Low-crystalline calcium phosphate-based antibacterial bone graft material and method for preparing same

Abstract

One embodiment of the present specification provides a method for preparing a low-crystalline calcium phosphate-based antibacterial bone graft material, the method comprising the steps of: (a) synthesizing calcium phosphate by batch-injecting a calcium precursor and a phosphate precursor; (b) reacting by injecting the calcium phosphate into an antibacterial material precursor solution; (c) freeze-drying the product from step (b); (d) preparing a molded body by mixing the product from step (c) and a shape control agent and pressure-molding; and (e) removing the shape control agent from the molded body in a solvent at a temperature higher than or equal to a boiling point, and then drying.

Description

Low-crystalline calcium phosphate-based antibacterial bone graft material and its manufacturing method

[0001] This specification relates to a low-crystalline calcium phosphate-based antibacterial bone graft material and a method for manufacturing the same.

[0002] Bone grafting materials are used to regenerate bone that has been absorbed or destroyed by factors such as disease, trauma, or aging. For example, bone grafting materials are sometimes used during dental procedures such as implant placement or periodontal surgery to create artificial alveolar bone.

[0003] Bone grafts stimulate bone growth, and their mechanisms of action can be broadly categorized into osteoconduction, osteoinduction, and osteogenesis. Osteoconduction is the mechanism by which osteoblasts migrate from surrounding bone tissue to form bone. Bone tissue or differentiated mesenchymal cells are essential at the graft site. Osteoinduction refers to the mechanism by which undifferentiated mesenchymal cells differentiate into osteoprogenitor cells, thereby forming bone tissue. Osteogenesis refers to the mechanism by which cells within the graft material generate bone tissue.

[0004] Bone grafts can be categorized by their origin into autogenous, allogenic, heterogeneous, and alloplastic bone grafts. Autogenous bone grafts utilize bone tissue harvested from the recipient. They possess osteogenic, osteoconductive, and osteoinductive properties, making them the only existing bone graft material with osteogenic properties. However, they require additional surgical procedures for bone tissue extraction, which are costly and time-consuming, and, most importantly, have limitations due to the limited amount of bone available. Allogeneic bone grafts are harvested from cadavers, typically donated from deceased individuals. Because they are derived from the same genetically identical species, they possess comparable healing properties, and their bone replacement effect and osteogenic properties are superior. Xenogeneic bone grafts are harvested from individuals of different species, most commonly bovine or porcine hydroxyapatite. Various components, including calcium phosphate ceramics, are used as synthetic bone grafting materials, but they are generally known to have inferior bone formation performance and bone quality compared to autologous bone or allogeneic bone grafting materials.

[0005] Calcium phosphate-based biomaterials commonly used as synthetic bone grafts include hydroxyapatite (HA) and tricalcium phosphate (TCP). However, hydroxyapatite is non-absorbable and persists in the body for long periods of time, while tricalcium phosphate suffers from an imbalance between bone formation and graft degradation rates.

[0006] In addition, if the bone grafting site becomes infected, bone recovery is difficult and bone loss may occur, requiring complex recovery methods. In particular, dental bone grafting is known to have a high risk of bacterial infection due to the oral environment.

[0007] Accordingly, there is a need to develop a synthetic bone graft material that has absorbability and bone formation performance similar to that of allogeneic bone, and that can actively control infection by inhibiting bacterial growth within the graft material itself.

[0008] The present specification is intended to solve the problems of the prior art described above, and one purpose of the present specification is to provide a low-crystalline calcium phosphate-based antibacterial bone graft material with excellent antibacterial properties and osteogenic properties and a method for manufacturing the same.

[0009] According to one aspect, a method for producing a low-crystallization calcium phosphate-based antibacterial bone graft material is provided, comprising: (a) a step of synthesizing calcium phosphate by simultaneously introducing a calcium precursor and a phosphate precursor; (b) a step of reacting the calcium phosphate by introducing the calcium phosphate into an antibacterial precursor solution; (c) a step of freeze-drying the product of step (b); (d) a step of mixing and press-molding the product of step (c) with a shape-controlling agent to produce a molded article; and (e) a step of removing the shape-controlling agent from the molded article in a solvent having a temperature higher than the boiling point and then drying the molded article.

[0010] In one embodiment, the calcium precursor may include one or more selected from the group consisting of calcium nitrate, calcium chloride, calcium fluoride, calcium iodide, calcium acetate, ammonium carbonate, and ammonium bicarbonate.

[0011] In one embodiment, the phosphate precursor may include at least one selected from the group consisting of ammonium phosphate, ammonium monophosphate, ammonium diphosphate, ammonium triphosphate, sodium monophosphate, sodium diphosphate, sodium triphosphate, and potassium phosphate.

[0012] In one embodiment, in step (a), the molar ratio of the calcium precursor and the phosphate precursor may be 1:1.5 to 3.

[0013] In one embodiment, step (a) may be a wet precipitation method in which each precursor is dissolved in distilled water and mixed.

[0014] In one embodiment, the antibacterial material may include one or more selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

[0015] In one embodiment, the reaction of step (b) can be performed at a temperature of 15 to 25°C for 10 to 20 hours.

[0016] In one embodiment, in step (d), the weight ratio of the product of step (c) and the shape-controlling agent may be 1:1 to 1.5.

[0017] In one embodiment, the average particle diameter of the shape control agent may be 100 to 500 μm.

[0018] In one embodiment, after step (e), the step (f) of heat-treating the molded body at a temperature of 250°C or lower may be further included.

[0019] In one embodiment, the antibacterial substance content of the low-crystalline calcium phosphate-based antibacterial bone graft material may be 0.2 to 1.8 wt%.

[0020] In one embodiment, the antibacterial substance content of the low-crystalline calcium phosphate-based antibacterial bone graft material may be 0.5 to 0.9 wt%.

[0021] According to another aspect, it comprises an antibacterial substance, comprises at least one of grooves and pores with an average diameter of 100 to 500 ㎛, and has a BET specific surface area of ​​100 m 2 / g or more, and includes both crystalline apatite and amorphous calcium phosphate, and has a crystallinity of 25% or less, and provides a low-crystalline calcium phosphate-based antibacterial bone graft material.

[0022] In one embodiment, the antibacterial material may include one or more selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

[0023] In one embodiment, the content of the antibacterial substance may be 0.2 to 1.8 wt% relative to the total elements constituting the bone graft material.

[0024] In one embodiment, the content of the antibacterial substance may be 0.5 to 0.9 wt% relative to the total elements constituting the bone graft material.

[0025] A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material according to one aspect of the present specification can be applied to producing a low-crystalline calcium phosphate-based antibacterial bone graft material having excellent antibacterial properties and osteogenic properties.

[0026] In addition, the low-crystalline calcium phosphate-based antibacterial bone graft material according to another aspect of the present specification has excellent antibacterial properties, thereby reducing the risk of infection due to bacterial proliferation, and at the same time has absorbability and osteoconductivity and can exhibit bone formation performance at the same level as allogeneic bone.

[0027] It should be understood that the effects of one aspect of this specification are not limited to the effects described above, but include all effects that can be inferred from the detailed description or claims of this specification.

[0028] Figure 1 shows the results of antibacterial evaluation of bone graft materials according to examples and comparative examples of the present specification.

[0029] Figure 2 shows the results of cytotoxicity evaluation of bone graft materials according to examples and comparative examples of the present specification.

[0030] Figures 3 and 4 are XRD analysis results of bone graft materials according to examples and comparative examples of the present specification.

[0031] Figure 5 is a graph showing the dispersion of EDS measurement values ​​of a bone graft material according to an embodiment of the present specification.

[0032] Figure 6 is an SEM image of a bone graft material according to one embodiment of the present specification.

[0033] Figure 7 shows the results of BET surface area analysis of bone graft materials according to examples and comparative examples of the present specification.

[0034] Hereinafter, aspects of this specification will be described with reference to the attached drawings. However, the contents of this specification may be implemented in various different forms and are therefore not limited to the embodiments described herein. Furthermore, in the drawings, irrelevant parts have been omitted to clearly illustrate aspects of this specification, and similar parts have been designated with similar drawing reference numerals throughout the specification.

[0035] Throughout the specification, when a part is said to be "connected" to another part, this includes not only "directly connected" but also "indirectly connected" with other elements intervening. Furthermore, when a part is said to "include" a component, this does not exclude other components, but rather implies that the other components may be included, unless otherwise specifically stated.

[0036] When a range of numerical values ​​is stated herein, unless the specific range is otherwise specified, the values ​​have the precision of the significant figures provided according to the standard rules in chemistry for significant figures. For example, the number 10 includes the range of 5.0 to 14.9, and the number 10.0 includes the range of 9.50 to 10.49.

[0037] Hereinafter, one embodiment of the present specification will be described in detail with reference to the attached drawings.

[0038] Method for manufacturing a low-crystalline calcium phosphate-based antibacterial bone graft material

[0039] A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material according to one aspect of the present disclosure comprises: (a) a step of synthesizing calcium phosphate by simultaneously introducing a calcium precursor and a phosphate precursor; (b) a step of reacting the calcium phosphate by introducing the calcium phosphate into an antibacterial precursor solution; (c) a step of freeze-drying the product of step (b); (d) a step of mixing and press-molding the product of step (c) with a shape-controlling agent to produce a molded article; and (e) a step of removing the shape-controlling agent from the molded article in a solvent having a temperature higher than the boiling point and then drying the molded article.

[0040] The term “low crystallinity” as used herein means that the proportion of the apatite structure, which is a crystalline phase, among the phases possessed by the components of the bone graft material is lower than the usual proportion. For example, the low crystallinity may mean that the crystallinity is less than half or less than 25%, but is not limited thereto, and may mean a low crystallinity component recognized within a typical technical range in the art. The “crystallization” can be obtained through X-ray diffraction (XRD) analysis according to the ISO 13779-3 standard.

[0041] The above step (a) is a step for preparing a raw material for a low-crystalline calcium phosphate-based bone graft material.

[0042] The above calcium precursor may include, but is not limited to, one or more selected from the group consisting of calcium nitrate, calcium chloride, calcium fluoride, calcium iodide, calcium acetate, ammonium carbonate, and ammonium bicarbonate.

[0043] The above phosphate precursor may include, but is not limited to, one or more selected from the group consisting of ammonium phosphate monobasic, ammonium phosphate dibasic, ammonium phosphate tribasic, sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic and potassium phosphate.

[0044] In the above step (a), the molar ratio of the calcium precursor and the phosphate precursor may be 1:1.5 to 3. For example, it may be 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, or a range between any two of these values. If it is out of the above range, the reaction may proceed inefficiently, or it may be difficult to manufacture a bone graft material of the desired composition.

[0045] The above step (a) may be a wet precipitation method in which each precursor is dissolved in distilled water and mixed, but is not limited thereto.

[0046] The above wet precipitation method can be performed by adding the precursor dropwise to maintain vacuum conditions, but since the above manufacturing method does not require vacuum conditions as a prerequisite, calcium phosphate can be simply synthesized by batch addition.

[0047] The above calcium phosphate can be used after aging for 1 to 24 hours prior to step (b), filtering, and washing.

[0048] The above step (b) is a step of imparting antibacterial properties to a low-crystalline calcium phosphate-based bone graft material.

[0049] The above antimicrobial substance precursor solution may include, but is not limited to, an antimicrobial substance precursor and distilled water.

[0050] The above antibacterial substance may include, but is not limited to, one or more selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

[0051] The reaction in step (b) may be an ion exchange reaction. For example, the antibacterial substance may be present as a substitute within the crystal of the calcium phosphate.

[0052] The reaction of step (b) above can be carried out at a temperature of 15 to 25°C for 10 to 20 hours. For example, it can be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or a temperature between any two of these temperatures, for 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours or a time between any two of these temperatures.

[0053] The product of step (b) can be used after being washed with ammonia and filtered before step (c).

[0054] The above step (c) is a step of manufacturing the product of the above step (b) into powder.

[0055] The freeze-drying described above may be a method of removing moisture by sublimation under reduced atmospheric pressure at a temperature below room temperature. These freeze-drying conditions are not limited to the temperature and pressure at which ice can sublimate.

[0056] The freeze-drying process can be performed for 36 to 48 hours. Freeze-drying can minimize damage or degeneration of the bone graft. High-temperature drying in step (c) increases the proportion of the crystalline apatite structure, making it difficult to achieve the desired properties.

[0057] The above step (d) is a step of forming a bone graft material.

[0058] The shape-controlling agent may be, but is not limited to, sodium chloride (NaCl). The shape-controlling agent may function as a template for forming the skeleton of the bone graft material during molding and hydrothermal treatment.

[0059] In the above step (d), the weight ratio of the product of the step (c) and the shape regulator may be 1:1 to 1.5. For example, the weight ratio may be 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, 1:1.5, or a weight ratio between any two of these. If the weight ratio of the product of the step (c) and the shape regulator is outside the above range, it may be difficult to remove the shape regulator, or pores or grooves of the bone graft material may not be properly formed, which may ultimately result in reduced bone regeneration and osteoconductivity.

[0060] The average particle diameter of the shape control agent may be 100 to 500 μm. For example, the average particle diameter may be 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, 500 μm, or a range between any two of these values. The area from which the shape control agent is removed may form grooves or pores of the bone graft material. Therefore, if the average particle diameter of the shape-controlling agent satisfies the above range, a low-crystalline calcium phosphate-based antibacterial bone graft material including at least one groove or pore having an average diameter of 100 to 500 ㎛ can be manufactured.

[0061] The above step (e) is a step of removing the shape control agent that acted as a mold and imparting the desired properties to the bone graft material.

[0062] The above step (e) may include, for example, a hydrothermal treatment process for removing a shape control agent present inside and outside the molded body in water having a temperature higher than the boiling point of 100°C. Thereafter, the hydrothermally treated molded body may be dried at a temperature of 250°C or less, 225°C or less, 200°C or less, 175°C or less, 150°C or less, 125°C or less, or 100°C or less to remove moisture.

[0063] After the step (e), the step (f) of heat-treating the molded body at a temperature of 250°C or lower may be further included. The heat treatment may be performed, for example, at a temperature of 250°C, 225°C, 200°C, 175°C, 150°C, 125°C, 100°C, 75°C, 50°C, or a temperature in a range between any two of these values. If the heat treatment temperature exceeds the above range, the surface area may decrease and the degree of crystallinity may increase due to densification caused by grain growth. As a result, the absorbability and osteogenic performance of the bone graft material may deteriorate.

[0064] That is, by controlling the temperature of the heat treatment process, the surface area of ​​the bone graft material can be increased, facilitating the binding and storage of proteins and growth factors. Furthermore, by increasing hemoglobin and clot formation, the adhesion of cells associated with osteogenesis can be enhanced. Ultimately, bone union between the graft material and bone tissue can be promoted.

[0065] Additionally, the absorption rate of the bone graft material can be controlled by adjusting the temperature of the heat treatment process. Performing a relatively high-temperature heat treatment increases the degree of crystallization, thereby slowing down the absorption rate.

[0066] The antimicrobial substance content of the above low-crystalline calcium phosphate-based antimicrobial bone graft material may be 0.2 to 1.8 wt%. For example, it may be 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, or a value between any two of these values. If the antimicrobial substance content is less than the above range, the antimicrobial property of the bone graft material may be reduced, thereby increasing the risk of infection due to bacterial growth. If the antimicrobial substance content exceeds the above range, a secondary phase may be formed in addition to the low-crystalline calcium phosphate, thereby reducing the physical properties.

[0067] In one example, the antimicrobial substance content of the low-crystalline calcium phosphate-based antimicrobial bone graft material may be, but is not limited to, 0.5 to 0.9 wt%. If the antimicrobial substance content is below the above range, the antimicrobial properties of the bone graft material may be reduced. If the antimicrobial substance content exceeds the above range, the antimicrobial substance may crystallize and exist in the form of particles, and an additional washing process may be performed to remove antimicrobial substance particles that have not been substituted into the lattice.

[0068] Low-crystalline calcium phosphate-based antibacterial bone graft material

[0069] According to another aspect of the present specification, a low-crystalline calcium phosphate-based antibacterial bone graft material comprises an antibacterial substance, comprises at least one of grooves and pores having an average diameter of 100 to 500 ㎛, and has a BET specific surface area of ​​100 m 2 / g or more, and contains both crystalline apatite and amorphous calcium phosphate, and may have a crystallinity of 25% or less.

[0070] The above antibacterial substance may include, but is not limited to, one or more selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

[0071] The content of the antibacterial substance may be 0.2 to 1.8 wt% based on the total elements constituting the bone graft material. For example, it may be 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, or a value between any two of these values. If the content of the antibacterial substance is less than the above range, the antibacterial property of the bone graft material may be reduced, thereby increasing the risk of infection due to bacterial growth, and if it exceeds the above range, a secondary phase may be generated in addition to low-crystalline calcium phosphate, thereby reducing the physical properties.

[0072] In one example, the content of the antimicrobial agent may be 0.5 to 0.9 wt% relative to the total elements constituting the bone graft material, but is not limited thereto. If the content of the antimicrobial agent is below the above range, the antimicrobial properties of the bone graft material may be reduced, and if it exceeds the above range, the antimicrobial agent may crystallize and exist in the form of particles.

[0073] The above low-crystalline calcium phosphate-based antibacterial bone graft material may have antibacterial properties against oral single bacteria such as Streptococcus oralis, Porphyromonas gingivalis, and Fusobacterium nucleatum, or a complex of bacteria including a combination thereof.

[0074] The above low-crystalline calcium phosphate-based antibacterial bone graft material can exhibit a bacterial growth inhibition effect of 90% or more compared to a low-crystalline calcium phosphate-based bone graft material that does not contain an antibacterial substance.

[0075] The above low-crystalline calcium phosphate-based antibacterial bone graft material may include at least one concave groove or pore in the center of the particle. These grooves or pores may allow blood cells, osteoblasts, osteoclasts, proteins, or growth factors to be loaded into the bone graft material particles, thereby promoting new blood vessel formation and new bone growth.

[0076] The grooves or pores of the above low-crystalline calcium phosphate-based antibacterial bone graft material may have an average diameter of 100 to 500 μm, and the average diameter may be, for example, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, 500 μm, or a range between two of these values. If the average diameter of the above-mentioned home or pores is outside the above range, the bone formation performance of the bone graft material may be reduced, or the specific surface area or crystallinity may be outside the desired range.

[0077] The term “specific surface area” used in this specification refers to the specific surface area obtained by the BET (Brunauer, Emmett and Teller theory) method, and is an estimated value obtained by measuring the amount of gas (e.g., nitrogen) physically adsorbed on the surface of a sample and calculating the area required for the gas to be adsorbed on a smooth surface.

[0078] The above low-crystalline calcium phosphate bone graft material has a BET specific surface area of ​​100 m 2 / g can be more than 100 m 2 / g, 102 m 2 / g, 104 m 2 / g, 106 m 2 / g, 108 m 2 / g, 110 m 2 / g, 112 m 2 / g, 114 m 2 / g, 116 m 2 / g, 118 m 2 / g, 120 m 2 / g, 122 m 2 / g, 124 m 2 / g, 126 m 2 / g, 128 m 2 / g, 130 m 2 / g, 130 m 2 / g, 132 m2 / g, 134 m 2 / g, 136 m 2 / g, 138 m 2 / g, 140 m 2 / g, 142 m 2 / g, 144 m 2 / g, 146 m 2 / g, 148 m 2 / g, 150 m 2 / g or a range between these two values, but is not limited thereto. BET surface area is 100 m 2 If it is less than 100g, it may be difficult for blood cells, osteoblasts, osteoclasts, proteins, and various growth factors to bind and store. On the other hand, if the BET specific surface area satisfies the above range, blood clot formation is easy, and blood wettability is excellent, so the adhesion of bone-forming cells is excellent, which can promote bone union between the graft material and bone tissue.

[0079] Conventional synthetic bone suffers from a small surface area due to particle growth and densification during the high-temperature sintering process. In contrast, natural bone, due to its microstructural characteristics, has a large surface area and is known to exhibit excellent osteosynthesis. A bone graft material according to one aspect of the present disclosure possesses a high surface area despite being synthetic bone, thereby exhibiting excellent osteosynthesis.

[0080] The above “crystalline apatite” may refer to a calcium phosphate mineral of the hexagonal crystal system. For example, the calcium phosphate mineral may be at least one selected from the group consisting of hydroxyapatite (HA), fluorapatite, chlorapatite, and carbonate apatite, but is not limited thereto.

[0081] For example, the above crystalline apatite may additionally include inorganic ions. The inorganic ions may be Mg 2+ , Sr 2+ , Ba 2+, Mn 2+ , Zn 2+ , Na + , K + , Ag + , Ga 3+ , VO4 3- , SO4 3- , CO3 2- , SiO4 3- , F - , Cl - It may be one or more selected from the group consisting of, but is not limited thereto. The above inorganic ion may be added during the process of synthesizing calcium phosphate from a calcium precursor and a phosphate precursor, but is not limited thereto.

[0082] The above bone graft material may have a crystallinity of 25% or less. For example, the crystallinity may be, but is not limited to, 25%, 24.5%, 24%, 23.5%, 23%, 22.5%, 22%, 21.5%, 21%, 20.5%, 20%, 19.5%, 19%, 18.5%, 18%, 17.5%, 17%, 16.5%, 16%, 15.5%, 15%, 14.5%, 14%, 13.5%, 13%, 12.5%, or a range between any two of these values. If the crystallinity of the bone graft material is outside the above range, the absorption rate may be excessively fast or the bone graft material may not have absorbability.

[0083] Hereinafter, the embodiments of this specification will be described in more detail. However, the experimental results below represent only representative experimental results among the above embodiments, and the scope and content of this specification cannot be interpreted as being reduced or limited by the embodiments, etc. The effects of each of the various implementation examples of this specification that are not explicitly presented below will be specifically described in the relevant sections.

[0084] Example 1

[0085] A calcium ion solution was prepared by dissolving 0.3 M calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), a calcium precursor, in 250 ml of distilled water (H2O). A phosphate ion solution was prepared by dissolving 0.6 M ammonium phosphate dibasic ((NH4)2HPO4), a phosphate precursor, in 500 ml of distilled water, the pH of which was adjusted to 9.5–11.5 with ammonia water. The calcium ion solution and the phosphate ion solution were mixed to prepare a hydroxyapatite precipitate.

[0086] After maturing at 22°C for 6 hours, the hydroxyapatite precipitate was filtered and washed using a suction filter, vacuum pump, and filter paper.

[0087] A silver precursor solution was prepared by dissolving silver nitrate (AgNO3), a silver (Ag) precursor, in 250 ml of distilled water (H2O) so that the silver ion content of the final product, a bone graft material, was 0.2 wt%. The filtered hydroxyapatite precipitate was added to the silver precursor solution, and an ion exchange reaction was performed at 19°C for 16 hours.

[0088] The product of the ion exchange reaction was washed with ammonia and then filtered using a suction filter, vacuum pump, and filter paper.

[0089] After freezing the filtered material, it was freeze-dried for 36 to 48 hours to produce silver-hydroxyapatite powder.

[0090] Sodium chloride (NaCl), a shape control agent, was sieved to a size of 100 to 500 μm, and the silver-hydroxyapatite powder and the sieved sodium chloride were dry mixed at a weight ratio of 1:1 to 1.5.

[0091] The mixed powder was placed in a mold and pressurized to produce a molded article. The manufactured molded article was placed in distilled water and heated for 2 to 4 hours to remove sodium chloride within the molded article.

[0092] The heat-treated molded body was dried at 100°C for 12 to 24 hours, and the dried molded body was pulverized into 0.25 to 2 mm to produce particles.

[0093] The particles were placed in a sieve, soaked in distilled water, and washed for 4 to 8 hours to remove impurities.

[0094] The washed particles were dried at 110°C for 12 to 24 hours to produce a bone graft material.

[0095] Example 2

[0096] A bone graft material was manufactured in the same manner as in Example 1, except that a silver precursor solution was manufactured by dissolving silver nitrate so that the silver ion content of the final product, the bone graft material, was 0.3 wt%.

[0097] Example 3

[0098] A bone graft material was manufactured in the same manner as in Example 1, except that a silver precursor solution was manufactured by dissolving silver nitrate so that the silver ion content of the final product, the bone graft material, was 0.4 wt%.

[0099] Example 4

[0100] A bone graft material was manufactured in the same manner as in Example 1, except that a silver precursor solution was manufactured by dissolving silver nitrate so that the silver ion content of the final product, the bone graft material, was 0.6 wt%.

[0101] Example 5

[0102] A bone graft material was manufactured in the same manner as in Example 1, except that a silver precursor solution was manufactured by dissolving silver nitrate so that the silver ion content of the final product, the bone graft material, was 0.7 wt%.

[0103] Example 6

[0104] A bone graft material was manufactured in the same manner as in Example 1, except that a silver precursor solution was manufactured by dissolving silver nitrate so that the silver ion content of the final product, the bone graft material, was 1.0 wt%.

[0105] Example 7

[0106] A bone graft material was manufactured in the same manner as in Example 1, except that a silver precursor solution was manufactured by dissolving silver nitrate so that the silver ion content of the final product, the bone graft material, was 1.5 wt%.

[0107] Comparative Example 1

[0108] A calcium ion solution was prepared by dissolving 0.3 M calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), a calcium precursor, in 250 ml of distilled water (H2O). A phosphate ion solution was prepared by dissolving 0.6 M ammonium phosphate dibasic ((NH4)2HPO4), a phosphate precursor, in 500 ml of distilled water, the pH of which was adjusted to 9.5–11.5 with ammonia water. The calcium ion solution and the phosphate ion solution were mixed to prepare a hydroxyapatite precipitate.

[0109] After maturing at 22°C for 6 hours, the hydroxyapatite precipitate was filtered and washed using a suction filter, vacuum pump, and filter paper.

[0110] The filtered hydroxyapatite precipitate was frozen and then freeze-dried for 36 to 48 hours to prepare hydroxyapatite powder.

[0111] Sodium chloride (NaCl), a shape control agent, was sieved to a size of 100 to 500 μm, and the hydroxyapatite powder and the sieved sodium chloride were dry mixed at a weight ratio of 1:1 to 1.5.

[0112] The mixed powder was placed in a mold and pressurized to produce a molded article. The manufactured molded article was placed in distilled water and heated for 2 to 4 hours to remove sodium chloride within the molded article.

[0113] The heat-treated molded body was dried at 100°C for 12 to 24 hours, and the dried molded body was pulverized into 0.25 to 2 mm to produce particles.

[0114] The particles were placed in a sieve, soaked in distilled water, and washed for 4 to 8 hours to remove impurities.

[0115] The washed particles were dried at 110°C for 12 to 24 hours to produce a bone graft material.

[0116] Comparative Example 2

[0117] A bone graft material was manufactured in the same manner as in Example 1, except that a silver precursor solution was manufactured by dissolving silver nitrate so that the silver ion content of the final product, the bone graft material, was 1.9 wt%.

[0118] Experimental Example 1: Antibacterial Evaluation

[0119] Streptococcus oralis (5%, 10%, respectively) was attached to each of the bone grafts manufactured in Examples 1 to 5 and Comparative Example 1 for 3 minutes and then removed. The bone grafts with attached bacteria were placed in culture medium and cultured at 37°C for 7 hours. Thereafter, the optical density at 595 nm (OD 595 ) was measured, and the bacterial growth inhibition rate was calculated based on the bone graft material of Comparative Example 1. The results are shown in Fig. 1.

[0120] Referring to Fig. 1, it was confirmed that the bone graft materials of Examples 1 to 5 with added silver exhibited a bacterial growth inhibition effect compared to the bone graft material of Comparative Example 1 without added silver. In particular, the bone graft materials of Examples 4 and 5, in which the silver ion content was 0.6 wt% and 0.7 wt%, respectively, relative to calcium ions, exhibited an excellent bacterial growth inhibition rate of 90% or more compared to the bone graft material of Comparative Example 1 without added silver.

[0121] Experimental Example 2: Cytotoxicity Evaluation

[0122] The bone graft effluents prepared in Examples 2 to 7 and Comparative Example 1 were dispensed onto fibroblast L929 cells and cultured for 24 hours. Cell viability was then confirmed using the CCK-8 assay. The results are shown in Fig. 2.

[0123] Referring to Fig. 2, the bone graft materials of Examples 2 to 7 and Comparative Example 1 all showed a high cell viability of 90% or more, confirming that they were free of cytotoxicity.

[0124] Experimental Example 3: XRD Analysis

[0125] In order to confirm the crystal structure of the bone graft materials manufactured in Examples 1 to 7 and Comparative Examples 1 and 2, X-ray diffraction (XRD) analysis was performed according to the ISO 13779-3 standard, and the results are shown in Fig. 3. In addition, the values ​​obtained by XRD analysis were substituted into the following calculation formula 1 to calculate the degree of crystallinity, and the results are shown in Fig. 4.

[0126] <Calculation Formula 1>

[0127]

[0128] Referring to FIG. 3, it was confirmed that the bone graft materials of Examples 1 to 7 had no significant difference in crystal structure from the bone graft material of Comparative Example 1 without silver addition. On the other hand, it was confirmed that the bone graft material of Comparative Example 2 with a silver ion content of 1.9 wt% formed a secondary phase in addition to low-crystalline calcium phosphate.

[0129] Referring to Fig. 4, it was confirmed that all of the bone graft materials of Examples 1 to 7 and Comparative Example 1 had a low crystallinity of 25% or less.

[0130] Experimental Example 4: SEM / EDS Analysis

[0131] In order to confirm whether the silver component exists in the form of particles or is substituted within the apatite crystal, the bone graft materials manufactured in Examples 1 to 6 were photographed using a scanning electron microscope (SEM), and energy-dispersive X-ray spectroscopy (EDS) was performed. The results of the EDS analysis are shown in Fig. 5 and Table 1 below. Fig. 6 is an SEM image of the bone graft material manufactured in Example 6.

[0132] Ag Content (at%) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 1000.06 0.11 0.07 3.9 2000.28 0.08 0.08 2.74 3000.09 0.10 07 1.35 4000.15 0.07 0.09 1.16 5000.10 0.07 0.12 0.14 60000.10 150.29 70000.07 3.73 0.23

[0133] Referring to FIG. 5, no silver particles were present in the bone graft materials of Examples 1 to 4, and a small amount of silver particles were present in the bone graft material of Example 5.

[0134] Referring to Table 1 above, it was confirmed that in Examples 1 to 4, no significant concentration of silver particles was observed, but in Example 5, some silver particles were crystallized outside the bone graft material, showing a high Ag Content, and this trend was more prominent in Example 6.

[0135] In particular, referring to FIGS. 5 and 6, crystallized silver particles were present in the bone graft material of Example 6, which had a high silver ion content of 1.0 wt%.

[0136] Through this, it was confirmed that the silver component added in Examples 1 to 6 existed as a substitute within the apatite crystal, and it was confirmed that when the amount of silver added increased, the saturated silver particles were not substituted within the lattice and were crystallized.

[0137] Experimental Example 5: ICP Analysis

[0138] In order to confirm the content of silver ions contained in the bone graft material, inductively coupled plasma (ICP) analysis was performed on the bone graft materials manufactured in Examples 1 to 7 and Comparative Examples 1 and 2. The results are shown in Table 2 below. The silver ion content (weight %) below is calculated by calculating the weight ratio of silver ions to the total elements constituting the bone graft material.

[0139] Ingredients Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Comparative Example 2 Silver ion content (wt%) 0.2 0.3 0.4 0.6 0.7 1.0 1.5 0 1.9

[0140] Referring to Table 2 above, it was confirmed that the bone graft materials of Examples 1 to 7 and Comparative Example 2 all exhibited the target silver ion content. The silver ion content included in the bone graft materials of Examples 1 to 7 increased in proportion to the amount of silver added.

[0141] Experimental Example 6: Surface Area Analysis

[0142] The specific surface area of ​​the bone graft material manufactured in Example 4 and Comparative Example 1 was analyzed according to the BET method using nitrogen gas, and the results are shown in Fig. 7.

[0143] Referring to Figure 7, both bone graft materials of Example 4 and Comparative Example 1 were 100 m 2 It was confirmed that it exhibited a high BET surface area of ​​ / g or more.

[0144] The description of this specification above is for illustrative purposes only, and those skilled in the art will readily appreciate that aspects of this specification can be readily modified into other specific forms without altering the technical concepts or essential features described herein. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0145] The scope of this specification is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included within the scope of this specification.

Claims

1. (a) A step of synthesizing calcium phosphate by simultaneously introducing calcium precursor and phosphate precursor; (b) a step of reacting the calcium phosphate by adding it to an antibacterial substance precursor solution; (c) a step of freeze-drying the product of step (b); (d) a step of mixing and pressurizing the product of step (c) and a shape control agent to produce a molded body; and (e) A method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material, comprising the step of removing the shape-controlling agent of the molded body from a solvent having a temperature higher than the boiling point and then drying it.

2. In paragraph 1, A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the calcium precursor comprises at least one selected from the group consisting of calcium nitrate, calcium chloride, calcium fluoride, calcium iodide, calcium acetate, ammonium carbonate, and ammonium bicarbonate.

3. In paragraph 1, A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the phosphate precursor comprises at least one selected from the group consisting of ammonium phosphate monobasic, ammonium phosphate dibasic, ammonium phosphate tribasic, sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, and potassium phosphate.

4. In paragraph 1, A method for producing a low-crystallization calcium phosphate-based antibacterial bone graft material, wherein in the step (a), the molar ratio of the calcium precursor and the phosphate precursor is 1:1.5 to 3.

5. In paragraph 1, The above step (a) is a wet precipitation method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material by dissolving and mixing each precursor in distilled water.

6. In paragraph 1, A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the antibacterial substance comprises at least one selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

7. In paragraph 1, A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the reaction of step (b) is performed at a temperature of 15 to 25°C for 10 to 20 hours.

8. In paragraph 1, A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material, wherein in the step (d), the weight ratio of the product of the step (c) and the shape-controlling agent is 1:1 to 1.

5.

9. In paragraph 1, A method for manufacturing a low-crystallization calcium phosphate-based antibacterial bone graft material, wherein the average particle size of the above-mentioned shape-controlling agent is 100 to 500 ㎛.

10. In paragraph 1, After step (e) above, (f) A method for manufacturing a low-crystalline calcium phosphate-based antibacterial bone graft material, further comprising a step of heat-treating the molded body at a temperature of 250°C or lower.

11. In paragraph 1, A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the antibacterial substance content of the low-crystalline calcium phosphate-based antibacterial bone graft material is 0.2 to 1.8 wt%.

12. In paragraph 1, A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the antibacterial substance content of the low-crystalline calcium phosphate-based antibacterial bone graft material is 0.5 to 0.9 wt%.

13. Contains antibacterial substances, Containing at least one groove and pore having an average diameter of 100 to 500 ㎛, BET surface area is 100 m 2 / g or more, Contains both crystalline apatite and amorphous calcium phosphate, A low-crystallization calcium phosphate-based antibacterial bone graft material with a crystallinity of 25% or less.

14. In paragraph 13, The above antibacterial substance is a low-crystalline calcium phosphate-based antibacterial bone graft material comprising at least one selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

15. In paragraph 13, A low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the content of the above antibacterial substance is 0.2 to 1.8 wt% of the total elements constituting the bone graft material.

16. In paragraph 13, A low-crystalline calcium phosphate-based antibacterial bone graft material, wherein the content of the above-mentioned antibacterial substance is 0.5 to 0.9 wt% relative to the total elements constituting the bone graft material.

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