KR102494197B1 - Cartilage component based bio ink for manufacturing microtia therapeutic construct and a method of making the same - Google Patents

Abstract

The present invention relates to a bioink composition for 3D (three dimensional, 3D) printing and a manufacturing method thereof.
The bio-ink composition according to the present invention contains a micronized cartilage component, so that cartilage tissue can be safely and effectively regenerated, and can be applied for fabricating a structure for the purpose of treating microtia.

Description

Cartilage component based bio ink for manufacturing microtia therapeutic construct and a method of making the same}

The present invention relates to cartilage component-based 3D printing bio-ink and a method for manufacturing a structure for microtia treatment using the bio-ink. More specifically, allogeneic and heterogeneous cartilage is decellularized to microparticulate cartilage tissue that minimizes the residual of immune response-inducing factors, and is prepared by physically mixing one or more biocompatible polymers and microparticulated polymers. It relates to a method of manufacturing a structure for microtia treatment using a 3D printing bio-ink composition.

Cartilage tissue reconstruction surgery is performed to restore patients with ear defects caused by microtia, which is a congenital anomaly, or trauma or burns. In the case of microtia, which is a congenital deformity with no ears or only traces from birth, surgical treatment is required because dissatisfaction with appearance during childhood or adolescence causes social phobia and makes it difficult to form social relationships such as relationships with the opposite sex and employment.

Currently, ear reconstruction surgery is performed by collecting autologous rib cartilage, making an ear shape with a knife, and connecting it with a wire. However, the above method requires surgery at around the age of 10 after the cartilage has grown, the rib cartilage that can be collected is limited, and it is difficult to express the shape of the ear with the rib cartilage. There are downsides.

There is a method of applying an artificial graft material such as allogeneic cartilage or synthetic material, but the method has limitations such as difficulty in supply and high cost, and in the case of an artificial graft material, side effects such as protrusion after implantation may occur.

In order to solve the limitations of the above-mentioned existing treatment methods, we intend to fabricate and apply a customized structure for microtia patients using 3D printing.

3D printing is a promising new technology that can produce customized tissues and organs by printing tissues and organs through patient information. In particular, when one ear is missing, such as in microtia, the same missing part can be implemented by mirroring the CT data of the normal ear. Moreover, since cartilage tissue such as the ear has few microvessels and has different curves and curves for each individual, 3D printing grafting can be considered suitable.

Bio-ink is a material that makes tissues or organs printable through 3D printing, and is one of the key elements of 3D printing. A variety of materials can be used, from synthetic polymers to biopolymers, and must be biocompatible. Biocompatible materials that can constitute bioink include agarose, alginate, chitosan, decellularized extracellular matrix, fibrin/fibrinogen, and gelatin. (gelatin), hyaluronic acid, polycaprolactone (PCL), polylactic acid (PLA), poly(DL-lactic-co-glycolic acid (poly-D, L-lactic-co- glycolic acid, PLGA) and pluronic (pluronic F 127), etc. Among them, alginate is crosslinked through chelation of divalent cations to form a gel, which The properties satisfy the printability of bioink and mechanical properties after printing, but alginate alone does not provide a suitable environment for cell attachment and growth due to its low cell affinity.

Micronized polymers are applied to medical fields such as drug delivery, precision detection, molecular imaging, and regenerative engineering. Polymers that can be micronized include polycaprolactone (PCL), poly(DL-lactic-co-glycolic acid (PLGA)), polyethylene glycol, PEG), etc. All of these materials are FDA-approved biodegradable polymers, and in particular, polycaprolactone (PCL) has little toxicity, is hydrolyzed by enzymes in the body, and is widely used clinically due to its high biocompatibility. In addition, because of its relatively high mechanical strength and excellent compatibility with other polymers, it is mixed with other materials for bone regeneration in the field of bone regeneration and used for bone formation induction research (non-patent literature [1] and [2]).

Accordingly, in the present invention, a method for fabricating a structure for the purpose of treating microtia using a cartilage component-based bioink composition is proposed.

[1] Malikammadov, Elbay, et al. "PCL and PCL-based materials in biomedical applications." Journal of Biomaterials science, Polymer edition 29.7-9 (2018): 863-893. [2] Xu, Ning, et al. "3D artificial bones for bone repair prepared by computed tomography-guided fused deposition modeling for bone repair." ACS applied materials & interfaces 6.17 (2014): 14952-14963.

An object of the present invention is to provide a bioink composition containing a micronized cartilage component and a method for fabricating a structure for the purpose of treating microtia using the bioink composition.

The present invention is a microparticled cartilage component; micronized polymers; and a bio-ink composition for 3D printing comprising a biocompatible polymer.

The present invention also provides a microparticled cartilage component; micronized polymers; and extruding a bio-ink composition for 3D printing containing a biocompatible polymer to prepare a structure.

The bio-ink composition according to the present invention contains micronized cartilage components, so that cartilage tissue can be safely and effectively regenerated, and can be easily used for constructing a structure for the purpose of treating microtia.

In the present invention, it is possible to improve the viscoelasticity and strength of a bioink composition by physically mixing a biocompatible polymer and a microparticled polymer together with the micronized cartilage component, thereby increasing the viscoelasticity and strength of the printed structure. can improve

In addition, the bio-ink composition of the present invention is in the form of a gel, and can solve the phenomenon of loss of shape due to failure to laminate 2 to 3 mm or more during conventional 3D printing.

FIG. 1 shows photographs of ear structures fabricated using 3D printing using (A) a bio-ink composition not containing a micro-particled polymer and (B) a bio-ink composition containing a micro-particled polymer. The bioink compositions of (A) and (B) were printed under the same conditions, and the printed products were viewed from above and from the side without a crosslinking process.
FIG. 2 shows (A) a scanning electron microscope (SEM) image of the inside of a bioink composition containing no micronized polymer and (B) a bioink composition including the micronized polymer.
Figure 3 shows the result of confirming cell viability by culturing cells in a construct made of a bioink composition containing (A) allogeneic or (B) allogeneic cartilage components. Specifically, in FIG. 3 , green indicates living cells and red indicates dead cells. (C) shows the result of analyzing cell proliferation in each condition using absorbance through CCK 8 assay.
Figure 4 shows the result of confirming cell viability by culturing cells in a structure made of a bioink composition containing a heterogeneous cartilage component.
Specifically, in (A) of FIG. 4, human skin-derived fibroblasts (human dermal This is the result of confirming cell survival by culturing fibroblast, HDF). Green represents live cells and red represents dead cells. (B) analyzed cell proliferation by period by measuring absorbance through CCK 8 assay.
5(A) shows human-derived chondrocytes cultured in a structure made of a bioink composition containing and not including a microparticled cartilage component in order to confirm the efficacy of the cartilage component. This is the result of confirming cell viability. (B) analyzed cell proliferation by period by measuring absorbance through CCK 8 assay.
6(A) shows an ear structure obtained by applying an ear structure model file to 3D printing and outputting a bioink composition containing homogeneous and heterogeneous cartilage components. (B) is an ear structure output in various ways with various ear structure model files and files in which the size of the ear structure is adjusted. This indicates that various outputs can be made by adjusting the file size according to the ear structure model.
7 shows a photograph of the fabricated structure directly inserted into an animal.

The present invention is a microparticled cartilage component; micronized polymers; and a bioink composition for 3D printing comprising a biocompatible polymer.

In an embodiment of the present invention, a microparticled cartilage component; micronized polymers; and a biocompatible polymer, and cell compatibility was confirmed by preparing a bioink composition and analyzing the cell adhesion of the bioink composition. In addition, an ear structure was fabricated with the bio-ink composition to confirm the applicability of the bio-ink composition to 3D bioprinting, and it was confirmed that the fabricated structure was transplanted to an animal to maintain the shape of the ear.

Hereinafter, the bio-ink composition for 3D printing of the present invention will be described in more detail.

The bio-ink composition (or may be referred to as bio-ink) according to the present invention includes a micronized cartilage component; micronized polymers; and biocompatible polymers.

In the present invention, the micronized cartilage component means cartilage pulverized to a size of 10 to 1000 μm. The micronized cartilage component can be used for the purpose of improving biocompatibility, cell adhesion, and physical properties of bioink. In particular, the micronized cartilage component provides conditions capable of creating a microenvironment similar to the original cartilage tissue. Since it is not an artificially produced material, but a structure formed through interaction with cells in the human body, it has components and phases specific to cartilage tissue, and can provide a favorable environment for cell growth and differentiation.

In one embodiment, the micronized cartilage component may be a homogeneous or heterogeneous micronized cartilage component. The allogeneic cartilage refers to human-derived cartilage, and heterologous cartilage may refer to cartilage derived from animals other than humans, that is, mammals such as pigs, cows, and horses.

In one embodiment, the micronized cartilage component may have an average particle diameter of 10 to 1000 um, 10 to 700 um or 10 to 500 um. In the above particle size range, the bioink can pass through the 3D printing nozzle in micro units to implement the input shape, and can impart strength capable of maintaining the structure of the structure. In addition, the original structure of the cartilage tissue can be preserved to create a microenvironment similar to the original cartilage tissue.

In one embodiment, the micronized cartilage component is (S1) a freeze-drying step of freeze-drying the cartilage;

(S2) grinding step of grinding the lyophilized cartilage; and

(S3) It can be prepared through a sieve separation step of sieving the pulverized cartilage.

In the present invention, a washing step may be performed before performing the lyophilization step, and sterile distilled water may be used as a washing solvent. Impurities in the cartilage may be removed through the above steps.

In addition, in the present invention, a step of removing soft tissue and perichondrium from cartilage may be performed before performing the lyophilization step.

In one embodiment, the removal of cartilage tissue and perichondrium can be performed using a blade and a longeur. Specifically, after cutting the interface between cartilage and cartilage vertically with a blade, the perichondrium can be removed by using a longer to wet the tissue surface with sterile distilled water so that it does not dry out and pulling by biting the edge of the cut surface.

In the present invention, the freeze-drying step (S1) is a step of freeze-drying the cartilage.

In one embodiment, the cartilage may be cartilage from which the above-described washing, and cartilage tissue and perichondrium have been removed.

In one embodiment, lyophilization is a method of rapidly cooling tissue (cartilage) in a frozen state and then absorbing moisture in a vacuum. The freeze-drying may be performed to control moisture in the cartilage.

In one embodiment, lyophilization may be performed at -50 to -80 ° C for 24 to 96 hours.

In the present invention, the grinding step (S2) is a step of grinding the freeze-dried cartilage.

In one embodiment, grinding may be performed using a tissue grinder. At this time, the grinding time may be 30 seconds to 5 hours. In one embodiment, the particle diameter of the cartilage after grinding may be 10 to 1000 μm.

The crushing may be performed one or more times.

In the present invention, the sieving step (S3) is a step of sieving the pulverized cartilage in the pulverizing step.

In one embodiment, sieving may be performed using a sieve having a grid of 100 to 1000 μm.

The sieving may be performed one or more times.

In the present invention, after performing the sieve separation step, (S4) a delipidation step of delipidating the pulverized microparticle cartilage component; and (S5) a decellularization step of decellularization may be further performed.

In the present invention, (S4) delipidation step is a step of removing lipid components from adipose tissue.

In one embodiment, delipidation refers to the removal of lipid components from tissue.

Removal of the lipid component may be performed by chemical treatment.

In one embodiment, the type of chemical treatment is not particularly limited and may be performed using a delipidating solution. The degreasing solution may include a polar solvent, a non-polar solvent, or a mixture thereof. Water, alcohol or a mixed solution thereof may be used as the polar solvent, and methanol, ethanol or isopropyl alcohol may be used as the alcohol. As the non-polar solvent, hexane, heptane, octane, or a mixed solution thereof may be used. Specifically, in the present invention, a mixed solution of isopropyl alcohol and hexane may be used as a degreasing solution. At this time, the mixing ratio of isopropyl alcohol and hexane may be 40:60 to 60:40.

The treatment time of the degreasing solution may be 1 to 30 hours, 1 to 20 hours, or 10 to 20 hours.

In the present invention, the decellularization step (S5) is a step of removing cells from the micronized cartilage components from which lipid components have been removed by the delocalization step.

In one embodiment, decellularization refers to removing other cellular components other than the extracellular matrix from tissues, such as nuclei, cell membranes, and nucleic acids.

In one embodiment, decellularization may be performed using a basic solution, and one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, calcium hydroxide and ammonia may be used as the basic solution. there is. In the present invention, sodium hydroxide (NaOH) may be used as a basic solution.

In one embodiment, the concentration of the basic solution may be 0.01 to 1 N, 0.06 to 0.45 N, 0.06 to 0.2 N, or 0.08 to 1.02 N. Removal of cells is easy in the above concentration range.

Also, in one embodiment, the decellularization step may be performed for 40 to 60 minutes, 70 to 200 minutes, or 90 to 150 minutes. It is easy to remove the cells in the above time range.

In the present invention, after performing the decellularization step, neutralization step of neutralizing with an acidic solution if necessary;

Washing step of washing the neutralized cartilage components of the microparticles;

Centrifuging the washed, finely granulated cartilage component; and

One or more steps selected from the group consisting of lyophilization may be further performed.

In one embodiment, impurities in the delipidation step and the decellularization step may be removed through the centrifugation step, and a high purity microparticled cartilage component (precipitate) may be obtained.

In addition, in the washing step, sterile distilled water and/or 70% ethanol may be used as a washing solution.

In one embodiment, the content of the micronized cartilage component may be 1 to 20 parts by weight based on the total weight (100 parts by weight) of the bioink composition. In addition, the micronized cartilage component may be present in an amount of 1 to 10% (w/v) in the bioink composition.

In the present invention, the micronized polymer controls the viscosity of the bio-ink composition and enables the realization of a more precise structure during 3D printing.

In one embodiment, the micronized polymer is granulated using a polymer capable of micronization, such as polycaprolactone (PCL), poly(DL-lactic-co-glycolic acid (poly- At least one selected from the group consisting of D, L-lactic-co-glycolic acid (PLGA) and polyethylene glycol (PEG) may be used In the present invention, polycaprolactone (PCL) is used as a polymer, and Polycaprolactone has the advantage of having little toxicity, being hydrolyzed by enzymes in the body, having high biocompatibility, and having high mechanical strength.

In one embodiment, the micronized polymer may have an average particle diameter of 1 nm to 200 um. In the above particle size range, the bioink can pass through the 3D printing nozzle in micro units to implement the input shape, and can impart strength capable of maintaining the structure of the structure.

In one embodiment, the micronized polymer is prepared by (A) preparing a polymer solution by solubilizing the polymer in an organic solvent; and

(b) mixing the polymer solution with a mixed solution of a surfactant and a biocompatible polymer, and then subjecting the polymer solution to ultrasonication.

In step (A), the polymer solution may be prepared by solubilizing the polymer in an organic solvent.

As the polymer, the aforementioned types may be used without limitation, and chloroform, chloromethane or acetone may be used as the organic solvent.

Step (B) is a step of mixing the polymer solution with a mixed solution of a surfactant and a biocompatible polymer, and then ultrasonicating the mixed solution. It can be made of polymers.

In the mixed solution, the surfactant reduces the surface tension of the solution to enable the production of fine particles and also enables the formation of stable particles. As the surfactant, polysorbates such as methyl cellulose (MC), polyvinyl alcohol (PVA), polyoxyethylene sorbitans, and Pluronics ™ may be used. Specifically, polyoxy Ethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate sorbitan tristearate), polyoxyethylene sorbitan monopalmitate, or polyoxyethylene sorbitan monolaurate (twin), including polyoxyethylene sorbitan monooleate , Tween 80 (TM)) and polyoxyethylene sorbitan monostearate (T ween 60 (TM)).

In addition, the biocompatible polymer may be used to increase the mixing rate with the compositions in the bioink and impart cell compatibility by imparting hydrophilicity to the micronized polymer. The type of biocompatible polymer is not particularly limited, and for example, agarose, alginate, chitosan, collagen, decellularized extracellular matrix, fibrin / At least one selected from the group consisting of fibrin/fibrinogen, gelatin, hyaluronic acid, and pluronic F 127 may be used.

In one embodiment, after mixing the polymer solution and the mixture solution, ultrasonication is performed to prepare spherical polymer particles. At this time, the spherical shape may be used as a meaning including a case in which a substantially spherical shape is included in addition to a complete spherical shape, and a case in which the cross section has an elliptical shape. In addition, ultrasonic conditions may be performed according to conditions known in the art.

The spherical polymer may be formed by extraction evaporation.

In one embodiment, the content of the micronized polymer may be 1 to 50% (w/v) of the total bioink composition. In the above range, desired physical properties may be satisfied during preparation of the support.

In the present invention, the biocompatible polymer imparts biocompatibility to the fabricated structure.

In one embodiment, the type of biocompatible polymer is not particularly limited, and for example, agarose, alginate, chitosan, collagen, and decellularized extracellular matrix. matrix), fibrin/fibrinogen, gelatin, hyaluronic acid, polycaprolactone (PCL), polylactic acid (PLA), poly(DL-lact-co- At least one selected from the group consisting of glycolic acid (poly-D, L-lactic-co-glycolic acid, PLGA) and pluronic (Pluronic F 127) may be used.

In one embodiment, alginic acid may be used as the biocompatible polymer. The alginic acid may include calcium, sodium, potassium, ammonium, or magnesium alginate. The alginic acid is characterized by excellent biodegradability, improved physical properties of bioink, and easy crosslinking. Specifically, since alginate is crosslinked and gelled through chelation of divalent cation, printability as a bioink and mechanical properties after printing can be satisfied through the gelation property. there is.

In one embodiment, the content of the biocompatible polymer may be 0.5 to 10 parts by weight based on the total weight of the bioink composition (100 parts by weight). The biocompatible polymer may be present in an amount of 1 to 12% (w/v) in the bioink composition. In the range of parts by weight, desired physical properties may be satisfied during preparation of the support.

In addition, the bioink composition of the present invention may further include water.

In one embodiment, the bio-ink composition according to the present invention can be used for 3D printing and applied to the fabrication of an ear structure for the purpose of treating microtia.

In addition, the present invention provides a method for preparing the aforementioned bio-ink composition.

The bio-ink composition according to the present invention includes the above-mentioned components, that is, a micronized cartilage component; micronized polymers; And it can be prepared by mixing a biocompatible polymer. Since the micronized cartilage component is in the form of nano- or micro-sized particles, it can be physically and evenly mixed with bio-derived polymers without a separate mixing process during hydration. In addition, in the present invention, the mechanical strength of the bioink can be adjusted according to the content of the micronized cartilage component and the micronized polymer.

In addition, the present invention is a microparticled cartilage component; micronized polymers; and extruding a bio-ink composition for 3D printing containing a biocompatible polymer to prepare a structure.

In the present invention, the micronized cartilage component; micronized polymers; And it is possible to manufacture an ear structure for the treatment of microtia through a bio-ink composition for 3D printing containing a biocompatible polymer.

In one embodiment, the bio-ink composition according to the present invention can be applied to 3D bio-printing.

The 3D bio-printing refers to a process of obtaining an image by scanning a target structure in three dimensions and making the scanned image into a three-dimensional structure through bio-ink. The present invention can be applied to extrusion-based processes.

In the present invention, the extruded product of the bio-ink composition for 3D printing may be referred to as a structure, an output product, or a scaffold. The bioink composition according to the present invention can serve as a support for cell growth and differentiation, and has biocompatibility and biocompatibility. Therefore, it can be easily transplanted into the body, and the growth and differentiation of cells can be controlled in an environment of in vitro culture or transplantation into the body.

In the present invention, the structure may be prepared by extruding a bio-ink composition using a 3D printer. Specifically, the structure may be manufactured by printing 3D drawing data according to a pre-designed program using a 3D computer.

In the present invention, the structure can be manufactured by appropriately adjusting the extrusion force and extrusion speed according to the purpose to be manufactured.

In the present invention, a step of crosslinking the extruded structure may be further included.

The crosslinking may be performed through a process commonly used in the art.

In one embodiment, when alginic acid is used as a biocompatible polymer, since the alginic acid is crosslinked and gelled through chelation of divalent cations, crosslinking can be performed using this characteristic.

In the present invention, by providing a bioink composition for 3D bioprinting, a structure completed through bioprinting, that is, an ear structure, can be used for the treatment of microtia. Specifically, a structure made of bioink has biocompatibility and can induce tissue regeneration by containing cells in a scaffold and attaching to a surface thereof, and thus can be usefully used in living tissue regeneration, tissue engineering, and regenerative medicine.

The present invention will be described in more detail through the following examples. However, those skilled in the art can understand that the scope of the present invention is not limited to the following examples, and that various changes, modifications, or applications are possible within a range that does not deviate from the technical details derived by the matters described in the appended claims. will be.

Example

Example 1. Preparation of bio-ink composition for fabrication of ear structure based on 3D printing and preparation of ear structure

(1) Manufacture of allogeneic microparticle cartilage components

Cartilage tissue (collected from a cadaver donated by a tissue bank for non-profit patient treatment) was pulverized using a micro grinder. During the grinding, cartilage tissue powder having a size of 1000 um or less was obtained by passing through a sieve having a diameter of 100 to 1000 um.

The obtained cartilage tissue powder was acellularized (decellularized).

First, cartilage tissue was subjected to delipidation using alcohol and nucleic acid. Thereafter, the cells were removed using sodium hydroxide, and washed using alcohol and distilled water. Freeze-drying was performed to remove moisture.

(2) Preparation of micronized polymer

The microparticleization process of the polymer was performed.

First, a polymer solution was prepared by dissolving polycaprolactone (PCL) in chloroform. Thereafter, the polymer solution was slowly dropped into a mixed solution of a surfactant and a biocompatible polymer and dispersed using ultrasonic waves.

Specifically, a 1 to 50% (10 mg/ml to 500 mg/ml (w/v)) polymer solution was prepared by dissolving the polymer in an organic solvent. Then, a mixed solution was prepared by mixing a surfactant solution of 10 mg/ml to 500 mg/ml and a biocompatible polymer solution of 10 mg/ml to 500 mg/ml. At this time, the surfactant solution and the biocompatible polymer solution were mixed at a volume ratio of 1:1.

A polymer solution was slowly dropped into the mixed solution, the organic solvent was volatilized from the dispersed solution by ultrasonic treatment, washed with distilled water and lyophilized to obtain a micronized polymer.

At this time, the average particle diameter of the obtained micronized polymer was 1 nm to 200 um.

(3) Preparation of bio-ink composition

A bioink composition in the form of a hydrogel was prepared by mixing the micronized cartilage component prepared in (1) with the micronized polymer prepared in (2) and an aqueous alginate solution.

A 1-12% (W/V) aqueous solution of alginate was prepared. Alginic acid aqueous solution was used after filtering with a 0.22 um syringe filter.

A 1-50% (W/V) aqueous solution of the micronized polymer was prepared.

1-10% (w/v) of the micronized cartilage component was hydrated in an aqueous solution of alginate and an aqueous solution of the micronized polymer. The composition was prepared by connecting two syringes to a luer adapter and hydrating them.

(4) Production of ear structure for microtia treatment using 3D printer

An STL file of an ear cartilage model to be printed was prepared and inputted to a 3D printer. 3D printing was performed by setting appropriate conditions for ink output.

Specifically, it was performed under conditions of a nozzle of 18-22G, a temperature of 4-40°C, a pressure of 10-200kPa, a printing speed of 1-5mm/s, and a printing height of 0.2-1.0mm. After printing according to the STL file, crosslinking was performed by immersing in an ionic solution (1% calcium chloride solution) for complete curing.

Example 2. Preparation of bioink composition for fabrication of ear structure based on 3D printing and preparation of ear structure

A bioink composition and an ear structure were prepared in the same manner as in Example 1, except that horse-derived cartilage (supplied from a farming association) was used instead of allogeneic cartilage in the preparation of the bio-ink composition.

Comparative Example 1.

In preparing the bioink composition, the bioink composition and the ear structure were prepared in the same manner as in Example 1, except that the micronized polymer prepared in (2) of Example 1 was not used.

Comparative Example 2.

In the preparation of the bioink composition, Example 1, except that the micronized cartilage component prepared in (1) of Example 1 and the micronized polymer prepared in (2) of Example 1 were not used. A bioink composition and an ear structure were prepared by the method of.

In the present invention, FIG. 1 shows (A) a bioink composition that does not contain the micronized polymer prepared in Comparative Example 1 and (B) a bioink composition that includes the micronized polymer prepared in Example 1 in 3D It shows the shape of the ear structure printed under the same conditions by attaching it to the printer. In the case of the ink composition of Comparative Example 1 not containing the micronized polymer, it was confirmed that the ear structure could not be implemented with the discharged bio-ink composition. On the other hand, it can be confirmed that the ink composition according to the present invention including the micronized polymer finely implements the shape of the ear.

In addition, FIG. 2 shows (A) an internal view of a bioink composition without the micronized polymer prepared in Comparative Example 1 and (B) a bioink composition including the micronized polymer prepared in Example 1 is shown using a scanning electron microscope. As shown in FIG. 2 , it can be confirmed that spherical microparticles are included in the bioink composition when the micronized polymer is included.

Experimental Example 1. Cytocompatibility analysis of bioink composition

In order to confirm compatibility with surrounding cells in the insertion environment when the structures manufactured in Examples and Comparative Examples are inserted, human-derived chondrocytes were cultured in the bioink compositions prepared in Examples and Comparative Examples to confirm cell compatibility. did

Experimental Example 1-1. 3.1 Cytocompatibility analysis of bioink compositions based on homogeneous and heterogeneous microparticles of cartilage components

(1) method

After mounting the bio-ink composition containing the homogeneous and heterogeneous micronized cartilage component, that is, the bio-ink composition prepared in Examples 1 and 2 to a 3D printer and fabricating a structure, 1 to 3 X 10 ⁵ The adipose-derived stem cells were sprayed and cultured on the construct. In order to confirm cell attachment and survival on the structure surface, Live/dead cell viability assay (Life Technology, USA) was performed after 7 days of culture. Specifically, the structure was immersed in a medium in which 0.5 μl/ml Calcein-AM and 2 ul/ml Ethidium homodimer-1 were dissolved and allowed to react for 30 minutes, and then confirmed using a confocal microscope.

In addition, after 1 day and 4 days of culture, the degree of cell proliferation was confirmed by measuring absorbance using a CCK-8 Kit.

(2) Results

The analysis results are shown in FIG. 3 .

3(A) is a bioink composition based on allogeneic microparticled cartilage component prepared in Example 1, and (B) in FIG. 3 is a bioink based on heterogeneous microparticled cartilage component prepared in Example 2 The results of analyzing the composition are shown. In the figure, green represents live cells and red represents dead cells.

As shown in FIG. 3, it can be confirmed that cells adhere to and proliferate on the surface of the structure in both allogeneic and heterogeneous microparticled cartilage component-based bioink compositions.

In addition, (C) is a result of analyzing the cell proliferation rate, and it can be confirmed that the cells are attached to the structure printed using the bioink composition and are able to survive and proliferate.

Experimental Example 1-2. Micronized cartilage component-based bioink composition for ear cartilage tissue-related cytocompatibility analysis

(1) method

In order to confirm the cell suitability of the ear cartilage tissue-related cells to the structure made of the microparticled cartilage component-based bioink composition prepared in Example 2, human skin-derived fibroblasts and cartilage cells were cultured and confirmed. .

Specifically, a cell suspension containing 1-3 X 10 ⁵ human-derived fibroblasts was sprayed onto the constructs prepared in Example 2 and Comparative Example 2 and cultured. In order to confirm cell attachment and proliferation on the surface of the construct, culture was performed in a cell culture medium for 1, 7, and 14 days.

Cytocompatibility analysis was performed as follows. After 1 day, 7 days, 14 days or 28 days, cell adhesion and survival were confirmed by a confocal microscope (LSM 700, Carl Zeiss, Germany) using a Live/dead cell viability assay (Life Technology, USA).

In addition, cells are cultured on the surface of the structure, and after 1, 7, and 14 days, absorbance is measured using a CCK-8 Kit to confirm the degree of cell proliferation.

(2) Results

The analysis results are shown in Figures 4 and 5.

4(A) shows human-derived bioink compositions containing micronized cartilage components (Example 2) and bioink compositions without micronized cartilage components (Comparative Example 2) according to culturing time. The results of attachment and proliferation of fibroblasts are shown. In the figure, green represents live cells and red represents dead cells.

As shown in FIG. 4(A), it can be confirmed that a large number of cells attach to, survive, and proliferate on the structure printed with bioink containing microparticle cartilage components from the beginning of culture.

In addition, FIG. 4(B) shows a structure made of a bioink composition containing a micronized cartilage component (Example 2) and a structure made of a bioink composition not containing a micronized cartilage component (Comparative Example). 2) shows the result of confirming the cell proliferation rate by culturing human skin-derived fibroblasts.

As shown in FIG. 4(B), it can be confirmed that the bioink composition containing the microparticled cartilage component has superior cell proliferation compared to the bioink composition without it from the beginning of cell culture to 2 weeks. .

On the other hand, FIG. 5(A) shows human-derived chondrocytes in a bioink composition containing micronized cartilage components (Example 2) and a bioink composition without micronized cartilage components (Comparative Example 2). The result of confirming cell adhesion and survival by culturing is shown.

As shown in FIG. 5(A), the cell proliferation rate increased in both groups with time, but the bioink containing the micronized cartilage component compared to the bioink composition without the micronized cartilage component. It can be confirmed that a large number of cells adhere, survive and proliferate in the composition.

In addition, FIG. 5(B) shows the bioink composition containing the micronized cartilage component (Example 2) and the bioink composition without the micronized cartilage component (Comparative Example 2) according to the culture time. It shows the proliferation rate of human-derived chondrocytes.

As shown in FIG. 5(B), the bioink composition containing the microparticled cartilage component from the beginning of culture has a cell proliferation rate that is more than twice as high as that of the bioink composition without the microparticled cartilage component. can confirm that

4 and 5, it can be confirmed that the bioink composition including the microparticled cartilage component has excellent cytocompatibility and cell affinity in ear cartilage-related cells.

Experimental Example 2. Fabrication of ear structure through 3D printer using bio ink composition

Ear structures were manufactured according to the manufacturing method of the ear structures disclosed in Examples 1 and 2.

In the present invention, Figure 6 shows the fabricated ear structure.

As shown in FIG. 6(A), the shapes of structures printed and fabricated with bioink compositions containing allogeneic (left photo) and heterogeneous (right photo) micronized cartilage components according to the same STL file are different from each other. can confirm the same.

In addition, FIG. 6(B) shows a structure fabricated by outputting a bioink composition including a heterogeneous microparticle cartilage component according to different STL files.

As shown in FIG. 6(B), it can be confirmed that structures of various STL files can be fabricated through the micronized cartilage component-based bioink composition, and even in the case of the same file, structures are fabricated by adjusting conditions such as size. You can confirm that this is possible.

Experimental Example 3. Implantation of fabricated structures into animals

After sterilizing the ear structure manufactured by 3D printing using the bio-ink composition prepared in Example 1, it was directly implanted under the skin of the animal.

7 is a view showing structures having a size of 70% of the actual ear size in (A) and 50% of the size of the actual ear in (B) inserted into the back of a nude mouse.

7, it can be confirmed that the structure inserted into the animal maintains the shape of the ear.

Claims (13)

micronized cartilage component; micronized polymers; And a biocompatible polymer,
The micronized cartilage component is 1 to 10% (w / v) of the total bioink composition,
The micronized polymer is 1 to 50% (w / v) relative to the total bioink composition,
The biocompatible polymer is 1 to 12% (w / v) of the total bioink composition,
The micronized polymer includes polycaprolactone (PCL),
The biocompatible polymer is a bio-ink composition for 3D printing comprising at least one selected from the group consisting of alginate, collagen and gelatin.
According to claim 1,
The average particle diameter of the micronized cartilage component is 10 to 1000 um bio-ink composition for 3D printing.
According to claim 1,
A lyophilization step of freeze-drying the micronized cartilage component;
Grinding step of pulverizing the lyophilized cartilage; and
A bioink composition for 3D printing prepared through a sieve separation step of sieving the pulverized cartilage.
delete delete According to claim 1,
The average particle diameter of the microparticled polymer is 1 nm to 200 um, the bio-ink composition for 3D printing.
According to claim 1,
preparing a polymer solution by solubilizing the micronized polymer in an organic solvent; and
A bio-ink composition for 3D printing that is prepared by mixing the polymer solution with a mixed solution of a surfactant and a biocompatible polymer and then ultrasonically treating the solution.
delete delete delete micronized cartilage component; micronized polymers; and extruding a bioink composition for 3D printing containing a biocompatible polymer to prepare a structure,
The micronized cartilage component is 1 to 10% (w / v) of the total bioink composition,
The micronized polymer is 1 to 50% (w / v) relative to the total bioink composition,
The biocompatible polymer is 1 to 12% (w / v) of the total bioink composition,
The micronized polymer includes polycaprolactone (PCL),
Wherein the biocompatible polymer comprises at least one selected from the group consisting of alginate, collagen and gelatin.
According to claim 11,
A method of making a structure further comprising the step of crosslinking the extruded structure.
According to claim 11,
A method of manufacturing a construct, wherein the construct is an auricular construct for the treatment of microtia.

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