Bone scaffold using 3D printing technology and method for manufacturing same

Abstract

The present invention relates to a method for manufacturing an osteoinductive scaffold having excellent osteoconductivity and osteoinductivity by using a 3D printing technology. According to the present invention, it is possible to provide an osteoinductive scaffold having improved biocompatibility, cell adhesion, cell proliferation ability, osteoconductivity, and osteoinductivity through a frame part formed of a mixture of a biodegradable polymer and an allogeneic bone-derived osteoinductive material having a lattice pattern and an osteoinductive part formed of an allogeneic bone-derived osteoinductive material.

Description

Bone scaffold using 3D printing technology and its manufacturing method

[0001] The present invention relates to a method for manufacturing an osteoinductive scaffold, and more particularly, to an osteoinductive scaffold using 3D printing technology for bone grafting having excellent osteoconductive and osteoinductive properties, including an osteoconductive material derived from homologous bone and an osteoinductive material, and a method for manufacturing the same.

[0002] Tissue engineering is a general term for technologies that utilize fundamental concepts and techniques from life sciences, medicine, and engineering to create substitutes for living tissue and transplant them into living organisms, enabling the maintenance, enhancement, and restoration of biological functions. It gained recognition as a new academic field in the 1980s with the first creation of artificial skin, and diverse and active research has continued to this day. While highly complex tissues like organs have not yet progressed far beyond the research stage, relatively simple tissues like skin and bone have advanced to the point of being applied to the human body.

[0003] Meanwhile, the demand and necessity for bone graft materials for bone regeneration are increasing due to factors such as the increase in the aging population, the increase in the surgical accident rate due to the diversification of industrial society, and the development of medical procedures. In addition, the demand for technology for bone graft materials that are safer, more efficient, and have superior bone regeneration capabilities is also increasing.

[0004] Typically, bone grafts can be categorized into autologous bone grafts, xenografts, synthetic bone grafts, and allografts. Autologous bone grafts (autografts) have the advantage of being relatively immune-free compared to non-autologous or synthetic bone grafts. Because autologous bone grafts transplant not only the patient's own bone matrix but also their own cells, they are relatively advantageous over other grafts in terms of osteogenic potential and possess excellent osteoinductivity and osteoconductivity. However, obtaining sufficient amounts of autologous bone for the procedure is difficult, and obtaining the desired type of autologous bone is also challenging. Furthermore, additional surgery at the bone donor site can result in incisions, bleeding, hematomas, fractures, and transfusions. This can also lead to problems such as nerve damage, sensory loss, chronic pain, and instability at the bone donor site.

[0005] Xenograft bone grafts, although lacking osteoinductive properties, were developed to address the shortcomings of autologous bone harvesting. However, these xenograft bone grafts have lower bone formation rates and new bone formation potential compared to autologous and allograft bone. Synthetic bone grafts, which lack osteoinductive properties, are made by extracting calcium carbonate from natural coral or chemically combining calcium carbonate and calcium phosphate. They are synthesized with a calcium to phosphorus ratio (Ca / P) of 1.67, similar to the Ca / P ratio of human bone tissue. While they offer the advantage of low toxicity in the body, synthetic materials still suffer from drawbacks such as high absorption and low strength.

[0006] Finally, unlike autologous bone grafts, allogeneic bone grafts lack bone-forming cells and therefore lack direct bone regeneration potential. Furthermore, they lack osteoinductive properties, which stimulate bone progenitor cells to differentiate into osteoblasts. Instead, they possess only osteoconductive properties, providing space for bone formation in adjacent tissues of the grafted area. However, allogeneic bone grafts offer the advantage of ensuring sufficient graft volume for use in procedures, eliminating the need for difficult autologous bone procurement.

[0007] The reason why allogeneic bone grafts possess only osteoconductive properties without osteoinductive ones is because bone morphogenetic proteins (BMPs) are trapped by calcium phosphate, an inorganic component of bone, and thus cannot function properly. Therefore, demineralized bone matrix (DBM) was developed to allow BMPs to participate appropriately in osteoinduction.

[0008] The present invention relates to a scaffold using an allogeneic bone graft material, and aims to provide a scaffold that has superior osteoinductive and osteoconductive properties compared to conventional technologies and is easy to manufacture to suit the type of bone to be regenerated.

[0009] The purpose of the present invention is to provide an osteoinductive scaffold having excellent osteoconductive and osteoinductive properties.

[0010] Another object of the present invention is to provide an osteoinductive scaffold having excellent osteoconductivity and osteoinduction using 3D printing technology, which allows cells to easily penetrate inside, allowing tissue formation to occur well, and which can also control the amount and location of an osteoinductive material within the scaffold to suit the type of bone to be regenerated, and a method for manufacturing the same.

[0011] In order to achieve the above object, the present invention provides an osteoinductive scaffold having excellent osteoconductivity and osteoinduction, and a method for manufacturing the same, comprising: a frame formed from a mixture of a biodegradable polymer and a bone-derived osteoconductive material and printed in a grid shape to form a plurality of empty spaces therein; and an osteoinductive portion containing an osteoinductive material and filling some of the empty spaces.

[0012] The biodegradable polymers that can be used include polylactic acid (PLA), polyglycolic acid (PGA) and its copolymer (PLGA), polycarprolactone (PCL), polydioxanone (PDO), and polyhydroxybutyrate (PHB).

[0013] The above bone conduction material may be hydroxyapatite or a mineral derived from biological bone.

[0014] It is preferable that the mixing ratio of the biodegradable polymer and the bone conductive material is 70 to 90:30 to 10.

[0015] It is preferable that the above-mentioned osteoinductive material is DBM (Demineralized Bone Matrix) derived from homologous bone.

[0016] The above-mentioned bone-inducing portion may further include a carrier material.

[0017] The carrier material may be CMC (Carboxy methyl cellulose), HA (Hyaluronic acid), HA-CMC, and Alginic acid.

[0018] The carrier material may be mixed with the bone-inducing material in an amount of 233 to 900 parts by weight based on 100 parts by weight of the bone-inducing material.

[0019] The above osteoinductive component may further comprise bone morphogenetic protein (BMP-2).

[0020] In addition, the method for manufacturing an osteoinductive scaffold with excellent osteoconductivity and osteoinduction is characterized by including a step (S1) of forming a frame portion forming a plurality of empty spaces in a grid shape by discharging a first ink containing a biodegradable polymer and a osteoconductive material, and a step (S2) of forming an osteoinductive portion filling a portion of the empty spaces by discharging a second ink containing an osteoinductive material.

[0021] The method for manufacturing the above-mentioned bone-inducing scaffold may further include a step (S0) of forming a support portion by discharging a third ink including at least one selected from the group consisting of PEG (Polyethylene glycol), Pluronic f-127, Glycerol, PCL (Polycarprolactone), and PEO (Polyethylene oxide).

[0022] The biodegradable polymers that can be used include polylactic acid (PLA), polyglycolic acid (PGA) and its copolymer (PLGA), polycarprolactone (PCL), polydioxanone (PDO), and polyhydroxybutyrate (PHB).

[0023] Hydroxyapatite may be used as the above bone conduction material.

[0024] It is preferable that the mixing ratio of the biodegradable polymer and the bone conductive material is 70 to 90:30 to 10.

[0025] It is preferable that the above-mentioned osteoinductive material is DBM (Demineralized Bone Matrix) derived from homologous bone.

[0026] The second ink may further include a carrier material, and the second ink may be manufactured by mixing the osteoinductive material and the carrier material.

[0027] The carrier material may be CMC (Carboxy methyl cellulose), HA (Hyaluronic acid), HA-CMC, and Alginate.

[0028] The carrier material may be mixed in an amount of 233 to 900 parts by weight based on 100 parts by weight of the bone-inducing material.

[0029] It is preferable that the above first ink is printed (discharged) at a temperature of 60 to 200°C and a pressure of 50 to 800 kPa.

[0030] It is preferable that the above second ink is printed (discharged) at a temperature of 10 to 25°C and a pressure of 20 to 800 kPa.

[0031] It is preferable that the content of the bone-inducing material in the second ink is 5 to 50%.

[0032] The second ink may be discharged in an amount of 10 to 50 wt% of the total weight of the scaffold.

[0033] The osteoinductive scaffold of the present invention is manufactured using a homologous bone-derived osteoconductive material and an osteoinductive material, and provides excellent osteoconductive and osteoinductive properties.

[0034] And a plurality of empty spaces can be formed within the scaffold through an osteoinductive portion formed of an osteoinductive material derived from homologous bone, and cells can easily penetrate into the empty spaces to facilitate tissue formation, thereby providing an osteoinductive scaffold with improved biocompatibility, cell adhesion, cell proliferation, osteoconductivity, and osteoinductive ability.

[0035] Additionally, the location and density of the bone-inducing portion within the empty space within the frame can be adjusted to suit the type of bone to be regenerated.

[0036] FIG. 1 is a schematic diagram schematically depicting a plane of a bone-inducing scaffold according to one embodiment of the present invention.

[0037] Figure 2 is a schematic diagram illustrating a cross-sectional side view of a bone-inducing scaffold according to one embodiment of the present invention.

[0038] Figure 3 is a photograph of a bone-inducing scaffold ((a), (c)) according to one embodiment and a photograph of its thickness measured ((b), (d)).

[0039] Figure 4 is a flowchart schematically illustrating a method for manufacturing a bone-inducing scaffold according to one embodiment.

[0040] FIG. 5 is a drawing showing an experiment to confirm the effectiveness of bone conduction of the frame portion of a bone-inductive scaffold according to one embodiment of the present invention and the results thereof, wherein (A) a microscope photograph of a PCL scaffold, (B) a microscope photograph of a PCL scaffold cultured with MC3T3-E1 for 24 hours, (C) a microscope photograph of a scaffold composed of 90% PCL and 10% HAp, and (D) a microscope photograph of a scaffold composed of 90% PCL and 10% HAp cultured with MC3T3-E1 for 24 hours.

[0041] Figure 6 is a graph showing the results of an experiment to confirm the effectiveness of cell proliferation of DBM and HAp.

[0042] Figure 7 is a photograph showing the results of an experiment to confirm the effectiveness of the osteoinductive ability of DBM and HAp. Naive, Ink 1, Ink 2, and Ink 1 + Ink 2 represent the undissolved medium, the frame dissolution medium, the second ink dissolution medium, and the osteoinductive scaffold dissolution medium, respectively. The photographs show cells stained after culturing each dissolution medium for 1 day, 1 week, and 2 weeks.

[0043] Figure 8 is a photograph showing the results of transplanting a scaffold into an animal model to evaluate the in vivo effectiveness of a bone-inductive scaffold according to the present invention. (a) is a photograph showing a PCL single-component scaffold, and (b) is a photograph showing a bone-inductive scaffold according to an embodiment of the present invention transplanted into an animal model, respectively, and staining cell nuclei and extracellular matrix one week later.

[0044] Figure 9 is a photograph of an experiment in which a scaffold was transplanted into an animal model to evaluate the in vivo effectiveness of the bone-inducing scaffold according to the present invention.

[0045] The present invention will be described in more detail with reference to the following examples and drawings. However, the following examples are intended only to specifically illustrate the present invention, and the scope of the present invention is not limited by the following examples.

[0046] The bone-inducing scaffold of the present invention is composed of a frame portion and a bone-inducing portion.

[0047] The above frame portion is made of a biodegradable polymer and a bone conductive material.

[0048] The biodegradable polymer may preferably be at least one selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA) and its copolymer (PLGA), and polycarprolactone (PCL). The bone conductive material may be HAp, and the mixing ratio thereof is preferably 70 to 90:30 to 10.

[0049] The above-mentioned osteoinductive component may include DBM, which is an osteoinductive substance.

[0050] The above frame portion is manufactured to form a number of empty spaces inside the frame, so that cells can easily penetrate into the scaffold, making it suitable for tissue formation.

[0051] The above-mentioned bone-inducing portion fills a portion of a plurality of empty spaces formed by the above-mentioned frame portion, and its position and density can be adjusted according to properties suitable for the physical environment of the bone into which the structure is to be implanted.

[0052] Figures 1 and 2 are schematic diagrams schematically depicting a plan view and a side cross-section of a bone-inducing scaffold according to one embodiment of the present invention, respectively.

[0053] As illustrated in FIGS. 1 and 2, a bone-inducing scaffold according to one embodiment of the present invention is characterized by including a frame portion that is printed in a grid shape to form a plurality of empty spaces therein, and a bone-inducing portion that fills some of the empty spaces.

[0054] The biodegradable polymers that can be used include polylactic acid (PLA), polyglycolic acid (PGA) and its copolymer (PLGA), polycarprolactone (PCL), polydioxanone (PDO), and polyhydroxybutyrate (PHB).

[0055] The above-mentioned bone conductive material may be hydroxyapatite (HAp), or other bone-derived minerals may be used.

[0056] The above hydroxyapatite may be derived from allogeneic or xenogeneic bone, or synthetic hydroxyapatite. The use of allogeneic bone-derived hydroxyapatite is most preferred.

[0057] It is preferable that the above-mentioned osteoinductive material is DBM (Demineralized Bone Matrix) derived from homologous bone.

[0058] The above-mentioned osteoconductive and osteoinductive materials may be obtained by separating them from the demineralization process of allogeneic bone. The demineralization process of the allogeneic bone may be performed using a known method, for example, the allogeneic bone demineralization method disclosed in Patent Registration No. 10-2416859 (June 30, 2022).

[0059] The carrier material may be CMC (Carboxy methyl cellulose), HA (Hyaluronic acid), HA-CMC, and Alginic acid.

[0060] The carrier material may be mixed in an amount of 233 to 900 parts by weight based on 100 parts by weight of the bone-inducing material.

[0061] The above osteoinductive component may further comprise bone morphogenetic protein (BMP-2).

[0062] One embodiment of a method for manufacturing a bone-inductive scaffold according to the present invention comprises a step (S1) of forming a frame portion forming a plurality of empty spaces in a grid shape by discharging a first ink containing a biodegradable polymer and a bone-conducting material; and a step (S2) of forming an bone-inductive portion filling a portion of the empty spaces by discharging a second ink containing a bone-inductive material.

[0063] The method for manufacturing the above-mentioned osteoinductive scaffold having excellent osteoconductivity and osteoinduction may further include a step (S0) of forming a support portion by discharging a third ink containing at least one selected from the group consisting of PEG (Polyethylene glycol), Pluronic f-127, Glycerol, PCL (Polycarprolactone), and PEO (Polyethylene oxide).

[0064] The above support portion may be formed by ejecting the third ink before the first ink. The above support portion may be at least one selected from the group consisting of PEG (Polyethylene glycol), Pluronic f-127, Glycerol, PCL (Polycarprolactone), and PEO (Polyethylene oxide).

[0065] The above support member can function to maintain the foundation of a complex scaffold, thereby ensuring a more stable scaffold shape. Furthermore, it can prevent bioink from leaking during the 3D printing process and prevent rapid dissolution of bioink into body fluids after insertion into the body.

[0066] The step (S0) of forming the support portion, the step (S1) of forming the frame portion, and the step (S2) of forming the bone guidance portion use an extrusion-type 3D printing method. The extrusion method is a method of ejecting a raw material by applying heat or pressure to a nozzle, and in particular, in the case of step S1, it is preferable to use the FDM (Fused Deposition Modeling) method.

[0067] The step (S0) of forming the above support portion can print the support portion by combining nozzles having a diameter of 100 to 1,000 μm and ejecting them.

[0068] It is preferable that the printing of the above step S0 is printed (discharged) at -10 to 200 ℃ and 10 to 800 kP.

[0069] The above support portion serves to support the three-dimensional shape so that it does not collapse when the first ink and the second ink are ejected, and may vary depending on the shape of the object to be printed, and may be removed after printing is complete.

[0070] The first to third inks are ejected through different nozzles, and three nozzles can be used to form the support part, the frame part, and the bone guidance part. It is preferable that the nozzles have a diameter of an outlet within a range of 200 μm to 1,200 μm.

[0071] The above-mentioned bone-conducting material may be hydroxyapatite (HAp). Hydroxyapatite may be derived from allogeneic or xenogeneic bone, or synthetic hydroxyapatite. The use of allogeneic bone-derived hydroxyapatite is most preferred.

[0072] It is preferable that the above-mentioned osteoinductive material is DBM (Demineralized Bone Matrix) derived from homologous bone.

[0073] The above-mentioned osteoconductive and osteoinductive materials may be obtained by separating them from the demineralization process of allogeneic bone. The demineralization process of the allogeneic bone may be performed using a known method, for example, the allogeneic bone demineralization method disclosed in Patent Registration No. 10-2416859 (June 30, 2022).

[0074] The above first ink can be manufactured by heating and mixing hydroxyapatite obtained by separation during the demineralization process of the homogeneous bone and a biodegradable polymer, and preferably, can be heated and mixed at 60 to 200°C.

[0075] The second ink may include DBM (Demineralized Bone Matrix) obtained by separating during the demineralization process of the homologous bone.

[0076] The carrier material may be CMC (Carboxy methyl cellulose), HA (Hyaluronic acid), HA-CMC, and Alginic acid.

[0077] The second ink can be prepared by mixing the bone-inducing material and the hydrated carrier material, and can be printed by injecting it into a syringe.

[0078] The carrier material may be mixed in an amount of 233 to 900 parts by weight based on 100 parts by weight of the bone-inducing material.

[0079] It is preferable that the above step S1 printing (discharging) is performed at a temperature of 60 to 200 ℃ and a pressure of 50 to 800 kPa.

[0080] It is preferable that the above step S2 printing is printed (discharged) at a temperature of 10 to 25°C and a pressure of 20 to 800 kPa.

[0081] It is preferable that the content of the bone-inducing material of the second ink is 5 to 50 wt%.

[0082] The above DBM contains osteoinductive components that help in early bone formation.

[0083] The scaffold of the present invention is in a form in which a portion of the empty space of the frame portion is filled with a second ink containing a bone-inducing material. If the entire empty space of the frame portion is filled with the second ink, the supply of nutrients to cells and the discharge of waste products may not be smooth, which may have a negative effect on initial cell attachment and growth.

[0084] FIGS. 3(a) and 3(c) are photographs of an osteoinductive scaffold manufactured according to one embodiment of the present invention, wherein a void exists within the frame where the osteoinductive portion is not filled, which can serve as a passage connecting the outside and inside of the scaffold. Since the void is formed in the osteoinductive scaffold manufactured according to one embodiment of the present invention, it is easy to supply nutrients and discharge waste products to the inside of the scaffold, and there is an advantage in that cells can attach and grow within the scaffold within a short period of time after implanting the scaffold in the body.

[0085] Figure 4 is a flowchart illustrating a method for manufacturing a bone-inducing scaffold according to one embodiment of the present invention.

[0086]

[0087] Example 1. Method for manufacturing a bone-inductive scaffold

[0088] 1.1 3D Printing Ink Manufacturing

[0089] (1) Homologous bone demineralization process

[0090] Allogeneic bone was demineralized to obtain hydroxyapatite (HAp) and demineralized bone matrix (DBM). The process of demineralizing allogeneic bone to obtain hydroxyapatite and DBM was referenced from the applicant's registered patent no. 10-2416859 (June 30, 2022).

[0091]

[0092] (2) First ink manufacturing

[0093] After adding 1 g of the hydroxyapatite obtained above to 9 g of PCL (Polycarprolactone), heating to 160°C and mixing, a first ink was prepared.

[0094] (3) Manufacturing of cross-linked hyaluronic acid (HA)-carboxymethylcellulose (CMC) carrier

[0095] A HA-CMC excipient (carrier) was prepared by mixing hyaluronic acid (Sodium Hyaluronate (HA) by Hyundai Bioland), a bio-derived polymer composed of N-acetylglucosamine and glucuronic acid, and carboxymethyl cellulose (Sodium carboxymethyl cellulose (CMC), Sigma Aldrich), with a cross-linking agent, BDDE (1,4-Butanediol diglycidyl ether, Sigma-Aldrich).

[0096] Specifically, 1 to 10 ml of BDDE was added to 100 ml of a 0. N sodium hydroxide aqueous solution heated to 50°C, and the solution was mixed with 1 to 10 g of HA and 1 to 10 g of CMC. The mixed solution was heated at 50°C for 3 hours to undergo a crosslinking process.

[0097] The cross-linked HA-CMC mixed solution was pulverized, washed with PBS, DW, etc., and then freeze-dried (-80°C, 0.01 mbar, 96 h).

[0098]

[0099] (4) Second ink manufacturing

[0100] The HA-CMC manufactured above was prepared by hydrating it in saline, and 1 g of the DBM (Demineralized Bone Matrix) obtained by separation was mixed with 9 g of the HA-CMC carrier to prepare a second ink.

[0101]

[0102] 1.2 Scaffold fabrication using 3D printing

[0103] The first and second inks manufactured using the method described in 1.1 above were each injected into a syringe and extruded using 3D printing. The first ink was produced using the fused deposition modeling (FDM) method, and the second ink was produced using the general extrusion method.

[0104]

[0105] (1) Frame printing

[0106] The frame part is printed on the surface of the support part, and a g-code for spraying the first ink to form a grid pattern with a spacing of 1,000 μm and a size of 10 mm x 10 mm was created and uploaded to the 3D printer. After mounting the syringe containing the first ink on the head, the first nozzle with a diameter of 500 μm was combined and used, and the first ink was sprayed according to the settings at a pressure of 500 kPa at 180°C.

[0107]

[0108] (2) Bone-inducing section printing

[0109] In the bone induction unit, a g-code was created to spray the second ink (bioink) so that it fills 74 of the 100 spaces (one space: 500 μm x 500 μm) of the internal space formed by the frame unit without overflowing, as shown in Fig. 3, and was uploaded to the 3D printer. After mounting the syringe containing the second ink on the head, a second nozzle with a diameter of 500 μm was combined and used, and the second ink was sprayed according to the settings while applying a pressure of 300 kPa at 25°C.

[0110]

[0111] Experimental Example 1. Experiment to confirm the validity of bone conduction

[0112] In order to confirm whether the bone conduction ability, i.e., cell adhesion ability, of the frame portion of the osteoinductive scaffold to which HAp is added according to the present invention is improved, an experiment was conducted to confirm the cell adhesion ability of a PCL single-component scaffold and a scaffold (90% PCL + 10% HAp) when HAp is added to PCL.

[0113] The scaffold with HAp added to PCL (90% PCL + 10% HAp) was prepared using the frame part manufactured in Example 1, and the PCL single component scaffold as a control group was prepared using a lattice-patterned frame manufactured under the same conditions as the frame part of Example 1 except that PCL single component was used. MC3T3-E1, which is a pre-osteoblast cell, was seeded at 4 x 10 4 Cells were cultured on each scaffold at a cell / scaffold concentration. After 24 hours, the nuclei of cells attached to each scaffold were stained with DAPI and their numbers were confirmed.

[0114] Figure 5 is a photograph showing an experiment to confirm the validity of bone conduction of a frame portion according to one embodiment of the present invention and the results thereof.

[0115] Referring to Fig. 5, a larger number of cells were observed in the experimental group in which pre-osteoblast MC3T3-E1 cells were cultured for 24 hours (scaffold with HAp added to PCL (90% PCL+10% HAp), Fig. 5 (D)) compared to the control group in which pre-osteoblast MC3T3-E1 cells were cultured for 24 hours (PCL single component scaffold, Fig. 5 (B)).

[0116] Through this, it was confirmed that the scaffold containing HAp had higher cell adhesion than the control group.

[0117]

[0118] Experimental Example 2. Experiment to confirm the effectiveness of cell proliferation

[0119] In order to confirm whether HAp, which is a main component of the first ink constituting the osteoinductive scaffold manufactured according to one embodiment of the present invention, improves the cell proliferation performance of the scaffold, a cell proliferation assay was performed on a PCL single-component scaffold and a scaffold (90% PCL + 10% HAp) in which HAp was added to PCL.

[0120] The scaffold with HAp added to PCL used the frame part manufactured in Example 1, and the PCL single component scaffold used a lattice-patterned frame manufactured under the same conditions as the frame part of Example 1, except that the PCL single component was used.

[0121] A scaffold containing HAp added to the above PCL was prepared by dissolving it in a cell culture medium (PCL+HAp dissolution medium), and a PCL single-component scaffold was also prepared by dissolving it in the same way (PCL dissolution medium).

[0122] MC3T3-E1, which are pre-osteoblast cells, were seeded at 4 x 10 in each of the PCL+HAp dissolution medium and PCL dissolution medium prepared above. 3The cells were cultured at a concentration of 1 cell / well for 2 weeks. The CCK-8 Assay, a cell proliferation assay that can check cell proliferation, was performed on day 1, week 1, and week 2.

[0123] Figure 6 is a graph showing the results of an experiment to confirm the effectiveness of cell proliferation of DBM. According to the experimental results of Figure 6, it was confirmed that cell proliferation was superior in the PCL+HAp dissolution medium (Figure 6 Ink 1) compared to the PCL dissolution medium (Figure 6 PCL only).

[0124] That is, it was confirmed that HAp according to one embodiment of the present invention has a positive effect of improving cell proliferation ability of the frame portion.

[0125]

[0126] Experimental Example 3. Experiment to confirm the effectiveness of bone induction

[0127] In order to confirm whether DBM, a main component of the second ink constituting the osteoinductive scaffold manufactured according to one embodiment of the present invention, improves the bone formation ability of the scaffold, a dissolution experiment was conducted.

[0128] The frame part and the second ink, and the bone-inducing scaffold (frame part and bone-inducing part) manufactured in Example 1 above were each prepared.

[0129] The above frame part and the second ink were each added to a cell culture medium to prepare the main components by dissolving them into the medium (frame part dissolution medium, second ink dissolution medium), and the osteoinductive scaffold was prepared by dissolving them in the same way (osteoinductive scaffold dissolution medium). For comparison, a cell culture medium in which nothing was dissolved (undissolved medium) was prepared.

[0130] MC3T3-E1, which are pre-osteoblasts, were seeded at 2 x 10 in each of the prepared frame dissolution medium, second ink dissolution medium, osteoinductive scaffold dissolution medium, and cosmetic dissolution medium. 4The cells were cultured at a concentration of 10 cells / well for 2 weeks. Alizarin Red S staining, which binds to calcium ions, an indicator of bone formation, and produces red staining proportional to the amount of calcium ions, was performed on day 1, week 1, and week 2.

[0131] Figure 7 is a photograph showing the results of an experiment to confirm the effectiveness of DBM's bone induction ability.

[0132] According to the experimental results of Fig. 7, bone formation was confirmed in each experimental group, but it was confirmed that the second ink dissolution medium (Fig. 7 Ink 2) was stained more red compared to the cosmetic dissolution medium (Fig. 7 Naive) and the frame dissolution medium (Fig. 7 Ink 1), and the staining in the osteoinductive scaffold dissolution medium (Fig. 7 Ink 1 + Ink 2) was the reddest.

[0133] This means that bone formation in the second ink elution medium is more excellent than in the non-elution medium and the frame-part elution medium, and it can be seen that this is due to the excellent osteoinductive ability of DBM, which is an osteoinductive material of one embodiment of the present invention.

[0134] In addition, the above experiment showed that bone formation was the most excellent in the osteoinductive scaffold dissolution medium compared to the cosmetic dissolution medium, frame dissolution medium, and second ink dissolution medium, and this confirmed that bone formation was promoted when the frame (first ink) and the osteoinductive portion (second ink) were used together.

[0135] That is, it was confirmed that the combination of HAp, a bone-conducting material of a bone-inductive scaffold according to one embodiment of the present invention, and DBM, a bone-inductive material, has the effect of improving bone formation performance.

[0136]

[0137] Experimental Example 4. Animal Experiment (in-vivo Test)

[0138] To evaluate the in vivo effectiveness of the bone-inducing scaffold according to the present invention, the scaffold was implanted into an animal model and cells were observed one week later.

[0139] Figures 8 (a) and (b) are photographs showing four 8 mm diameter defects formed in the calvaria of an animal model (3 kg New Zealand white rabbit) and implanted with a PCL single component scaffold and an osteoinductive scaffold manufactured according to an embodiment of the present invention, one week later, after staining the cell nuclei and extracellular matrix using H&E stain (hematoxylin and eosin stain).

[0140] In the above H&E stain, hematoxylin stained the cell nucleus purple and blue, and eosin stained the extracellular matrix and cytoplasm pink.

[0141] The PCL single-component scaffold (Fig. 8 (a)) was stained red overall, but upon magnification, it was confirmed to be red blood cells. This is believed to be the result of bleeding that occurred during tissue extraction after transplantation.

[0142] In the case of the bone-inducing scaffold manufactured according to one embodiment of the present invention, the entire scaffold was stained purple, which confirmed that cells were attached and cell nuclei were stained purple.

[0143] As a result of the above in-vivo experiment, the PCL single-component scaffold had very few purple-stained areas, which can be judged to have a low degree of cell attachment. The osteoinductive scaffold manufactured according to one embodiment of the present invention had relatively many purple-stained areas, which can be judged to have a high degree of cell attachment.

[0144] Since the experimental results are from one week after each scaffold was transplanted into a rabbit, it is a short time for self-organization to occur, but it can be judged that the bone-inductive scaffold manufactured according to one embodiment of the present invention is advantageous for cell attachment at the early stage of transplantation.

Claims

1. A frame portion formed from a mixture of a biodegradable polymer and a bone-derived osteoconductive material of the same type, and printed in a grid shape to form a plurality of empty spaces inside; and An osteoinductive portion comprising a bone-inductive material and filling a portion of the empty space; characterized by including Bone-inducing scaffold.

2. In paragraph 1, The biodegradable polymer is characterized in that it is at least one selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA) and its copolymer (PLGA), polycarprolactone (PCL), polydioxanone (PDO), and polyhydroxybutyrate (PHB). Bone-inducing scaffold.

3. In paragraph 1, The above bone conduction material is characterized in that it is hydroxyapatite. Bone-inducing scaffold.

4. In paragraph 1, The biodegradable polymer and the bone conductive material are characterized in that they are mixed in a mass ratio of 70 to 90:30 to 10. Bone-inducing scaffold.

5. In paragraph 1, The above osteoinductive material is characterized in that it is a DBM (Demineralized Bone Matrix) derived from homologous bone. Bone-inducing scaffold.

6. In paragraph 1, The above bone induction part is characterized in that it further comprises a carrier material. Bone-inducing scaffold.

7. In paragraph 6, The carrier material is characterized in that it is at least one selected from the group consisting of CMC (Carboxy methyl cellulose), HA (Hyaluronic acid), HA-CMC and Alginic acid. Bone-inducing scaffold.

8. In paragraph 6, The carrier material is characterized in that 233 to 900 parts by weight are mixed with the bone-inducing material based on 100 parts by weight of the bone-inducing material. Bone-inducing scaffold.

9. In paragraph 1, The above-mentioned osteoinduction part is characterized in that it further comprises bone morphogenetic protein (BMP-2). Bone-inducing scaffold.

10. In paragraph 9, The above bone formation protein (BMP-2) is characterized in that 5 to 50 parts by weight are mixed with the above bone induction material based on 100 parts by weight of the above bone induction material. Bone-inducing scaffold.

11. In paragraph 1, The above-mentioned bone-inducing portion is characterized in that it is 10 to 50 wt% of the total weight of the scaffold. Bone-inducing scaffold.

12. A step (S1) of forming a frame portion that forms a plurality of empty spaces in a grid shape by discharging a first ink containing a biodegradable polymer and a bone conductive material; and A step (S2) of forming a bone-inducing portion by discharging a second ink containing a bone-inducing material to fill a portion of the empty space; characterized by including Method for manufacturing a bone-inductive scaffold.

13. In paragraph 12, The above manufacturing method is characterized in that it further includes a step (S0) of forming a support portion by discharging a third ink before the first ink is discharged. Method for manufacturing a bone-inductive scaffold.

14. In paragraph 13, The third ink is characterized in that it contains at least one selected from the group consisting of PEG (Polyethylene glycol), Pluronic f-127, Glycerol, PCL (Polycarprolactone), and PEO (Polyethylene oxide). Method for manufacturing a bone-inductive scaffold.

15. In paragraph 13, The above first to third inks are characterized in that they are ejected through three nozzles. Method for manufacturing a bone-inductive scaffold.

16. In paragraph 12, The biodegradable polymer is characterized in that it is at least one selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA) and its copolymer (PLGA), polycarprolactone (PCL), polydioxanone (PDO), and polyhydroxybutyrate (PHB). Method for manufacturing a bone-inductive scaffold.

17. In paragraph 12, The above bone conduction material is characterized in that it is hydroxyapatite. Method for manufacturing a bone-inductive scaffold.

18. In paragraph 12, The biodegradable polymer and the bone conductive material are characterized in that they are mixed in a mass ratio of 70 to 90:30 to 10. Method for manufacturing a bone-inductive scaffold.

19. In paragraph 12, The second ink is characterized in that it further comprises a carrier material. Method for manufacturing a bone-inductive scaffold.

20. In paragraph 19, The carrier material is characterized in that it is at least one selected from the group consisting of CMC (Carboxy methyl cellulose), HA (Hyaluronic acid), HA-CMC and Alginic acid. Method for manufacturing a bone-inductive scaffold.

21. In paragraph 20, The carrier material is characterized in that 233 to 900 parts by weight are mixed with the bone-inducing material based on 100 parts by weight of the bone-inducing material. Method for manufacturing a bone-inductive scaffold.

22. In paragraph 12, The above first ink is characterized in that it is 3D printed under a temperature of 60 to 200 ℃ and a pressure of 50 to 800 kPa. Method for manufacturing a bone-inductive scaffold.

23. In paragraph 12, The second ink is characterized in that it is 3D printed under a temperature of 10 to 25°C and a pressure of 20 to 800 kPa. Method for manufacturing a bone-inductive scaffold.

24. In paragraph 12, The bone-inducing material in the second ink is characterized in that it is 5 to 50 wt%. Method for manufacturing a bone-inductive scaffold.

25. In paragraph 12, The second ink is characterized in that it is discharged in an amount of 10 to 50 wt% of the total weight of the scaffold. Method for manufacturing a bone-inductive scaffold.

Images