WO2017192004A1

WO2017192004A1 - Method for supplying inks for three-dimensional printing, and three-dimensional printing method using same

Info

Publication number: WO2017192004A1
Application number: PCT/KR2017/004687
Authority: WO
Inventors: 안근선; 진송완; 심진형; 윤원수; 강동구
Original assignee: 주식회사 티앤알바이오팹; 한국산업기술대학교산학협력단
Priority date: 2016-05-03
Filing date: 2017-05-02
Publication date: 2017-11-09

Abstract

The present invention relates to a method for filling two or more different kinds of multiple inks in an ink discharge member for three-dimensional printing and to a three-dimensional printing method using the filled inks, the three-dimensional printing method using multiple inks comprising the steps of: producing an ink discharge by allowing the accommodated multiple inks to be discharged under pressure from a single discharge port of a discharge unit; and printing using the ink discharge.

Description

【Specification】

[Name of invention]

3D printing ink supply method and 3D printing method using the same

Technical Field

The present invention relates to a method for supplying ink to an ink receiving portion of a three-dimensional printing apparatus, a three-dimensional printing method including the method, and a three-dimensional printing apparatus to which the method is applied, and more particularly, to an ink receiving portion provided in the three-dimensional printing apparatus. The present invention relates to a method of providing ink by a three-dimensional printing method and a three-dimensional printing apparatus to which the method is applied. Using the method according to the invention, it is possible to print a highly structured biological tissue shape with high precision. Background Art

Regenerative medicine and tissue engineering mainly use the method of making three-dimensional tissue structures as a field for repairing or replacing damaged organs, and extrusion printing based bioprinting is one of the most suitable methods for making such three-dimensional tissue structures. . There is an active research to regenerate the functional tissues required by the production of cell structures similar to the tissues constituting the human body using 3D bioprinting, technology. _,

Head models used in bioprinting technology are largely divided into ink jet-based printing and extrusion-based printing. In addition, various methods using laser, ultrasound, and the like have been proposed, but the first two models are most widely used.

The physical properties of the bio inks required by such bio printing techniques vary greatly depending on the printing head models used. Discharge-based printing systems that use fine nozzles and syringes are more effective than jet-based technologies. Viscosity is not limited. As a result, the range of applicable biomaterials becomes much wider than that of inkjet-based printing technology. In addition, it is easy to process the thick layer, it is easy to manufacture the cell structure of the size required by the clinical. However, the resolution of bioprinting techniques based on extrusion laminated molding developed to date is hundreds of micrometers, but the basic structure of tissues and organs in the human body is tens of micrometers or less. In particular, the diameter of the capillaries that supply nutrients to cells constituting organs or tissues is 3 to 4 micrometers, so it is difficult to implement the current bioprinting technique.

In addition, multiple inks may be used for bioprinting. For example, when the hydrogel and the curing agent are cured together, or when co-culture of several kinds of cells, the function of the cells is improved when the division between heterogeneous cells is used rather than the method of simply mixing and spraying the cells. As it is known, bioprinting should be performed by spraying multiple inks. According to this conventional method, this problem has been solved by using multiple heads containing heterogeneous materials, but the printing process time increases, which adversely affects cell viability and complicates the system. In order to increase the resolution of the printing technique, a nozzle with a small diameter should be used. When using a nozzle with a small diameter, a shear stress is generated between the material and the wall discharged from the inside of the nozzle during discharging. have. In addition, since the problem of cell death due to shear force occurs frequently, it is also difficult to miniaturize the diameter of the nozzle.

[Detailed Description of the Invention]

[Technical problem]

According to the present invention, at least one ink is supplied to an ink receiving portion by a three-dimensional printing method and three-dimensional by using one ejection, in order to produce a highly precise biological tissue shape of a complex structure using a three-dimensional printing method. An ink ejecting member for producing a printed matter having a pattern, and a printing method using the same.

The present invention relates to a printing apparatus for producing a printed matter having a three-dimensional pattern by using one ejection containing two or more different inks and a printing method using the same.

An object of the present invention is to provide an apparatus for printing a biological tissue shape having a complex structure with high precision and resolution, and a printing method using the same. The present invention is to provide a printing apparatus and printing method that can significantly reduce the shear stress of the cell while heterogeneously printing the desired shape.

Technical Solution

The present invention relates to a three-dimensional printing method comprising an ink providing step of providing at least one type of ink as an ink printed matter printed by a three-dimensional printing method to a three-dimensional printing ink ejecting member including an ink receiving portion and an ejecting portion. The printing method may be a bioprinting apparatus and method used for manufacturing artificial tissues, organs, and the like.

Another example of the present invention, the step of providing at least one type of ink in the space of the ink discharge member including the ink containing portion and the discharge portion, provided in the three-dimensional printing apparatus, is applied by applying a physical force to the received ink Discharging ink to a single discharge port to produce an ink discharge, and printing the ink discharge on a substrate.

The providing of the ink may include providing at least one ink as an ink substrate printed by a three-dimensional printing method, and when using two or more different inks, another at least one ink may be used in addition to the three-dimensional printing method. It may be an ink layer provided by the providing method. Specifically, the ink filling and the at least one second ink filling the at least one first ink in the ink receiving portion It can be provided as an ink printed matter which is printed and injected by a three-dimensional printing method.

The ink printed matter provided to the ink receiving member in the ink providing step may itself have a two-dimensional or three-dimensional pattern, and a fragment of the ink receiving member may also have a pattern. It is particularly useful when the printed material is an artificial organ, because it consists of one or more structures of various materials.

In addition, in one embodiment of the present invention, there is provided a three-dimensional printing method of a printed matter having a cross-sectional pattern, the ink ejecting member may include an ink filling and an ink ejection to have a cross-sectional pattern of the same shape as the three-dimensional printed matter. Applying a physical force to the ink contained in the space, discharging ink through a discharge portion and a nozzle connected thereto to produce an ink discharge product having a cross-sectional pattern having the same shape as a print, and printing the ink discharge onto a substrate. It relates to a three-dimensional printing method of a printed matter having a cross-sectional pattern, comprising the step of. In the three-dimensional printing method of the printed matter having the cross-sectional pattern, it is pressed into the ink provided in the receiving portion is printed as a single discharge through the discharge portion or the nozzle, the discharge may have a cross-sectional pattern of the same shape as the final printed matter Preferably, the ratio (= B / A) of the cross-sectional pattern (B) of the discharge or final printed matter to the cross-sectional pattern (A) of the ink provided in the receiving portion is, for example, the diameter ratio of the cross-section, or the cross-sectional area. The ratio can be reduced to a ratio of 100: 99 to 100: 0.1, or 100: 50 to 100: 1, 100: 18 to 100: 1. Further, the cross-sectional diameter ratio of the primary ink prints provided by the three-dimensional printing method, or the cross-sectional area ratio, or the cross-sectional diameter ratio of the secondary prints of the primary ink prints, may be 100: 99 to 100: 0.1. Or, it may be reduced in a ratio of 100: 50 to 100: 1 and 100: 18 to 100: 1.

Another example of the present invention is a three-dimensional printing apparatus of a printed matter having a cross-sectional pattern, and includes an accommodating part for receiving ink for printing in an inner space, and having a single passage positioned below the accommodating part and passing the ink therein and accommodated in the accommodating part. An ink ejecting member comprising an ejecting portion for ejecting ink,

A nozzle connected to the discharging end; And a pressurizing member for applying a physical force to the contained ink, wherein the ink is printed to have a cross-sectional pattern having the same shape as a printed matter through a discharge portion having a single passage. The printing apparatus according to the present invention may include a means for applying a physical force to the received ink, and for example, may apply a force using a pressure device or a screw. In order to apply the pressure under the same conditions, The pressure may be performed using a single pressing member, or may be performed by pressing the same pressure using two or more pressing members. By pressing with the pressing member, different inks may be printed so as to have a cross-sectional pattern having the same shape as a printed matter through a discharge part having a single passage. Therefore, when using the printing apparatus according to the present invention, it is possible to produce a printed matter having a variety of cross-sectional patterns, in particular can be produced by a three-dimensional printing method by printing a biological tissue shape having a complex cross-sectional structure with high precision and resolution, In the case of including the cells in the biological tissue, there is an advantage that can significantly reduce the shear stress of the cell while heterogeneously printing the desired shape. Hereinafter, the present invention will be described in more detail.

In the three-dimensional printing method according to the present invention, an ink providing step of providing a three-dimensional printing ink ejecting member including an ink receiving portion and the ejecting portion, at least one ink as an ink printed matter printed by a three-dimensional printing method, to the ink ejecting member The ink contained by applying a physical force is discharged to the discharge portion to produce an ink discharge, and printing the ink discharge on the substrate. According to the three-dimensional printing method according to the invention it can be produced a printed matter having a cross-sectional pattern.

In the ink providing step, at least one ink may be further provided as an ink layered product by a method other than a three-dimensional printing method. In the present specification, unlike the ink prints provided on the ink receiving member by the three-dimensional printing method, the ink laminated material is provided by the layered method rather than the three-dimensional printing method. As an example of the layering method by a method other than the three-dimensional printing method, there is a method of layering using a tube or a syringe and the like, and is not particularly limited. Applying the physical force is Im, and a means for pushing the ink supplied to the ink receiving member as part ejection can be performed using a ^", can be used, for example the pressure member and the screw or the like. Specifically, applying the pressure may be performed by using a single pressing member or by using two or more pressing members. For example, the pressure may apply the pressure under the same conditions, and the pressure under the same conditions is a pressure condition under which ink can be discharged to a single discharge port to form a single ink discharge, preferably two or more different inks, or The ink printed matter provided by the three-dimensional printing method and the ink layered material provided by the method other than the three-dimensional printing are ejected to a single ejection opening to form a single ink ejection. It means a pressure condition to have the same shape.

The three-dimensional printing method of a printed matter having a cross-sectional pattern according to the present invention can be performed using the ink ejecting member or the three-dimensional printing apparatus including the ink ejecting member according to the present invention.

According to another embodiment of the present invention, a three-dimensional printing apparatus of a printed matter having a cross-sectional pattern includes an accommodation part accommodating ink so as to have a cross-sectional pattern having the same shape as a printed matter, and a single passage located below the accommodation part. And an ink discharge member including an ejection portion for ejecting ink contained in the accommodation portion, and further comprising a nozzle connected to the discharge portion, and a pressurizing member for applying pressure to the ink contained in the partitioned space. Can be.

In addition, the three-dimensional printing apparatus is a substrate for printing the discharge

Additional devices for printing and printing may include components included in conventional three-dimensional printing devices.

Specifically, three-dimensional of a print having a cross-sectional pattern according to the present invention A printing method, providing an ink of an accommodating part of the ink ejecting member, applying pressure to the ink contained in each partitioned space, ejecting ink through the ejecting part, and forming an ink ejection having a cross-sectional pattern having the same shape as a printed matter. Manufacturing, and printing the ink discharge on a substrate. In the ink providing step, at least one ink may be provided as an ink printed matter printed by a three-dimensional printing method, and at least one ink may be further provided as an ink layered product by a method other than the three-dimensional printing method.

The method of manufacturing a printed matter having a cross-sectional pattern using an ink ejecting member or a three-dimensional printing apparatus including the ink ejecting member, and a three-dimensional printing apparatus including the ink ejecting member or the ink ejecting member is a component of the apparatus. The steps of the star and method will be described in detail below.

An ink ejecting member according to the present invention has a receiving portion in which ink is received, and a single passage positioned below the receiving portion so as to have a cross-sectional pattern having the same shape as a printed matter, and having a single passage through which the ink passes, and ejecting the ink contained in the receiving portion. And a discharge part.

When the ink ejecting member according to the present invention is used, the shear force is hardly applied to the ink or the cells contained in the ink because the bio ink is ejected because the diameter of the printing portion is commercially available. In addition, compared with the prior art of using multiple heads in which multiple materials are heterogeneously contained, the present invention can print by discharging two or more different inks using one ink ejecting member, so that a single printing head can be used. Printing process time is reduced and little shear force is applied to the ink or cells contained therein. Therefore, when using the ink containing the cells using the printing method or apparatus according to the present invention, there is an advantage that the cell viability is high and the system is simple.

The discharge member is a component for supplying ink by pushing the ink under pressure from the outside, which is commonly used in a three-dimensional printing apparatus. Cartridge or syringe (Syr inge). In order to discharge and print the ink, the pressure applied to the ink receiving member may vary depending on the concentration of the ink and the size of the nozzle, for example, 0.1 to 700 kPa, 1 to 500 kPa or 1 to 700 kPa. have. If the printing pneumatic is high, too much material is discharged, and if the pneumatic pressure is low, subsequent material may not print and slip into the supporting material.

The ink ejecting member has a single passage located below the accommodating portion and has a single passage through which the multiple inks pass, and includes an ejecting portion for ejecting the ink contained in the accommodating portion. The discharge part may have a single passage, and may discharge ink contained in the accommodation part divided into a plurality of spaces by a single channel control method instead of multi channel control.

The inner diameter of the discharge port is formed very small. The ink contained in the accommodation portion through the discharge port can be discharged to the outside of the accommodation portion. The ink discharge discharged from the discharge portion has a cross-sectional pattern that is the same as the cross-sectional pattern of the accommodation portion, but may be reduced in size.

The three-dimensional printing apparatus according to the present invention may further include a nozzle connected to the discharge end of the discharge member, the ink is discharged through the exposure, the plate is located under the nozzle is discharged from the ^' Printing ink is laminated on the plate to produce printed matter.

The printed matter having a cross-sectional pattern according to the present invention is preferably a human simulated tissue. For example, muscle tissue (bundle structure), bone tissue (lamel lae & canal structure), neural tissue (per ineurium structure), vascular tissue (mul t i_layer structure), spinal cord (spinal cord) tissue and the like.

In order to increase the resolution of the extrusion-based bioprinting technique, it is necessary to reduce the diameter of the printing nozzle. As the diameter of the nozzle becomes smaller, the cells are killed by the shear force generated between the material discharged from the nozzle and the wall. Is a problem that occurs frequently, it is also difficult to blindly miniaturize the diameter of the nozzle. Thus According to the present invention, a housing having the same cross-sectional pattern and a discharge part for discharging the same into a single passage may be provided to realize high resolution.

The method for printing a living tissue according to the present invention provides a printing ink having an enlarged cross-sectional shape having the same shape and proportion as a printed matter in the accommodating portion, while maintaining a same cross-sectional pattern of the accommodating portion, The pressure can be controlled to pass through the discharge section having a small cross section. It is preferable that the cross-sectional pattern of the discharged ejection is at a level capable of maintaining the same shape as the cross-sectional pattern of the accommodation portion.

According to the present invention, it is possible to easily print a complicated and very small microstructure using a printing ink of a large size that can be produced relatively easily.

In the present specification, the meaning of 'same' is defined as a meaning including not only 100% identical but also identical enough to perform substantially the same function. In the specification, "the cross-section maintains the same shape" means that only the size of the cross section is reduced but the shape of the original cross-section remains intact. After fabrication, printing of the desired size, ie the actual tissue cells and the tissue cells that correspond to the size, is possible.

The viscosity of the printing ink is preferably such that the cross section of the discharged discharged through the nozzle can maintain the same shape as the cross section of the printing material.

The cross-section of the printed result is a small size that cannot be achieved with current bioprinting techniques or a microstructure that can significantly reduce survival when printed. As described above, muscle tissue (bundle structure), bone tissue (lamel) lae & canal structure), nerve tissue (per ineurium structure), vascular tissue (mul ti-layer structure), spinal cord (spinal cord) tissue, and the like.

The cross section of the printing apparatus accommodating portion according to the present invention has a cross-sectional pattern having the same shape as the printed matter, and the cross-sectional pattern of the accommodating portion is the discharge or The ratio of the cross-sectional pattern of the printed matter may be represented by various methods such as the area ratio of the cross section, the diameter ratio, and the like. The ratio (= B / A) of the ejection or the final printed matter to the cross-sectional pattern (A) with respect to the cross-sectional pattern (A) of the ink provided in the accommodation portion is, for example, the diameter ratio of the cross section or the cross-sectional area ratio to 100: 99 to 100: 0.1, or 100: 50 to 100: 1, 100: 18 to 100: 1 ratio can be reduced. Further, the cross-sectional diameter ratio of the primary ink prints provided by the three-dimensional printing method, or the cross-sectional area ratio of the secondary prints of the primary ink prints, or the cross-sectional area ratio based on the cross-sectional area, is 100: 99 to 100: 0.1. Or 100: 50 to 100: 1, 100: 18 to 100: 1 ratio. The reduction ratio of the secondary prints is that the ink prints provided on the ink receiving member are subjected to two printing processes, and when the reduction ratio is expressed based on the ink prints printed first in the syringe, the syringes are three-dimensional printing. Equation 1 is obtained by subtracting the diameter of the secondary printed matter or ejection of the printing ink ejected through the nozzle from the diameter of the primary ink printed matter provided by the ratio divided by the diameter of the primary ink printed matter provided to the syringe by the three-dimensional printing method. It can be expressed as

[Equation 1]

Ink Substrate and Reduction Ratio = -8) / 八 X100 (%)

In Equation 1,

A is the diameter of the primary ink substrate provided in the syringe by three-dimensional printing

B is the diameter of the secondary print of the printing ink,

A and B are the same length unit.

The reduction ratio of the secondary printed matter is directly affected by the cross-sectional diameter of the receiving portion, the fragment diameter of the discharge portion, or the diameter of the nozzle, and can be variously designed by appropriately adjusting to the cross-sectional pattern size of the desired printed matter. The cross-sectional diameter of the printout varies depending on the size of the nozzle, which ranges from 0.1 mm to lmm, and is usually used for printing, such as material properties, pressure, speed of the printing head, and the location of the printing result (printing bed). According to the process Can change.

According to an embodiment of the present invention, the ratio can be downsized to 99% or 98.7% (200 μιη) from the total diameter (15 μs) of a particular shape (eg Lobule). In one embodiment of the present invention, the reduction ratio (= -8) / 八 X 100 of the primary printing ink represented by Equation 1 is 0.1% or more, 10% or more, 20% or more, 30% or more, 4W or more, 50 More than 60%, more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, more than 92%, more than 95%, or more than 99%, for example, from 0.01 to 99.9%, 50% To 99% or 45% to 99%. The ink provided to the ink ejecting member according to the present invention is preferably a bio ink capable of producing artificial organs or the like. Specifically, printing may be performed by providing ink to the ink containing portion so as to have a cross-sectional pattern having the same shape as the printed matter. The different inks mean that the inks are different from one or more selected from the group consisting of constituents, constituent contents and physical properties.

In the present specification, the term 'bio ink' includes living cells or biomolecules, and is a term used to collectively refer to a material capable of fabricating a structure required for a bioprinting technology. The bio ink of the present invention comprises a liquid, semisolid, or solid composition comprising a plurality of cells.

Therefore, the bio ink should provide a physical environment for three-dimensional processing and a biological environment for the cells to perform the intended function. When the printing process is lengthened, it is preferable that the supply of nutrients and oxygen necessary for the survival of cells in the ink ejecting member is appropriately performed. It should also be able to protect the cells from the physical stresses that occur during the printing process. In addition, the bio ink should have the physical properties required in the printing process such as repeatability of 3D patterning, productivity, and no clogging of the nozzle.

The ink according to the present invention is preferably a hydrogel, and thus The ink may include a gelling polymer and may include, for example, one or more selected from the group consisting of gelling polymers, cells, growth factors, and extracellular substrates.

. The bioink used in the present invention may be, for example, a mixed / non-mixed hydrogel of desired cells, a hydrogel containing a specific growth factor, a hydrogel containing a cell and a growth factor, a cytokine (cytokine). ) Containing hydrogels, different types of hydrogels. Hydrogels include collagen, matrigel, alginate, gelatin and agarose. Decellularized tissue-derived cell ink, hyaluronic acid, fibrin gel, or the like or a compatible hydrogel is suitable.

In addition, since bio-inks diffuse faster at lower viscosities, they are thicker than water (1 cp) and have a viscosity measured at 25 ^° C, ranging from 2 cp to 1,000,000 cp, for example 2 cp to 10,000 cp, 5 cp to 1,000,000 cp, 2 to Materials on gels having a viscosity of 500 cps, 5 to 300 cps and the like are suitable. When the viscosity of the ink is too low, the shape of the ink printed matter printed by the three-dimensional printing method may collapse or be deformed, and the printed matter and the ink packing may be mixed with the boundary to be broken. The viscosity of the gel-type material used in the method of the present invention preferably has a suitable viscosity so that the printing material can be discharged in the discharge process described later. According to one embodiment of the present invention, it is preferable to provide an ink having a relatively high viscosity compared to the inkjet method since the ejection type three-dimensional printing method is applied. In one example, the inks applicable to the present invention may use various viscosity enhancers to provide a viscosity suitable for ejection. Viscosity of the printing material is such that the cross section of the discharged discharged through the nozzle can maintain the same shape as the cross section of the printing material.

The ink viscosity difference between the ink layer and the ink printed matter, the viscosity difference measured at 25 ^° C is less than 5,000cp, for example 0 to 5,000cp, less than 1,000cp, less than 500cp, less than 200cp, less than 150cp, less than lOOcp It may be less than 50cp. If the difference in viscosity of the ink is very large, the shape of the ink printed matter may be deformed by the molecular force of different materials, the same if you want to three-dimensional printing by ejecting When pressure is applied to the ink or the ink receiving member, the ink pattern accommodated in the ink receiving member may collapse due to the difference in viscosity. Therefore, different from each other

When using two or more types of inks, the smaller the difference in viscosity, the better.

The difference between the elastic values of the ink filling and the ink printing may be 10,000 Pa or less, for example, 0 to 10,000 Pa. It is preferable that the ink layered matter and the ink printed matter have similar trends in viscosity and elasticity change according to the shear rate and similar viscosity and elasticity values.

In addition, when the gelling polymer used in the different inks is different, for example, the gelling polymer is temperature sensitive like collagen and gelatin, and the other is not temperature sensitive like alginate and fibrin gel, it is necessary to adjust the temperature inside the syringe. There can be. For example, the temperature of the ink accommodating member can be appropriately adjusted within the 4 ^S C to 37 ^C C temperature range.

Naturally derived or synthetic hydrogels in the field of three-dimensional bio printing. Although bio inks have been developed and are currently in use, biogels based on hydrogels have been used for physical and biological reasons such as biocompatibility, printing suitability, geometrical precision, and precision.

The ^first extrudable "or" ejection possible 'is the discharge portion, the nozzle (nozzle) or an orifice (for example, one or more holes or tubes) means that can be molded by passing (e.g., under pressure). Furthermore, Densification of the bio ink is derived from growing cells at a suitable density. The cell density required for the bio ink depends on the cell to be used and the tissue or organ to be produced.

The present invention also provides a bio ink composition, wherein the bio ink composition further comprises a tissue-derived component. Tissue-derived component means that specific tissues of animals such as cartilage, kidney, heart, liver, muscle, etc. are decellularized and gelled of a substance mainly composed of extracellular matrix. Can be included to enhance. In the present invention ^" the bio ink composition is a cell culture medium It may further include. The cell culture medium is a concept including any medium suitable for the cells of interest.

The ink according to the present invention may include a gelling polymer, and the gelling polymer solution used for such printing may be used in various kinds. The conditions that the polymer solution should have are as follows. First, in order to achieve a three-dimensional printing well, it should be easy to eject to the nozzle with a suitable viscosity, and should not cause problems such as crushing the shape of the object made by rapid curing after discharge. In addition, for manufacturing purposes, it should be possible to create a cell culture environment similar to the tissue in the human body. Examples of the gelling polymer include fucoidan, collagen alginate, chitosan, hyaluronic acid, silk, polyimides, polyamix acid, polycarprolactone, polyetherimide, nylon (nylon) , Polyaramid, polyvinyl alcohol, polyvinylpyrrolidone,

Poly-benzy 卜 glutamate,

Polyphenylene terephthalamide (po 1 ypheny leneterephthalamide),

Polyaniline, polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethylmethacrylate ), Polylact ic acid (PLA), polyglycol ic acid (PGA), copolymers of polylactic acid and polyglycolic acid (PLGA), poly

{Poly (ethylene oxide) terephthalate-co-butyleneterephthalate KPE0T / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyortho Ester {poly (ortho ester; P0E), poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylates {poly (ethylene glycol) diacrylate; Out of the group consisting of PEG-DA} It may be one or more selected or a combination of the above materials. However, the material is not limited to this embodiment. In addition, the gelling polymer may be a chemically modified natural polymer, for example, the binding si of GelMA, Alginate / Gel at in, Alginate that combines the chemical at the gel at in and methacrylate (MA) and the photoini ti ator The stock price may include pentapeptide sequencing, Tyr-I le-Gly-Ser-Arg (YIGSR) and alginate in combination with EDC / NHS.

In particular, the hydrogels including polyethylene glycol, alginate, nitrate, collagen and gelatin have high water content, have excellent biocompatibility, can control mechanical properties, and have excellent biodegradability, and thus are widely used in the preparation of cellular-embedded carriers. Has been used. For this reason, hydrogels are well suited for the manufacture of cell-loaded structures and can be printed directly to obtain various types of tissue regeneration backbones.

The gelatin is particularly suitable as the cell carrier because it exhibits temperature-sensitive properties. That is, gelatin has the property of liquefying at 37 ^° C and solidifying below room temperature.

The gelling polymer may be formed using a physical treatment or a chemical treatment to form a crosslinking ol, the crosslinking solution may be used for the chemical treatment, and a crosslinking solution may be appropriately selected according to the selected gelling polymer. For example, the crosslinking solution may be gypsum; Or hydroxyapatite, apatite carbonate, apatite fluoride, apatite chloride, a-TCP, β-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium pyrophosphate, calcium carbonate, calcium sulfate, EDC {l-ethyl- (3-3-dimethylaminopropyl) carbodi imide hydrochlor ide} or a solution of one or more combinations thereof selected from salts thereof.

Ink containing the gelling polymer solution, it is preferable that the ratio of collagen concentration in the collagen solution of the liquid form is usually in the range of 0.01 to 30% by weight. The method for producing hydrogels uses ink for normal three-dimensional printing. It can be carried out by applying the manufacturing method used when manufacturing, but is not particularly limited,

The bioink according to the present invention may include cells, and applicable cells or tissues are not particularly limited, and may be animal cells or plant cells, or tissues of animals or plants. The cells are stem cells, osteoblasts, myoblasts, myocytes, tenocytes, neuroblasts, fibroblasts, glioblasts, germ cells ( germcells, hepatocytes, renal cells, sertoli cells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells cells, cardiomyocytes, glial cells, endothelial cells, hormone secreting cells, cotton cells, pancreatic islet cells and neurons. It may be any one or more. The cell type used in the prepared artificial tissue of the present invention may be cultured in any manner known in the art. Cell and tissue culture methods are known in the art.

The cells may also be incubated with cell differentiation materials that induce differentiation of the cells along the desired cell line. For example, stem cells are incubated in contact with differentiation medium to produce a range of cell types. Many types of differentiation media are suitable. The stem cells include, but are not limited to, osteogenic differentiation medium, chondrogenic differentiation medium, adipogenic differentiation medium, neuronal differentiation medium, cardiomyocyte differentiation medium, and enterocyte differentiation medium (e.g., intestine). Incubated in contact with the differentiation medium).

In addition, the cells may be cultured with growth factors, cytokines and the like. Growth factor refers to a protein, polypeptide, or polypeptide complex comprising a cytokine, produced by a cell and capable of affecting itself and / or various other adjacent or isolated cells. Usually, the growth factor is In response to developmental or multiple biochemical or environmental stimuli, it affects the growth and / or differentiation of certain types of cells. Some, but not all, growth factors are hormones. Exemplary growth factors include fibers including insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), and basic FGF (bFGF) Platelet-derived growth factor (PDGF) including blast growth factor (FGF), PDGFM and PDGF-AB, bone morphogenic proteins (BMP), hepatocyte growth factor (HGF), traits including BMP-2 and 腿 P-7 Transforming growth factor beta (TGF-β), epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM) including transforming growth factor alpha (TGF-α), TGF | 3-1 and TGF13-3 -CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, and the like.

In the present invention, 'bioprinting' refers to three-dimensional accurate cell deposition (eg, cell solutions, cell-containing gels, cell suspensions, cells) through a methodology compatible with automated computer-assisted, three-dimensional prototyping devices (eg bioprinters). Concentrate, multicellular aggregates, multicellular bodies, etc.). 3D printing can be performed by extruding a biodegradable polymer from a nozzle using a bio-plotter and laminating it on a stage.

Various kinds of tissue analogues can be generated by the methods described above. The pattern or stacking arrangement for stacking the bio ink composition may be determined by the size and diameter of the tissue-like organ to be manufactured. In addition, the number of cells included in the bio ink used to manufacture tissue-like organs may be controlled according to the type of cells, the content of cell nutrients contained in the bio ink composition, and the like. In addition, the type of cells included in the bio ink composition may be variously changed according to the type of tissue-like organ to be prepared according to the above method. Those skilled in the art to which the present invention pertains will be able to select and apply appropriate cells according to the type of tissue-like organs to be prepared through three-dimensional bioprinting.

Bio ink composition is sprayed by a three-dimensional bio printer laminated Thereafter, it is possible to promote crosslinking of the bio ink ≤ composition by heating it, exposing it to ultraviolet rays or adding a crosslinking solution. This crosslinking allows the laminated bio ink composition to be completed into a more rigid structure. An optical initiator may be used to promote the crosslinking.

In order to manufacture an ink ejecting member having the same cross-sectional pattern as the printed matter but having the same or different cross-sectional pattern, at least one ink provided by a three-dimensional printing method is accommodated in a space of the ink ejecting member accommodating portion, It is provided with a single passage through which the ink passes and controls the pressure, for example the piston, to pass through the discharge portion for discharging the ink. After the bio ink has been layered in the container, it is preferable to put a small amount of hydrogel as a support material in the amount of 0.01 mL to 2 mL in advance so as not to pour the bio ink into the container. Thereafter, or without adding hydrogel to the barrel. When printing again, the filled hydrogel is ejected at the beginning of printing, and then the desired shape is printed. After printing the printed material of the desired shape, the hydrogel filled above will come out. The reason for adding support material (support mater al) at the beginning and the end is to make printing stable.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1, 2, 3, 4, and 5 schematically illustrate a process of providing ink to a ink receiving member by a three-dimensional printing method according to an example of the present invention.

In FIG. 1, the inside of the ink discharge member 10 is divided into a plurality of spaces for accommodating the respective inks 11, 12, 13, and 14. 9 shows a bioprinting apparatus which can be applied to an example of discharging five inks (1, 2, 3, 4, 5) according to one embodiment of the present invention. The cell liquid substance contained in the ink ejecting member 10 is pressed in the A direction and printed through the ejecting portion 20 or the nozzle 80. The ink is ejected to complete the final object 50. At this time, the piston 60 is preferably controlled to pass through the nozzle 20 is reduced in size while maintaining the same shape of the cross section of the printing material, the base may be a container containing a liquid material in some cases.

If the pressure is too high, the load on the nozzle may be increased and damage may occur, or the hydrogel may not be discharged in the form of a thread smoothly and may be discharged in an unbalanced shape, and the pressure may be too weak. In this case, due to the resistance due to the viscosity of the hydrogel may not be smooth discharge from the nozzle. On the other hand, if the diameter is too small, the discharge pressure is increased, so that the risks when the pressure is strong may occur the same, and if the diameter is too large, the precision of the three-dimensional shape when manufacturing the scaffold may be reduced. The above-described pressure range and diameter range are all considered as described above, so that the discharge of the hydrogel can be made smoothly and easily, and at the same time, to achieve a desired level of accuracy of the manufactured scaffold shape. It was determined experimentally as a range to do.

For example, the printing method according to the present invention includes the steps of accommodating ink in the accommodating part, applying a pressure within the range of 0.1 to 700 kPa, 1 to 500 kPa or 1 to 700 kPa, for example, 0. Discharging the ink to the nozzle having an outlet diameter within a range of 1 to 1 mm, and printing the ink while the nozzle moves at a speed within a range of 1 to 700 mm / min by a moving part of a printing apparatus. Can be done.

Next, the cell liquid substance contained in the ink discharge member 10 is pressed in the A direction to discharge the printing material to the base 100 through the discharge portion 20 to complete the final object 50. At this time, the piston is preferably controlled to pass through the discharge portion 20 is reduced in size only while maintaining the same shape of the cross section of the printing material, in some cases the base may be a container containing a liquid material. In the case of bioprinting by spraying one material as in the prior art, there is a limit to the volume reduction of the material because there is a limit to the size reduction of the nozzle inner diameter. However, according to the present invention, it is possible to reduce the volume of the ink ejected in proportion to the number of multiply inks, so that precise jetting is possible in comparison with the prior art. When the multiple inks pass through the passage of the ejection section 20, the area of contact between each ink and the inner surface of the ejection section passage is reduced, so that the shear force generated is reduced than when ejecting a single material. . Therefore, there is an advantageous effect compared to the prior art in terms of cell activity.

【Effects of the Invention】

According to the present invention, the volume of material discharged in proportion to the number of multiple inks can be reduced, so that precise jetting is possible compared to the prior art. In addition, when multiple inks pass through the discharge section or the passage of the nozzle, the area of contact between each material and the inner surface of the nozzle passage is reduced, and the shear force generated is also reduced than when discharging a single material. Therefore, in terms of improving cell activity and printing accuracy, there is an advantageous effect compared to the prior art. [Brief Description of Drawings]

1 is a schematic diagram illustrating a process of manufacturing a printed matter of a second ink and a layered ink of a first ink according to an embodiment of the present invention.

FIG. 2 is a schematic view showing an ink ejecting member into which a first ink layered product and a spiral second ink printed matter are injected and an ejection obtained therefrom according to an example of the present invention.

3 is a schematic view showing an ink ejecting member into which a print made of three kinds of ink of crab 1 ink and a spiral shape is injected, and an ejection obtained therefrom according to an example of the present invention.

4 is ink ejected by a three-dimensional printing method according to an embodiment of the present invention A diagram schematically showing an example of injecting ink into a member.

5 is a view schematically illustrating a method of injecting the second to seventh inks by a three-dimensional printing method in addition to the filling of the first ink in the ink ejection member according to an embodiment of the present invention.

FIG. 6 is a photograph of a syringe and a discharging part provided at the end of the syringe showing a process of manufacturing a print of the crab 1 ink and the crab 2 ink according to Example 1

FIG. 7 is a confocal micrograph showing the result of printing using the ejection obtained from the ink ejecting member in which the filling of the first ink and the printed matter of the spiral second ink were injected according to Example 1. FIG.

FIG. 8 is a confocal micrograph showing the result of printing using a syringe in which a layered material of crab 1 ink and a printed material of a second ink are laminated according to Example 2, and an ejection obtained from the syringe.

9 is a view schematically showing an ink ejecting member having a receiving portion in which five different inks are layered according to an example of the present invention. 10 is a view schematically showing an ink ejecting member according to an embodiment of the present invention. ^,

[Form for implementation of invention]

The present invention will be described in more detail with reference to the following examples, but the scope of protection of the present invention is not intended to be limited to the following examples. Example 1: A syringe including an ink substrate and an ink layer

A 1-gel hydrogel for layering of 3 w / v% sodium alginate was injected into a syringe, which is an ink receiving portion of a 3D printing apparatus. In a syringe into which the first hydrogel was injected, a 3D printing apparatus having a long length nozzle was used. Then, 3 w / v% sodium alginate containing green fluorescent particles was injected as a 12 hydrogel by three-dimensional printing. Of concrete manufacturing process One example was performed similar to the method illustrated in FIG. 1. The ink shape printed on the syringe was photographed and shown in FIG. 2.

The first hydrogel for filling the syringe and the three-dimensional printed giant 12 hydrogel were printed by a three-dimensional printing method using a discharge obtained through a discharge unit by applying pressure, and the nozzle size (nozz le I. D) was 1.0 mm. Printed in three lines. Printed section length is 30 micrometers when using a nozzle with a nozzle size (nozz le I .D) of 1 mm and printed section length when using a nozzle with a nozzle size (nozzl e I .D) of 2 mm Was 70 micrometers.

As a result of fluorescence observation by the confocal microscope, the printed matter was confirmed by confocal microscopy of the hydrogel containing green fluorescent particles. The micrograph is shown in FIG. 3. FIG. 6 shows a photograph in which a first hydrogel for supporting and a second hydrogel having a specific shape formed therein by a three-dimensional printing method are formed using a discharge member including an ink receiving unit according to Example 1. FIG. FIG. 7 shows the results of confocal microscopy observation of a printed matter produced by a three-dimensional printing method by ejecting the ink containing layer having the first hydrogel and the crab hydrogel layered in accordance with Example 1 under pressure conditions. That is, when the discharged product is observed by fluorescence with a confocal microscope, it is as shown in FIG. The method according to the invention indicates that the ink is printed with high resolution.

"

Example 2 three-dimensional printing using nozzles of various sizes

Using a three-dimensional printing apparatus using the same ink ejecting member as Example 1, it was confirmed by confocal microscopy that the hydrogel is printed in the nozzle size of 18, 20, 22, 25 and 27 Gauge. Specifically, the nozzle inner diameters of 18, 20, 22, 25, and 27 Gauge were 0.82 mm, 0.63 mm, 0.41 mm, 0.28 mm, and 0.1 mm. The confocal micrograph is shown in FIG. 8.

FIG. 8 shows the first hydrogel layered product and the second hydrogel printed matter of the size of the nozzle using the discharge member including the ink containing portion according to Example 2. FIG. Confocal microscopy results showing the printing results with changes. As shown in the cross-sect ional view, it was possible to miniaturize to the same shape as the cross-sectional shape of the ink discharge member. According to an embodiment of the present invention, the ratio could be downsized to 98.7% (200 μηι) from the total diameter (15 mm 3) of the specific shape (eg Lobule). It was calculated according to the above equation.

[Equation 1]

Reduction ratio of ink printed matter =-^ / 八 X100 (%)

In Equation 1,

A is the diameter of the primary ink substrate provided in the syringe by three-dimensional printing method B is the diameter of the secondary substrate of the printing ink,

A and B are the same length unit.

TABLE 1

Example 3 Ink Prints Containing Two or More Inks

1 hydrogel was injected into the same syringe as in Example 1. The hydrogel is ^injected, the syringe, using the future a three-dimensional printing, comprising a long length of the nozzle, the ink printed print each Green, Blue, Red fluorescent particle is a 3 w / v% sodium alginate comprising sequentially It was injected into a syringe, and it was confirmed by confocal microscopy that the obtained RGB hydrogel was printed. A schematic diagram of a syringe filled with ink prints prepared with the three inks and a manufacturing process thereof are shown in FIG. 7.

According to the method of the present invention, in addition to preparing an ink print with one ink inside the syringe, three-dimensional printing is performed with two or more inks. It can be seen that ink prints can be produced, and thus, various types of tissue can be simulated. Example 4 Fabrication of Artificial Vessels with Lumen Structure

3 w / v% sodium alginate was injected into the same syringe as in Example 1, and 3% gelatin, a temperature-sensitive hydrogel, was injected into the alginate pre-injected using a long nozzle using a printing method. Printing the prepared complex hydrogel in 200 Mm calcium chloride induces only alginate gelation and gelatin is present in the ungelled body. When the printing structure was placed in a liquid of 37 ^a C, the gelled alginate remained intact but gelatin melted to form a lumen structure. Example 5 Blood Vessel Fabrication with Cells and Multiple Lumen Structures In the same manner as in Example 4, 3 w / v% sodium alginate was injected into the syringe and smooth muscle was used, using a three-dimensional printing apparatus equipped with a long nozzle. 3% alginate in which the cells are contained at a concentration of at least IX 10 ⁷ Cel ls / mL is injected into the filled 3% alginate, and 3% gelatin embedded at a concentration of at least 1 X 10 ⁷ Cel ls / mL is used for smooth muscle cells. Vascular structures were simulated by a continuous method of injecting the intrinsic alginate.

In particular, the aorta and the vena cava of the blood vessels are stacked in a four-cylindrical structure. This method enables easy hand printing of quadrilateral blood vessels, as well as size control. In addition, the double structure of the cleansing vein and the single structure of the microvessel can be similarly simulated.

[Description of the code]

10: ink discharge member

20: discharge part

40, 50 ： Ink discharge

80 ： Ink discharge port (nozzle) 100: plate

Although the present invention has been described above with reference to the accompanying drawings, the scope of the present invention is determined by the claims below and is not intended to be limited to the embodiments and / or drawings described above.

Claims

[Range of request]

[Claim 1]

An ink providing step of providing a three-dimensional printing ink ejecting member including an ink containing portion and an ejecting portion, with at least one ink as an ink printed matter printed by a three-dimensional printing method;

And applying the physical force to the ink ejecting member to eject the ink into the ejecting portion to produce an ink ejection, and printing the ink ejection on the substrate.

[Claim 2]

The method of claim 1, wherein the ink print in the ink providing step has a two-dimensional or three-dimensional pattern, three-dimensional printing method. ^'

[Claim 3]

The three-dimensional printing method according to claim 1 ^, wherein in the ink providing step, at least one ink is further provided as an ink layered material by a method other than the three-dimensional printing method.

[Claim 4]

The three-dimensional printing method according to claim 1, wherein a cross section of the ink layered on the ink ejecting member and a cross section of the ink ejection have the same pattern.

[Claim 5]

The method of claim 5, wherein the ink has a viscosity measured at 25 ^° C. of 2 cps to 1,000 cps.

[Claim 6]

4. The method of claim 3 wherein the ink viscosity difference between the ink deposits and ink prints is between 0 and 5,000 cp.

[Claim 7]

The method according to claim 1, wherein the difference in elasticity of the ink layered product and the ink printed matter is within 0 to 10,000 Pa.

[Claim 8] The method according to claim 13, wherein in the ink providing step, the ink layered matter and the ink printout are provided to the ink ejecting member simultaneously or sequentially.

[Claim 9]

The method according to claim 8, wherein in the ink providing step, after providing the ink layered product, an ink printout of printing one ink by three-dimensional printing is provided.

[Claim 10]

The ink ejecting member of claim 1, wherein the ink ejecting member comprises an ink receiving portion for receiving ink in an inner space, a discharge portion for discharging ink contained in the receiving portion, having a single passage through which the ink passes and positioned below the receiving portion; How to.

[Claim 11]

The method according to claim 1, wherein the ratio of the cross-sectional diameter of the ink provided to the ink ejecting member and the cross-sectional diameter of the ejection is 100: 99 to 100: 0.1.

[Claim 12]

The method of claim 1, wherein the printed material is artificial tissue.

[Claim 13]

The method of claim 1, wherein producing the ink ejection is performed simultaneously or sequentially with receiving the ink.

[Claim 14]

The method according to claim U, wherein the ink is one or more selected from the group consisting of constituents, constituent contents and physical properties.

[Claim 15]

The method of claim 1, wherein the ink comprises a gelling polymer. [Claim 16]

The method of claim 1, wherein the ink comprises one or more selected from the group consisting of gelling polymers, cells, growth factors and extracellular matrix.