 is the coolant, and 3-3 is the outer wall.

[0073] Figure 4 This is a schematic diagram of the heating cylinder; where 4-1 is the gas interface.

[0074] Figure 5 This is a cross-sectional view of the heating cylinder; where 5-1 is the cylinder fixing lock, 5-2 is the heating cylinder body, 5-3 is the bio-ink, and 5-4 is the needle.

[0075] Figure 6 This is a simulation diagram of the internal temperature distribution of the refrigeration cycle group in Example 1.

[0076] Figure 7 The image shows the morphology and cross-sectional SEM micrograph of the COLD-CMC / TCP prepared in Example 2.

[0077] Figure 8 The image shows the morphology of the COLD-CMC / TCP-Scaffold prepared in Example 3.

[0078] Figure 9 The image shows the morphology of the COLD-CMC / TCP-High Scaffold prepared in Example 4.

[0079] Figure 10 Morphology of the CMC / TCP-Scaffold prepared for Comparative Example 1.

[0080] Figure 11Morphology of CMC / TCP-Scaffold-P prepared for Comparative Example 2. Detailed Implementation

[0081] The structure of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0082] In the description of this invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0083] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0084] Example 1

[0085] A cryogenic bio-3D printing molding device includes an extrusion module, a freezing module, and a molding platform 3-1. The extrusion module is disposed above the molding platform 3-1, and the freezing module is used to provide a freezing environment for the molding platform 3-1. The extrusion module includes molding control components and dispensing jet components.

[0086] The freezing module includes a freezing field, which includes a freezing chamber and a freezing component. The freezing component is used to provide a low-temperature environment for the freezing chamber. The freezing chamber includes a supporting bottom and a freezing outer wall. The freezing outer wall is wrapped around the supporting bottom and has a height greater than 1 cm. The forming platform 3-1 is set on the supporting bottom. The ink extruded by the extrusion module is deposited, cooled and shaped on the forming platform 3-1.

[0087] The refrigeration assembly includes a refrigeration unit and a refrigeration circulation unit. The refrigeration unit is used to provide a refrigerant to the refrigeration chamber. The refrigerant can circulate between the refrigeration chamber and the refrigeration unit. The refrigeration circulation unit surrounds the refrigeration chamber and provides a refrigeration environment for the refrigeration chamber. The refrigeration chamber includes a refrigeration chamber and a refrigeration circulation unit surrounding the refrigeration chamber. The top of the refrigeration circulation unit has a recessed open structure, and the recess is the refrigeration chamber.

[0088] Specifically, see Figure 1 As shown, the refrigeration module includes a refrigeration unit 1-1, a refrigeration cycle group 1-2, a temperature measurement module, and an external control system. The refrigeration unit 1-1 is used to provide the refrigeration medium to the refrigeration cycle group 1-2. The refrigeration medium can circulate between the refrigeration cycle group 1-2 and the refrigeration unit 1-1. The external control system includes a computer, etc. The temperature measurement module includes several temperature detectors for detecting the temperature inside the refrigeration unit 1-1, the refrigeration cycle group 1-2, and the refrigeration chamber.

[0089] The top of the freezing cycle group 1-2 is a grooved open structure, and the groove forms a freezing chamber. The freezing chamber is used to provide a cooling space for the ink to be cooled. In this embodiment, the ink is stored in a barrel. In order to prevent the ink from freezing when it is not in operation, the barrel is placed in a heating cylinder 1-3. The heating cylinder 1-3 can be inserted into the groove of the freezing cycle group.

[0090] See Figure 1 As shown, in order to facilitate the adjustment of the position of the heating cylinder 1-3, such as in the X, Y, Z or other directions, the heating cylinder 1-3 is connected to a position adjustment mechanism. The position adjustment mechanism includes a Y-axis linear motion mechanism 1-4, a dual Z-axis linear motion mechanism 1-5 and an X-axis linear motion mechanism 1-6, and may also include a rotation mechanism. The rotation mechanism can drive the needle at the bottom of the heating cylinder 1-3 to move to any position in the freezing chamber, thereby realizing printing at any position.

[0091] In practical use, the position adjustment mechanism can also be a multi-axis robotic arm. The multi-axis robotic arm can be 4-axis, 6-axis, 8-axis or other structures. The multi-axis robotic arm includes a clamping component and a moving component. The clamping component is used to clamp the heating cylinders 1-3, and the moving component is used to drive the clamping component to move.

[0092] Specifically, the position adjustment mechanism also includes a base frame, a Y-axis linear motion mechanism 1-4 located at the bottom of the base frame, and a freezing circulation group 1-2 located at the top of the Y-axis linear motion mechanism 1-4. The Y-axis linear motion mechanism 1-4 includes a sliding rail, on which the freezing circulation group 1-2 can slide. A double Z-axis linear motion mechanism 1-5 is located on opposite sides of the base frame, and an X-axis linear motion mechanism 1-6 is located above the double Z-axis linear motion mechanism 1-5. The heating cylinder 1-3 is connected to the X-axis linear motion mechanism 1-6. When the X-axis linear motion mechanism 1-6 moves up and down on the double Z-axis linear motion mechanism 1-5, it can change the distance between the heating cylinder 1-3 and the forming platform 3-1. When the freezing circulation group 1-2 slides on the Y-axis linear motion mechanism 1-4, it can also change the relative position between the heating cylinder 1-3 and the inner wall of the freezing chamber.

[0093] Among them, the Y-axis linear motion mechanism 1-4, the double Z-axis linear motion mechanism 1-5, and the X-axis linear motion mechanism 1-6 are driven by cylinders to perform corresponding actions.

[0094] An air pressure adjustment mechanism is also provided on the outside of the heating cylinder 1-3. The air pressure adjustment mechanism is connected to the material cylinder inside the heating cylinder 1-3 and is used to adjust the air pressure in the material cylinder, thereby applying pressure to the ink in the material cylinder and squeezing out the ink. The air pressure adjustment mechanism includes an air compressor 1-10. The air compressor 1-10 is connected to the heating cylinder 1-3 through a connecting pipe. A temperature control system 1-7 is provided between the air compressor 1-10 and the heating cylinder 1-3. The temperature control system 1-7 is provided with a temperature adjustment knob 1-8 and an air pressure adjustment knob 1-9, which are used to adjust the pressure and temperature of the gas entering the heating cylinder 1-3.

[0095] See Figure 2 and Figure 3 As shown, the refrigeration circulation group 1-2 is a space for containing coolant. The outer wall 3-3 of the refrigeration circulation group 1-2 is provided with a coolant circulation outlet 2-1 and a coolant circulation inlet 2-2. An insulation layer is provided on the outer wall 3-3. The insulation layer can be any material that can provide insulation, such as foam.

[0096] The refrigeration cycle group 1-2 is connected to the refrigeration unit 1-1 through a low-temperature insulated pipe. Its structure can be set according to actual needs, such as a quadrangular prism structure. In this embodiment, the forming platform 3-1 is set in the middle of the refrigeration cycle group 1-2. The upper surface of the cavity is a grooved open structure, which can better provide a low-temperature freezing field. The forming platform 3-1 is embedded in the refrigeration cycle group 1-2, and the coolant 3-2 circulates continuously inside the cavity to continuously provide a low-temperature printing environment.

[0097] The structure of the cooling chamber can be designed according to actual needs, for example, as follows: Figure 3The cuboid shown has a rectangular longitudinal section. In order to avoid the air at the top of the cooling cavity interfering with the temperature of the upper part of the cooling cavity, it can also be set as a cavity structure with a top area smaller than the bottom area, wherein the area of ​​the forming platform 3-1 is smaller than the top area.

[0098] See Figure 4 , Figure 5 As shown, the heating cylinder 1-3 includes a heating cylinder body 5-2. The interior of the heating cylinder body 5-2 is used to accommodate a material cylinder. A printing needle 5-4 is provided at the bottom of the material cylinder. The material cylinder is used to contain bio-ink 5-3. A material cylinder fixing lock 5-1 is provided at the top of the heating cylinder body 5-2. The material cylinder fixing lock 5-1 is used to fix the material cylinder. A gas interface 4-1 is provided on the material cylinder fixing lock 5-1 to achieve good airtightness. Under the action of air pressure, the bio-ink is extruded from the needle 5-4 and then deposited on the sample stage 3-1. Under the action of the freezing field, it is rapidly frozen and solidified.

[0099] The heating cylinder 5-2 is provided with an insulation layer on the outside to prevent the heat on the surface of the heating cylinder 5-2 from affecting the temperature inside the cooling chamber. Specifically, the heating cylinder 5-2 may include a heating layer and at least two insulation layers arranged sequentially from the inside to the outside. The heating layer generates heat through built-in heating wires, etc., and the heating layer is connected to a power source to provide the electrical energy required for heating.

[0100] The gas supplied by the air compressor 1-10 is connected to the heating cylinder through interface 4-1. After the bio-ink 5-3 is loaded into the barrel, it is placed into the heating cylinder 5-2 for heating, ensuring that the ink will not freeze prematurely at the needle tip or inside the printing syringe and thus cannot be squeezed out.

[0101] Flow control valves can be installed on connecting pipes and low-temperature insulated pipes to control the flow rate of the corresponding liquids or gases.

[0102] The temperature detection module, flow control valve, and cylinder are connected to an external control system, such as a wired or wireless connection, to control and collect relevant information such as position and temperature.

[0103] The temperature distribution of the cooling circulation chamber of the cryogenic biological 3D printing molding device was simulated using ANASY software, and the results are as follows: Figure 6 As shown, the cold plate temperature is preset to -70℃. It can be seen that the center temperature of the molding platform 3-1 placed in the groove of the freezing cycle group 1-2 is slightly higher than that of the surrounding area. The groove depth is 4.5cm, and the actual printed bracket height is 2cm, which is sufficient for the application. The measured temperature of the upper layer is about -32℃. The simulated temperature is in the yellow area, which is between -35℃ and -30℃. The actual temperature is very close to the simulation result, which can realize the rapid molding of the upper bracket.

[0104] Example 2

[0105] A 3D printing method for inorganic-organic composite bioscaffold filaments using the aforementioned cryogenic field.

[0106] The method for printing a 3D scaffold using carboxymethyl cellulose and tricalcium phosphate as the main materials of bio-ink and deionized water as the solvent includes the following steps:

[0107] Step 1: Weigh 3g of tricalcium phosphate powder and 2g of carboxymethyl cellulose powder into a 100mL beaker and stir well. Then add 50mL of deionized water and stir continuously with a magnetic stirrer until the carboxymethyl cellulose is completely dissolved and the carboxymethyl cellulose and tricalcium phosphate are mixed evenly. The resulting suspension is the 3D printing bio-ink.

[0108] Step 2: Use the modeling software Solidworks to construct a cylindrical model with a diameter of 2×20mm. Use the slicing software Ultimaker to obtain a slicing file that matches the printer and set the nozzle diameter to 2mm.

[0109] Step 3: Turn on the refrigeration system, set the temperature to -80℃, and place the molding platform 3-1 into the groove of the freezing circulation group 1-2 for pre-cooling.

[0110] Step 4: Load the uniform bio-ink obtained in Step 1 into the syringe (i.e., the ink cartridge), insert the printing needle 5-4 into the bottom of the ink cartridge, close the ink cartridge fixing lock 5-1, insert the air tube connected to the air pressure and temperature control system, turn on the heating switch of the heating cylinder 5-2, set the temperature to 40℃, and preheat the printing ink.

[0111] Step 5: Use an external low-temperature temperature recorder to measure the temperature of the sample stage in the freezing cycle group. Once it approaches the preset temperature, printing can begin.

[0112] Step Six: Start the printer, adjust the air pressure and speed to match, then select the set slice file to start the straight printing process of the filament. Under the action of the freezing field, it will be quickly cured to obtain a filament with a diameter of 2mm and a length of 20mm.

[0113] Step 7: Take out the filament obtained in Step 6 along with the forming platform 3-1 and put it into a freeze dryer for drying. Set the freeze dryer parameters to freeze temperature -55℃ and freeze time 48h. Finally, name it COLD-CMC / TCP.

[0114] Microscopic morphology of the cross-section of COLD-CMC / TCP filament is shown in the figure. Figure 7 As shown, the cross-section is a uniform circular structure, arranged in an orderly radial pattern from bottom to top.

[0115] Example 3

[0116] A 3D printing method for inorganic-organic composite porous biological scaffolds using the aforementioned cryogenic field.

[0117] Using carboxymethyl cellulose and tricalcium phosphate as the main materials for bio-ink and deionized water as the solvent, the printing of its 3D scaffold includes the following steps:

[0118] Step 1: Weigh 3g of tricalcium phosphate powder and 2g of carboxymethyl cellulose powder into a 100mL beaker and stir well. Then add 50mL of deionized water and stir continuously with a magnetic stirrer until the carboxymethyl cellulose is completely dissolved and the carboxymethyl cellulose and tricalcium phosphate are mixed evenly. The resulting suspension is the 3D printing bio-ink.

[0119] Step 2: Use the modeling software Solidworks to construct a cylindrical model with a diameter of 15×5mm. Use the slicing software Ultimaker to obtain a slicing file that matches the printer. Set the nozzle diameter to 0.6mm, the trace width to 0.6mm, the layer height to 0.6mm, and the fill density to 50%.

[0120] Step 3: Turn on the refrigeration system, set the temperature to -80℃, and place the molding platform inside the freezing chamber for pre-cooling.

[0121] Step 4: Load the uniform bio-ink obtained in Step 1 into the syringe (i.e., the ink cartridge), insert the printing needle 5-4 into the bottom of the syringe, close the ink cartridge locking buckle 5-1, insert the air tube connected to the air pressure and temperature control system, turn on the heating switch of the heating cylinder 5-2, set the temperature to 40℃, and preheat the printing ink.

[0122] Step 5: Use an external low-temperature temperature recorder to measure the temperature of the forming platform 3-1 in the freezing cycle group. Once the temperature is close to the preset temperature, printing can begin.

[0123] Step Six: Start the printer, adjust the air pressure and speed to match, then select the set slice file and start printing layer by layer. Under the action of the freezing field, each layer is cured to obtain a scaffold with a porous structure.

[0124] Step 7: Remove the scaffold obtained in Step 6 along with the molding platform 3-1 and place it in a freeze dryer for drying. Set the freeze dryer parameters to -55℃ and 48h. The resulting scaffold is named COLD-CMC / TCP-Scaffold. Figure 8 It can be seen that the support has uniform pores and a good structure.

[0125] Example 4

[0126] A 3D printing method for inorganic-organic composite porous biological scaffolds using the aforementioned cryogenic field.

[0127] Except for the cylindrical model, which has dimensions of Φ15×20mm, everything else is identical to Example 3, and it is named COLD-CMC / TCP-High Scaffold. From Figure 9 It can be seen that the upper layer of the support has a good morphology and clear side holes, achieving a good shape control effect.

[0128] Comparative Example 1

[0129] A 3D printing method for inorganic-organic composite bioscaffolds that uses freeze drying to replace the aforementioned freezing process.

[0130] Using carboxymethyl cellulose and tricalcium phosphate as the main materials for bio-ink and deionized water as the solvent, the printing of its 3D scaffold includes the following steps:

[0131] Step 1: Weigh 3g of tricalcium phosphate powder and 2g of carboxymethyl cellulose powder into a 100mL beaker and stir well. Then add 50mL of deionized water and stir continuously with a magnetic stirrer until the carboxymethyl cellulose is completely dissolved and the carboxymethyl cellulose and tricalcium phosphate are mixed evenly. The resulting suspension is the 3D printing bio-ink.

[0132] Step 2: Use the modeling software Solidworks to construct a cylindrical model with a diameter of 15×5mm. Use the slicing software Ultimaker to obtain a slicing file that matches the printer. Set the nozzle diameter to 0.6mm, the trace width to 0.6mm, the layer height to 0.6mm, and the fill density to 50%.

[0133] Step 3: Load the uniform bio-ink obtained in Step 1 into a syringe, insert the needle, close the syringe locking buckle, and connect the air tube to the air pressure and temperature control system.

[0134] Step 4: Start the printer, adjust the air pressure and speed to match, then select the set slice file and start printing layer by layer to obtain the bracket.

[0135] Step 5: Remove the scaffold obtained in Step 5 along with the sample stage and place it in a freeze dryer (Shanghai Bilang Instrument Manufacturing Co., Ltd., VFD-1000) for drying. Set the freeze dryer parameters to -55℃ and 48h. The resulting scaffold is named CMC / TCP-Scaffold. Figure 10 It can be seen that the pores of the support have collapsed, and there is no interconnected porous structure. Figure 8 , Figure 9 The comparison further demonstrates the role of the cryogenic module in maintaining the pre-set porous structure of the support.

[0136] Comparative Example 2

[0137] A 3D printing method for inorganic-organic composite bioscaffolds using existing planar cooling to provide a cooling field.

[0138] Using carboxymethyl cellulose and tricalcium phosphate as the main materials for bio-ink and deionized water as the solvent, the printing of its 3D scaffold includes the following steps:

[0139] Step 1: Weigh 3g of tricalcium phosphate powder and 2g of carboxymethyl cellulose powder into a 100mL beaker and stir well. Then add 50mL of deionized water and stir continuously with a magnetic stirrer until the carboxymethyl cellulose is completely dissolved and the carboxymethyl cellulose and tricalcium phosphate are mixed evenly. The resulting suspension is the 3D printing bio-ink.

[0140] Step 2: Use the modeling software Solidworks to construct a cylindrical model with a diameter of 15×20mm. Use the slicing software Ultimaker to obtain a slicing file that matches the printer. Set the nozzle diameter to 0.6mm, the trace width to 0.6mm, the layer height to 0.6mm, and the fill density to 50%.

[0141] Step 3: Turn on the refrigeration system, place the molding platform on the flat refrigeration plate, set the refrigeration temperature to -80℃, and pre-cool.

[0142] Step 4: Load the uniform bio-ink obtained in Step 1 into a syringe, insert the needle, close the syringe locking buckle, and connect the air tube to the air pressure and temperature control system.

[0143] Step 5: When the temperature drops to the preset temperature, start the printer, adjust the air pressure and speed to match, then select the set slice file and start printing layer by layer to obtain the bracket.

[0144] Step Six: Remove the scaffold obtained in Step Five along with the sample stage and place it in a freeze dryer for drying. Set the freeze dryer parameters to -55℃ and 48h. The resulting scaffold is named CMC / TCP-Scaffold-P. Figure 11 It can be seen that the bottom layer of the support structure can maintain the preset shape well due to the low temperature, but as the height of the support structure increases, the temperature of the upper layer is insufficient to allow the slurry to solidify quickly, resulting in shrinkage and central collapse. Figure 8 , Figure 9 The comparison further demonstrates the role of the freezing module in maintaining the pre-set porous structure of the support at a certain height.

[0145] In Comparative Example 2, the conditions were the same as in Example 3, except that the cold space used was provided by an existing planar refrigeration system.

[0146] The specific embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above exemplary embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A freeze field biological 3D printing forming device, comprising an extrusion module, a freezing module and a forming platform, characterized in that, The extrusion module is positioned above the molding platform, and the freezing module provides a freezing environment for the molding platform. The freezing module includes a freezing field, which includes a freezing chamber and a freezing component. The freezing component is used to provide a low-temperature environment for the freezing chamber. The freezing chamber includes a supporting bottom and a freezing outer wall. The outer wall is wrapped around the supporting bottom and the height of the freezing outer wall is greater than 1 cm. The forming platform is set on the supporting bottom. The ink extruded by the extrusion module is deposited, cooled and shaped on the forming platform. The refrigeration assembly includes a refrigeration unit and a refrigeration circulation unit. The refrigeration unit is used to provide a refrigeration medium to the refrigeration chamber. The refrigeration medium can circulate between the refrigeration chamber and the refrigeration unit. The refrigeration circulation unit surrounds the refrigeration chamber and provides a refrigeration environment for the refrigeration chamber. The extrusion module includes a barrel and a heating cylinder, the barrel being placed inside the heating cylinder, and the heating cylinder being able to be inserted into the freezing chamber.

2. The freeze-to-field biological 3D printing forming device according to claim 1, characterized in that, The height of the frozen outer wall is greater than or equal to 3 cm.

3. The freeze-to-field biological 3D printing forming device according to claim 1, characterized in that, The freezing field includes a freezing chamber and a freezing circulation group surrounding the freezing chamber. The top of the freezing circulation group has a groove-type open structure, and the groove is the freezing chamber.

4. The freeze-to-field biological 3D printing forming device according to any one of claims 1-3, characterized in that, The molding apparatus also includes a temperature measurement module, which includes several temperature detectors used to detect the temperature at different locations.

5. The freeze-to-field biological 3D printing forming device according to claim 4, characterized in that, The heating cylinder is connected to a position adjustment mechanism, which includes a Y-axis linear motion mechanism, a double Z-axis linear motion mechanism, and an X-axis linear motion mechanism.

6. The freeze-to-field biological 3D printing forming device according to claim 5, characterized in that, The position adjustment mechanism also includes a base frame, a Y-axis linear motion mechanism is located at the bottom of the base frame, and a refrigeration circulation group is located at the top of the Y-axis linear motion mechanism. The Y-axis linear motion mechanism includes a sliding track, and the refrigeration circulation group can slide on the sliding track.

7. The freeze-to-field biological 3D printing forming device according to claim 5, characterized in that, The position adjustment mechanism further includes a rotary motion mechanism for changing the position of the heating cylinder.

8. The freeze-to-field biological 3D printing forming device according to claim 4, characterized in that, The heating cylinder includes a heating cylinder body, the interior of which is used to house the material cylinder, the bottom of which is provided with a printing needle, and the material cylinder is used to hold bio-ink.

9. The application of the molding apparatus according to any one of claims 1-8 in 3D printing.

10. A method for 3D printing using the molding apparatus according to any one of claims 1-8, comprising the following steps: Add bio-ink to the barrel, insert the heating cylinder, adjust the position of the heating cylinder in the freezing chamber, run the 3D printer with the pre-set slicing program, and obtain a solidified scaffold under the action of the freezing zone. Then freeze-dry the solidified scaffold.

11. The printing method according to claim 10, characterized by, The bio-ink comprises inorganic phase solid powder, organic phase solid powder and solvent, wherein the inorganic phase solid powder and organic phase solid powder are solutes, and the mass ratio of solute to solvent is 5-30%.

12. The printing method according to claim 11, characterized by, The mass ratio of solute to solvent is 10-20%.

13. The printing method of claim 11, wherein, The mass ratio of the inorganic phase solid powder to the organic phase solid powder is (4-7):(3-6).

14. The printing method of claim 11, wherein, The bio-ink also contains at least one of cells, drugs, growth factors, and peptides, and the inorganic-organic composite scaffold has a filling density of 40-90%.

Images