Method for producing three-dimensional shaped article and ink set for

Abstract

Provided are: a method for producing a three-dimensional molded article, which is capable of producing a three-dimensional molded article with good molding accuracy, even when producing a three-dimensional molded article having a complicated shape, for example, comprising a soft material; and an ink set for producing a three-dimensional molded article. The present invention pertains to a method for producing a three-dimensionally shaped article, the method comprising: a step for discharging (a) a model material ink containing a crosslinkable polymer and (b) a support material ink containing a support material, respectively; and a step for removing the molded article of the support material ink after the crosslinking of the crosslinkable polymer, in which either the model material ink (a) or the support material ink (b) further contains a hydrogen peroxide decomposition agent and the other further contains hydrogen peroxide or a hydrogen peroxide donor, the discharged hydrogen peroxide decomposition agent decomposes the discharged hydrogen peroxide, and the hydrogen peroxide donor decomposes the discharged hydrogen peroxide. A three-dimensional molded article is molded by crosslinking the crosslinkable polymer.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing a three-dimensional object and an ink set for making a three-dimensional object, and in particular, to a method for manufacturing a three-dimensional object of a complex shape made of soft materials in the field of regenerative medicine, etc. Background Art

[0002] The technology of manufacturing three-dimensional objects using a modeling device called a three-dimensional printer has been studied in various fields with the goal of practical application. For example, in the field of regenerative medicine, in order to be used as artificial organs, it is required to manufacture three-dimensional objects with complex shapes and bridges or holes made of soft materials. When such three-dimensional objects are manufactured from bio-ink using a three-dimensional printer, the bio-ink will flow out (liquid dripping) and disintegrate before gelation, or deform due to gravity, so it is required to improve the modeling accuracy.

[0003] Patent document 1 discloses the following: With the goal of applying to fields such as regenerative medicine, by adding silk nanofibers to 3D printing system ink containing natural polymers and / or synthetic polymers, the viscosity is low when discharged from the nozzle, the pressure during discharge can be reduced, and the viscosity is high on the printed substrate, which can fully prevent the deformation of the modeled object. On the other hand, it is hoped to develop a new method for making three-dimensional models with a further expanded range of materials.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2020-156357 Summary of the invention

[0007] Problems to be solved by the invention

[0008] An object of the present invention is to provide a method for manufacturing a three-dimensional object and an ink set for manufacturing a three-dimensional object, which can manufacture the three-dimensional object with good modeling accuracy even when manufacturing a three-dimensional object of a complex shape, such as one made of a soft material.

[0009] Means for solving problems

[0010] The present inventors have conducted various studies and found that when a model material ink containing a crosslinkable polymer and a hydrogen peroxide decomposing agent and a support material ink containing a support material and hydrogen peroxide or a hydrogen peroxide donor are discharged, the discharged hydrogen peroxide decomposing agent decomposes the discharged hydrogen peroxide to crosslink the crosslinkable polymer, and then the molded object of the support material ink is removed to mold a three-dimensional molded object, even when a three-dimensional molded object of a complex shape, such as one made of a soft material, is produced, a three-dimensional molded object can be manufactured with good molding accuracy. Furthermore, the present inventors have found that the hydrogen peroxide decomposing agent can be contained in the support material ink, and the hydrogen peroxide or the hydrogen peroxide donor can be contained in the model material ink, thereby completing the present invention.

[0011] That is, the present invention (1) relates to a method for manufacturing a three-dimensional object, which includes: the steps of respectively discharging (a) a model material ink containing a cross-linkable polymer and (b) a support material ink containing a support material; and the step of removing the object of the support material ink after the cross-linking of the cross-linking polymer, wherein either the model material ink (a) or the support material ink (b) further contains a hydrogen peroxide decomposer, and the other further contains hydrogen peroxide or a hydrogen peroxide donor, and the discharged hydrogen peroxide decomposer decomposes the discharged hydrogen peroxide to cross-link the cross-linking polymer, thereby shaping the three-dimensional object.

[0012] The present invention (2) is the method for producing a three-dimensional object according to the present invention (1), wherein the hydrogen peroxide decomposing agent is a cross-linking enzyme.

[0013] The present invention (3) is the method for producing a three-dimensional object according to the present invention (1) or (2), wherein the hydrogen peroxide donor is a hydrogen peroxide generating enzyme.

[0014] The present invention (4) is a method for producing a three-dimensional object in combination with any one of the present inventions (1) to (3), wherein the crosslinkable polymer has a phenolic hydroxyl group.

[0015] The present invention (5) is a method for producing a three-dimensional object in combination with any one of the present inventions (1) to (4), wherein the support material is a water-soluble polymer or a water-dispersible polymer.

[0016] The present invention (6) is a method for manufacturing a three-dimensional object in any combination with any one of the present inventions (1) to (5), wherein the above-mentioned water-soluble polymer or water-dispersible polymer is a water-soluble polymer or water-dispersible polymer that forms an aqueous gel that agglomerates or has a reduced viscosity when reacted with an ionic compound, and in the process of removing the object of the support material ink, the ionic compound is allowed to act to remove the above-mentioned support material ink (b) from the object.

[0017] The present invention (7) is an ink set for making three-dimensional objects, which comprises: (a) a model material ink containing a cross-linked polymer; and (b) a support material ink containing a support material, wherein either the model material ink (a) or the support material ink (b) further comprises a hydrogen peroxide decomposer, and the other further comprises hydrogen peroxide or a hydrogen peroxide donor.

[0018] The present invention (8) is the ink set for producing a three-dimensional object according to the present invention (7), wherein the hydrogen peroxide decomposing agent is a cross-linking enzyme.

[0019] The present invention (9) is the ink set for producing a three-dimensional object according to the present invention (7) or (8), wherein the hydrogen peroxide donor is a hydrogen peroxide generating enzyme.

[0020] The present invention (10) is an ink set for producing a three-dimensional object in combination with any one of the present inventions (7) to (9), wherein the crosslinkable polymer has a phenolic hydroxyl group.

[0021] The present invention (11) is an ink set for making three-dimensional objects in any combination with any one of the present inventions (7) to (10), wherein the above-mentioned supporting material ink (b) contains a water-soluble polymer or a water-dispersible polymer that forms an aqueous gel that agglomerates or reduces viscosity by reacting with ionic compounds.

[0022] Effects of the Invention

[0023] According to the present invention, a method for manufacturing a three-dimensional object can be provided, which can manufacture the three-dimensional object with good modeling accuracy even when manufacturing a three-dimensional object of a complex shape made of a soft material, for example. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram for explaining the process of manufacturing a three-dimensional object having a bridge portion by the method for manufacturing a three-dimensional object of the present invention.

[0025] Figure 2 This is a graph showing the viscosity of the supporting material ink produced in Production Example 2 with respect to the shear rate.

[0026] Figure 3 This is a photograph showing how a white precipitate is generated when Projelly (gel for ultrasonic inspection) used in Production Example 2 is allowed to react with an ionic compound.

[0027] Figure 4 The Projelly used in Production Example 2 was mixed with DMEM medium (Double's Modified Eagle's Medium), and cells were grown and cell growth was observed.

[0028] Figure 5These are photographs of the three-dimensional objects produced in Example 1 and Comparative Example 1.

[0029] Figure 6 These are photographs of the three-dimensional objects produced in Examples 1 to 3.

[0030] Figure 7 These are photographs of the three-dimensional objects produced in Examples 4 and 5.

[0031] Figure 8 These are photographs of the three-dimensional objects produced and cultured in Examples 6 to 7.

[0032] Fig. 9 These are photographs of cells recovered and cultured from a three-dimensional structure in Example 8.

[0033] Fig.10 This is a schematic diagram for explaining a step of further using a second supporting material ink to produce a three-dimensional object in the method for producing a three-dimensional object of the present invention.

[0034] Fig.11 It is used to illustrate Fig.10 A schematic diagram of another example of the process shown.

[0035] Fig.12 It is used to illustrate Fig.10 A schematic diagram of another example of the process shown. DETAILED DESCRIPTION

[0036] <<Method for producing three-dimensional object>>

[0037] The method for manufacturing a three-dimensional object of the present invention is characterized in that it includes: the steps of respectively discharging (a) a model material ink containing a cross-linkable polymer and (b) a support material ink containing a support material; and the step of removing the modeled object of the support material ink after cross-linking of the above-mentioned cross-linkable polymer, wherein either the above-mentioned model material ink (a) or the above-mentioned support material ink (b) further contains a hydrogen peroxide decomposer, and the other further contains hydrogen peroxide or a hydrogen peroxide donor, and the discharged hydrogen peroxide decomposer decomposes the discharged hydrogen peroxide to cross-link the cross-linkable polymer, thereby shaping a three-dimensional object.

[0038] Below, refer to Figure 1 The method for producing a three-dimensional object of the present invention will be briefly described. Figure 1 This is a schematic diagram for explaining the process of manufacturing a three-dimensional object having a bridge portion by the method for manufacturing a three-dimensional object of the present invention. Figure 1 (a) schematically shows a state where the supporting material ink 1 is discharged from a nozzle 3a of a 3D printer. Figure 1(b) schematically shows the discharge of model material ink 2 from nozzle 3b of a 3D printer. It should be noted that nozzle 3a and nozzle 3b may be the same nozzle, but are preferably different nozzles. Here, support material ink 1 may be discharged, and then model material ink 2 may be discharged at a position surrounded by support material ink 1 to form a layer, and this operation may be repeated to form a layered structure.

[0039] like Figure 1 (a) and Figure 1 As shown in (b), the discharged support material ink 1 contacts the discharged model material ink 2. For example, when the support material ink 1 contains a support material and hydrogen peroxide or a hydrogen peroxide donor, and the model material ink 2 contains a cross-linking polymer and a hydrogen peroxide decomposing agent, hydrogen peroxide is supplied from the discharged support material ink 1 to the discharged model material ink 2, and the hydrogen peroxide decomposing agent in the discharged model material ink 2 decomposes the hydrogen peroxide to cross-link the cross-linking polymer, thereby gelling the model material ink 2. Figure 1 (c) shows the following process: after the gelation of the model material ink 2 is completed, the obtained object is immersed in a solution 5 for removing the object of the support material ink 1 to remove the object of the support material ink 1. By removing the object of the support material ink 1, a three-dimensional object 2A (an object of the model material ink) having a complex shape having bridges 2a or holes 2b can be produced.

[0040] In the method for manufacturing a three-dimensional object of the present invention, it is also possible to discharge model material ink and support material ink into cylindrical containers such as beakers and culture dishes, square tube-shaped containers, and containers of other various shapes to make a three-dimensional object. The inner wall of the container becomes a support for the model material ink and / or the support material ink, so a higher-height modeling object can be made. It should be noted that the material of the container is not particularly limited as long as it becomes a material that becomes a support for the model material ink and / or the support material ink, and a container made of glass, resin, etc. can be used. In addition, the container can also be manufactured by three-dimensional modeling.

[0041] <Step of Separately Discharging (a) Model Material Ink Containing a Crosslinkable Polymer and (b) Support Material Ink Containing a Support Material>

[0042] In the discharge process, for example, the model material ink (a) and the support material ink (b) containing the support material are discharged from the nozzle of the 3D printer to the target part such as on the substrate. When a 3D printer is used in the ink discharge process, the 3D printer is not particularly limited and can be any one of inkjet, laser, extrusion, etc., and an extrusion 3D printer is more preferred. The nozzle for discharging the model material ink (a) is preferably different from the nozzle for discharging the support material ink (b).

[0043] ((a) Model material ink containing cross-linkable polymer)

[0044] The model material ink (a) contains a cross-linking polymer, which is cross-linked with the decomposition of hydrogen peroxide to form a gel. The cross-linking polymer is not particularly limited, and examples thereof include polymers obtained by combining a polymer substrate with a compound having a phenolic hydroxyl group, amino-containing polysaccharides, polyphenols, and the like.

[0045] The polymer substrate is not particularly limited, and examples thereof include polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides, poly(α-hydroxy acids), polylactones, polyamino acids, polyanhydrides, polyorthoesters, polyanhydride-imide copolymers (poly(anhydride-co-imides)), polyorthocarbonates, poly(α-hydroxyalkanoates), polydioxanone, polyphosphates, polylactic acid, poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolic acid, polyglycolide (PGA), polyglycolide (PLGA), L-lactide-D,L-lactide copolymers, D,L-lactide-trimethylene carbonate copolymers, Polyhydroxybutyrate (PHB), poly(ε-caprolactone), poly(δ-valerolactone), poly(γ-butyrolactone), polycaprolactone, poly(meth)acrylic acid, polycarboxylic acid, polyallylamine hydrochloride, polydiallyldimethylammonium chloride, polyalkyleneimines such as polyethyleneimine, polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone, polyolefins such as polyethylene (polyalkylenes), polymethyl methacrylate, carbon fiber, polyalkylene glycols such as polyethylene glycol, polyalkylene oxides such as polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyethyloxazoline, polyethylene oxide-polypropylene oxide block copolymers, poly(ethylene terephthalate) polyamide, salts of these polymer substrates, etc.

[0046] Examples of polysaccharides include cellulose, hemicellulose, dextran, hyaluronic acid, chitin, chitosan, alginic acid, chondroitin sulfate, starch, pullulan, carrageenan, curdlan, fucoidan, amylose, amylopectin, pectin, salts thereof such as alkali metal salts, derivatives thereof, etc. Examples of polysaccharide derivatives include carboxymethylcellulose, methylcellulose, deacetylated chitins, deacetylated chitosans, etc.

[0047] Examples of proteins include simple proteins such as albumin, globulin, prolamin, gluten, histone, protamine, and sclerostin; complex proteins such as nucleoprotein, glycoprotein, chromoprotein, and phosphoprotein; gelatin, Proteins derived from peptone, etc.

[0048] The salt in the salt of the polymer substrate is not particularly limited, and examples thereof include acid addition salts, metal salts, ammonium salts, organic amine salts, and the like. Examples of acid addition salts include inorganic acid salts such as hydrochlorides, sulfates, and phosphates; organic acid salts such as acetates, maleates, fumarates, tartrates, and citrates. Examples of metal salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts, magnesium salts, aluminum salts, and zinc salts. Examples of organic amine salts include salts of morpholine, piperidine, and the like.

[0049] As the polymer substrate, polysaccharides and proteins are preferred because they are easy to supply and the resulting three-dimensional objects have good properties, and polysaccharides are more preferred. The polymer substrate may be used alone or in combination of two or more.

[0050] The compound having a phenolic hydroxyl group is not particularly limited, but is preferably a compound of the following general formula (1):

[0051] [Chemistry 1]

[0052]

[0053] (Where X 1 represents a hydroxyl group, an amino group or a carboxylate group. 2 ~X 6 One or two of them represent hydroxyl groups, and the rest represent hydrogen atoms or alkoxy groups having 1 to 6 carbon atoms. 2 ~X 6 When two of the hydroxyl groups represent hydroxyl groups, it is preferred that the two hydroxyl groups are in an ortho or para position. R represents a divalent hydrocarbon group having 1 to 10 carbon atoms which may or may not have a substituent. The compound represented by ). The cross-linkable polymer formed by introducing phenolic hydroxyl groups into the polymer substrate is easily cross-linked by the action of a hydrogen peroxide decomposing agent such as a cross-linking enzyme. From the perspective of easily introducing phenolic hydroxyl groups into the polymer substrate, X 1 Preferably, a carboxylic acid (salt) group or an amino group is used, and an amino group is more preferred. It should be noted that the amino group is preferably a primary amino group or a secondary amino group. As the carboxylic acid (salt) group, a carboxylic acid group, an alkali metal salt of a carboxylic acid group, or an alkaline earth metal salt is preferred. In addition, the number of carbon atoms of the divalent hydrocarbon group represented by R is preferably 1 to 8, and more preferably 1 to 6. The divalent hydrocarbon group represented by R is not particularly limited, and examples thereof include alkylene groups, alkyl alkylene groups, and the like. In addition, examples of substituents that R may have include amide groups, ester groups, ether groups, and the like. It should be noted that X 1 In the case of a hydroxyl group, it is an alcoholic hydroxyl group.

[0054] Examples of the compound having a phenolic hydroxyl group include compounds having one phenolic hydroxyl group such as tyramine, homovanillic acid, and derivatives thereof; and compounds having two phenolic hydroxyl groups such as catecholamines such as dopamine, norepinephrine, and epinephrine. Among these, compounds having one phenolic hydroxyl group are preferred from the perspective of the crosslinking reactivity of the resulting crosslinkable polymer, and tyramine and tyramine derivatives are more preferred.

[0055] As the compound having a phenolic hydroxyl group, a commercially available product may be used, and examples of the commercially available product include tyramine hydrochloride (4-(2-aminoethyl)phenol hydrochloride, product number: T2879, manufactured by Sigma-Aldrich Co., Ltd.).

[0056] The method for combining the polymer substrate with the compound having a phenolic hydroxyl group is not particularly limited, and examples include: a method of using a condensation agent to condense functional groups such as carboxyl, amino or alcoholic hydroxyl groups that the compound having a phenolic hydroxyl group may have with functional groups such as amino, carboxyl or thiol groups that the polymer substrate may have; a method of using glutamyl transferase for condensation; etc.

[0057] The condensing agent is not particularly limited, and examples thereof include 1,1-carbonyldiimidazole, dicyclohexylcarbodiimide, 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (water-soluble carbodiimide: WSCD·HCl), etc. In the method of condensing the polymer substrate and the compound having a phenolic hydroxyl group using a condensing agent, and the method of condensing using glutamyl transferase, the pH, the concentration of the polymer substrate, the reaction temperature, the solvent, etc. can be appropriately selected according to the properties of the polymer substrate, the compound having a phenolic hydroxyl group, the condensing agent, or the glutamyl transferase used.

[0058] The introduction rate (modification rate) of the compound having a phenolic hydroxyl group into the polymer substrate is the ratio of the (modified) condensation reaction groups that have undergone condensation reaction among all the condensation reaction groups possessed by the polymer substrate. From the aspect of balancing the gelation speed and hardness (not too hard), it is preferably 0.3 mol% to 20 mol%, and more preferably 1 mol% to 10 mol% relative to 100 mol% of the condensation reaction groups possessed by the polymer substrate.

[0059] As the cross-linking polymer, amino-containing polysaccharides and polyphenols may also be used. Examples of amino-containing polysaccharides include acetylated chitin and chitosan. In addition, examples of polyphenols include catechin compounds, anthocyanidin compounds, rutin, and natural pigments.

[0060] The concentration of the cross-linked polymer in the model material ink (a) is usually 0.1% to 30% by weight, preferably 0.5% to 20% by weight, and more preferably 0.75% to 10% by weight. If the concentration of the cross-linked polymer is less than 0.1% by weight, the strength of the three-dimensional object may become insufficient. If it exceeds 30% by weight, the viscosity of the model material ink becomes too high, and the pressure when discharged from the nozzle becomes high, which may affect the material of the model material ink (a). In addition, the concentration of the cross-linked polymer in the model material ink (a) is usually 0.1 to 30 w / v% (g / 100mL), preferably 0.5 to 20 w / v% (g / 100mL), and more preferably 0.75 to 10 w / v% (g / 100mL).

[0061] In the production method of the present invention, the model material ink (a) further contains a hydrogen peroxide decomposing agent, or hydrogen peroxide or a hydrogen peroxide donor. Hereinafter, the case where the model material ink (a) further contains a hydrogen peroxide decomposing agent will be described in detail.

[0062] As long as the hydrogen peroxide decomposing agent is a substance that decomposes hydrogen peroxide and crosslinks the crosslinkable polymer in the model material ink (a), it is preferred to use a crosslinking enzyme, complex, protein, etc. As a crosslinking enzyme, it is not particularly limited. From the aspect that the reaction is carried out at room temperature and atmospheric pressure and the safety is also high, for example, peroxidase, laccase, tyrosinase, catalase, etc. can be cited as preferred examples. There is no particular limitation on the source of the crosslinking enzyme. As a peroxidase, it is preferably a peroxidase from horseradish. As a complex, for example, from the aspect that the reaction is carried out at room temperature and atmospheric pressure and the safety is also high, an iron porphyrin complex, etc. can be cited. As a protein, hemoglobin, etc. can be cited.

[0063] In the case where the model material ink (a) includes a cross-linking enzyme, the concentration of the cross-linking enzyme in the model material ink (a) is not particularly limited, and is preferably 0.1U / mL or more, more preferably 1U / mL or more, from the perspective of further accelerating gelation. In addition, the concentration of the cross-linking enzyme is generally 10000U / mL or less, preferably 2000U / mL or less, more preferably 1000U / mL or less, and further preferably 500U / mL or less. In the case where the model material ink (a) includes a hydrogen peroxide decomposition agent other than the cross-linking enzyme, from the perspective of further accelerating gelation, the concentration of the hydrogen peroxide decomposition agent other than the cross-linking enzyme in the model material ink (a) is preferably 0.1% by weight or more, more preferably 1% by weight or more. In addition, the concentration of the hydrogen peroxide decomposition agent other than the cross-linking enzyme is generally 20% by weight or less, preferably 10% by weight or less.

[0064] When the model material ink (a) contains hydrogen peroxide or a hydrogen peroxide donor, specific examples of the hydrogen peroxide donor and preferred concentrations of the hydrogen peroxide or the hydrogen peroxide donor in the model material ink (a) are the same as specific examples of the hydrogen peroxide donor contained in the support material ink (b) and preferred concentrations of the hydrogen peroxide or the hydrogen peroxide donor in the support material ink (b) described later.

[0065] In addition to the cross-linking polymer, hydrogen peroxide decomposing agent, or hydrogen peroxide or hydrogen peroxide donor, the model material ink (a) may also contain other components. As other components, various cells, solvents, polymerization initiators, chain transfer agents, plasticizers, pH adjusters, buffers, preservatives, solvents, ultraviolet absorbers, colorants, surfactants, etc. can be cited. As solvents, for example, water, phosphate buffered saline (PBS), a mixture of water and an organic solvent, etc. can be cited. As organic solvents, there are no particular restrictions, and for example, ethanol, glycerol, dimethyl sulfoxide, etc. can be cited.

[0066] As the source animal of the cell, mammals, birds, reptiles, etc. can be mentioned, preferably mammals. As mammals, humans, pigs, cattle, sheep, goats, rabbits, mice, rats, dogs, cats, chickens, etc. can be mentioned, preferably humans. As the cell type, epithelial cells, fibroblasts, chondrocytes, osteoblasts, smooth muscle cells, neural cells, stem cells, etc. can be mentioned.

[0067] The concentration of cells in the model material ink is not particularly limited, but is preferably 1×10 3 Cells / mL~1×10 9 cells / mL, more preferably 1×10 4 Cells / mL~1×10 7 cells / mL.

[0068] The model material ink (a) is preferably in liquid form at room temperature. By being in liquid form at room temperature, it can be easily discharged using a 3D printer or the like. The viscosity of the model material ink (a) at 20°C is not particularly limited, and is preferably 10 mPa·s or more, more preferably 20 mPa·s or more, and further preferably 50 mPa·s or more. In addition, the above-mentioned viscosity is preferably 100000 mPa·s or less, more preferably 10000 mPa·s or less, and further preferably 2000 mPa·s or less. The manufacturing method of the present invention can fully prevent the outflow (liquid sagging) of the model material ink (a) before gelation, so that a low-viscosity model material ink (a) can be appropriately used. It should be noted that the above-mentioned viscosity is measured using a cone-plate E-type viscometer at a rotation speed of 20 rpm.

[0069] The model material ink (a) can be manufactured by mixing the above-mentioned cross-linking polymer, hydrogen peroxide decomposing agent, or hydrogen peroxide or hydrogen peroxide donor, and other components as needed. The conditions during mixing are not particularly limited, but are preferably 4°C to 45°C, and more preferably 10°C to 40°C, from the perspective of enzyme activity. In addition, during mixing, the pH is usually 2 to 11, and preferably 5 to 9, from the perspective of enzyme activity. The obtained model material ink is usually preferably stored at -80°C to 20°C. In addition, the model material ink can also be freeze-dried after production. The freeze-drying method can adopt a known method.

[0070] ((b) Support material ink including support material)

[0071] The support material ink (b) can be formed into a modeled object by maintaining its shape through cross-linking after being discharged, but an ink that can maintain its shape and directly form a modeled object is preferred because of its simplicity. The modeled object of the support material ink (b) maintains the shape of the model material ink (a) until the model material ink (a) is fully gelled, thereby preventing the model material ink (a) from flowing out (liquid dripping) and deformation of the model material ink (a) due to gravity. After the model material ink (a) is gelled, the modeled object of the support material ink (b) is removed by the method described below.

[0072] The support material contained in the support material ink (b) is a natural polymer, a synthetic polymer or other polymer, and as the polymer, for example, polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides, polyamino acids, polylactic acid, polyalkylene oxides such as polyethylene oxide, polyalkylene imines such as polyethylene imine, polyvinyl pyrrolidone, polyalkylene glycols such as polyethylene glycol, polyacrylic acid, polymethacrylic acid, polyglutamic acid, carboxyvinyl polymers, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, albumin, gelatin, collagen, silk fibroin, polyvinyl alcohol, salts of these polymers, etc. As the above-mentioned polymer, polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides, polyamino acids, polylactic acid, polyethylene oxide, polyethylene imine, polyvinyl pyrrolidone, polyethylene glycol, polyacrylic acid, polyglutamic acid, albumin, gelatin, collagen, polyvinyl alcohol, salts of these polymers are preferred. The salts of the polysaccharide, protein, and polymer salts are the same as the salts of the polysaccharide, protein, and polymer salts described above in the polymer base material. In addition, the support material may be a material obtained by cross-linking the polymer using a cross-linking agent.

[0073] Examples of the polysaccharides include cellulose, hemicellulose, dextran, hyaluronic acid, chitin, chitosan, alginic acid, chondroitin sulfate, starch, pullulan, carrageenan, curdlan, fucoidan, amylose, amylopectin, pectin, and salts thereof such as alkali metal salts, and derivatives thereof.

[0074] The supporting material is preferably a water-soluble polymer or a water-dispersible polymer. As a result, as described later, in the process of removing the shaped object of the supporting material ink (b), the shaped object can be easily removed under mild conditions using water or an aqueous solution. Here, in this specification, the solubility of the water-soluble polymer in 100 g of water at 20° C. is not particularly limited, but is preferably 1 g or more, and more preferably 5 g or more.

[0075] The water-soluble polymer or water-dispersible polymer forms a polymer aqueous gel, and the formed polymer aqueous gel is preferably a polymer aqueous gel that condenses or has a reduced viscosity by the action of an ionic compound. The water-soluble polymer or water-dispersible polymer that forms a polymer aqueous gel that condenses or has a reduced viscosity by the action of an ionic compound can include a polymer having a hydrocarbon chain (such as polyacrylic acid) having an acid group such as a carboxylic acid group as the main chain and cross-linked by a cross-linking agent. The cross-linking agent is not particularly limited, and allyl sucrose, pentaerythritol, etc. can be mentioned. The ionic compound is described below.

[0076] From the perspective of further improving the pseudoplasticity of the support material ink (b), the concentration of the polymer in the support material ink (b) is preferably 0.1% by weight or more, more preferably 0.3% by weight or more, more preferably 0.5% by weight or more, and particularly preferably 1.0% by weight or more. In addition, the concentration of the polymer is preferably 30.0% by weight or less, more preferably 15.0% by weight or less, and even more preferably 10.0% by weight or less. It should be noted that when the support material ink (b) contains two or more polymers, the above concentration refers to the total concentration of all polymers contained in the support material ink.

[0077] The supporting material ink (b) further contains hydrogen peroxide or a hydrogen peroxide donor, or a hydrogen peroxide decomposing agent. Hereinafter, the case where the supporting material ink (b) further contains hydrogen peroxide or a hydrogen peroxide donor will be described in detail.

[0078] When the support material ink (b) contains hydrogen peroxide, the concentration of hydrogen peroxide in the support material ink (b) is not particularly limited, but is preferably 0.01 mM or more and 1 M or less. The concentration is more preferably 0.1 mM or more, further preferably 1 mM or more, and particularly preferably 5 mM or more. In addition, the concentration of hydrogen peroxide is more preferably 100 mM or less, and from the perspective of achieving a more excellent molding accuracy of a three-dimensional molded object, it is further preferably 30 mM or less, and particularly preferably 10 mM or less.

[0079] The hydrogen peroxide donor is not particularly limited as long as it can generate hydrogen peroxide, and examples thereof include hydrogen peroxide generating enzymes and photocatalysts, preferably hydrogen peroxide generating enzymes. Examples of hydrogen peroxide generating enzymes include choline oxidase, amino acid oxidase, alcohol oxidase, pyruvate oxidase, cholesterol oxidase, glucose oxidase, and the like.

[0080] In the case where the support material ink (b) contains a hydrogen peroxide generating enzyme, from the aspect of reactivity, the concentration of the hydrogen peroxide generating enzyme in the support material ink (b) is preferably 0.1mU / mL or more, more preferably 1mU / mL or more, and further preferably 2.5mU / mL or more. In addition, the concentration of the hydrogen peroxide generating enzyme is preferably 20000mU / mL or less, more preferably 10000mU / mL or less, and more preferably 1000mU / mL or less, and particularly preferably 200mU / mL or less. In the case where the support material ink (b) contains a hydrogen peroxide donor other than the hydrogen peroxide generating enzyme, from the aspect of reactivity, the concentration of the hydrogen peroxide donor other than the hydrogen peroxide generating enzyme in the support material ink (b) is preferably 0.1% by weight or more, and more preferably 1% by weight or more. In addition, the concentration of the hydrogen peroxide donor other than the hydrogen peroxide generating enzyme is generally 20% by weight or less, preferably 10% by weight or less.

[0081] When the support material ink (b) contains a hydrogen peroxide decomposition agent, specific examples of the hydrogen peroxide decomposition agent and preferred concentrations of the hydrogen peroxide decomposition agent in the support material ink (b) are the same as the specific examples of the hydrogen peroxide decomposition agent contained in the model material ink (a) and preferred concentrations of the hydrogen peroxide decomposition agent in the model material ink (a).

[0082] In addition to the polymer and hydrogen peroxide or hydrogen peroxide donor, or hydrogen peroxide decomposer as the supporting material, the supporting material ink (b) may also contain other components. As other components, humectants such as glycerol, alkali metal hydroxides, pH adjusters such as organic amines, preservatives, colorants, solvents, plasticizers, ultraviolet absorbers, surfactants, etc. can be cited. As solvents, for example, water, phosphate buffered saline (PBS), mixtures of water and organic solvents, etc. can be cited. As organic solvents, there are no particular restrictions, and for example, ethanol, glycerol, etc. can be cited.

[0083] The ink (b) for supporting material is preferably in liquid or gel form at room temperature, more preferably in gel form. By being in gel form at room temperature, it can be easily molded using a 3D printer or the like. The viscosity of the ink (b) for supporting material at 20°C and a shear rate of 0.01 (1 / s) is preferably 10000 mPa·s or more, and from the perspective of being able to maintain its own shape, it is more preferably 100000 mPa·s or more, and further preferably 500000 mPa·s or more. In addition, the upper limit of the viscosity is not particularly limited, and is usually 10000000 mPa·s or less.

[0084] The viscosity of the support material ink (b) at 20°C and a shear rate of 10 (1 / s) is preferably 10 mPa·s or more, more preferably 100 mPa·s or more, and further preferably 1000 mPa·s or more. In addition, the upper limit of the viscosity is not particularly limited, but is usually less than 10000 mPa·s. It should be noted that the above viscosity is a value measured using a viscosity / viscoelasticity measuring device manufactured by Thermo Fisher Scientific.

[0085] The ratio of the viscosity of the support material ink (b) at 20°C and a shear rate of 0.01 (1 / s) to the viscosity at a shear rate of 10 (1 / s) is preferably 5 or more, more preferably 10 or more, further preferably 20 or more, and particularly preferably 50 or more. As a result, the pseudoplasticity of the support material ink (b) becomes more excellent.

[0086] The support material ink (b) can be produced by mixing a polymer with hydrogen peroxide or a hydrogen peroxide donor, or a hydrogen peroxide decomposing agent, and other components as required. The conditions for mixing are not particularly limited.

[0087] As described above, in the manufacturing method of the present invention, a three-dimensional printer is usually used to discharge the model material ink (a) and the supporting material ink (b) respectively to form a layer, and this operation is repeated to perform lamination. The discharged model material ink (a) is contacted with the discharged supporting material ink (b), and the discharged hydrogen peroxide decomposition agent decomposes the discharged hydrogen peroxide, crosslinking the crosslinked polymer, thereby molding a modeling object of the model material ink (a). Here, the hydrogen peroxide or hydrogen peroxide donor or hydrogen peroxide decomposition agent contained in the discharged supporting material ink (b) is supplied to the discharged model material ink (a). It should be noted that in the manufacturing method of the present invention, in addition to the crosslinking of the crosslinked polymer by the decomposition of hydrogen peroxide, ionic crosslinking, photocrosslinking, enzyme crosslinking, temperature changes, etc. can also contribute to the crosslinking of the crosslinked polymer.

[0088] (Ink for second supporting material including second supporting material)

[0089] In the manufacturing method of the present invention, the second supporting material ink can be discharged together with the model material ink (a) and the supporting material ink (b) (hereinafter also referred to as the first supporting material ink) as a supporting material for the model material ink and the first supporting material ink for manufacturing. The second supporting material ink can be discharged in advance to produce a three-dimensional modeled object (container or outer frame), or only a part of the three-dimensional modeled object (container or outer frame) can be produced and the remaining part can be discharged together with the model material ink and the first supporting material ink. In addition, all can be produced at the same time. The three-dimensional modeled object (container or outer frame) made of the second supporting material ink becomes a support body for the model material ink and the first supporting material ink, so that the collapse of the model material ink and the first supporting material ink can be more fully prevented, and a three-dimensional modeled object with a higher height can be produced as desired. In addition, such a second supporting material ink may not contain a hydrogen peroxide decomposer, or hydrogen peroxide or a hydrogen peroxide donor. By replacing the first supporting material ink with such a second supporting material ink, the discharge amount of the first supporting material ink is reduced, and the amount of hydrogen peroxide decomposer, or hydrogen peroxide or a hydrogen peroxide donor contained in the first supporting material ink can also be saved. It should be noted that the second supporting material contained in the second supporting material ink is not particularly limited as long as it becomes a support body for the model material ink and / or the supporting material ink. The supporting material or glass, resin, etc. used in the above-mentioned supporting material ink (b) can be used. In addition, as described later, the shape of the second supporting material ink can be removed in the same way as the shape of the first supporting material ink by selecting an appropriate material. The model material ink can be partially or completely in contact with the shape composed of the second supporting material.

[0090] Fig.10 Schematic diagram for explaining the process of further using a second supporting material ink to produce a three-dimensional object in the method for producing a three-dimensional object of the present invention. Figure 1 Different parts. Fig.10 (a) schematically shows a state where the second supporting material ink 11s is discharged from the nozzle 13a of the 3D printer. Fig.10 (b) schematically shows a state where the first supporting material ink 11 is discharged from the nozzle 13b of the three-dimensional printer. Fig.10 (c) schematically shows a state where the modeling material ink 12 is discharged from the nozzle 13c of the three-dimensional printer. It should be noted that the nozzle 13a, the nozzle 13b, and the nozzle 13c may be the same nozzle, but are preferably different from each other. Fig.10 In the process, the second supporting material ink 11s is discharged, the first supporting material ink 11 is discharged to the position surrounded by the second supporting material ink 11s, and the model material ink 12 is discharged to the position surrounded by the first supporting material ink 11, thereby forming one layer, and this operation is repeated to form a layered structure.

[0091] like Fig.10 (a)~ Fig.10 As shown in (c), the inner and outer peripheries of the discharged second supporting material ink 11s are both flat and thin-walled, and they are in contact with and support the discharged first supporting material ink 11, and also indirectly support the discharged modeling material ink 12. By using a material that can form a hard modeling object as the second supporting material ink 11s, it is possible to more fully prevent the modeling material ink 12 and the first supporting material ink 11 from collapsing during the gelation process, etc. In addition, a three-dimensional modeling object with a higher height can be produced as desired.

[0092] Fig.10 (d) with Figure 1 (c) also shows a step in which, after the gelation of the model material ink 12 is completed, the obtained object is immersed in a removal solution 15 to remove the object formed by the first support material ink 11 and the object formed by the second support material ink 11s. This step will be described below.

[0093] Fig.11 In addition to changing the discharge range of the first supporting material ink 21 and the second supporting material ink 21s to Fig.11 In addition to the Fig.10 The following only describes the Fig.10 Different parts. Fig.11 In the embodiment, the second supporting material ink is discharged in a manner that the inner peripheral portion becomes a step-like shape extending downward, and Fig.10 Compared with the case of the first supporting material ink, a part of the first supporting material ink is replaced with the second supporting material ink. By using a material that can form a hard modeling object and does not contain a hydrogen peroxide decomposer, hydrogen peroxide, or a hydrogen peroxide donor as the second supporting material ink 21s, it is possible to more fully prevent the model material ink 22 and the first supporting material ink 21 from collapsing during the gelation process, etc., and at the same time reduce the discharge amount of the first supporting material ink 21, which can save the amount of hydrogen peroxide or a hydrogen peroxide donor, or a hydrogen peroxide decomposer.

[0094] Fig.12 In addition to changing the discharge range of the second supporting material ink 31s to Fig.12 In addition to the Fig.10 The following only describes the Fig.10 Different parts. Fig.12 In the process, the second supporting material ink is discharged in a manner that the outer periphery is formed into a step-like shape that expands upward in accordance with the shape of the discharged model material ink 32 and the discharged first supporting material ink 31, and the second supporting material ink is discharged in a manner that the outer periphery is formed into a step-like shape that expands upward, and Fig.10 In comparison, the discharge amount of the second supporting material ink can also be reduced.

[0095] <Step of Removing the Modeled Object of the Support Material Ink after Crosslinking the Crosslinkable Polymer>

[0096] The removal process is not particularly limited as long as the object of the support material ink (b) can be removed to obtain the object of the model material ink (a). For example, it is preferably carried out by immersing the object of the support material ink (b) in a removal solution for the object of the support material ink (b) and / or washing. From the perspective of reducing the impact on the object (three-dimensional object) of the model material ink (a), the removal process is preferably carried out under mild conditions, for example, preferably at a temperature of 10°C to 40°C. In addition, from the perspective of enzyme activity, the pH of the removal solution at 20°C is preferably 2 to 10, more preferably 5 to 9. It should be noted that the object of the support material ink is composed of the remaining components such as the support material (polymer) after hydrogen peroxide or a hydrogen peroxide decomposing agent is supplied from the support material ink to the model material ink.

[0097] When the second supporting material ink is further used together with the supporting material ink (b), the shaped object of the supporting material ink (b) and the shaped object of the second supporting material ink can be removed by different methods respectively, but it is preferred to remove the shaped object of the second supporting material ink together with the shaped object of the supporting material ink (b) through the above-mentioned removal process.

[0098] When the supporting material is a water-soluble polymer or a water-dispersible polymer, water or an aqueous solution can be used as a removal solution. In addition, when the water-soluble polymer or the water-dispersible polymer forms a polymer aqueous gel, and the formed polymer aqueous gel acts on an ionic compound to condense or reduce viscosity, an aqueous solution of an ionic compound can be particularly preferably used as a removal solution. Regarding the action of the water-soluble polymer or the water-dispersible polymer on the ionic compound, as long as the water-soluble polymer or the water-dispersible polymer contacts the ionic compound, the polymer aqueous gel condenses or the viscosity is reduced, there is no particular limitation, for example, reaction (chemical reaction) with the ionic compound can be cited. In the case where the polymer aqueous gel is a material decomposed by enzyme treatment, a decomposing enzyme can be used, and by using culture fluid, buffer, physiological saline and water after enzyme treatment, the shape of the supporting material ink can be removed. In the case where the supporting material is a material that undergoes phase change due to temperature, it can be caused to undergo phase change by heating or cooling, and by using water or an aqueous solution after the phase change, the shape of the supporting material ink can be removed.

[0099] Examples of the ionic compound include polyvalent metal salts of calcium, zinc, magnesium, barium, strontium, copper, iron, aluminum, cobalt, etc. Among them, calcium chloride, calcium hydroxide, calcium carbonate, and calcium lactate are preferred, and calcium chloride is more preferred.

[0100] The concentration of the ionic compound in the removal solution is preferably 1 mM or more, more preferably 10 mM or more, and preferably 2 M or less, more preferably 0.2 M or less.

[0101] Examples of the degrading enzyme include cellulase, amylase, glucanase, carrageenase, alginate lyase, hyaluronidase, chitinase, and chitosanase, but are not limited thereto.

[0102] The concentration of the degrading enzyme in the removal solution is preferably 0.1% by weight to 5% by weight, more preferably 0.1% by weight to 1% by weight.

[0103] <Other Processes>

[0104] The manufacturing method of the present invention may include a step of leaving the discharged model material ink and support material ink to stand after the step of discharging the model material ink and the support material ink separately and before the step of removing the shaped object of the support material ink to allow the model material to be fully gelled.

[0105] The three-dimensional object obtained by the manufacturing method of the present invention can be made into a three-dimensional object of a complex shape, for example, made of soft materials, and can be used as an artificial organ, etc., particularly in the field of regenerative medicine. It should be noted that the shape of the three-dimensional object is not particularly limited, and is also suitable for a shape having a bridge portion and / or a hole. The size of the three-dimensional object can be set appropriately.

[0106] <<Ink set for making three-dimensional objects>>

[0107] The present invention also relates to an ink set for making a three-dimensional object, which comprises: (a) a model material ink comprising a cross-linked polymer; and (b) a support material ink comprising a support material, wherein either the model material ink or the support material ink further comprises a hydrogen peroxide decomposer, and the other further comprises hydrogen peroxide or a hydrogen peroxide donor. The other components contained in the cross-linked polymer, the support material, the hydrogen peroxide decomposer, hydrogen peroxide, the hydrogen peroxide donor, the model material ink (a) or the support material ink (b) are as described above. Using the ink set for making a three-dimensional object of the present invention, the method for making a three-dimensional object of the present invention can be appropriately implemented.

[0108] Example

[0109] The present invention will be described below with reference to Examples, but the present invention is not limited to the following Examples. Hereinafter, "parts" or "%" means "parts by weight" or "% by weight" respectively, unless otherwise specified.

[0110] Manufacturing Example 1 (Preparation of ink for model material)

[0111] Sodium hyaluronate (manufactured by Fujimedica Co., Ltd.) 10 g was dissolved in 1 L of 0.1 M 2-Morpholinoethanesulfonic acid monohydrate (2-Morpholinoethanesulfolic acid, monohydrate, MES) buffer (pH 5.8). A solution prepared by dissolving 0.56 g of tyramine hydrochloride, 1.43 g of water-soluble carbodiimide (WSCD·HCl) and 0.43 g of N-hydroxysuccinimide (NHS) in 10 mL of purified water was further added, and the mixture was stirred at room temperature for 22 hours. The obtained reaction mixture was filtered by ultrafiltration and freeze-dried in a vacuum to prepare HYA-Ph (modification rate of phenolic hydroxyl group: 4.8 mol%). The prepared HYA-Ph was dissolved in a solvent (phosphate buffered saline without calcium / magnesium) at a concentration of 1.0 w / v% (g / 100 mL) and a hydrogen peroxide decomposition agent (HRP [peroxidase from horseradish]) at a concentration of 10 U / mL to prepare ink for a model material.

[0112] Manufacturing Example 2 (Preparation of Support Material Ink)

[0113] Hydrogen peroxide was dissolved in Projelly (a high viscosity type manufactured by JEX INC.) at concentrations of 0.1 mM, 1.0 mM, 10 mM, and 100 mM to prepare a support material ink.

[0114] The viscosity of the Projelly used in the support material ink relative to the shear rate is as follows Figure 2 As shown, it can be seen that even if hydrogen peroxide is added as a cross-linking agent, the viscosity does not change. In addition, since the viscosity of Projelly is very high at a low shear rate, the support material ink obtained in Manufacturing Example 2 containing it as a main component can form a molded object that can maintain a shape with a single substance after being discharged.

[0115] like Figure 3 As shown, Projelly is mixed with calcium ions Ca in a 100 mM calcium chloride aqueous solution. 2+ The reaction produces a white precipitate, and the viscosity is greatly reduced. It can be seen that Projelly reacts with the ionic compound to aggregate or reduce the viscosity, so the ionic compound can be used appropriately when removing the shape of the support material ink containing Projelly.

[0116] Figure 4It is shown in the figure that in the DMEM medium (Double's modified Eagle's medium) containing Projelly, the cells proliferate and expand as usual in the same manner as in the DMEM medium without Projelly. The cells used are fibroblasts from fetal mice (10T1 / 2 cells). Therefore, Projelly is essentially non-cytotoxic. In summary, Projelly is suitable as a raw material for ink used as a support material.

[0117] Manufacturing Example 3 (Preparation of ink for model material)

[0118] A solution of modified polyvinyl alcohol (AF-17 manufactured by JAPAN VAM & POVAL CO., LTD.) dissolved at 3.3 (w / v)% in 0.1 M 2-morpholinoethanesulfonic acid monohydrate (2-Morpholinoethanesulfolic acid, monohydrate, MES) buffer (pH 6.0) was heated at 120°C for 15 minutes. After cooling to room temperature, tyramine hydrochloride, water-soluble carbodiimide (WSCD·HCl) and N-hydroxysuccinimide (NHS) were added in such a manner that they became 3.3, 3.3 and 1.0 (w / v)% respectively, and then stirred at 40°C for 20 hours. The obtained reaction solution was added to ethanol to precipitate it, and the precipitate was washed with 90% ethanol aqueous solution and ethanol, and dried under reduced pressure to prepare PVA-Ph (modification rate of phenolic hydroxyl group: 2.4×10 -4 A model material ink was prepared in the same manner as in Production Example 1 so that the concentration of the prepared PVA-Ph was 1.0 w / v% (g / 100 mL).

[0119] Manufacturing Example 4 (Preparation of ink for model material)

[0120] 10 g of gelatin (manufactured by JELLICE Co., Ltd.) was dissolved in 500 mL of 0.05 M 2-morpholinoethanesulfonic acid monohydrate (2-Morpholinoethanesulfolic acid, monohydrate, MES) buffer (pH 6.0). A solution of 1.84 g of tyramine hydrochloride, 1.85 g of water-soluble carbodiimide (WSCD·HCl) and 0.57 g of N-hydroxysuccinimide (NHS) dissolved in 5 mL of purified water was further added, and the mixture was stirred at room temperature for 20 hours. The obtained reaction mixture was filtered by ultrafiltration and freeze-dried in a vacuum to prepare Gela-Ph (modification rate of phenolic hydroxyl group: 2.7×10 -4 A model material ink was prepared in the same manner as in Production Example 1 so that the concentration of the prepared Gela-Ph was 3.0 w / v% (g / 100 mL).

[0121] Example 1

[0122] As a three-dimensional object, two types of hexagonal cylinders of the following sizes were produced according to the following steps: Using a three-dimensional printer (product name: BIO X, manufacturer name: Cellink), the support material ink with a hydrogen peroxide concentration of 10 mM produced in Production Example 2 and the model material ink produced in Production Example 1 were respectively discharged onto a substrate to form layers, and this operation was repeated to perform lamination. After the discharge was completed, the ink was allowed to stand at 25° C. for 10 minutes to fully gel the model material ink. Then, the object was immersed in a calcium chloride solution (concentration 100 mM) to remove the support material ink.

[0123] Model 1 (thickness: 1mm, height: 5mm, length of one side: 6mm)

[0124] Model 2 (thickness: 2mm, height: 5mm, length of one side: 6mm)

[0125] Comparative Example 1

[0126] A three-dimensional object was produced in the same manner as in Example 1 except that the supporting material ink was not used and the conditions for exposure to hydrogen peroxide vapor were set.

[0127] like Figure 5 As shown, in Example 1, the support material ink was used, and the model material ink was gelled while the modeled object was held by the support material ink, thereby making it possible to produce a modeled object with very good modeling accuracy. On the other hand, in Comparative Example 1, no support material ink was used, and the modeled object was exposed to hydrogen peroxide vapor, resulting in deformation, and only a modeled object with low modeling accuracy was obtained.

[0128] Example 2

[0129] A three-dimensional object was produced in the same manner as in Example 1 except that the hydrogen peroxide concentration of the supporting material ink was 1 mM.

[0130] Example 3

[0131] A three-dimensional object was produced in the same manner as in Example 1 except that the hydrogen peroxide concentration of the supporting material ink was changed to 100 mM.

[0132] like Figure 6 As shown, in Example 2, three-dimensional objects with good modeling accuracy were obtained. On the other hand, in Example 3, although a three-dimensional object with good modeling accuracy was obtained, the modeling accuracy was slightly lower than that of the three-dimensional objects obtained in Examples 1 and 2 due to the high concentration of hydrogen peroxide and the large number of bubbles.

[0133] The above results show that, when manufacturing a complex-shaped object made of a soft material (soft material), a three-dimensional object can be obtained with good molding accuracy by applying the manufacturing method of the present invention.

[0134] Example 4

[0135] A three-dimensional object was produced in the same manner as in Example 1 except that the ink produced in Production Example 3 was used as the model material ink.

[0136] Example 5

[0137] A three-dimensional object was produced in the same manner as in Example 1 except that the ink produced in Production Example 4 was used as the model material ink.

[0138] like Figure 7 As shown, in Example 4 using the model material ink containing modified polyvinyl alcohol and Example 5 using the model material ink containing gelatin, similar to Example 1 using the model material ink containing hyaluronic acid, it is possible to produce a modeled object with very good modeling accuracy.

[0139] Example 6

[0140] Mouse fibroblasts 10T1 / 2 were cultured at 1×10 5 The cells / mL were dispersed in the model material ink prepared in Production Example 1, and a three-dimensional object was prepared in the same manner as in Example 1 using the obtained cell-containing model material ink and the support material ink prepared in Production Example 2.

[0141] The obtained three-dimensional structures were cultured in DMEM medium containing 10 vol% fetal bovine serum. Before the start of culture, on the 7th day and the 14th day after the start of culture, the structures containing cells were stained by immersion in phosphate buffered saline (pH 7.4) containing 1 μg / mL of live cell staining reagent Calcein-AM and dead cell staining reagent PI for 10 minutes. The photos taken of the results are shown in Figure 8 A large number of cells survived even after 14 days of culture.

[0142] Example 7

[0143] Human hepatoma-derived HepG2 cells were cultured at 1×10 5 The cells / mL were dispersed in the model material ink prepared in Production Example 1, and a three-dimensional object was prepared in the same manner as in Example 1 using the obtained cell-containing model material ink and the support material ink prepared in Production Example 2.

[0144] The obtained three-dimensional structures were cultured in DMEM medium containing 10 vol% fetal bovine serum. Before the start of culture, on the 7th day and the 14th day after the start of culture, the structures containing cells were stained by immersion in phosphate buffered saline (pH 7.4) containing 1 μg / mL of live cell staining reagent Calcein-AM and dead cell staining reagent PI for 10 minutes. The photos taken of the results are shown in Figure 8 A large number of cells survived even after 14 days of culture, and the green region indicating live cells in the structure increased in area, confirming the proliferation of HepG2 cells.

[0145] Example 8

[0146] The three-dimensional structure produced in Example 6 was immersed in a DMEM medium containing 1 mg / mL hyaluronidase for 60 minutes immediately after production to decompose the structure, and the cells were recovered. The recovered cells were plated at 1×10 4 Cells / cm 2 The cells were inoculated in a culture dish (Corning, 60 mm diameter) and cultured. The results were photographed and shown in Fig. 9 The cells maintained the same shape, adhesion and proliferation abilities as the untreated cells. Based on the results of Examples 6 to 8, it is estimated that the production method of the present invention has little adverse effect on cells.

[0147] Description of Reference Numerals

[0148] 1: Ink for support material

[0149] 2, 12, 22, 32: Ink for model materials

[0150] 2A, 12A, 22A, 32A: Three-dimensional objects

[0151] 2a, 12a, 22a, 32a: Bridge area

[0152] 2b, 12b, 22b, 32b: Hole

[0153] 3a, 3b, 13a, 13b, 13c, 23a, 23b, 23c, 33a, 33b, 33c: Nozzle

[0154] 4, 14, 24, 34: substrate

[0155] 5, 15, 25, 35: Remove solution

[0156] 11, 21, 31: Ink for the first supporting material

[0157] 11s, 21s, 31s: ink for the second supporting material.

Claims

1. A method for manufacturing a three-dimensional object, comprising: A step of separately discharging (a) a model material ink containing a crosslinkable polymer and (b) a support material ink containing a support material; as well as A step of removing the shaped object of the support material ink after cross-linking the cross-linkable polymer, Either one of the model material ink (a) or the support material ink (b) further contains a hydrogen peroxide decomposing agent, and the other further contains hydrogen peroxide or a hydrogen peroxide donor. The discharged hydrogen peroxide decomposing agent decomposes the discharged hydrogen peroxide to cross-link the cross-linkable polymer, thereby forming a three-dimensional object.

2. The method for manufacturing a three-dimensional object according to claim 1, wherein: The hydrogen peroxide decomposer is a cross-link forming enzyme.

3. The method for producing a three-dimensional object according to claim 1 or 2, wherein: The hydrogen peroxide donor is a hydrogen peroxide generating enzyme.

4. The method for producing a three-dimensional object according to any one of claims 1 to 3, wherein: The cross-linkable polymer has a phenolic hydroxyl group.

5. The method for producing a three-dimensional object according to any one of claims 1 to 4, wherein: The supporting material is a water-soluble polymer or a water-dispersible polymer.

6. The method for manufacturing a three-dimensional object according to claim 5, wherein: The water-soluble polymer or water-dispersible polymer is a water-soluble polymer or water-dispersible polymer that forms an aqueous gel that agglomerates or reduces its viscosity when reacting with an ionic compound. In the process of removing the shaped object of the support material ink, the ionic compound is allowed to act to remove the shaped object of the support material ink (b).

7. An ink set for making a three-dimensional object, comprising: (a) a model material ink comprising a cross-linkable polymer; and (b) a support material ink comprising a support material, Either one of the model material ink (a) and the support material ink (b) further contains a hydrogen peroxide decomposing agent, and the other further contains hydrogen peroxide or a hydrogen peroxide donor.

8. The ink set for producing a three-dimensional object according to claim 7, wherein: The hydrogen peroxide decomposer is a cross-link forming enzyme.

9. The ink set for producing a three-dimensional object according to claim 7 or 8, wherein: The hydrogen peroxide donor is a hydrogen peroxide generating enzyme.

10. The ink set for producing a three-dimensional object according to any one of claims 7 to 9, wherein The cross-linkable polymer has a phenolic hydroxyl group.

11. The ink set for producing a three-dimensional object according to any one of claims 7 to 10, wherein The supporting material ink (b) contains a water-soluble polymer or a water-dispersible polymer that forms an aqueous gel that aggregates or reduces its viscosity when reacting with an ionic compound.

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