Bioink composition derived from fibrous cartilage, bone graft composition containing the same, and method for producing the same

Abstract

The present invention relates to a bioink composition derived from fibrocartilage, a bone graft composition containing the same, and a method for producing the same. More specifically, it was confirmed that the inner, middle, and outer sides of the meniscus cartilage tissue partitioned by anatomical position each have different physicochemical properties, and a bioink composition for 3D printing having different physicochemical properties using the extracellular matrix derived from each partitioned tissue and a bone graft are provided, and these can be utilized by adjusting the physicochemical properties according to the purpose.

Description

Technical Field

[0001] The present invention relates to a tissue differentiation technique containing components derived from a composite tissue, and more particularly, to a bioink composition derived from fibrocartilage, a bone graft composition containing the same, and a method for producing the same.

Background Art

[0002] Rupture of the meniscus is one of the most common orthopedic diseases, showing a morbidity rate of about 20% in the general middle-aged population. Rupture of the meniscus induces knee pain, loss of function, and most importantly, acceleration of osteoarthritis of the knee joint. Similar to articular cartilage defects, meniscal injuries are not spontaneously curable, especially due to the limited blood supply restricted to the inner 2 / 3 of the tissue. Current surgical treatments such as surgery, partial meniscectomy, and allograft are insufficient to fully restore the function of the meniscus and prevent osteoarthritis. As a result, the interest and clinical need for meniscal cartilage engineering using stem cells, biomaterials, and tissue engineering techniques are increasing.

[0003] Problems associated with meniscal cartilage engineering can be attributed to the complex morphology and functional requirements of the meniscus. The meniscus resists stress not only by compressive force but also by tensile and shear forces within the knee joint, mainly due to the anisotropy and composite characteristics of the meniscal tissue. However, the results of current meniscal cartilage engineering are insufficient to achieve the biomimicry and functionality of the native meniscus, and it is difficult to reproduce the composite tissue that reflects the innate characteristics of each region.

[0004] The use of a biomaterial based on an extracellular matrix (ECM) derived from decellularized tissue represents a promising strategy for meniscus cartilage engineering. The decellularized ECM not only retains the biochemical properties of the donor-site tissue but also provides tissue-specific factors that guide cell behavior towards the donor-site tissue, thereby regulating specific cellular mechanisms such as proliferation and differentiation. In relation to the meniscus cartilage, various studies have been actively conducted on the biocompatibility and regenerability of the decellularized meniscus extracellular matrix (DMECM), but the exact composition and respective properties of the DMECM still remain unclear.

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a bioink for 3D printing that forms a composite tissue having different physicochemical properties using a substance derived from a compartmentalized living tissue, and a method for producing the same.

[0006] Another object of the present invention is to provide a bone graft obtained by crosslinking the bioink to a bone support and a method for producing the same.

Means for Solving the Problems

[0007] To achieve the above object, the present invention provides a bioink composition for 3D printing, which contains an extracellular matrix derived from a fibrocartilage composite tissue, and the fibrocartilage composite tissue is one or more selected from tissues compartmentalized into inner, mid, and outer regions of the meniscus cartilage tissue according to anatomical position, wherein the inner region has cartilaginous properties, the outer region has fibrous properties, and the mid region has properties in which cartilaginous and fibrous properties are transformed, and the physicochemical properties are changed by the compartmentalized tissues.

[0008] The present invention provides a bone graft composition comprising a decalcified bone matrix support, characterized in that the bioink composition is contained in and crosslinked within the support.

[0009] The present invention includes the steps of obtaining and pulverizing a fibrous cartilage composite tissue; decellularizing the pulverized tissue to obtain an extracellular matrix and pulverizing it; and subjecting the pulverized extracellular matrix to enzymatic treatment to solubilize it. The fibrous cartilage composite tissue is one or more selected from tissues partitioned into inner, middle, and outer portions of the meniscus cartilage tissue according to anatomical position. The inner portion has cartilage characteristics, the outer portion has fibrous characteristics, and the middle portion has characteristics where cartilage and fibrous characteristics are transformed, and the physicochemical properties change depending on the partitioned tissue. A method for manufacturing a bioink for 3D printing is provided.

[0010] The present invention includes the steps of manufacturing a decalcified bone matrix support; manufacturing a bioink containing an extracellular matrix derived from a fibrous cartilage composite tissue by the above manufacturing method; and injecting and crosslinking the manufactured bioink into the manufactured decalcified bone matrix support. A method for manufacturing a bone graft is provided.

[0011] Furthermore, the present invention provides a method for manufacturing an artificial tissue including the step of three-dimensionally printing the above bioink composition, and an artificial tissue manufactured thereby.

Advantages of the Invention

[0012] By confirming that the inner, middle, and outer portions of the meniscus cartilage tissue partitioned according to anatomical position each have different physicochemical properties, the bioink composition according to the present invention can provide a bioink composition for 3D printing having different physicochemical characteristics using extracellular matrices derived from each partitioned tissue.

[0013] The bone graft composition according to the present invention can provide a bone graft having physicochemical characteristics according to each partition by crosslinking the above bioink to a decalcified bone matrix support.

[0014] The bioink composition and bone graft material according to the present invention have different characteristics by compartment, such as cartilage tissue, fibrous tissue, angiogenesis, and anti-angiogenic properties, and thus can be utilized by adjusting their physicochemical properties according to the purpose.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0016] Hereinafter, the present invention will be described in detail.

[0017] The present inventors found that the inner, middle, and outer tissues partitioned by the anatomical position of the meniscus cartilage have significantly different physicochemical properties, and when the extracellular matrix derived from the partitioned tissues is manufactured as a bioink and a bone graft material containing the same, it can be adjusted and utilized by the desired mechanical properties and biochemical properties. Thus, the present invention was completed.

[0018] The present invention provides a bioink composition for three-dimensional (3D) printing containing an extracellular matrix derived from a fibrocartilage composite tissue.

[0019] As used herein, "3D printing" or "Three-Dimensional Printing" refers to a technology that can generate a 3D cell model or tissue structure with a complexity similar to that of tissue and an anatomically shaped substrate, which can be precisely controlled.

[0020] As used herein, "Bio-ink" is used in 3D bioprinting technology to embody a three-dimensional artificial tissue structure using cells and biomaterials, and must have both physicochemical properties capable of retaining a fine 3D structure and properties related to biological safety at a level that can be used with living cells.

[0021] Preferably, the fibrous cartilage composite tissue is a meniscus cartilage tissue, and the meniscus cartilage tissue is partitioned into an inner side, an intermediate side, and an outer side according to its anatomical position.

[0022] According to an embodiment of the present invention, in the porcine meniscus cartilage tissue, a tissue with a width of 2-3 mm from the inner side is defined as the inner side, a tissue with a width of 4-6 mm from the outer side is defined as the outer side, and an intermediate site with a width of 7-9 mm between the inner side and the outer side is defined as the intermediate side, but it is not limited thereto.

[0023] In addition to the anatomical position, the meniscus cartilage tissue can be partitioned by differences in tissue physical properties, blood vessel penetration degree, microstructure differences, biochemical component content differences, cell distribution, and cell type differences, but it is not limited thereto.

[0024] The meniscus cartilage tissue can have more cartilage characteristics and less fibrous characteristics from the inner side to the outer side, and may have significant biochemical property differences.

[0025] More specifically, since the inner side has cartilage characteristics, the outer side has fibrous characteristics, and the intermediate side has characteristics where cartilage and fibrous characteristics are converted, the bio-ink composition containing the extracellular matrix derived from the partitioned tissue can have different physicochemical properties depending on the partitioned tissue.

[0026] According to one embodiment of the present invention, in the bioink containing the extracellular matrix derived from the inner tissue of the meniscus cartilage tissue, the expression of type II collagen and aggrecan, which are representative markers of the cartilage-derived extracellular matrix, was relatively strongly expressed compared to the middle and outer sides. However, it was confirmed that in the bioink containing the extracellular matrix derived from the middle and outer tissue, the expression of type I collagen, which is a representative marker of fibrous tissue, was strongly expressed compared to the inner side.

[0027] Further, according to another embodiment of the present invention, it can be confirmed that the inner tissue of the meniscus cartilage tissue has the highest content of sulfated glycosaminoglycan (sGAG), the middle tissue has a high total collagen content, and the outer tissue has the highest elastin content.

[0028] That is, since the tissues partitioned as described above can be selectively and differently selected according to the purpose to adjust the physicochemical properties, the bioink composition according to the present invention can have compartment-specific properties including the extracellular matrix derived from the inner tissue, the middle tissue, the outer tissue, or a combination thereof.

[0029] The bioink composition according to the present invention can promote the migration, adhesion, and proliferation of fibrocartilage cells, and can be differentiated into fibrocartilage composite tissue in vitro or in vivo to induce the formation of artificial tissue.

[0030] In addition, the bioink composition according to the present invention may have angiogenesis induction or anti-angiogenic properties.

[0031] According to an embodiment of the present invention, it has been confirmed that a bioink composition derived from inner or intermediate tissue exhibits anti-angiogenic properties, such as suppressing cell migration and cell adhesion of vascular endothelial cells, and that a bioink composition derived from outer tissue has chemotaxis for vascular endothelial cells, significantly promotes cell adhesion, and cell proliferation occurs in a reticular structure similar to a capillary network, and it can be confirmed that it has pro-angiogenic properties.

[0032] Supply of nutrients and induction of stem cell migration through angiogenesis are very important for meniscus reconstruction. In particular, the synovial membrane adjacent to the outer part of the meniscus is a rich source of blood, nutrients and stem cells, and induction of angiogenesis into the meniscus tissue at the adjacent site can play an important role in the regeneration of the entire meniscus tissue. Therefore, the bioink composition according to the present invention can meet such characteristics, which is desirable.

[0033] That is, the bioink composition according to the present invention has fibrous tissue, cartilage tissue, angiogenesis-inducing or anti-angiogenic properties by compartment, and is useful for the regeneration of fibrocartilage composite tissue using such compartment-specific properties.

[0034] In addition, the bioink composition according to the present invention may have a temperature sensitivity that can be gelled at a temperature of 37°C or higher, preferably at a temperature of 37 - 45°C, while existing in a liquid state at room temperature and can maintain a specific shape.

[0035] As described above, the bioink for 3D printing must have physicochemical properties such as printability extruded from a 3D printer and rheological properties that can maintain a 3D structure even after printing. Since it must serve as a scaffold for cell growth and differentiation, it must have cell affinity and biocompatibility, and also have properties that can regulate cell growth and differentiation in an in vitro culture or in vivo transplantation environment. Therefore, the bioink composition according to the present invention can meet such properties, which is desirable.

[0036] The present invention provides a bone graft composition containing a demineralized bone matrix support.

[0037] As used herein, "demineralized bone matrix (DBM)" is an allogeneic bone graft from which inorganic substances have been removed. When it is transplanted alone, it has problems such as difficulty in maintaining its shape, insufficient durability, and compressive strength.

[0038] The demineralized bone matrix support is composed of cancellous bone, cortical bone, or a combination thereof. Desirably, it can include both cancellous bone and cortical bone, but is not limited thereto.

[0039] The demineralized bone matrix support has a form in which cortical bone and cancellous bone are bonded in a layered structure. The cortical bone and cancellous bone are bonded at an average thickness ratio of 1:(2 - 5), but are not limited thereto.

[0040] According to an embodiment of the present invention, it can be confirmed that the demineralized bone matrix support containing both cancellous bone and cortical bone has a significantly increased compressive strength and tensile strength compared to the demineralized bone matrix support containing only cancellous bone.

[0041] Desirably, the bone graft composition contains the above bioink composition in the demineralized bone matrix support.

[0042] The bioink composition has a concentration of 0.1 to 10 mass / volume (w / v) percent, desirably a concentration of 2 to 5 mass / volume (w / v) percent, but is not limited thereto.

[0043] The decalcified bone matrix support is formed with a porous structure, and the bioink composition is contained in the porous structure of the support and crosslinked into a hydrated gel state by a temperature change.

[0044] Desirably, the bioink composition is 3D printed on the decalcified bone matrix support.

[0045] The bioink composition has different biochemical properties depending on the compartment tissue of the semilunar cartilage tissue and can regulate mechanical physical properties. Therefore, the compartment tissue is selectively regulated according to the purpose and contained and crosslinked in the decalcified bone matrix support.

[0046] According to an embodiment of the present invention, the bioink composition derived from the inner tissue has excellent compressive strength compared to other compartments, so this is selectively used in tissue regeneration that must maintain physical compressive strength biomechanically.

[0047] In addition, the bone graft composition can be utilized for 3D bioprinting and can further improve physicochemical properties through physical or chemical crosslinking.

[0048] The present invention provides a method for manufacturing a bioink for three-dimensional (3D) printing including an extracellular matrix derived from a fibrocartilage composite tissue.

[0049] The method for manufacturing a bioink according to the present invention may include the steps of obtaining and pulverizing a fibrocartilage composite tissue; decellularizing the pulverized tissue to obtain an extracellular matrix and pulverizing it; and subjecting the pulverized extracellular matrix to enzymatic treatment to solubilize it.

[0050] The step of obtaining and pulverizing the fibrous cartilage composite tissue is performed by obtaining the meniscus cartilage tissue. Desirably, it is performed by obtaining and pulverizing the tissue obtained by partitioning the meniscus cartilage tissue into inner, middle, and outer parts according to the anatomical position.

[0051] In addition to the anatomical position, the partitioning of the tissue can be performed according to differences in the physical properties of the tissue, the degree of blood vessel penetration, differences in the microstructure, differences in the content of biochemical components, and differences in cell distribution and types, but is not limited thereto.

[0052] The pulverization is performed by washing each of the obtained tissues or a mixed tissue thereof with distilled water three or more times, and then performing freeze-drying and freeze-crushing.

[0053] The step of decellularizing the pulverized tissue to obtain the extracellular matrix and pulverizing. In the decellularization process, cells and dielectric substances present in the tissue are removed to obtain a pure extracellular matrix. First, the previously pulverized tissue is placed in a hypotonic solution and treated at room temperature for 3 to 5 hours, and then placed in a surfactant solution and treated for 1 to 3 hours. In order to remove the surfactant, it is washed with distilled water, and then, in order to remove the dielectric substances present in the extracellular matrix, it is treated with a DNA degrading enzyme solution for 10 to 15 hours and washed, whereby decellularization can be completed.

[0054] The step of enzymatically treating and solubilizing the pulverized extracellular matrix can solubilize the pulverized extracellular matrix through enzymatic treatment with an enzyme such as pepsin in an acidic solution. After neutralizing it again, it is diluted with a buffer solution to adjust the concentration of physiological ions. The solubilized extracellular matrix can be produced at 0.1 to 10% (w / v), and desirably, it can be produced at 2 to 5% (w / v), but is not limited thereto.

[0055] The bioink produced by the above manufacturing method can have a temperature sensitivity that allows it to gel at a temperature of 37°C or higher while existing in a liquid state at room temperature and retain a specific shape.

[0056] The present invention provides a method for manufacturing a bone graft material including a decalcified bone matrix support containing the above-described bioink composition.

[0057] The method for manufacturing a bone graft material according to the present invention may include the steps of manufacturing a decalcified bone matrix support; manufacturing a bioink containing an extracellular matrix derived from a fibrocartilage composite tissue; and injecting and crosslinking the manufactured bioink into the manufactured decalcified bone matrix support.

[0058] The step of manufacturing the decalcified bone matrix support may include the steps of obtaining a bone-cartilage composite tissue and removing inorganic substances from the composite tissue; and removing cartilage tissue from the composite tissue from which the inorganic substances have been removed.

[0059] The step of removing the inorganic substances is performed by treating with an acidic solution to remove minerals or calcium, etc.

[0060] After removing the cartilage tissue, the step of washing for removing cells and foreign substances may further be included.

[0061] The decalcified bone matrix support manufactured by the above method is composed of cancellous bone, compact bone, or a combination thereof, and preferably may include both cancellous bone and compact bone, but is not limited thereto.

[0062] The step of manufacturing the bioink may be replaced by the method for manufacturing a bioink for 3D printing described above.

[0063] The step of injecting and crosslinking the manufactured bioink into the manufactured decalcified bone matrix support is performed by injecting the bioink into the porous structure inside the decalcified bone matrix support and then crosslinking it into a hydrated gel state at a temperature of preferably 37°C or higher due to a temperature change. Or, it is performed by 3D printing the bioink onto the manufactured decalcified bone matrix support.

[0064] The method for manufacturing the bone graft material may further include a step of additionally crosslinking the bioink crosslinked in the porous structure inside the demineralized bone matrix support by one or more physical or chemical crosslinking methods.

[0065] The physical crosslinking method is performed by irradiating with gamma rays, and the chemical crosslinking method is performed by using a crosslinking agent such as glutaraldehyde.

[0066] According to an embodiment of the present invention, it can be confirmed that as the concentration of the crosslinking agent increases or as the irradiation dose of gamma rays increases, the compressive strength of the demineralized bone matrix support crosslinked with the bioink increases significantly.

[0067] Also, it can be confirmed that as the concentration of the bioink injected into the demineralized bone matrix support increases, the compressive strength of the support increases significantly, and the porosity and pore size are shown to decrease.

[0068] That is, with the manufacturing method according to the present invention, it is possible to adjust physicochemical properties such as mechanical properties, porosity, and functionality according to the purpose, and by such property adjustment, it is possible to manufacture a bone graft material whose cell seeding efficiency can also be adjusted.

[0069] The present invention also provides a method for manufacturing an artificial tissue by three-dimensionally printing a bioink composition for 3D printing containing the extracellular matrix derived from the fibrous cartilage composite tissue.

[0070] More specifically, the three-dimensional printing is performed at a pressure of less than 80 kPa and a temperature of 37°C at a speed of 1 to 2 mm / sec using a nozzle having an average diameter of 300 to 600 μm.

[0071] The present invention provides an artificial tissue obtained by three-dimensionally printing the bioink composition.

[0072] The artificial tissue described above may have the characteristics of the above-mentioned fibrous cartilage composite tissue.

[0073] The artificial tissue is an artificial meniscus tissue, but is not limited thereto.

[0074] Hereinafter, in order to facilitate the understanding of the present invention, examples will be given and described in detail. However, the following examples illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more fully explain the present invention to those skilled in the art.

[0075] <Example 1> Production of extracellular matrix (DMECM) bioink derived from compartment-specific temperature-sensitive meniscus Porcine meniscus tissue has different tissue characteristics in the inner, middle and outer regions as a fibrous cartilage composite tissue ((A) in FIG. 1).

[0076] Referring to (B) of FIG. 1, in order to produce extracellular matrix (DMECM) bioink derived from compartment-specific temperature-sensitive meniscus, meniscus tissue was harvested from the knees of pigs using a surgical blade and saw first. Subsequently, based on the anatomical characteristics of the meniscus, a tissue with a width of 2-3 mm from the inner side of the meniscus was defined as the inner zone, a tissue with a width of 4-6 mm from the outer side was defined as the outer zone, and an intermediate site with a width of 7-9 mm between the inner and outer zones was defined as the mid zone.

[0077] The compartment-specific meniscus cartilage tissue was washed three times with distilled water and then obtained in powder form through freeze-drying and freeze-crushing. The obtained meniscus cartilage tissue powder was placed in a hypotonic solution and treated at room temperature for 4 hours, and then treated with a surfactant solution containing SDS (sodium dodecyl sulfate) for 2 hours to promote decellularization. Thereafter, it was washed six times with distilled water to remove SDS. Finally, to remove the dielectric substances present in the tissue extracellular matrix powder, it was treated with a solution containing DNAase (DNAase) for 12 hours. Thereafter, it was additionally washed six times with distilled water to complete the decellularization process. Subsequently, the powdered extracellular matrix was solubilized through pepsin treatment with a 0.1M HCl solution, the pH was neutralized with NaOH, and high-concentration phosphate buffered saline (PBS) was appropriately diluted to adjust the concentration of physiological ions.

[0078] Finally, the meniscus cartilage-derived extracellular matrix bioink (DMECM-bioink) produced showed the characteristics of a temperature-sensitive hydrated gel that gels at a temperature of 37°C or higher and 45°C or lower while showing liquid properties at room temperature (25°C or lower), as shown in (B) of Figure 1.

[0079] <Example 2> Analysis of the biochemical properties of compartment-specific temperature-sensitive DMECM bioink 2-1. Analysis of the contents of dsDNA, collagen, sGAG, and elastin in the compartment-specific DMECM-bioink The dsDNA content of the inner, middle, and outer DMECM-bioinks was analyzed through the Picogreen assay. As a result, as shown in (A) of Figure 2, all bioinks showed a dsDNA value of 50 ng or less per mg. Through this, it can be confirmed that decellularization was successfully advanced in the above three types of tissues.

[0080] (B) to (D) of Fig. 2 are the results of quantitatively analyzing the contents of total collagen, sulfated glycosaminoglycan (sGAG), and elastin in the compartment-specific DMECM-bioink through biochemical analysis. Referring to it, the collagen content was shown to be significantly higher in the Mid group compared to the Inner and Outer groups (Mid: 935.02 ± 21.3 μg / mg, Inner: 821.01 ± 23.8 μg / mg, Outer: 786.05 ± 26 μg / mg). The sGAG content was the highest in the Inner group compared to the other groups, and then the sGAG quantification values decreased in the order of the Mid and Outer group DMECM-bioink (Inner: 33.4 ± 4 μg / mg, Mid: 20.9 ± 1.3 μg / mg, Outer: 15.9 ± 1.2 μg / mg). In the case of elastin, it was confirmed that the content increased from the inner tissue to the outer tissue (Inner: 19.1 ± 0.8 μg / mg, Mid: 24.2 ± 1.01 μg / mg, Outer: 27.7 ± 1.5 μg / mg).

[0081] 2-2. Protein analysis of the compartment-specific DMECM-bioink To analyze the differences in the protein cargo profiles of the compartment-specific DMECM-bioink, SDS-PAGE analysis was carried out. As a result, as shown in (E) of Fig. 2, it was confirmed that the position of the collagen chain band in the inner group was higher compared to the other groups.

[0082] Subsequently, as a result of comparative analysis of the relative expression patterns of type I collagen, type II collagen, and aggrecan contained in the compartment-specific DMECM-bioink through western blot, as shown in (F) of Figure 2, it was confirmed that Inner DMECM-bioink relatively strongly expressed type II collagen and aggrecan, which are representative markers of cartilage-derived extracellular matrix, compared to Mid and Outer DMECM-bioink. On the other hand, in the case of Mid and Outer DMECM-bioink, the expression of type I collagen, which is a representative marker of fibrous tissue, was stronger than that of Inner DMECM-bioink.

[0083] Through this, it can be confirmed that the Inner, Mid, and Outer groups of DMECM-bioink separated by the anatomical position of the meniscus cartilage show significant differences in biochemical properties.

[0084] <Example 3> Analysis of the Proteomic Properties of Compartment-Specific Temperature-Sensitive DMECM Bioink Table 1 below shows the results of proteomics analysis of the compartment-specific temperature-sensitive DMECM bioink, representing the relative expression levels of the proteins present in Inner, Mid, and Outer DMECM-bioink numerically.

[0085]

Table 1

[0086] Referring to Table 1 above, it can be confirmed that in protein analysis through Proteomics, the expressions of type II collagen (COL2A1), aggrecan (ACAN), tenascin (TNC), Cartilage intermediate layer protein 1 (CLIP), etc., which represent the extracellular matrix (ECM) of chondrocytes, are dominantly expressed in Inner DMECM-bioink compared to Mid and Outer DMECM-bioink.

[0087] On the other hand, proteins such as type I collagen (COL1A2), vimentin (VIM), annexin A2 (ANXA2), tubulin alpha chain (TUBA1C), etc., showed dominant expression in Mid and Outer DMECM-bioink compared to Inner DMECM-bioink.

[0088] <Example 4> 3D Printing of Temperature-Sensitive DMECM Bioink To examine the rheological properties of temperature-sensitive DMECM-bioink due to temperature changes, the shear rate was maintained at 1 s -1 and the temperature of the analytical instrument was changed from 4°C to 45°C to measure the gelation kinetics.

[0089] As a result, as shown in Figure 3, it was confirmed that the elastic modulus of DMECM-bioink increased significantly at temperatures above 20°C due to the thermal gelation of DMECM-bioink rich in collagen (Figure 3(A)). It was confirmed that DMECM-bioink has temperature sensitivity and can be freely administered in the desired amount at room temperature and can maintain a specific shape at 37°C (Figure 3(B)).

[0090] To fabricate a structure of a desired shape by utilizing the temperature-responsive DMECM-bioink in a 3D printer, the printing conditions were set as follows. Considering the temperature responsiveness, viscosity, and printability of the DMECM-bioink, the size of the nozzle was 300 to 600 μm or less, an air pressure of less than 80 kPa and a printing speed of 1 mm / sec were used, and the temperature of the lower plate of the 3D printer was set at 37°C.

[0091] As a result, as shown in the right figure of (C) in Fig. 3, it was confirmed that not only a fine cross-shaped structure with a size of 500 μm but also a structure with a size of 1 cm or more and a complex cartilage-shaped structure could be fabricated.

[0092] <Example 5> Cell Affinity of Compartment-Specific Temperature-Responsive DMECM Bioink To evaluate the difference in the effect of the compartment-specific temperature-responsive DMECM-bioink on cell behavior, analyses regarding cell proliferation and survival migration adhesion were performed.

[0093] 5-1. Analysis of the migration ability of fiber-chondrocytes in the compartment-specific temperature-responsive DMECM-bioink To confirm whether the compartment-specific temperature-responsive DMECM-bioink is biochemically chemotactic and promotes the migration of cells derived from the lunar cartilage (lunar cells), a Boyden chamber assay was performed.

[0094] As a result, as shown in (A) of Fig. 4, it was confirmed that all DMECM-bioinks significantly promoted the migration of lunar cells compared to the control group (cell culture medium: 100%). In particular, Inner DMECM-bioink showed the most significant chemotaxis for lunar cells among all groups, followed by Mid and Outer DMECM-bioinks in that order.

[0095] 5-2. Analysis of the adhesion ability of fiber-chondrocytes in the compartment-specific temperature-responsive DMECM-bioink For the evaluation of cell adhesion, 5% DMECM-bioink was dispensed into a culture dish and incubated at 37 °C for 30 minutes to coat the surface of the culture dish. Subsequently, the meniscus cells were inoculated, and after 2 hours, the culture dish was washed twice with PBS, and the cells attached to the bioink surface were stained with Calcein-AM and the absorbance was measured.

[0096] As a result, as shown in (B) of Figure 4, it was confirmed that all DMECM-bioink treatment groups significantly promoted cell adhesion compared to the control group (culture dish: 100%), and in particular, the Inner DMECM-bioink group showed the highest cell adhesion rate.

[0097] 5-3. Analysis of the proliferation ability of fiber-chondrocytes in the compartment-specific temperature-responsive DMECM-bioink To confirm the cell proliferation ability of the compartment-specific DMECM-bioink on meniscus cells, 1×10 4 meniscus cells per 200 μl of 5% DMECM-bioink were suspended and then inoculated into a 96-well plate and cultured at 37 °C with cell culture medium, and the cell proliferation on days 1, 4, 7, and 10 was analyzed by WST assay.

[0098] As a result, as shown in (C) of Figure 4, it was confirmed that cell proliferation increased in all DMECM-bioink treatment groups compared to the untreated control group.

[0099] In addition, to analyze the long-term cell affinity of the compartment-specific DMECM-bioink for meniscus cells, a LIVE & DEAD assay was performed 4 weeks after cell culture.

[0100] As a result, as shown in (D) of Figure 4, it was confirmed that the cells survived (Green) inside and on the surface of the (Red) bioink without the death of meniscus cells.

[0101] <Example 6> Inductive Characteristics of Compartment-Specific Temperature-Sensitive DMECM Bioink for In Vitro Fibrocartilage Differentiation RT-PCR analysis was performed to evaluate how zonal-specific DMECM-bioink treatment affects the differentiation of human synovium-derived mesenchymal stem cells (hMSCs).

[0102] As a result, as shown in Fig. 5, it was confirmed that the expressions of collagen type II (COL2), aggrecan (ACAN), and SOX9, which are known as representative markers of cartilage tissue, were relatively higher in the Inner DMECM-bioink treatment group compared to the Mid and Outer DMECM-bioink groups. On the other hand, it was confirmed that the expression of collagen type I (COL1) was the lowest in the Inner DMECM-bioink group and increased in the order of Mid and Outer DMECM-bioink.

[0103] <Example 7> Evaluation of in vivo fibrocartilage differentiation ability of zonal-specific temperature-responsive DMECM bioink 7-1. Histological evaluation of the artificial tissue formed subcutaneously in nude mice

[0104] To evaluate the in vivo artificial tissue formation ability, hMSCs and 2% zonal-specific DMECM-bioink were suspended and injected subcutaneously into nude mice at 1×10 6 / 100 μl each. Four weeks after subcutaneous injection, macroscopic observation and histological evaluation were performed to sacrifice the nude mice and evaluate the degree of tissue formation and differentiation.

[0105] As a result, as shown in Fig. 6(A), it was confirmed that a single homogeneous artificial tissue was formed at the position where cell-zonal DMECM-bioink was injected. Also, as a result of H&E staining, it was confirmed that the cytoplasm was homogeneously formed and the cell distribution was also made homogeneous. In addition, it was confirmed that the safranin-O staining pattern and the expression pattern of collagen type, which are similar to the anatomical position of extracellular matrix-derived fibrocartilage, were also represented in the artificial tissue.

[0106] 7-2. Confirmation of the component content of the artificial tissue formed subcutaneously in nude mice To confirm the component content of the artificial tissue formed in vivo by hMSC and compartment-specific DMECM-bioink injection, the analysis of collagen and sGAG was performed.

[0107] As a result, as shown in (B) of Fig. 6, the collagen content was measured to be the highest in the Outer DMECM-bioink group and the lowest in the Inner DMECM-bioink group (Inner: 225.8 ± 17.4 μg / mg, Mid: 280.8 ± 20.6 μg / mg, Outer: 300.8 ± 12.2 μg / mg).

[0108] On the other hand, in the case of sGAG, the highest value was shown in the Inner DMECM-bioink group, and no significant difference was shown between Mid and Outer DMECM-bioink (Inner: 12.5 ± 1.94 μg / mg, Mid: 7.6 ± 1.2 μg / mg, Outer: 7.8 ± 0.9 μg / mg).

[0109] <Example 8> Evaluation of in vitro angiogenesis induction of compartment-specific temperature-responsive DMECM bioink To evaluate the difference of compartment-specific DMECM-bioink on the induction of neovascularization, the analysis related to the migration, adhesion, proliferation and angiogenesis of vascular endothelial cells (Human umbilical vein endothelial cells: HUVEC) was performed.

[0110] 8-1. Analysis of the migration ability of vascular endothelial cells in the compartment-specific DMECM-bioink To confirm whether the compartment-specific DMECM-bioink is biochemically chemotactic and promotes or inhibits the migration of vascular endothelial cells (HUVEC), the Boyden chamber analysis was performed.

[0111] As a result, as shown in (A) and (B) of Fig. 7, it can be confirmed that Inner and Mid DMECM-bioink significantly inhibit the migration of vascular endothelial cells compared with the control group (cell culture medium containing 10% FBS: 100%) (compared with the control group, Inner: decreased by about 39.2%, Mid: decreased by about 26%).

[0112] On the one hand, among all the groups, Outer DMECM-bioink showed the most significant chemotaxis towards vascular endothelial cells (an increase of 39.3% for Outer compared to the control group).

[0113] 8-2. Analysis of the adhesion ability of vascular endothelial cells in the compartment-specific DMECM-bioink For the evaluation of cell adhesion, 5% DMECM-bioink was dispensed into culture dishes and incubated at 37 °C for 30 minutes to coat the surface of the culture dishes. Subsequently, vascular endothelial cells were inoculated, and after 2 hours, the culture dishes were washed twice with PBS, and the cells attached to the bioink surface were stained with Calcein-AM to measure the absorbance.

[0114] As a result, as shown in (C) and (D) of Figure 7, the Inner and Mid DMECM-bioink treatment groups significantly suppressed cell adhesion compared to the control group (culture dish: 100%), and it was confirmed that Outer DMECM-bioink significantly promoted the adhesion of vascular endothelial cells (Inner: 53.8 ± 7.02%, Mid: 74.8 ± 7.9%, Outer: 137.3 ± 12.9%).

[0115] 8-3. Analysis of the proliferation ability of vascular endothelial cells in the compartment-specific temperature-responsive DMECM-bioink To confirm the cell proliferation ability of compartment-specific DMECM-bioink on vascular endothelial cells, 200 μl of 5% DMECM-bioink was coated on a 96-well plate and incubated at 37 °C. Then, 1×10 4 vascular endothelial cells per well were inoculated together with cell culture medium, and the cell proliferation on days 1, 3, and 7 was analyzed by WST assay, and the cell morphology on day 7 was stained with Calcein-AM.

[0116] As a result, as shown in (E) of Fig. 7, it was observed that the control group without any treatment and the Inner and Mid DMECM-bioink treatment groups were attached and cultured in a circular or oval form on the cell culture dish and the surface of each DMECM-bioink. On the other hand, the morphology of vascular endothelial cells in the Outer DMECM-bioink treatment group was characterized by being relatively elongated compared to other groups, and it was observed that they were arranged in a network structure similar to a capillary network.

[0117] In the analysis of cell proliferation through the WST assay, as shown in (F) of Fig. 7, significant proliferation of vascular endothelial cells was observed in the control group and the Outer DMECM-bioink treatment group over the culture period. On the other hand, in the case of the Inner and Mid DMECM-bioink treatment groups, the WST absorbance for early vascular endothelial cells was maintained during the culture period, and no significant change in cell proliferation was observed.

[0118] 8-4. Analysis of the ability to induce in vitro angiogenesis of vascular endothelial cells in the compartment-specific temperature-responsive DMECM-bioink To confirm the in vitro angiogenesis-inducing ability of compartment-specific DMECM-bioink on vascular endothelial cells, Matrigel and 5% DMECM-bioink were suspended at a ratio of 9:1, and then 100 μl of each was coated on a 96-well plate and incubated at 37°C. Thereafter, 4×10 4 vascular endothelial cells per well were inoculated together with the cell culture medium, and after 24 hours, the tube formation of vascular endothelial cells was stained with Calcein-AM and observed under a microscope. The newly formed vascular network generated in vitro was quantitatively analyzed for the total length of the newly formed blood vessels and the number of branch points through image analysis software (ImageJ) to evaluate the degree of promotion or inhibition of angiogenesis.

[0119] As a result, as shown in (G) to (I) of FIG. 7, it was confirmed that a tube structure similar to the vascular network structure was formed in the control group treated with only Matrigel. On the other hand, in the case of the Outer DMECM-bioink treatment group, it was confirmed that the total tube length and the number of branch points were significantly increased compared to the control group. On the other hand, in the case of Inner and Mid DMECM-bioink, it was observed that the tube structure was not correctly formed compared to the control group, and it was confirmed that the branch points and the tube length were significantly decreased compared to the control group.

[0120] Therefore, Inner and Mid DMECM-bioink exhibit anti-angiogenic properties, Outer DMECM-bioink exhibits pro-angiogenic properties, and it was confirmed that the DMECM-bioink has the potential to regulate the promotion or suppression of angiogenesis at the desired transplantation site depending on the compartment of the tissue from which it originated.

[0121] <Example 9> Production of Demineralized Bone Matrix (DBM) Support Demineralized bone matrix (DBM) tissue can be obtained from animal tissues including humans, and preferably, it is separated from bone-cartilage tissue obtained from cadavers, patients who have undergone joint replacement surgery, or pigs. The obtained bone-cartilage tissue was treated with a 5% nitric acid solution at 4°C for 48 hours for the removal of minerals and calcium. The nitric acid solution was replaced every 24 hours. After the removal of minerals and calcium, the cartilage tissue part was removed from the bone-cartilage tissue using a surgical blade, and the separated bone tissue was washed with a 15% triton-x 100 solution at 4°C for 72 hours for the removal of cells and foreign substances. The Triton-x 100 solution was replaced every 24 hours. After washing, it was stirred with tertiary water twice for 24 hours each, for a total of 48 hours, for the removal of residual Triton-x 100 (FIG. 8).

[0122] The demineralized and decellularized DBM can be cut and shaped according to the desired form and size. When fabricating a DBM support with a three-dimensional curved surface, the wet DBM can be fixed to a curved mold and then freeze-dried to fabricate the shape into the desired form.

[0123] In addition, the bone matrix has a structure in which spongy bone and compact bone are combined. Spongy bone has a high porosity, excellent tensile strength and elasticity, and is easy to form into a support. In the case of compact bone, it has relatively low porosity, is hard, and has good physical strength. Depending on the intended use of the support, either only spongy bone can be selectively used, or both spongy bone and compact bone can be used.

[0124] <Example 10> Manufacture of a Demineralized Bone Matrix (DBM) Support Loaded with a Compartment-Specific Temperature-Sensitive DMECM Bioink Referring to FIG. 9, the DMECM-bioink 3D printed on the surface of the DBM support penetrates into the porous structure of the DBM support and is then crosslinked into a hydrated gel state by a lower temperature control device set at 37°C. The concentration of the DMECM-bioink used for the bioprinting is 0.1 to 10% (w / v), more preferably 2 to 5%, but is not limited thereto. The DBM used for the bioprinting is spongy bone or spongy bone containing a layer of compact bone. The DMECM-bioink crosslinked in the internal porous structure of the DBM support at a temperature of 37°C or higher can improve the physicochemical properties of the support through additional physical or chemical crosslinking methods.

[0125] <Example 11> Microstructure and Biochemical Properties of a DBM Support Loaded with a Compartment-Specific Temperature-Sensitive DMECM Bioink To analyze the structure of the DBM support by printing with compartment-specific DMECM-bioink, the DBM-DMECM support was fabricated under the condition of manufacturing and printing each compartment-specific DMECM-bioink at 5% (w / v) on a cylindrical DBM support with a diameter of 5 mm and a height of 2 mm.

[0126] As a result of the image analysis, as shown in Fig. 10(A), it was confirmed that the compartment-specific DMECM-bioink was effectively loaded into the porous structure of the DBM support by the said process technology.

[0127] In order to analyze the difference in the component content of the DBM support loaded with the compartment-specific DMECM-bioink, the content analysis of collagen, sulfated glycosaminoglycan, and elastin was carried out. Collagen was measured using S1000 (Biocolor, UK), sGAG was measured using B1000 (Biocolor, UK), and elastin was measured using Fastin assay (Biocolor, UK).

[0128] As a result, as shown in Fig. 10(B), in the case of collagen, it showed the highest proportion when loaded with Mid DMECM-bioink, for sGAG, it occupied the highest proportion when loaded with Inner DMECM-bioink, and for elastin, it showed the highest proportion when loaded with Outer DMECM-bioink. By confirming this, it was confirmed that the biochemical composition of the finally produced DBM support can be adjusted according to the type of the selected compartment-specific DMECM-bioink.

[0129] <Example 12> Physicochemical properties of the DBM support loaded with the compartment-specific temperature-responsive DMECM bioink depending on the crosslinking conditions The DBM support loaded with the compartment-specific DMECM-bioink fabricated according to an embodiment of the present invention can improve its physicochemical properties through additional physical or chemical crosslinking methods. Representative physical crosslinking methods include, but are not limited to, irradiation with ionization energy such as gamma rays or electron beams. As chemical crosslinking methods, glutaraldehyde or EDC / NHS are mainly used, but it is not limited thereto.

[0130] In this example, glutaraldehyde was used as a typical example of the chemical crosslinking method to additionally crosslink the DMECM-DBM support fabricated above. The crosslinking method was to add the DMECM-DBM support to a 0.01 - 1% (w / v) glutaraldehyde solution, and then stir at 100 rpm for about 1 hour at room temperature. The chemically crosslinked DMECM-DBM support was washed three times repeatedly with PBS solution for 30 minutes each time, and then continuously washed three times repeatedly with triple distilled water.

[0131] On the other hand, the gamma-ray irradiation method was used as a typical example of the physical crosslinking method to additionally crosslink the DMECM-DBM support fabricated above. The crosslinking method was to put the wet DMECM-DBM support after the printing was completed into an EP tube, wrap it with silver foil, and then perform gamma-ray irradiation at intensities of 10, 25, and 50 kGy respectively. In order to analyze the changes in the physicochemical properties of the DMECM-DBM support due to the additional crosslinking process, the compressive strength was analyzed through a universal physical property measuring instrument. As a result of the analysis, as shown in (A) of FIG. 11, it was confirmed that the compressive strength increased significantly with the increase in the concentration of the crosslinking agent and the gamma-ray irradiation dose.

[0132] Next, the changes in the mechanical properties of the DBM-support due to the concentration of the printed DMECM-bioink were observed. Referring to (B) of FIG. 11, it can be confirmed that the compressive strength of the DBM support increased significantly with the increase in the concentration of the DMECM-bioink in both the glutaraldehyde and gamma-ray irradiation crosslinking methods.

[0133] Subsequently, as a result of analyzing the porosity and pore size changes of the DBM-support according to the concentration of the DMECM-bioink, as shown in (C) of FIG. 11 and (D) of FIG. 11, it was observed that the porosity and pore size decreased as the concentration of the DMECM-bioink increased. Therefore, it was confirmed that the above manufacturing technique enables the adjustment of the production of the DBM support according to the desired mechanical properties and pore characteristics.

[0134] Specifically, when the bioinks manufactured at 1, 2, and 5% (w / v) were injected into the demineralized bone support, the porosities were 93.7 ± 2.07%, 90.1 ± 2.1%, and 84.6 ± 5.7% respectively, and the average diameters of the pores were represented as approximately 295.1 ± 97.7 μm, approximately 137.9 ± 27.6 μm, and approximately 111.2 ± 18.5 μm respectively.

[0135] It was reported that the porosity and the pore size affected the water content (Swelling ratio) of the support and the cell seeding efficiency (Cell seeding ratio) (Figs. 11(E) and 11(F)).

[0136] Specifically, when the bioinks manufactured at 1, 2, and 5% (w / v) were injected into the demineralized bone support, the water contents were 437.17 ± 65.8, 362.4 ± 59.7, and 247.5 ± 44.1 respectively, and the cell seeding efficiencies of the support were represented as approximately 74.9 ± 6.6, 86.3 ± 8.3, and 80.9 ± 7.4 respectively.

[0137] <Example 13> Biomechanical properties due to the dense bone content of the DBM support The DBM support according to an embodiment of the present invention can use cancellous bone or cancellous bone containing cortical bone. Cancellous bone is observed from the central part of the bone or flat bone, has relatively high porosity and wide pore size compared to cortical bone, is excellent in elasticity and resilience, and is easy to process and form as a biological support. On the other hand, cortical bone is observed from the peripheral surface of cancellous bone, is denser than cancellous bone, and is mechanically stronger. The DMECM-DBM support produced from the above can be applied to both cancellous bone and DBM composed of cancellous bone + cortical bone.

[0138] Referring to Fig. 12, the difference in physical properties due to the structure of the DBM support can be confirmed. The DBM support containing cortical bone and cancellous bone in a ratio of 1:3 had significantly increased compressive strength and tensile strength compared to the DBM support containing only cancellous bone (Figs. 12(B) and 12(C)).

[0139] Therefore, it can be confirmed that the manufacturing technique according to the present invention can selectively use the presence or absence of the spongy bone or compact bone layer of DBM according to the desired mechanical properties and porous characteristics to adjust the production of the DMECM-DBM support.

[0140] <Example 14> Biomechanical properties of a DBM support loaded with compartment-specific temperature-responsive DMECM bioink The DMECM-DBM support according to an embodiment of the present invention can adjust the physical strength characteristics according to the type of compartment-specific DMECM-bioink.

[0141] Referring to FIG. 13, the compressive strength of all DMECM-bioink treatment groups increased compared to the DBM only support. In particular, the DBM support loaded with Inner DMECM-bioink showed the best compressive strength among all groups.

[0142] Therefore, it can be confirmed that the manufacturing technique according to the present invention can selectively use DMECM-bioink according to the desired mechanical properties to adjust the production of the DMECM-DBM support.

[0143] <Example 15> Cell affinity of a DBM support loaded with compartment-specific temperature-responsive DMECM bioink To evaluate the difference in the effect of the compartment-specific DMECM-DBM support on cell behavior, analysis related to cell proliferation and survival migration was performed.

[0144] 15-1. Analysis of the migration ability of fiber-chondrocytes in the compartment-specific DMECM-DBM support To confirm whether the compartment-specific DMECM-DBM support is biochemically chemotactic and promotes the migration of cells derived from the meniscus cartilage (meniscus cells), Boyden chamber analysis was performed.

[0145] As a result, as shown in Fig. 14(A), it can be confirmed that all DMECM-DBM supports significantly promote the migration of the lunate cells compared to the control group (cell culture medium: 100%). In particular, the Inner DMECM-DBM support showed the most significant chemotaxis to the lunate cells among all groups, followed by the Mid and Outer DMECM-DBM supports in that order.

[0146] 15-2. Analysis of the seeding rate of fiber-chondrocytes in the compartment-specific DMECM-DBM support To evaluate the cell adhesion efficiency of the compartment-specific DMECM-DBM supports, the seeding rate was analyzed.

[0147] After inoculating 1×10 6 cells onto the DMECM-DBM supports and culturing them at 37°C for 2 hours on the culture dish, the number of cells that did not adhere to the inside of the support and escaped onto the culture dish was counted, and the number of cells that adhered to the support among all the cells was calculated.

[0148] As a result, as shown in Fig. 14(B), it was confirmed that all DMECM-DBM support treatment groups significantly promoted cell adhesion compared to the control group (DBM only scaffold), and in particular, the Inner DMECM-DBM support group showed the highest cell seeding rate.

[0149] 15-3. Analysis of the proliferation ability of fiber-chondrocytes in the compartment-specific temperature-responsive DMECM-bioink To confirm the cell proliferation ability of the compartment-specific DMECM-DBM supports against the lunate cells, 1×10 6 lunate cells were inoculated per support, and then cultured with the cell culture medium at 37°C, and the cell proliferation on days 1, 3, and 7 was analyzed by the WST assay.

[0150] As a result, as shown in Fig. 14 (C), it was confirmed that cell proliferation increased in the DMECM-DBM support group of all groups compared to the DBM only group. In particular, it was observed that the Outer DMECM-DBM support group significantly increased cell proliferation more than any other group.

[0151] In addition, to analyze the long-term cell affinity of the compartment-specific DMECM-DBM support for the meniscus cells, a LIVE & DEAD assay was performed 4 weeks after cell culture.

[0152] As a result, as shown in Fig. 14 (D), it was confirmed that cells were surviving (Green) inside the porous interior of the (Red) DBM scaffold without meniscus cell death.

[0153] <Example 16> Inductive characteristics of in vitro fibrocartilage differentiation of a DBM support loaded with compartment-specific temperature-sensitive DMECM bioink RT-PCR analysis was performed to evaluate the effect of culturing in the compartment-specific DMECM-DBM scaffold on the differentiation of human synovium-derived mesenchymal stem cells (hMSCs).

[0154] As a result, as shown in Fig. 15, it was observed that the compartment-specific DMECM-DBM support of all groups significantly increased the gene expression of cartilage and fibrotic differentiation markers compared to the DBM only support group. In particular, it was confirmed that the Inner DMECM-DBM support significantly improved the gene expression of cartilage differentiation markers, type II collagen (COL2), aggrecan (ACAN), and SOX9.

[0155] On the one hand, in the case of type I collagen (COL1), which is a fibrotic differentiation marker, the highest gene expression was observed in the Outer DMECM-DBM support. In the case of the Mid DMECM-DBM support, the expression levels of the fibrotic and chondrogenic differentiation marker genes were observed at intermediate levels between the Inner and Outer DMECM-DBM supports.

[0156] <Example 17> Evaluation of the in vivo fibrochondrogenic differentiation ability of a DBM support loaded with a compartment-specific temperature-responsive DMECM bioink 17-1. Histological evaluation of the compartment-specific DMECM-DBM support transplanted subcutaneously in nude mice To evaluate the in vivo artificial tissue formation ability, hMSCs were seeded onto the compartment-specific DMECM-DBM supports and injected subcutaneously into nude mice (1×10 6 / support). Four weeks after subcutaneous transplantation, in order to sacrifice the nude mice and evaluate the degree of tissue formation and differentiation, histological evaluation was performed through Alcian blue staining.

[0157] As a result, as shown in (A) of FIG. 16, it was confirmed that the cytoplasm was homogeneously formed inside the porous structure of the DBM support and the cell distribution was also made homogeneous. In particular, it was judged that differentiation into cartilage tissue occurred because the strongest sGAG expression was observed in the Inner DMECM-DBM support group.

[0158] 17-2. Confirmation of the component content of the compartment-specific DMECM-DBM support transplanted subcutaneously in nude mice To confirm the component content of the artificial tissue formed in vivo by transplantation of hMSCs and the compartment-specific DMECM-DBM support, analysis of collagen and sGAG was performed.

[0159] As a result, as shown in (B) and (C) of FIG. 16, it was confirmed that all DMECM-DBM support groups significantly improved the collagen and sGAG content compared to the DBM only support. The collagen content was higher in the Mid and Outer DMECM-DBM scaffold groups compared to the Inner group (DBM only: 9.5±2.7, Inner: 46.9±9.3, Mid: 55.5±12.5, Outer: 55.4±8.3 μg / mg).

[0160] On the other hand, in the case of sGAG, the Inner group showed the highest sGAG content, followed by the Mid and Outer groups in that order (DBM only: 4.5±1.8, Inner: 30.6±5.8, Mid: 27.6±4.6, Outer: 18.9±3.9 μg / mg).

[0161] Therefore, it is judged that the Inner DMECM-DBM support shows chondrogenic tissue differentiation characteristics, the Outer DMECM-DBM support shows fibrous tissue differentiation characteristics, and the Mid DMECM-DBM support shows the differentiation ability of the fibrous-cartilage intermediate of the two supports.

[0162] <Example 18> Manufacture of an artificial semilunar cartilage support using the manufacturing technology of a DBM support loaded with a compartment-specific temperature-sensitive DMECM bioink The temperature-sensitive DMECM-bioink printing technology inside the porous DBM support enables the reproduction of the compartment specificity of the semilunar cartilage. In the present invention, after binding three other fluorescent dyes to Inner (Blue), Mid (Red), and Outer (Green) DMECM-bioink respectively, 3D printing was performed on a DBM support fabricated in the shape of a semilunar cartilage, and fluorescence imaging analysis and internal microstructure analysis were carried out.

[0163] As a result, as shown in FIG. 17, it was confirmed that the compartment-specific DMECM-bioink was well loaded inside the DBM support in the shape of a semilunar cartilage.

[0164] Overall, the DBM support loaded with bioink derived from compartment-specific meniscus cartilage can have its physicochemical properties adjusted by the structure of the DBM support and the type, concentration of DMECM-bioink, and additional crosslinking processes. It was confirmed that the DMECM-DBM support can not only act as a chemoattractant that draws in cells, but also promote cell adhesion and proliferation, and induce compartment-specific differentiation into fibrous-cartilage tissue. It was confirmed that the said technology can mimic not only the compartment-specific biochemical properties where meniscus cartilage tissue can be seen, but also the fibrous-cartilage differentiation specificity, and can be utilized for the reconstruction of various fibrous-cartilage tissues including meniscus cartilage.

[0165] As described in detail above for specific parts of the content of the present invention, it is clear to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention thereby. That is, the substantial scope of the present invention is defined by the claims and their equivalents.

Claims

1. It contains an extracellular matrix derived from a fibrous cartilage composite tissue, wherein the fibrous cartilage composite tissue is one or more selected from tissues partitioned into an inner side, an intermediate side, and an outer side of the meniscus cartilage tissue according to an anatomical position, the inner side has cartilage characteristics, the outer side has fibrous characteristics, and the intermediate side has characteristics in which cartilage and fibrous characteristics are transformed, and the physicochemical properties are changed by the partitioned tissues. A bioink composition for 3D printing.

2. The composition is characterized by promoting the migration, adhesion, and proliferation of fibrous cartilage cells. The bioink composition for 3D printing according to Claim 1.

3. The composition is characterized by differentiating into a fibrous cartilage composite tissue in vitro or in vivo and inducing artificial tissue formation. The bioink composition for 3D printing according to Claim 1.

4. The bioink composition containing an extracellular matrix derived from the tissue partitioned on the outer side is characterized by inducing angiogenesis. The bioink composition for 3D printing according to Claim 1.

5. The bioink composition containing an extracellular matrix derived from the tissue partitioned on the inner side or the intermediate side is characterized by suppressing angiogenesis. The bioink composition for 3D printing according to Claim 1.

6. The composition is characterized by being gelled at a temperature of 37°C or higher while existing in a liquid state at room temperature. The bioink composition for 3D printing according to Claim 1.

7. It contains a demineralized bone matrix support, and the bioink composition according to any one of Claims 1 to 6 is contained in the support and crosslinked. A bone graft composition.

8. The bioink composition is characterized by having a concentration of 0.1 to 10 mass / volume (w / v) percent. The bone graft composition according to Claim 7.

9. The demineralized bone matrix support is characterized by being composed of cancellous bone, compact bone, or a combination thereof. The bone graft composition according to Claim 7.

10. Obtaining a fibrous cartilage composite tissue and pulverizing it; Decellularizing the pulverized tissue to obtain an extracellular matrix and pulverizing it; Enzymatically treating the pulverized extracellular matrix to solubilize it, and including The fibrous cartilage composite tissue is one or more selected from tissues partitioned into an inner side, an intermediate side, and an outer side of the meniscus cartilage tissue according to anatomical position. The inner side has cartilage characteristics, the outer side has fibrous characteristics, and the intermediate side has characteristics in which cartilage and fibrous characteristics are transformed, and the physicochemical properties are changed by the partitioned tissue. A method for manufacturing a bioink for 3D printing, characterized in that.

11. The step of subjecting the powdered extracellular matrix to enzymatic treatment to solubilize it is The method for manufacturing a bioink for 3D printing according to claim 10, characterized in that it is carried out by subjecting the extracellular matrix to pepsin enzymatic treatment with an acidic solution.

12. The step of manufacturing a demineralized bone matrix support, and The step of manufacturing a bioink containing an extracellular matrix derived from a fibrous cartilage composite tissue by the manufacturing method according to claim 10 or 11, and The step of injecting the manufactured bioink into the manufactured demineralized bone matrix support and crosslinking it, A method for manufacturing a bone graft material, comprising.

13. The step of manufacturing the demineralized bone matrix support is Obtaining an osteochondral composite tissue and removing inorganic substances from the composite tissue, and Removing cartilage tissue from the composite tissue from which the inorganic substances have been removed, The method for manufacturing a bone graft material according to claim 12, characterized in that it comprises.

14. The step of injecting the manufactured bioink into the manufactured demineralized bone matrix support and crosslinking it is The method for manufacturing a bone graft material according to claim 12, characterized in that after outputting the bioink by 3D printing and injecting it into the porous structure inside the demineralized bone matrix support, crosslinking is carried out at a temperature of 37 ° C or higher.

15. The method for manufacturing the bone graft material is The method for manufacturing a bone graft material according to claim 12, further comprising a step of performing additional crosslinking by one or two or more physical or chemical methods after the step of injecting the manufactured bioink into the manufactured demineralized bone matrix support and crosslinking it.

16. The physical method is The method for manufacturing a bone graft material according to claim 15, characterized in that it is a method of irradiating with ionization energy selected from gamma rays or electron beams.

17. The chemical method is The method for manufacturing the bone graft material according to claim 15, characterized in that a crosslinking agent selected from glutaraldehyde or EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide) is used.

18. A method for manufacturing an artificial tissue, comprising the step of three-dimensionally printing the bioink composition according to any one of claims 1 to 6.

19. The step of three-dimensionally printing is performed at a speed of 1 to 2 mm / sec at a pneumatic pressure of less than 80 kPa and a temperature of 37°C or higher using a nozzle having an average diameter of 300 to 600 μm, the method for manufacturing an artificial tissue according to claim 18.

20. An artificial tissue, manufactured by the manufacturing method according to claim 18, and having the characteristics of a fibrous cartilage composite tissue.

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