Method of manufacturing free-standing organic-inorganic complex 3D printing structure

Abstract

The present invention relates to a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, specifically comprising the steps of: 3D printing a paste containing printing inorganic powder and a collagen solution in a coagulation bath to form a molded body; a step of primary curing in the coagulation bath; and a step of secondary curing the molded body; wherein the coagulation bath is a hydrogel containing a gelling agent, ethanol, and a pH adjuster.

Description

Technology Field

[0001] The present invention relates to a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure. Background Technology

[0003] Bio 3D printing refers to a technology that uses a 3D printer to create human tissue and transplant it into patients with damaged areas. Recently, active research is being conducted to 3D print scaffolds for regenerating hard tissues, such as bones and teeth, using bio 3D printing technology.

[0004] Such scaffolds for hard tissue regeneration can be fabricated using materials that provide a suitable environment for cell attachment, differentiation, growth, and migration; as such materials, natural and synthetic polymers, bioceramics, and polymer-ceramic composites are being studied.

[0005] In this regard, a method for manufacturing a 3D structure using ceramics has been disclosed, wherein a molded body is obtained by 3D printing with a paste containing ceramic powder, and subsequently cured by hydration or a cement reaction by acid-base reaction, thereby enabling the production of a three-dimensional ceramic structure without a high-temperature sintering process. Since this method does not undergo a high-temperature sintering process of 1000°C or higher, it has the advantage of not causing unexpected crystallization problems due to high-temperature heat treatment, problems of reduced biodegradability and bioactivity, and problems of shrinkage and cracking caused by heat treatment.

[0006] Meanwhile, when forming a molded body using general 3D printing, in order to 3D print a complex molded body having an overhang structure (a structure that becomes unstable as the amount of overlap between the lower and upper layers decreases), supports have been printed together with the structure to prevent the structure from collapsing. However, this has problems such as the long production time required, the consumption of a large amount of ceramic material for making supports, and the risk of damage to the structure when removing supports because additional post-processing steps must be performed to remove the supports.

[0007] To solve this, Korean registered patent No. 10-2474673 disclosed a method for manufacturing a self-supporting ceramic 3D printed structure, wherein a 3D printed structure is manufactured by 3D printing a paste containing ceramic powder in a coagulation bath containing a hydrogel to form a molded body, and then curing it through the cement reaction, thereby enabling the production of a complex structure having an overhang structure without support.

[0008] Meanwhile, hard tissues are composed of not only inorganic materials such as calcium phosphate-based ceramics but also extracellular matrix materials such as collagen; therefore, the ultimate goal of developing scaffolds for hard tissue regeneration is to develop such organic-inorganic composites.

[0009] In this regard, conventional methods for manufacturing 3D printed structures containing collagen and ceramics by crosslinking collagen using chemical crosslinking agents such as glutaraldehyde, genipin, and EDC / NHS have been disclosed, but the use of such chemical crosslinking agents has the problem of posing a risk due to residual crosslinking agents.

[0010] Accordingly, the inventors have developed a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure having an overhang structure and capable of manufacturing a structure for hard tissue regeneration containing collagen and inorganic materials without support, by 3D printing a paste containing collagen and inorganic materials in a coagulation bath to form a molded body, wherein the coagulation bath includes ethanol and a pH adjuster to induce collagen hardening, and includes a gelling agent that supports the 3D printed paste and can be mixed with the pH adjuster, thereby completing the present invention. Prior art literature

[0012] Republic of Korea Registered Patent No. 10-2474673 The problem to be solved

[0013] The purpose in one aspect is...

[0014] The invention provides a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure. means of solving the problem

[0016] In order to achieve the above objective,

[0017] In terms of work

[0018] A step of forming a molded body by 3D printing a paste containing printing inorganic powder and a collagen solution in a coagulation bath;

[0019] A step of primary curing in the above-mentioned coagulation bath; and

[0020] The method includes a step of secondarily curing the above-mentioned molded body; and

[0021] A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure is provided, wherein the above-mentioned coagulation bath is a hydrogel comprising a gelling agent, ethanol, and a pH adjuster.

[0023] The above first curing step

[0024] It includes a step of hardening collagen through self-assembly by the above-mentioned pH adjuster and a dehydration reaction induced by ethanol.

[0026] The above collagen solution is a solution in which collagen is dissolved in an acidic solvent.

[0028] The above pH adjuster is one or more of triethanolamine (TEA), NaOH, KOH, arginine, tromethamine, and aminomethyl propanediol.

[0030] The above coagulation bath contains the pH adjuster in an amount of 1 to 30 weight percent relative to the total weight of the hydrogel.

[0031] In addition, the above coagulation bath contains less than 10 to 80 weight percent of the ethanol relative to the total weight of the hydrogel.

[0033] The above gelling agent is one or more of Carbopol, laponite RD, laponite EP, laponite XLG, gelatin, p123, F127, hydroxyethyl cellulose, methyl cellulose, xanthan gum, and polyquaternium.

[0035] The above coagulation bath contains the gelling agent in an amount of 0.6 to 40 weight percent relative to the total weight of the hydrogel.

[0037] The above first curing step is performed at a temperature of 1℃ to 40℃.

[0039] The above-mentioned printing inorganic powder includes ceramic powder, and

[0040] The above secondary curing step is,

[0041] The step may be to treat the molded body with a ceramic hardening solution to harden the ceramic contained in the molded body.

[0043] Here, the ceramic powder may be a calcium phosphate-based ceramic powder.

[0045] In addition, the ceramic hardening solution may be one or more selected from the group consisting of DSP (disodium phosphate dihydrate, Na2HPO4·2H2O), MSP (monosodium phosphate dihydrate, NaH2PO4·2H2O), PBS (Phosphate buffer saline), MCPM (Monocalcium phosphates monohydrate, Ca(H2PO4)2·H2O), and cell culture medium.

[0047] The ceramic curing liquid may be at a temperature of 20°C to 40°C.

[0049] The method for manufacturing the above-mentioned self-supporting organic-inorganic composite 3D printed structure is

[0050] The method may further include the step of separating the molded body from the coagulation bath and treating it with a buffer solution.

[0052] In addition, the method may further include the step of treating the molded body treated with the above buffer solution with ethanol.

[0054] The above paste may further include biofunctional materials.

[0056] The above manufacturing method can produce a collagen-containing ceramic 3D printed structure that includes an overhang structure in at least a part.

[0058] The above manufacturing method can produce a scaffold for hard tissue regeneration. Effects of the invention

[0060] The present invention is a method for manufacturing an organic-inorganic composite 3D printed structure, wherein a 3D printed structure comprising collagen and an inorganic material can be manufactured, and a 3D printed structure for a bone regeneration scaffold comprising collagen and a bioceramic can be manufactured.

[0061] In addition, the present invention is a method for manufacturing a self-standing 3D printed structure, which allows a structure having an overhang structure to be manufactured in a self-standing or free-standing manner without printing on supports, thereby saving process time and costs, and since no post-processing step for removing supports is required, the process is simplified and the risk of damage to the structure is reduced.

[0062] In addition, the present invention provides a method for manufacturing a scaffold for hard tissue regeneration, wherein the scaffold for hard tissue regeneration can be manufactured by an organic-inorganic composite material containing collagen and having a complex structure including an overhang structure, and has the advantage of not causing problems caused by residual chemical crosslinking agents by not using a chemical crosslinking agent to crosslink the collagen.

[0063] The scaffold for hard tissue regeneration produced by the manufacturing method of the present invention has the advantage of excellent bone differentiation gene expression.

[0064] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by a person skilled in the art from the description below. Brief explanation of the drawing

[0066] FIG. 1 is a schematic diagram showing a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. FIG. 2 is a photograph schematically showing the steps after forming a molded body in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. FIG. 3 is a graph of storage modulus and loss modulus according to printing temperature in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. FIG. 4 is a graph evaluating viscosity characteristics according to the concentration of ethanol in a coagulation bath in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. FIG. 5 is a graph evaluating viscosity characteristics according to the concentration of a gelling agent in a coagulation bath in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. Figure 6 is a photograph of the result of printing using a coagulation bath containing ethanol according to the method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. Figure 7 is a comparative example of Figure 6, showing a photograph of the result printed using a coagulation bath that does not contain ethanol. FIG. 8 is a photograph evaluating printing characteristics according to the concentration of a pH adjuster in a coagulation bath in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. FIG. 9 is a graph evaluating mechanical strength according to the concentration of a pH adjuster in a coagulation bath in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. Figure 10 is a graph showing the highest value among the modulus values ​​measured in Figure 9. FIG. 11 is a photograph showing a vertical ring structure having an overhang structure manufactured according to an embodiment. FIG. 12 is a photograph showing a zygomatic bone structure having an overhang structure manufactured according to an embodiment. FIG. 13 is a photograph evaluating the resolution according to printing speed and nozzle size in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. FIG. 14 is a graph evaluating the line width according to pneumatic pressure and nozzle size in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. FIG. 15 is a graph evaluating the spacing between lines (pore size) according to pneumatic pressure and nozzle size in a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment. Figure 16 is a graph of XRD results for a molded body manufactured according to an example, a molded body treated with a buffer solution and ethanol, and a structure cured with a curing solution. Figure 17 is a graph of the results of measuring a structure manufactured according to an example using FT-IR. FIG. 18 is a graph showing the compressive strength of 3D printed structures manufactured according to the example and comparative example. FIGS. 19 to 25 are graphs confirming the bone regeneration ability of 3D printed structures prepared according to the examples and comparative examples, FIG. 19 is an absorbance graph for a wavelength of 450 nm, FIG. 20 is a DNA concentration graph calculated based thereon, and FIGS. 21 to 25 are graphs showing the results of measuring the expression of OCN (Osteocalcin), OPN (Osteopontin), ALP (Alkaline phosphatase), COL 1 (Collagen type 1), and RUNX 2 (Runt-related transcription factor 2), respectively. Specific details for implementing the invention

[0067] Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, embodiments of the present invention may be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. Furthermore, the following embodiments are provided to more completely explain the present invention to those with average knowledge in the art. Accordingly, the shapes and sizes of elements in the drawings may be exaggerated for clearer explanation, and elements indicated by the same reference numerals in the drawings are the same elements. In addition, the same reference numerals are used throughout the drawings for parts having similar functions and operations. Furthermore, throughout the specification, the term "comprising" a component means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0069] In one aspect,

[0070] A step of forming a molded body by 3D printing a paste containing printing inorganic powder and a collagen solution in a coagulation bath;

[0071] A step of primary curing in the above-mentioned coagulation bath; and

[0072] The method includes a step of secondarily curing the above-mentioned molded body; and

[0073] A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure is provided, wherein the above-mentioned coagulation bath is a hydrogel comprising a gelling agent, ethanol, and a pH adjuster.

[0075] A 3D printed structure manufactured by the manufacturing method according to one embodiment is preferably a scaffold for hard tissue regeneration.

[0076] A 3D printed structure manufactured by a manufacturing method according to one embodiment may include an overhang structure in at least a portion.

[0077] A 3D printed structure manufactured by a manufacturing method according to one embodiment comprises collagen and inorganic materials, preferably comprises collagen and bioceramics, and may further comprise biofunctional materials.

[0079] Hereinafter, a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment will be described in detail step by step.

[0081] A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment includes the step of forming a molded body by 3D printing a paste containing printing inorganic powder and a collagen solution in a coagulation bath.

[0083] At this time, the collagen solution included in the paste is a solution in which collagen is dissolved in an acidic solvent. For example, the collagen solution may be a solution in which 0.3 weight% of collagen is dissolved in 0.5M acetic acid.

[0084] In this case, the collagen may be atellocollagen, fibrillar collagen of collagen type I, III, V, or VI, decellularized extracellular matrix, fish collagen, recombinant collagen, etc., but is not limited thereto.

[0085] In addition, the printing inorganic powder may be a ceramic powder, and preferably a bioceramic powder.

[0086] The above ceramic powder is a calcium phosphate-based ceramic powder and may be one or more selected from the group consisting of α-TCP (α-Tricalcium phosphate), ββphosphate), hydroxyapatite, DCPD (Dicalcium phosphate dihydrate), MCPM (Monocalcium phosphate monohydrate), DCPA (Dicalcium phosphate anhydrous), and BCP (Biphasic Calcium Phosphate).

[0088] The above paste may contain 0.1 to 5 weight percent of the collagen relative to the total weight and 1 to 70 weight percent of the printing inorganic powder.

[0090] Meanwhile, the above paste may further include a biofunctional material.

[0091] At this time, the above-mentioned biofunctional substance may be one or more selected from the group consisting of growth factors, proteins, protein drugs, antiproliferative agents, antithrombin, immunosuppressants, lipids, anti-lipids, liposomes, anti-inflammatory agents, antitumor agents, antiplatelet agents, angiogenic agents, anti-angiogenic agents, vitamins, aptamers, antimitotic agents, metalloproteinase inhibitors, NO donors, estradiol, antisclerotic agents, vasoactive agents, beta-blockers, AZ-blockers, hormones, statins, antioxidants, membrane stabilizers, calcium channel blockers, retinoids, peptides, lipoproteins, polypeptides, polynucleotide-encoding polypeptides, enzymes, genetic material, chemical solvents, energy-activators, lymphocyte inhibitors, macrophage inhibitors, and mixtures thereof.

[0093] A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment allows the paste to be obtained as a molded body with a three-dimensional structure through 3D printing technology. The 3D printing technology is a technology that manufactures a product by continuously reconstructing a digitized three-dimensional product design into a two-dimensional cross-section and then printing raw materials layer by layer.

[0094] The column thickness of the support can be controlled by using nozzles of various sizes applied to the 3D printer, and can be molded into various shapes (column spacing, pore size, pore shape, shape of the support, etc.) through a computer program.

[0096] A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment has the advantage of being able to form a self-supporting structure having an overhang structure without printing a separate support together by forming a molded body in a solidification bath.

[0097] An overhang structure refers to a structure where the 3D printing becomes unstable as the amount of overlap between the lower and upper layers decreases. For example, the slope of the overhang section can be 30° or more, and more specifically, 45° or more.

[0098] In the case of the conventional process, namely manufacturing a ceramic 3D printed structure with an overhang structure in air, supports must be printed together to support the unstable structure and then removed later. This consumes time and cost to print the supports, requires a post-processing step to remove them, and also poses a risk that the structure may be damaged during support removal.

[0099] On the other hand, the method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment can be formed as a self-supporting structure without printing separate supports together, thereby saving time and costs, and has the advantage of not requiring the above post-processing process and not having the risk of damage to the structure due to the removal of supports.

[0101] Here, the coagulation bath is characterized as being a hydrogel comprising a gelling agent, ethanol, and a pH adjuster.

[0102] Here, the gelling agent is included in a coagulation bath to form a hydrogel and support a molded body formed by 3D printing.

[0103] The viscosity of the above hydrogel is 10 2 It may be Pa·s or higher, preferably 10 3 It is maintained in the form of a gel in a coagulation bath at a level of Pa·s or higher.

[0105] Meanwhile, the above coagulation bath preferably has a pH of 8 or higher, preferably 9 to 11, for hardening the collagen.

[0106] Accordingly, the gelling agent is preferably a gelling agent capable of forming a gel at a pH of 8 or higher, and, for example, may be a gelling agent synthesized from polyacrylic acid known as carbopol, and H known as laponite 12 Li2Mg 16 Na2O 72 Si 24 As lithium magnesium silicate sodium represented by, it may be laponite RD, laponite EP, or laponite XLG, or one or more of gelatin, P123, F127, hydroxyethyl cellulose, methyl cellulose, xanthan gum, and polyquaternium.

[0108] The above coagulation bath may contain the gelling agent in an amount of 0.6 to 40 weight% relative to the total weight of the hydrogel, preferably 0.6 to 10 weight%, and more preferably 0.6 to 5 weight%.

[0110] This is intended to serve the role of supporting the molded body through the gelling agent. If the gelling agent is included in an amount of less than 0.6 weight%, the viscosity of the formed hydrogel is low, so it may not be able to support the printed paste and the structure may collapse. If the gelling agent is included in an amount exceeding 40 weight%, the rigidity of the formed hydrogel increases and interferes with the movement of the 3D printing nozzle, which may result in the ceramic paste not being printed smoothly.

[0112] In addition, the above ethanol and pH adjuster are included in the coagulation bath to cure the collagen contained in the molded body.

[0113] The above ethanol can induce dehydration of collagen, thereby causing collagen hardening.

[0114] At this time, the ethanol may be included in an amount of 10 to less than 80% by weight relative to the total weight of the hydrogel, and preferably, it may be included in an amount of 10% or more, 20% or more, 30% or more, 40% or more, 45% or more, 50% or more, or 60% or more by weight, and may be included in an amount of 75% or less, 70% or less, 68% or less, 65% or less, 62% or less, or 60% or less by weight.

[0116] If the above ethanol is included in an amount of less than 10% by weight, the dehydration reaction of collagen by the above ethanol is insufficient, and thus the problem of collagen hardening not being properly achieved may occur. If the above ethanol is included in an amount of 70% by weight or more, the viscosity capable of supporting the paste is low, and thus the problem of the structure collapsing may occur.

[0118] In addition, the above pH adjuster may cause a rapid pH change in the collagen, inducing self-assembly and causing collagen hardening.

[0119] At this time, the pH adjuster may be one or more of triethanolamine (TEA), NaOH, KOH, arginine, tromethamine, and aminomethyl propanediol, and preferably may be triethanolamine (TEA).

[0121] At this time, the pH adjuster may be included in an amount of 1 to 30% by weight relative to the total weight of the hydrogel, and preferably, it may be included in an amount of 3% or more by weight, 5% or more by weight, 7% or more by weight, 10% or more by weight, 13% or more by weight, 15% or more by weight, and 18% or less by weight.

[0123] If the above pH adjuster is included in an amount of less than 1% by weight, a problem may arise in which collagen hardening by the above pH adjuster does not occur, and if the above pH adjuster is included in an amount of 30% by weight or more, a problem may arise in which it is difficult to control the appropriate viscosity of the hydrogel, making it difficult to fabricate the structure.

[0125] Meanwhile, it may be more preferable for the above coagulation bath not to contain a buffer solution such as PBS in order to induce hardening of the collagen by causing a rapid pH change in the collagen.

[0127] In the step of forming the molded body, the temperature of the paste during 3D printing is preferably 1 to 25℃, and more preferably 25℃ or lower, 20℃ or lower, 15℃ or lower, 10℃ or lower, and 5℃ or lower.

[0129] This is intended to prevent changes in the physical properties of the formed molded body. If the temperature is 25°C or higher, the paste may harden and cause problems with printing not working properly, and if the paste temperature is less than 1°C, the viscosity of the paste may increase rapidly and cause problems with ejection.

[0131] Next, a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment includes a step of first curing in a solidification bath after forming the molded body.

[0133] The above first curing step may be a step of curing collagen through self-assembly by the pH adjuster and a dehydration reaction induced by ethanol.

[0134] The above first curing step can be performed simultaneously with the step of forming the molded body. That is, collagen curing can occur while printing in a coagulation bath.

[0136] The above first curing step is preferably performed at a temperature of 40°C or lower, and can be performed at a temperature of 1°C to 40°C.

[0138] If the above first curing is performed at a temperature exceeding 40°C, denaturation of the collagen may occur, and the structure may collapse.

[0140] Meanwhile, since biofunctional materials may be destroyed or bioactivity may be reduced at temperatures exceeding 37℃, if the paste further contains biofunctional materials, it is preferable to perform the first curing step at 37℃ or lower, preferably 20 to 37℃, and preferably 30 to 37℃.

[0142] The above first curing step may be performed for 30 minutes or more.

[0143] If the above first curing is performed for less than 30 minutes, the collagen curing reaction is insufficient, resulting in the 3D ceramic printed structure having low mechanical properties, which may cause the structure to collapse when removed from the coagulation bath.

[0144] In addition, if the paste contains additional biofunctional materials, it is preferable to perform the first curing for 30 minutes to 1 hour, as a decrease in biofunctionality may occur if cured for more than 1 hour.

[0145] For example, the step of primary curing in the above-mentioned coagulation bath can be performed at 37°C for 30 minutes.

[0147] Next, a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment includes a step of secondarily curing the molded body.

[0148] The above secondary curing step may be a step of curing the inorganic material contained in the molded body.

[0149] Here, the printing inorganic powder comprises ceramic powder, and

[0150] The above secondary curing step is,

[0151] The step may be to treat the molded body with a ceramic hardening solution to harden the ceramic contained in the molded body.

[0152] FIG. 1 is a schematic diagram illustrating a method for manufacturing a self-supporting organic-inorganic composite 3D printing structure according to one embodiment. As shown in FIG. 1, a paste (10) is printed in a coagulation bath (20) to form a molded body (20), and the molded body (20) is treated with a curing liquid (40) to manufacture a 3D printing structure (50).

[0154] At this time, the ceramic hardening liquid is a hardening liquid that induces a cement reaction of the ceramic.

[0155] The above-mentioned curing solution may be one or more selected from the group consisting of DSP (disodium phosphate dihydrate, Na2HPO4·2H2O), MSP (monosodium phosphate dihydrate, NaH2PO4·2H2O), PBS (Phosphate buffer saline), MCPM (Monocalcium phosphates monohydrate, Ca(H2PO4)2·H2O), and cell culture medium.

[0157] The ceramic curing solution can be performed at a temperature of 20°C to 40°C.

[0158] If the above ceramic hardening solution is below 20°C, the cement reaction of the ceramic may be insufficient, resulting in a problem of low mechanical properties of the manufactured structure. Additionally, if the above primary hardening is performed at a temperature exceeding 40°C, the collagen may be denatured, causing the structure to collapse.

[0160] Meanwhile, since the bioactivity of the biofunctional material may decrease at temperatures exceeding 37℃, if the paste further contains the biofunctional material, it is preferable to perform the ceramic curing solution at 37℃ or lower, preferably 20 to 37℃, and preferably 30 to 37℃.

[0161] The above secondary curing step can be performed for 20 to 30 hours.

[0162] For example, the above secondary curing can be performed by treating the molded body in a PBS curing solution at 37°C for 24 hours.

[0164] Meanwhile, a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment may further include the step of separating the molded body from a coagulation bath and treating it with a buffer solution.

[0165] The step of treating with the above buffer solution may be a step for washing the molded body to remove the gelling agent, etc. remaining on the molded body.

[0166] Here, the above buffer solution may be PBS (Phosphate buffer saline).

[0167] Meanwhile, the step of treating with the buffer solution is performed prior to the step of hardening the ceramic powder, and in the step, the cement hardening reaction of the ceramic is carried out by the buffer solution so that ceramic hardening can be achieved.

[0169] In addition, the method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment may further include the step of treating the molded body treated with the buffer solution with ethanol.

[0170] A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment can perform cleaning of the molded body through the step of treating with the ethanol, and can further harden the collagen by additionally inducing a dehydration reaction of the collagen with the ethanol.

[0172] The present invention will be explained in detail below through embodiments and experimental examples.

[0173] However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0174] <Manufacturing Example 1>

[0175] A hydrogel containing carbopol as a gelling agent, ethanol, and triethylamine (TEA) as a pH adjuster in amounts of 0.6 wt%, 60 wt%, and 15 wt%, respectively, was prepared as a coagulation bath.

[0177] <Manufacturing Example 2>

[0178] A hydrogel containing carbopol as a gelling agent, ethanol, and triethylamine (TEA) as a pH adjuster in amounts of 0.6 wt%, 60 wt%, and 10 wt%, respectively, was prepared as a coagulation bath.

[0180] <Manufacturing Example 3>

[0181] A hydrogel containing carbopol as a gelling agent, ethanol, and triethylamine (TEA) as a pH adjuster in amounts of 0.6 wt%, 60 wt%, and 5 wt%, respectively, was prepared as a coagulation bath.

[0183] <Manufacturing Example 4>

[0184] A hydrogel containing carbopol as a gelling agent, ethanol, and triethylamine (TEA) as a pH adjuster in amounts of 0.6 wt%, 60 wt%, and 1 wt%, respectively, was prepared as a coagulation bath.

[0186] <Manufacturing Example 5>

[0187] A coagulation bath was prepared by performing the same method as in Preparation Example 1, except that it was different in that it did not contain ethanol.

[0189] <Manufacturing Example 6>

[0190] A hydrogel containing PBS and P-123 as a gelling agent, wherein P-123 is contained in an amount of 25 to 30 weight percent relative to the total weight of PBS, was prepared as a coagulation bath.

[0192] <Example 1>

[0193] According to FIG. 2, a 3D printed structure was manufactured by the following method.

[0194] Step 1: A collagen solution containing 3% by weight of collagen in 0.5M acetic acid was prepared, and α-TCP was added as a ceramic powder to prepare a paste containing collagen and ceramic (containing 69% by weight of ceramic).

[0195] Step 2: The above paste was placed in an extrusion container, and a molded body was formed in the coagulation bath prepared in Preparation Example 1 using a 3D printing device.

[0196] Step 3: The temperature of the coagulation bath was set to 37°C and maintained for 30 minutes to harden the collagen component in the molded body (see Fig. 2(a)).

[0197] Step 4: The molded body was separated from the coagulation bath and treated with a PBS solution at 37°C for about 3 hours to remove the residual gelling agent contained in the molded body (see Fig. 2(b)).

[0198] Step 5: After separating the PBS-treated molded body from the PBS solution (see Fig. 2(c)), the molded body was washed by treating it with ethanol at 37°C for about 3 hours (see Fig. 2(d)).

[0199] Step 7: The molded body treated with the above ethanol was immersed in a PBS solution for about 24 hours to cement-harden the ceramic components within the molded body, thereby producing a 3D printed structure (see Figure 2 (e)).

[0201] <Comparison Example 1>

[0202] A 3D printed structure was manufactured by performing the same method as in Example 1, except that the coagulation bath in Example 1 above was changed to use the coagulation bath of Example 5.

[0204] <Comparison Example 2>

[0205] A 3D printed structure was manufactured by performing the same method as in Example 1, except that the coagulation bath in Example 1 was different from the coagulation bath in Example 6.

[0207] <Comparison Example 3>

[0208] Step 1: A solution was prepared by dissolving 1 wt% of HPMC (hydroxypropyl methylcellulose) in a 30 wt% aqueous ethanol solution, and α-TCP was added as a ceramic powder to prepare a paste (containing 69 wt% ceramic).

[0209] Step 2: The above paste was placed in an extrusion container and a molded body was formed using a 3D printing device.

[0210] Step 3: After drying the above molded body, it was immersed in a PBS solution and treated for about 24 hours to cement-harden the ceramic components within the molded body, thereby manufacturing a 3D printed structure.

[0212] <Experiment Example 1>

[0213] In order to compare the physical properties of 3D printed structures according to the temperature of the paste when printing the paste during the formation of the molded body, the storage modulus and loss modulus were measured for 3D printed structures manufactured by varying the paste temperature to 4°C and 25°C in Step 2 of Example 1, and the results are shown in FIG. 3.

[0214] As shown in Figure 3, it was confirmed that when a paste at 25°C is printed in a coagulation bath, changes in physical properties occur over time, whereas when printed at 4°C, the physical properties are maintained. Through the above results, it can be confirmed that it is desirable to print a paste at a somewhat lower temperature of 1 to 5°C, specifically 4°C, to increase the structural stability of the manufactured 3D printed structure.

[0216] <Experiment Example 2>

[0217] In order to determine the solution viscosity according to the ethanol content of a coagulation bath, a hydrogel comprising ethanol, carbopol, and triethylamine (TEA) is prepared, wherein the carbopol and TEA are included in amounts of 0.6 wt% and 15 wt%, respectively, relative to the total weight of the hydrogel, and hydrogels are prepared with varying ethanol contents of 50, 60, 70, and 80 wt%, and the viscosity thereof is measured. texture The results are shown in Figure 4.

[0218] As shown in Figure 4, it can be confirmed that when ethanol is included in the amount of 70 to 80 weight%, the viscosity of the coagulation bath is very low, whereas when it is included in the amount of 50 to 60 weight%, it has a viscosity suitable for printing.

[0219] Accordingly, it can be confirmed that in order to form a molded body by 3D printing in the above-mentioned coagulation bath, it is preferable to include ethanol in an amount of less than 70% by weight and 60% by weight or less.

[0221] <Experiment Example 3>

[0222] In order to determine the viscosity of the hydrogel according to the gelling agent content of the coagulation bath, a hydrogel containing ethanol, carbopol, and triethylamine (TEA) was prepared, wherein the ethanol and TEA were included in amounts of 60% by weight and 15% by weight, respectively, relative to the total weight of the hydrogel, and the hydrogels were prepared with varying amounts of carbopol, such as 0.2%, 0.4%, 0.6%, and 0.8% by weight, and the viscosity thereof was measured and the results are shown in FIG. 5.

[0223] As shown in Figure 5, it can be confirmed that when Carbopol is included in an amount of 0.2 to 0.4 weight%, the viscosity of the coagulation bath is very low, whereas when it is included in an amount of 0.6 to 0.8 weight%, it has a viscosity suitable for printing.

[0224] Accordingly, it can be confirmed that it is more preferable to include at least 0.6% by weight of a gelling agent in order to form a molded body by 3D printing in the above-mentioned coagulation bath.

[0226] <Experiment Example 4>

[0227] To determine the difference depending on whether the coagulation bath contains ethanol, 3D printed molded bodies cured in Step 3 of Example 1 (containing ethanol) and Comparative Example 1 (not containing ethanol) were compared and observed, and the results are shown in Figures 6 and 7.

[0228] FIG. 6 shows a molded body manufactured according to Example 1, having a line width of approximately 0.435 ± 0.015, whereas FIG. 7 shows a molded body manufactured according to Comparative Example 1, having a line width of approximately 0.512 ± 0.023, resulting in a relatively thicker molded body, and it can be confirmed that severe swelling occurs. Accordingly, the structure manufactured according to Comparative Example 1 is expected to have low mechanical strength.

[0229] Through the above results, it can be confirmed that it is desirable to include ethanol in the coagulation bath to manufacture 3D printed structures with high resolution and mechanical strength.

[0231] <Experiment Example 5>

[0232] In order to confirm the difference in the coagulation bath according to the content of the pH adjuster, the molded body cured in step 3 in Examples 1 to 4 is shown in FIG. 8, and the modulus of the structure prepared in step 7 was measured and the results are shown in FIG. 9 and 10.

[0233] As shown in Figure 7, it can be confirmed that as the TEA content increases to 1, 5, 10, and 15 weight%, curing in the d-coagulation bath is achieved more effectively. On the other hand, it was found that when TEA is not included, curing in the coagulation bath is not properly achieved.

[0234] In addition, through Fig. 9, it can be seen that the modulus value of the structure increases as the TEA content increases to 1, 5, 10, and 15 wt%, and through Fig. 10, it can be seen that the maximum modulus value is 4.19 to 4.92 Pa.

[0235] From the above results, it can be seen that when TEA is not included, the self-assembly reaction of collagen due to rapid pH changes through TEA does not occur, and thus collagen hardening is not properly achieved, whereas when TEA is included in the coagulation bath in an amount of 1 to 15 weight%, preferably 5 to 15 weight%, collagen hardening by TEA is achieved more effectively, and through this, it can be confirmed that a 3D printed structure with higher mechanical strength can be manufactured.

[0237] <Experiment Example 6>

[0238] In order to determine whether a 3D printed structure having an overhang structure without support can be manufactured by the method according to the example, a vertical ring structure and a zygomatic bone structure having an overhang structure were printed by the method of Example 1, and the results are shown in FIGS. 11 and 12.

[0239] As shown in FIGS. 11 and 12, it can be confirmed that the method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to one embodiment can manufacture a 3D printed structure having an overhang structure without support.

[0241] <Experiment Example 7>

[0242] A 3D printed structure was manufactured using the method according to the embodiment, and in order to verify the differences according to nozzle size, pneumatic pressure, and printing speed during 3D printing, the nozzle size was varied to 23G, 24G, and 25G, the pneumatic pressure was varied from 100KPa to 350KPa in increments of 50KPa, and the printing speed was varied from 100mm / s to 700mm / s in increments of 100mm / s, and measurements were taken, and the results are shown in FIGS. 13 to 15.

[0243] FIG. 13 is a photograph of the result of printing with a pneumatic pressure of 250 Kpa but with different nozzle sizes and printing speeds, and FIG. 14 and FIG. 15 are graphs showing the line width and pore size of a molded body printed with a printing speed of 200 mm / s but with different pneumatic pressure and nozzle sizes.

[0244] Through FIGS. 13 to 15, it can be seen that a 3D printed structure manufactured by the method according to the embodiment can form a molded body having a line width of 160 μm to 900 μm by adjusting the nozzle size, pneumatic pressure, and printing speed.

[0246] <Experiment Example 8>

[0247] In order to confirm the phase of the 3D printed structure manufactured by the method according to the example, X-ray diffraction (XRD) analysis was performed on the molded body manufactured in Step 2 of Example 1 (as printed), the molded body before cement hardening in Step 5 (immersion in PBS 3h and ethanol 3h), and the structure manufactured after cement hardening (cementation in PBS 1d), and Fourier-transform infrared spectroscopy (FT-IR) analysis was performed on the manufactured structure, and the results are shown in FIGS. 16 and 17.

[0248] Figure 16 shows the XRD analysis results. As shown in Figure 16, it can be confirmed that only the α-TCP phase appears in the molded body printed in the coagulation bath, whereas the α-TCP and CDHA (Calcium-deficient hydroxyapatites) phases appear in the molded body treated with PBS and ethanol solution after separation from the coagulation bath, and that only CDHA (Calcium-deficient hydroxyapatites) appears after cement hardening.

[0249] Through the above results, it can be confirmed that the ceramic components were not hardened in the solidification bath, but a partial phase transition occurred through PBS treatment, and a complete phase transition occurred during the secondary hardening stage.

[0250] Figure 17 is the result of FT-IR analysis, and as shown in Figure 17, it can be confirmed that the manufactured structure (CDHA / Collagen) contains CDHA and collagen.

[0252] <Experiment Example 9>

[0253] In order to verify the mechanical strength of the 3D printed structures manufactured by the methods according to the examples and comparative examples, a compressive strength test was performed on the structures manufactured in Example 1 and Comparative Example 2 (with fill density set to 50% and 70% during printing), and the results are shown in FIG. 18.

[0254] As shown in FIG. 18, FIG. 18 shows the results of measuring compressive strength when the fill density is set to 50% and 70% during the printing of the example. As shown in FIG. 18, it can be confirmed that the mechanical strength of the structure manufactured by the method of Example 1 is improved by more than 1.9 times compared to the structure manufactured by the method of Comparative Example 2.

[0255] From the above results, it can be confirmed that the structure manufactured according to the example has high mechanical strength.

[0257] <Experiment Example 10>

[0258] To confirm the bone regeneration performance of the structure prepared according to the example, osteoblasts were seeded in the structure prepared in Example 1, Comparative Example 2 (p123 bath), and Comparative Example 3 (open-air), and the cell proliferation rate and bone differentiation gene expression rate were confirmed, and the results are shown in FIGS. 19 to 25.

[0259] Figure 19 is a graph of absorbance at a wavelength of 450 nm for 1 to 7 days after osteoblast seeding, and Figure 20 is a graph of DNA concentration calculated based on this. As shown in Figures 19 and 20, it can be confirmed that the cell proliferation rate in the structure prepared in Example 1 is significantly higher than the cell proliferation rate in the structures prepared in Comparative Example 1 and Comparative Example 2.

[0260] In addition, FIGS. 21 to 25 are graphs showing the results of measuring the expression of OCN (Osteocalcin), OPN (Osteopontin), ALP (Alkaline phosphatase), COL 1 (Collagen type 1), and RUNX 2 (Runt-related transcription factor 2), respectively. As shown in FIGS. 21 to 25, it can be confirmed that the bone differentiation gene expression rates of OCN, OPN, ALP, COL 1, and RUNX 2 are significantly higher in the structure prepared in Example 1 than in the structures prepared in Comparative Example 1 and Comparative Example 2.

[0261] Through the above results, it can be confirmed that the structure manufactured according to the example has significantly superior bone regeneration performance. Explanation of the symbols

[0263] 10: Paste 20: 3D printed molded body 30: Coagulation bath 40: Hardening solution 50: 3D printed structure

Claims

Claim 1 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, comprising: a step of 3D printing a paste containing printing inorganic powder and a collagen solution in a coagulation bath to form a molded body; a step of first curing the collagen contained in the molded body in the coagulation bath; and a step of secondarily curing the printing inorganic powder contained in the firstly cured molded body; wherein the coagulation bath is a hydrogel containing a gelling agent, ethanol, and a pH adjuster, and the first curing step is a step of curing uncured collagen through self-assembly by the pH adjuster and a dehydration reaction induced by ethanol. Claim 2 delete Claim 3 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein, in claim 1, the collagen solution is a solution in which collagen is dissolved in an acidic solvent. Claim 4 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein the pH adjuster is one or more of triethanolamine (TEA), NaOH, KOH, arginine, tromethamine, and aminomethyl propanediol. Claim 5 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein the coagulation bath comprises a pH adjuster in an amount of 1 to 20 weight percent relative to the total weight of the hydrogel. Claim 6 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein the coagulation bath comprises less than 10 to 80 weight percent of the ethanol relative to the total weight of the hydrogel. Claim 7 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure according to claim 1, wherein the gelling agent is one or more of Carbopol, laponite, gelatin, p123, F127, hydroxyethyl cellulose, methyl cellulose, xanthan gum, and polyquaternium. Claim 8 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein the coagulation bath comprises the gelling agent in an amount of 0.6 to 40 weight% relative to the total weight of the hydrogel. Claim 9 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein the first curing step is performed at a temperature of 1°C to 40°C. Claim 10 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein, in claim 1, the printing inorganic powder comprises ceramic powder, and the secondary curing step is a step of treating the molded body with a ceramic curing liquid to cure the ceramic contained in the molded body. Claim 11 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein, in claim 10, the ceramic powder is a calcium phosphate-based ceramic powder. Claim 12 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein the ceramic curing solution is one or more selected from the group consisting of DSP (disodium phosphate dihydrate, Na2HPO4·2H2O), MSP (monosodium phosphate dihydrate, NaH2PO4·2H2O), PBS (Phosphate buffer saline), MCPM (Monocalcium phosphates monohydrate, Ca(H2PO4)2·H2O), and cell culture medium. Claim 13 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, wherein, in claim 10, the ceramic curing solution is at a temperature of 20°C to 40°C. Claim 14 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, further comprising the step of separating the molded body from a coagulation bath and treating it with a buffer solution in claim 10. Claim 15 A method for manufacturing a self-supporting organic-inorganic composite 3D printed structure, further comprising the step of treating the molded body treated with the buffer solution with ethanol in claim 14. Claim 16 A method for manufacturing a ceramic 3D printed structure according to claim 1, wherein the paste further comprises a thickening agent. Claim 17 A method for manufacturing a ceramic 3D printed structure according to claim 1, wherein the paste further comprises a biofunctional material. Claim 18 In claim 1, the manufacturing method comprises manufacturing a collagen-containing ceramic 3D printed structure comprising an overhang structure in at least a portion thereof, a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure. Claim 19 In claim 1, the manufacturing method is a method for manufacturing a self-supporting organic-inorganic composite 3D printed structure for manufacturing a support for hard tissue regeneration.

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