Method and apparatus for manufacturing a printout having a cross-sectional pattern

Abstract

This invention relates to a printing apparatus using multiple inks and a printing method using the same apparatus. More specifically, the invention relates to a printing apparatus using multiple inks, the printing apparatus comprising an ink extrusion member, the ink extrusion member comprising: a storage member divided into a plurality of receiving members for respectively receiving multiple inks; and an extrusion member having a single channel through which the multiple inks received in the storage members pass. The invention also relates to a three-dimensional printing method for producing a printed output with a cross-sectional pattern, the method comprising the steps of: using the printing apparatus to supply different inks to the divided spaces and applying the same pressure conditions to the received inks, so as to extrude the inks received in two or more divided spaces through a single extrusion port, thereby producing an ink extrusion product and printing.

Description

Technical Field

[0001] This invention relates to an apparatus for preparing printed products having cross-sectional patterns and a method for preparing such printed products using the apparatus. More specifically, this invention relates to a printing apparatus capable of preparing printed products having cross-sectional patterns using extruded products obtained from two or more different inks, and a printing method using the printing apparatus. Background Technology

[0002] Human organs or tissues can be constructed from biomaterials that make up various cells and the extracellular matrix. Research is actively underway to regenerate desired functional tissues by using three-dimensional (3D) bioprinting technology to create cellular structures similar to those that make up human tissues.

[0003] Head modules used in bioprinting technology can be broadly categorized into inkjet-based and extrusion-based printing modules. In addition, various methods using lasers, ultrasound, etc., have been proposed, but these two types of modules are the most widely used.

[0004] The physical properties of the bioink required for such bioprinting technology vary significantly depending on the printhead module. In extrusion-based printing systems, there are no significant limitations on material viscosity compared to jet-based technologies. Therefore, the range of applicable biomaterials becomes much wider compared to inkjet-based printing technologies. Furthermore, the ease of fabricating thick layers facilitates the preparation of cell structures of the sizes required for clinical applications.

[0005] However, the resolution of bioprinting technologies developed to date based on fused deposition modeling is only a few hundred micrometers, while the basic structure of organs or tissues in the human body is vastly different, ranging from tens of micrometers or smaller. In particular, the diameter of capillaries that supply nutrients to the cells that make up organs or tissues is 3 to 4 micrometers, making them difficult to achieve with existing bioprinting technologies.

[0006] Furthermore, there are cases where multiple inks are used for bioprinting. For example, since cell function is improved when cells are partitioned between different types of cells rather than simply mixed and ejected, it is necessary to eject multiple inks for bioprinting when the hydrogel and curing agent are cured together or when various cells are co-cultured. Conventional methods attempt to address this by using multiple heads that non-uniformly contain multiple materials, but this results in drawbacks such as increased printing processing time, adverse effects on cell viability, and increased system complexity.

[0007] To improve the resolution of printing technology, nozzles with small diameters should be used. However, when using nozzles with small diameters, there is a side effect: the material extruded from inside the nozzle during extrusion generates shear stress between the material and the nozzle wall, thus reducing cell viability. Furthermore, due to the problem of cells being frequently killed by shear stress, it is difficult to blindly reduce the nozzle diameter. Summary of the Invention

[0008] Technical issues

[0009] The present invention relates to an ink extrusion member and a printing method using the ink extrusion member, wherein the ink extrusion member is used to prepare a printed product having a cross-sectional pattern by using an extruded ink product containing two or more different inks.

[0010] The present invention relates to a printing apparatus for preparing a printed product having a cross-sectional pattern by using an extruded ink product containing two or more different inks, and a printing method using the printing apparatus.

[0011] The purpose of this invention is to provide an apparatus for printing biological tissue shapes with complex cross-sectional structures with high precision and high resolution, as well as a printing method using the apparatus.

[0012] The present invention provides a printing apparatus and a printing method that can non-uniformly print desired shapes and significantly reduce the shear stress of cells.

[0013] Technical solution

[0014] This invention relates to an ink extrusion member, a printing apparatus comprising the ink extrusion member, and a printing method using the printing apparatus. The ink extrusion member prepares a printed product with a cross-sectional pattern by using an extruded ink product comprising two or more different inks. Preferably, the printing apparatus can be a bioprinting apparatus and method for preparing artificial tissues, organs, etc.

[0015] Another embodiment of the present invention relates to a method for three-dimensional printing a printed product having a cross-sectional pattern. The method includes: providing different inks to each compartment of an ink extrusion member, the ink extrusion member being configured in a three-dimensional printing apparatus and including an ink receiving component for receiving ink, an ink extrusion member, and a partition member providing two or more compartments; applying the same pressure conditions to the ink received in each compartment, thereby extruding the ink received in the two or more compartments into a single extrusion nozzle to prepare an extruded ink product; and printing the extruded ink product onto a plate. The ink extrusion member can provide two or more compartments to have the same cross-sectional pattern as the three-dimensional printed product through the partition members.

[0016] Specifically, the method for preparing a printed product having a cross-sectional pattern according to the present invention relates to a three-dimensional printing method for a printed product having a cross-sectional pattern, the three-dimensional printing method comprising:

[0017] The step of providing different inks to each compartment of an ink extrusion component, the ink extrusion component including an ink receiving component and an ink extrusion component, the ink receiving component including a separating member providing two or more compartments having the same cross-sectional pattern as the printed product, and the ink receiving component receiving each different ink in the compartments separated by the separating member, the ink extrusion component being located below the ink receiving component and equipped with a single channel for ink passage and extrusion.

[0018] The steps of applying the same pressure conditions to the ink received in each compartment, thereby extruding the ink received in two or more compartments into a single extrusion port to prepare an extruded ink product, and

[0019] The step of printing the extruded ink product onto a plate.

[0020] In a three-dimensional printing method for a printed product with a cross-sectional pattern, each different ink for printing is provided to each space separated by a partition member in an ink receiving component, and pressure is applied simultaneously by a single pressure member or by two or more pressure members to print the ink through an ink extrusion member or nozzle into an extruded product, and the extruded product or printed product has the same cross-sectional pattern as the printed product. Preferably, the ratio of the cross-sectional pattern of the ink receiving component to the cross-sectional pattern of the extruded ink product or printed ink product, for example, the ratio of the diameter of the cross section, can be reduced to a ratio of 100:99 to 100:0.1, 100:50 to 100:1, or 100:18 to 100:1.

[0021] Other embodiments of the present invention relate to a three-dimensional printing apparatus for a printed product having a cross-sectional pattern, the three-dimensional printing apparatus comprising:

[0022] An ink extrusion component includes an ink receiving component and an ink extrusion component. The ink receiving component includes a separating member that provides two or more partitioned spaces having the same cross-sectional pattern as the printed product. The ink receiving component receives each different type of ink in the spaces partitioned by the separating member. The ink extrusion component is located below the ink receiving component and is equipped with a single channel through which the ink contained in the ink receiving component is extruded.

[0023] A nozzle, the nozzle being connected to the end of the ink extrusion component, and

[0024] Pressure member, which applies pressure to the ink received in each compartment.

[0025] Each different ink is printed through an ink extrusion component equipped with a single channel so that it has the same cross-sectional pattern shape as the printed ink product.

[0026] The printing apparatus according to the invention can apply pressure using a single pressure member, or by using two or more pressure members to apply pressure at the same pressure, so as to apply the same pressure conditions to the ink received in each compartment. By applying pressure using pressure members, each different ink can be printed by an ink extrusion component equipped with a single channel to have the same cross-sectional pattern shape as the printed product. Therefore, when using the printing apparatus according to the invention, there is the advantage of being able to prepare printed products with various cross-sectional patterns, and in particular, biological tissues with complex cross-sectional structures can be prepared by printing with high precision and high resolution using a three-dimensional method, and when cells are included in the biological tissue, the desired shape can be printed non-uniformly, and the shear stress on the cells can be greatly reduced.

[0027] The invention will be described in more detail below.

[0028] The three-dimensional printing method for a printed product having a cross-sectional pattern according to the present invention relates to a three-dimensional printing method for a printed product having a cross-sectional pattern, the three-dimensional printing method comprising: providing different inks to each compartment of an ink extrusion member, the ink extrusion member being equipped in a three-dimensional printing apparatus and including an ink receiving member for receiving ink, an ink extrusion member, and a compartment member for providing two or more compartments; applying the same pressure conditions to the ink received in each compartment, thereby extruding the ink received in the two or more compartments into a single extrusion port to prepare an extruded ink product; and printing the extruded ink product onto a plate.

[0029] Applying the same pressure conditions can be performed by using a single pressure member, or by using two or more pressure members to apply pressure at the same pressure. The same pressure conditions are those under which ink received in two or more compartments is extruded through a single extrusion port to form a single extruded ink product, and preferably, this pressure condition allows ink received in two or more compartments to be extruded through a single extrusion port to form a single extruded ink product, and the extruded ink product and the printed product prepared therefrom have the same shape as the cross-sectional pattern of the target printed product.

[0030] The three-dimensional printing method for printed products with cross-sectional patterns according to the present invention can be performed using the ink extrusion member according to the present invention or a three-dimensional printing apparatus including the ink extrusion member according to the present invention.

[0031] A three-dimensional printing apparatus for a printed product with a cross-sectional pattern according to another embodiment of the present invention may include an ink extrusion member comprising an ink receiving member and an ink extrusion member. The ink receiving member includes a separating member providing two or more partitioned spaces to have the same cross-sectional pattern shape as the printed product, and the ink receiving member receives each different ink in the spaces partitioned by the separating member. The ink extrusion member is located below the ink receiving member and is equipped with a single channel through which ink passes, and the ink extrusion member extrudes the ink received in the ink receiving member. Furthermore, the three-dimensional printing apparatus may also include a nozzle connected to the ink extrusion member, and a pressure member applying pressure to the ink received in each partitioned space.

[0032] Additionally, a 3D printing apparatus may include a plate for printing extruded products and components included in a conventional 3D printing apparatus as additional devices for printing.

[0033] Specifically, the three-dimensional printing method for a printed product having a cross-sectional pattern according to the present invention includes: the step of providing different inks to each partition space of an ink extrusion member; the step of applying pressure to the ink received in each partition space, thereby extruding the ink through an extrusion port to prepare an extruded ink product having the same cross-sectional pattern shape as the printed product; and the step of printing the extruded ink product on a plate.

[0034] The following will describe in detail the composition of the apparatus and the steps of the method, including the ink extrusion member or the three-dimensional printing apparatus including the ink extrusion member, and the method for preparing a printed product with a cross-sectional pattern using the ink extrusion member or the three-dimensional printing apparatus including the ink extrusion member.

[0035] The ink extrusion component according to the invention includes an ink receiving component and an ink extrusion component. The ink receiving component includes a separating member that provides two or more partitioned spaces having the same cross-sectional pattern shape as the printed product. The ink receiving component receives each different ink used for printing into the spaces partitioned by the separating member. The ink extrusion component is located below the ink receiving component and is equipped with a single channel for ink passage. The ink extrusion component extrudes the ink received in the ink receiving component.

[0036] When using the ink extrusion member according to the invention, the bio-ink is extruded due to the very large diameter of the printable and commercially viable ink receiving component, and therefore shear stress is hardly applied to the ink or the cells contained therein. Furthermore, compared to prior art using multiple heads that unevenly contain various materials, the present invention can use a single ink extrusion member to extrude two or more different inks together for printing, thus allowing the use of a single printhead, resulting in reduced printing processing time and almost no shear stress applied to the ink or the cells contained therein. Therefore, when using cell-containing ink with the printing method or apparatus according to the invention, there are advantages of high cell viability and a simplified system.

[0037] An extrusion component is a component that supplies ink by pushing it through pressure applied from the outside, and can be a box or syringe commonly used in 3D printing devices.

[0038] The ink receiving component includes a separating member that provides two or more compartments having the same cross-sectional pattern shape as the extruded product, and each different ink can be received in the compartments separated by the separating member. An extruded ink product having the same cross-sectional pattern as the printed product can be prepared through an extrusion orifice equipped with a single channel by applying pressure under the same pressure conditions to the two or more inks provided in each compartment.

[0039] The separator included in the ink extrusion member is integrally formed with the ink receiving member, but it is detachable from the ink extrusion member. Furthermore, the separator can be housed within the ink receiving member itself, or mounted by further including a hollow portion that can be installed within the ink receiving member to provide two or more spaces within the ink receiving member. When a hollow portion is included, the separator can be integrally or detachably disposed with the hollow portion. The separator can be fabricated by various methods, such as injection molding, extrusion, or 3D printing, but preferably by 3D printing.

[0040] The size of the separator can be inserted inside the ink receiving component, and any size and shape, such as cylindrical, square, triangular pyramidal, etc., is possible. The cross-sectional pattern of the separator can be prepared in various shapes; for example, it can have a pattern with the same cross-sectional area as the artificial tissue to be prepared. Examples of various cross-sectional shapes of the separator are shown in... Figure 5 It can include shapes similar to the shape of the urethra, shapes including multiple concentric circles, etc. Figure 5 This is a photograph showing a separator used in an ink extrusion component according to an example of the invention.

[0041] Figure 3 and Figure 4An embodiment of the hollow portion according to the invention is shown. The hollow portion (70) can be installed in the ink extrusion member (10) by combining with a separator member (30) prepared in an integral or detachable form.

[0042] For the materials of the partition components, thermoplastic resins and photocurable resins that can be FDM printed, such as ABS (acrylonitrile butadiene styrene), PCL (polycaprolactone), ASA (acrylonitrile styrene acrylate), SAN (styrene-acrylonitrile copolymer), PS (polystyrene), PPSF / PPSU (polyphenylsulfone), polyetherimide, PLA (polylactic acid), PDL (poly-d-lysine), etc., as well as solid materials such as iron-free / iron-free alloy materials that can be machined, are suitable.

[0043] The ink extrusion component is located below the ink receiving component and includes a single channel for passing through various inks and an ink extrusion component for extruding the ink received in the ink receiving component. The ink extrusion component equipped with a single channel can extrude the ink received in the ink receiving component divided into multiple spaces in a single-channel controlled manner rather than a multi-channel controlled manner.

[0044] The inner diameter of the extrusion orifice is very small. The ink received in the ink receiving component can be extruded to the outside of the ink receiving component through the extrusion orifice. The extruded ink product discharged from the ink extrusion component has the same cross-sectional pattern as the ink receiving component, but with a reduced size.

[0045] The three-dimensional printing apparatus according to the invention may further include a nozzle connected to the end of an ink extrusion member of an extrusion member, through which ink is discharged, and a plate is located below the nozzle. The ink discharged from the nozzle for printing is deposited on the upper part of the plate, thereby producing a printed ink product.

[0046] Preferably, the printing ink product with a cross-sectional pattern according to the present invention is artificial human tissue. For example, it includes muscle tissue (bundle structure), bone tissue (sheet and tube structure), nerve tissue (nerve perithemnium structure), vascular tissue (multilayer structure), spinal cord tissue, etc.

[0047] While reducing the nozzle diameter is necessary to improve the resolution of bioprinting based on fused deposition modeling, smaller nozzle diameters lead to a higher frequency of cell death caused by shear stress between the material extruded from the nozzle and the nozzle wall surface. Therefore, blindly reducing the nozzle diameter is not feasible. Thus, high resolution can be achieved by using an ink receiving component with the same cross-sectional pattern as the ink product being printed and an ink extrusion component that extrudes ink through a single channel.

[0048] The method for printing biological tissue according to the invention allows for pressure control such that the ink contained in the ink receiving component has the same proportions as the pattern of the printed product, but provides an enlarged cross-sectional shape for the ink receiving component, and the cross-sectional pattern of the ink receiving component is maintained by the ink extrusion component having a cross-section smaller than that of the ink receiving component, while retaining the pattern. Preferably, the cross-sectional pattern of the extruded ink product is able to remain the same as the cross-sectional pattern of the ink receiving component.

[0049] According to the present invention, by using large-size printing ink that can be prepared relatively easily, complex microstructures with very small dimensions can be easily printed.

[0050] In this specification, "identical" means not only 100% identical, but also includes the degree to which the same function can be performed. In this specification, "the cross-section maintains the same shape" means that only the size of the cross-section is reduced, while the original shape of the cross-section itself is maintained. Therefore, after manufacturing a printing material with a pre-formed, larger cross-section, tissue cells corresponding to the desired size, i.e., the actual size, can be printed.

[0051] Preferably, the viscosity of the printing ink is such that the cross-section of the ink product extruded through the nozzle can maintain the same shape as the cross-section of the printing material.

[0052] The printed ink products have cross-sections that are too small to be achieved with current bioprinting technology, or microstructures with very low survival rates during printing. As mentioned above, the printed products can be muscle tissue (bundle structures), bone tissue (sheet and tube structures), nerve tissue (nerve perithecia structures), vascular tissue (multilayer structures), spinal cord tissue, etc.

[0053] The cross-section of the ink receiving component in the printing apparatus according to the present invention can have the same cross-sectional pattern as the printed product, and the ratio of the cross-sectional pattern of the ink receiving component to the cross-sectional pattern of the extruded ink product or the printed ink product can be expressed by various methods, such as area ratio and cross-sectional diameter ratio. For example, the ratio of the cross-sectional pattern of the ink receiving component to the cross-sectional pattern of the extruded ink product or the printed ink product, for example, the ratio of the diameter of the cross-sectional pattern, can be reduced to a ratio of 100:99 to 100:0.1, 100:50 to 100:1, or 100:18 to 100:1. However, the reduction ratio is directly affected by the cross-sectional diameter of the ink receiving component, the cross-sectional diameter of the ink extrusion component, or the diameter of the nozzle, and it can be designed differently by appropriately adjusting it according to the cross-sectional pattern size of the printed ink product. The cross-sectional diameter of the printed ink product varies according to the size of the nozzle and is generally used in the range of 0.1 mm to 1 mm, and can be changed according to the printing process such as the properties of the ink material, pressure, printhead speed, and the position of the printed product (print bed).

[0054] According to an example of the invention, this ratio can be reduced by 98.7% (200 μm) from the total diameter (15 mm) of a particular shape (e.g., leaflet). For example, the reduction ratio can be calculated according to the following Equation 1.

[0055] [Equation 1]

[0056] Reduction percentage = 100 - (diameter of ink receiving component / diameter of printed product) × 100 (%)

[0057] Preferably, the ink supplied to the ink extrusion member according to the invention is bio-ink capable of producing artificial organs, etc. Specifically, printing can be performed by supplying different inks to each compartment of an ink receiving member equipped with a separating member, the separating member providing two or more compartments having the same cross-sectional pattern shape as the printed product. Different inks refer to inks in which one or more selected from components, component content, and physical properties are different.

[0058] In this specification, "bioink" includes living cells or biomolecules, and is a material that can be used to construct desired structures through bioprinting technology. The bioink of this invention includes liquid, semi-solid, or solid compositions containing multiple cells.

[0059] Therefore, bio-ink can provide physical properties for 3D processing and biological environments, enabling cells to perform target functions. Preferably, during prolonged printing processes, the ink extrusion component appropriately supplies the nutrients and oxygen required for cell survival. Furthermore, cells should be protected from the physical stresses that occur during printing. In addition, bio-ink should possess the physical properties required for the printing process, namely, the repeatability and productivity of the 3D pattern, and should not clog nozzles, etc.

[0060] Preferably, the ink of the present invention is a hydrogel, and therefore may include a gelling polymer. For example, it may include one or more selected from gelling polymers, cells, growth factors, and extracellular matrix.

[0061] The bio-ink used in this invention is, for example, a hydrogel containing or not containing the desired cells. The hydrogel can be a hydrogel containing growth factors, a hydrogel containing cells and growth factors, a hydrogel containing cytokines, or hydrogels of different types. Preferably, the hydrogel is a collagen, matrix gel, alginate, gelatin, agarose, cellular ink derived from cellular tissues, hyaluronic acid, fibrin gel, or a mixture thereof.

[0062] Furthermore, bio-ink diffuses more quickly due to its lower viscosity. The viscosity of bio-ink, measured at 25°C, is higher than that of water (1 cp), and for example, bio-ink is a gel phase material with a viscosity of 2 cp to 1,000,000 cp, such as 2 cp to 10,000 cp or 5 cp to 1,000,000 cp. The viscosity of the gel phase material used in the method of the present invention is preferably a suitable viscosity for printing materials that can be extruded in the extrusion method described below. According to one example of the invention, due to the use of an extrusion-type 3D printing method, a higher ink viscosity is preferably provided compared to inkjet methods. In one example, various thickeners can be used for the ink suitable for the present invention to provide a suitable extrusion viscosity. The viscosity of the printing material is at a level where the cross-section of the extruded ink product formed through the nozzle maintains the same shape as the cross-section of the printing material in the ink receiving component.

[0063] Currently, natural or synthetic hydrogel bio-inks have been developed and used in the field of 3D bioprinting. However, hydrogel-based bio-inks are used because they are superior in physical and biological aspects, such as biocompatibility, printability, geometric accuracy, and precision.

[0064] "Extrudable" means capable of being molded through an ink extrusion component, nozzle, orifice (e.g., one or more holes or tubes) (e.g., under pressure). Additionally, densification is induced by growing cells to an appropriate density. The cell density required for bio-ink differs from the cells to be used and the tissue or organ to be prepared.

[0065] Furthermore, the present invention provides a bioink composition, wherein the bioink composition further comprises a tissue-derived component. The tissue-derived component refers to a gelling material made by decellularizing certain animal tissues such as cartilage, kidney, heart, liver, and muscle, and having an extracellular matrix as its main component; it can be included to enhance the tissue specificity of the bioink composition.

[0066] In this invention, the bioink composition may additionally include a cell culture medium. The cell culture medium is a concept encompassing any culture medium suitable for the target cells.

[0067] The ink according to the present invention may contain a gelling polymer, and various gelling polymer solutions for printing can be used, and the conditions of the polymer solution should be as follows. First, to ensure good 3D printing, it should be easily sprayed into the nozzle by having an appropriate viscosity, and problems such as the distortion of the object's shape due to rapid solidification after discharge should not occur. Furthermore, it is essentially necessary to construct a cell culture environment similar to human tissue for the purpose of preparation.

[0068] Examples of gelling polymers may be selected from one or more of the following: fucoidan, collagen, alginate, chitosan, hyaluronic acid, silk, polyimide, polyamic acid, polyacrylonitrile, polyetherimide, nylon, polyaramid, polyvinyl alcohol, polyvinylpyrrolidone, poly-benzylglutamic acid, poly(p-phenylene terephthalamide), polyaniline, polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethyl methacrylate, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid and polyglycolic acid copolymer (PLGA), poly(ethylene oxide) terephthalate (PEOT / PBT), polyphosphate (PPE), polyphosphazene (PPA), polyanhydride (PA), poly(orthoester) (POE), poly(fumarate acrylate) diacrylate (PPF-DA), and poly(ethylene glycol) diacrylate (PEG-DA), or combinations thereof. However, the materials are not limited to the examples. In addition, the gelling polymer can be a chemically modified natural polymer, for example, it can contain: GelMA, in which gelatin and methacrylate (MA) are chemically bonded and a photoinitiator is bound thereto; alginate, in which pentapeptide sequencing Tyr-Ile-Gly-Ser-Arg (YIGSR) and EDC / NHS are combined to add binding sites for alginate / gelatin, alginate, etc.

[0069] In particular, hydrogels such as polyethylene glycol, alginate, collagen, and gelatin have been widely used to prepare carriers in which cells are embedded because of their high water content, excellent biocompatibility, controllable mechanical properties, and excellent biodegradability. For these reasons, hydrogels are well-suited for preparing structures in which cells are embedded, and they can be directly printed to obtain various types of tissue regeneration frameworks.

[0070] Gelatin exhibits temperature sensitivity, making it particularly suitable as a cell delivery material. In other words, gelatin liquefies at 37°C and has the property of curing at or below room temperature.

[0071] The gelling polymer can be cross-linked using physical or chemical treatments, and the cross-linking solution can be used for chemical treatment. The cross-linking solution can be appropriately selected and used depending on the selected gelling polymer. For example, the cross-linking solution can be a solution of one or more of the following: gypsum; or hydroxyapatite, carbonate apatite, fluorapatite, chloroapatite, α-TCP, β-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium pyrophosphate, calcium carbonate, calcium sulfonate, EDC{1-ethyl-(3-3-dimethylaminopropyl)carbodiimide hydrochloride} or its salts.

[0072] The ink containing the gelling polymer is preferably configured such that the collagen concentration in the liquid collagen solution is typically in the range of 0.1% to 30%. The preparation of the hydrogel can be carried out using, but is not limited to, commonly used methods for preparing inks for 3D printing.

[0073] The bio-ink according to the present invention may contain cells, and there are no particular limitations on the applicable cells or tissues; it may be animal cells or plant cells, or animal or plant tissues. The cells may be selected from one or more of the following: stem cells, osteoblasts, myoblasts, tendon cells, nerve cells, fibroblasts, glial cells, germ cells, hepatocytes, kidney cells, supporting cells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells, cardiomyocytes, glial cells, endothelial cells, hormone-secreting cells, immune cells, pancreatic islet cells, and neurons.

[0074] The cell types used in the preparation of artificial tissues for this invention can be cultured in any manner known in the art. Methods for culturing cells and tissues are well known in the art.

[0075] Additionally, cells can be cultured together with cell differentiation material, which induces cell differentiation according to a desired cell line. For example, stem cells can generate a range of cell types by contacting and incubating with a differentiation medium. Multiple types of differentiation media are suitable. Stem cells can be incubated by contacting differentiation media including osteogenic differentiation media, chondrogenic differentiation media, adipogenic differentiation media, neuronal differentiation media, cardiomyocyte differentiation media, and intestinal cell differentiation media (e.g., intestinal epithelial cells).

[0076] Additionally, cells can be cultured together with growth factors, cytokines, etc. "Growth factors" refer to proteins, polypeptides, or polypeptide complexes, including cytokines, that are produced by cells and can affect themselves and / or various other adjacent or distant cells. Typically, growth factors influence the growth and / or differentiation of certain cell types naturally or through responses to a variety of biochemical or environmental stimuli. Some (but not all) growth factors are hormones. Exemplary growth factors include insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factor (bFGF) including basic FGF, platelet-derived growth factor (PDGF) including PDGFAA and PDGF-AB, bone morphogenetic protein (BMP) including BMP-2 and BMP-7, transforming growth factor-β (TGF-β) including TGFβ1 and TGFβ3, epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, etc.

[0077] In this invention, "bioprinting" refers to the precise deposition of cells in three dimensions (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, etc.) using a method typically employed in conjunction with automated, computer-aided three-dimensional prototyping devices (e.g., bioprinters). 3D printing is performed by extruding a biodegradable polymer from a nozzle using a bioplotter and laminating it onto a platform.

[0078] Various tissue-like organs can be generated using this method. The pattern of the laminated bio-ink composition or the laminated arrangement can be determined by the size and diameter of the tissue-like organ to be prepared. Furthermore, the number of cells contained in the bio-ink used to prepare the tissue-like organ can be adjusted according to the cell type and the content of cellular nutrients in the bio-ink composition. In addition, the cell type contained in the bio-ink composition can be varied depending on the type of tissue-like organ prepared according to this method. Those skilled in the art will be able to select suitable cells and apply them to the cells according to the type of tissue-like organ to be prepared by 3D bioprinting.

[0079] After the bioink composition is sprayed and laminated using a 3D bioprinter, cross-linking of the bioink composition can be promoted by heating it, exposing it to ultraviolet light, or adding a cross-linking solution. This cross-linking allows the laminated bioink composition to achieve a stiffer structure. Photoinitiators can be used to promote cross-linking.

[0080] To prepare an ink extrusion member having the same cross-sectional pattern as the printed product or a cross-sectional pattern with the same or different dimensions, different inks are received in each compartment of the ink receiving component of the ink extrusion member, and pressure is controlled, for example by a piston, to pass through an extrusion orifice equipped with a single channel through which the ink passes and is extruded. Preferably, after a small amount of hydrogel (0.1 mL to 2 mL) is pre-added as a support material, bio-ink is placed into the ink receiving component so that the bio-ink is not poured out after it is filled. Hydrogel may or may not be added to the container. When printing resumes, the filled hydrogel is expelled at the start of printing, and then the desired shape is printed. When the printed product of the desired shape is completed, the filled hydrogel is expelled. The reason for adding support material at the beginning and later is to ensure stable printing.

[0081] The invention will now be described in detail with reference to the accompanying drawings.

[0082] Figure 1 The diagram schematically illustrates an ink extrusion component of a bioprinting apparatus according to an example of the invention, the ink extrusion component having an ink receiving component divided into four spaces.

[0083] exist Figure 1In the ink extrusion member (10), multiple spaces for receiving each type of ink (11, 12, 13, 14) are separated. The ink receiving components of the ink extrusion member can be integrally formed to create a number of spaces, or they can be separated into multiple spaces by partition members (30) that are detachable from the ink receiving components. An advantage is that when using partition members detachable from the ink extrusion member, various patterns can be printed by changing only the partition portions in a single ink receiving component (10). The size of the partition spaces can be controlled according to the area ratio of each type of ink. Figure 2 The image shows an example of a bioprinting apparatus according to an embodiment of the invention, suitable for extruding five types of ink (1, 2, 3, 4, 5).

[0084] In such Figure 1 and Figure 2 In the case of an example using a removable separator member that can be detached from the ink extrusion component, the separator member (30) can be inserted into the ink receiving component. In cases such as... Figure 3 and Figure 4 In the example case, the separator can be inserted into the ink receiving component by assembling the hollow parts together.

[0085] Then, the cellular liquid material received in the ink extrusion member (10) is pushed along the A direction, and the printing ink is extruded through the ink extrusion member (20) or nozzle (80) to obtain the final object (50). Then, preferably, the piston (60) is controlled so that the cross-section of the printing material remains the same shape and only decreases in size to pass through the nozzle (20), and the base can be a container in which the liquid material is contained in some cases.

[0086] When the pressure is too high, there is a risk of increased load on the nozzle, leading to damage, or the hydrogel may not be discharged smoothly in a linear form, instead becoming clumped and discharged in an unbalanced shape. Conversely, when the pressure is too low, the viscous resistance of the hydrogel prevents smooth release from the nozzle. On the other hand, when the diameter is too small, the release pressure becomes high, potentially posing a risk under high pressure, while when the diameter is too large, the accuracy of the three-dimensional shape during scaffold fabrication may decrease. The aforementioned pressure and diameter ranges were determined experimentally, taking all the above considerations into account, to ensure that the release of the hydrogel is appropriately smooth and easy to achieve, while simultaneously achieving the desired level of accuracy in the shape of the scaffold to be fabricated.

[0087] For example, the printing method according to the present invention can be performed as follows: including the steps of receiving ink into an ink receiving member, extruding ink with a nozzle having a release port diameter (e.g., in the range of 0.1 to 500 kPa) by applying pressure to the ink receiving member in the range of 0.1 to 500 kPa, and printing ink when the nozzle moves through a moving member of the printing device at a speed in the range of 1 to 700 mm / min.

[0088] Then, the cellular liquid material received in the ink extrusion member (10) is pushed along the A direction, and the printing material is extruded through the nozzle (80) to the base (100) to obtain the final object (50). Then, preferably, the piston (60) is controlled so that the cross-section of the printing material maintains the same shape and only decreases in size to pass through the nozzle (20), and the base can be a container in which the liquid material is contained in some cases.

[0089] In bioprinting using a single material as described in the prior art, there are limitations on reducing the material volume due to restrictions on the reduction of the nozzle inner diameter of the ink extrusion component. However, since the volume of extruded ink can be reduced proportionally to the quantity of the various inks according to the present invention, precise extrusion can be achieved compared to the prior art. Moreover, since the contact area between each ink and the inner surface of the channel of the ink extrusion component (20) is reduced when the various inks pass through it, the shear stress generated is reduced compared to when extruding a single material. Therefore, it has a favorable effect on cell viability compared to the prior art.

[0090] [Beneficial Effects]

[0091] According to the present invention, since the volume of the extruded material can be reduced proportionally to the amount of various inks, more precise injection can be achieved compared to the prior art. Furthermore, when various inks pass through the channels of the ink extrusion component or nozzle, the contact area between the various materials and the inner surface of the nozzle channels is reduced, and the shear stress generated is also reduced compared to extruding a single material. Therefore, it has beneficial effects on improving cell activity and printing accuracy compared to the prior art. Attached Figure Description

[0092] Figure 1 This is a schematic diagram illustrating an ink extrusion member according to an example of the present invention, the ink extrusion member having an ink receiving component divided into four spaces.

[0093] Figure 2 The diagram schematically illustrates an ink extrusion member according to an example of the invention, which has an ink receiving component divided into five spaces.

[0094] Figure 3and Figure 4 These are schematic combined and exploded views of an ink extrusion member equipped with a hollow portion and a separator, according to an example of the invention.

[0095] Figure 5 This is a photograph showing a separator member for use in an ink extrusion member according to an example of the present invention.

[0096] Figure 6 The results of using confocal microscopy to observe the printing of RGB hydrogels using an extrusion member, which includes an ink receiving component divided into four sections, are based on Example 1.

[0097] Figure 7 The results of confocal microscopy observations according to Example 2 are shown, illustrating the printing results of RGB hydrogel using an extrusion member that varies with nozzle size, the extrusion member comprising an ink receiving component divided into 4 zones.

[0098] Figure 8 and Figure 9 The results of printing RGB hydrogels using an extrusion member equipped with various shaped separators are shown in Example 3, observed using a confocal microscope. Detailed Implementation

[0099] The invention will be described in more detail with reference to the following examples, but the scope of the invention is not intended to be limited to the following examples.

[0100] Example 1

[0101] In order to 3D print an extruded component including an ink receiving part divided into 4 partitions, a partitioned component with 4 partitions was prepared by 3D printing using polylactic acid (PLA) as the material.

[0102] For observing fluorescence using a confocal microscope, 3 w / v sodium alginate containing green, blue, and red fluorescent particles was placed into a syringe equipped with a separator having four partitions, and 3 w / v sodium alginate without fluorescent particles was placed into another syringe. Then, RGB hydrogels were printed in each partition containing fluorescent particles.

[0103] Figure 6 The image shows observations of printing an RGB hydrogel using an extrusion member according to Example 1, the extrusion member comprising an ink receiving component divided into four sections. In other words, fluorescence observations of the extrusion results using confocal microscopy are shown. Figure 6 The left image is shown, and the right image is a cross-sectional view of the extrusion result (both scale bars are 100 μm). The image below shows the analysis of the material passing through... Figure 6The cross-sectional diagram on the right shows the result of a cross-section of the center of this diameter, and also shows the distribution of fluorescence intensity values ​​in the diagram. It demonstrates that high resolution is possible with the printing ink divided into four zones according to the invention.

[0104] Example 2

[0105] The printing of RGB hydrogels using a 3D printing apparatus was confirmed using a confocal microscope. This apparatus used the same ink extrusion component as in Example 1, with nozzle sizes of 18, 20, 22, 25, and 27. Figure 6 The results of confocal microscopy observations are shown, illustrating the printing results of an RGB hydrogel using an extrusion member comprising an ink receiving component divided into four sections, according to Example 2, as the nozzle size varies. As shown in the cross-sectional view, the size can be reduced to a shape identical to the cross-sectional shape of the ink extrusion member.

[0106] According to an example of the invention, the size can be reduced by 98.7% (200 μm) from the total diameter (15 mm) of a certain shape (e.g., leaflet). This is calculated based on an equation.

[0107] [Equation 1]

[0108] Reduction ratio = 100 - (Ink receiver diameter / Printer diameter) × 100 (%)

[0109] [Table 1]

[0110]

[0111] Figure 7 The results of confocal microscopy observations are shown, illustrating the printing results of RGB hydrogels according to Example 2 using an extrusion member comprising an ink receiving component divided into four sections, as the nozzle size varies.

[0112] Example 3

[0113] To enable 3D printing of ink extrusion components equipped with various shaped separators, separators of various shapes are prepared by using polylactic acid (PLA) as the material via a 3D printing method.

[0114] For each partition, RGB hydrogels were printed by placing 3 w / v % sodium alginate containing green, blue, and red fluorescent particles into syringes equipped with separators.

[0115] Figure 8 and Figure 9The results of printing RGB hydrogels using an extrusion member equipped with various shaped partitions are shown in Example 3, observed using a confocal microscope. Based on the experimental results, it can be confirmed that the ink receiving component of the ink extrusion member can replicate not only four partitions but also various forms of tissue. Figure 9 Shown from Figure 8 Fluorescence intensity graphs of extruded products of various shapes, the left graph showing fluorescence intensity from... Figure 8 The two-dimensional fluorescence intensity map with the yellow dashed line is shown on the right. Figure 8 The three-dimensional fluorescence intensity map of the surface within the white box. For Figure 9 The first image, starting from the top, is using... Figure 8 The analysis of the two-dimensional light intensity of the first dividing member in the 3D printing result, and the results from... Figure 8 The first image shows the one-dimensional fluorescent RGB light intensity along the yellow dashed line; the second image uses... Figure 8 The second separator component is used to analyze the three-dimensional light intensity of the 3D printing result, and the light intensity from the second separator component is shown. Figure 8 The three-dimensional fluorescent RGB light intensity of the two-dimensional surface of the white frame.

[0116] Although the invention has been described with reference to the accompanying drawings, the scope of the invention is defined by the appended claims and is not intended to be limited to the foregoing examples and / or drawings.

Claims

1. A method for 3D printing a printed product with a cross-sectional pattern using a 3D printing apparatus. in, The 3D printing apparatus includes: an ink extrusion component comprising an ink receiving component and an ink extrusion component; the ink receiving component including a separating component providing two or more separating spaces having the same cross-sectional pattern as the printed product, and receiving different inks in the separating spaces; the ink extrusion component being located below the ink receiving component and equipped with a single channel through which the ink contained in the ink receiving component is extruded. A nozzle, the nozzle being connected to the end of the ink extrusion component, and Pressure member, which applies pressure to the ink received in each compartment. The method includes: The steps of supplying different inks into each compartment of the ink extrusion component and forming the cross-sectional pattern. The steps of applying the same pressure conditions to the ink contained in each compartment and extruding the ink from two or more compartments into the single channel of the ink extrusion component to prepare an extruded ink product having the same cross-sectional pattern as the ink receiving component and having a reduced size, and The step of printing the extruded ink product onto the plate. The ink includes one or more selected from the group consisting of gelling polymers, cells, growth factors and extracellular matrix.

2. The method according to claim 1, wherein, The printed product is artificial tissue.

3. The method according to claim 1, wherein, The ratio of the cross-sectional diameter of the ink receiving component to the cross-sectional diameter of the printed product is 100:99 to 100:0.

1.

4. The method according to claim 1, wherein, The separating component is prepared using a 3D printing method.

5. The method according to claim 1, wherein, The steps of preparing the extruded ink product and receiving the ink are performed simultaneously or sequentially.

6. The method according to claim 1, wherein, The ink has one or more different characteristics selected from ink components, the content of ink components, and the physical properties of ink components.

7. The method according to claim 1, wherein, The viscosity of the ink, measured at 25°C, ranges from 2 cp to 1,000,000 cp.

8. The method according to claim 1, wherein, The ink contains a gelling polymer.

9. The method according to claim 1, wherein, The same pressure conditions can be applied by using a single pressure member or by using two or more pressure members.

10. A three-dimensional printing apparatus for a printed product having a cross-sectional pattern, comprising: An ink extrusion component includes an ink receiving component and an ink extrusion component. The ink receiving component includes a separating member that provides two or more separating spaces having the same cross-sectional pattern as the printed product, and receiving different inks in the separating spaces to form the cross-sectional pattern. The ink extrusion component is located below the ink receiving component and is equipped with a single channel through which the ink contained in the ink receiving component is extruded. A nozzle, the nozzle being connected to the end of the ink extrusion component, and Pressure member, which applies pressure to the ink contained in each compartment. Different inks are printed through the ink extrusion component equipped with the single channel to achieve the same cross-sectional pattern as the printed product. The extruded ink product discharged from the ink extrusion component has the same cross-sectional pattern as the ink receiving component, and has a reduced size. The ink includes one or more selected from the group consisting of gelling polymers, cells, growth factors and extracellular matrix.

11. The printing apparatus according to claim 10, wherein, By applying pressure to the ink received in each compartment using a single pressure member, the ink received in each compartment is extruded together.

12. The printing apparatus according to claim 10, wherein, The separator can be detached from the ink extrusion member.

13. The printing apparatus according to claim 10, wherein, The separating member also includes a hollow portion that can be installed in the ink receiving component.

14. The printing apparatus according to claim 10, wherein, The separating component is prepared using a 3D printing method.

15. The printing apparatus of claim 10, further comprising an ink supply member connected to the ink extrusion member and supplying ink to each compartment of the ink extrusion member.

16. The printing apparatus according to claim 15, wherein, The ink supply component includes an ink storage container and an ink supply control component, the ink supply control component controlling the supply of ink from the storage container to the ink receiving component through a supply tube.

17. The printing apparatus according to claim 10, wherein, The printed product is artificial tissue.

18. The printing apparatus according to claim 10, wherein, The ink has one or more different characteristics selected from ink components, the content of ink components, and the physical properties of ink components.

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