Low-crystallinity calcium phosphate-based antibacterial bone graft material and method for producing the same

Abstract

One embodiment of this specification provides a method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material, comprising: (a) a step of synthesizing calcium phosphate by adding a calcium precursor and a phosphate precursor together; (b) a step of adding the calcium phosphate to an antibacterial substance precursor solution and reacting it; (c) a step of freeze-drying the product of step (b); (d) a step of mixing the product of step (c) with a shape control agent and press-molding to produce a molded body; and (e) a step of removing the shape control agent from the molded body with a solvent at a temperature above its boiling point and then drying it.

Description

[Technical Field]

[0001] This specification relates to a low-crystallinity calcium phosphate-based antibacterial bone graft material and a method for producing the same. [Background technology]

[0002] Bone graft material is a material used to regenerate bone that has been absorbed or destroyed due to factors such as disease, trauma, or aging. For example, during dental treatments such as implant placement, surgery, or periodontal surgery, bone graft material may be used to artificially create alveolar bone in a bone grafting procedure.

[0003] Bone graft materials play a role in stimulating bone growth, and their mechanisms of action can be broadly classified into osteoconduction, osteoinduction, and osteoogenesis. Osteoconduction is the mechanism by which osteoblasts migrate from surrounding bone tissue to form bone, and bone tissue or differentiated mesenchymal cells are essential at the graft site. Osteoinduction refers to the mechanism by which undifferentiated mesenchymal cells influence the differentiation of bone progenitor 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 classified into autogenous bone, allogenic bone, heterogeneous bone, and alloplastic bone grafts based on their origin. Autogenous bone grafts utilize bone tissue harvested from the recipient and possess all three properties: osteogenic, osteoconductive, and osteoinductive. They are the only bone grafts currently available that possess osteogenic properties. However, they require additional surgical procedures for bone tissue harvesting, which incurs costs and time, and most importantly, the amount of bone that can be used is limited. Allogenic bone grafts are typically obtained by harvesting bone tissue from cadavers or by receiving bone donations from deceased individuals. Because they are derived from genetically identical species, they possess equivalent levels of healing ability and excel in bone replacement due to their absorbability and osteogenic properties. Heterogeneous bone grafts are harvested from individuals of other species, mainly using hydroxyapatite obtained from cattle (bovine) or pigs (porcine). While various components, including calcium phosphate ceramics, are used as synthetic bone graft materials, they are generally known to have inferior bone formation ability and bone quality compared to autogenous bone or allogeneic bone graft materials.

[0005] Hydroxyapatite (HA) and tricalcium phosphate (TCP) are the main calcium phosphate-based biomaterials used as synthetic bone graft materials. However, hydroxyapatite has the disadvantage of being non-absorbable and remaining in the body for a long period of time, and tricalcium phosphate has the problem of an imbalance between the rate of bone formation and the rate of degradation of the graft material.

[0006] Furthermore, if the site of bone grafting becomes infected, bone recovery can be difficult and may result in bone loss, requiring complex recovery methods. In particular, dental bone grafts are known to have a high risk of bacterial infection due to the oral environment.

[0007] Therefore, there is a need to develop a synthetic bone graft material that is absorbable yet possesses a similar level of osteogenesis as allogeneic bone, and that can actively control infection by suppressing bacterial growth within the graft material itself. [Overview of the project]

Problems to be Solved by the Invention

[0008] The description in this specification is for solving the problems of the above-mentioned prior art. One object of this specification is to provide a low-crystalline calcium phosphate-based antibacterial bone graft material excellent in antibacterial property and bone-forming ability, and a method for producing the same.

Means for Solving the Problems

[0009] According to one aspect, a method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material is provided, including: (a) a step of synthesizing calcium phosphate by batch charging a calcium precursor and a phosphate precursor; (b) a step of charging the calcium phosphate into an antibacterial substance precursor solution and reacting; (c) a step of freeze-drying the product of step (b); (d) a step of mixing and pressure-molding the product of step (c) and a shape control agent to produce a molded body; and (e) a step of removing the shape control agent of the molded body with a solvent at a temperature above the boiling point and then drying.

[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 one or more selected from the group consisting of phosphorus oxide, monoammonium phosphate, diammonium phosphate, triammonium phosphate, monosodium phosphate, disodium phosphate, trisodium phosphate, and potassium phosphate.

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

[0013] In one embodiment, the 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 substance can 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 in the step (b) can be carried out at a temperature of 15 to 25 °C for 10 to 20 hours.

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

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

[0018] In one embodiment, after the step (e), it can further include (f) a step of heat-treating the formed body at a temperature of 250 °C or lower.

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

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

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

[0022] In one embodiment, the antibacterial substance can 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 antimicrobial substance may be 0.2 to 1.8% by weight relative to the total elements constituting the bone graft material.

[0024] In one embodiment, the content of the antimicrobial substance may be 0.5 to 0.9% by weight relative to the total elements constituting the bone graft material. [Effects of the Invention]

[0025] One aspect of the method for producing a low-crystallinity calcium phosphate antibacterial bone graft material described herein can be applied to the production of a low-crystallinity calcium phosphate antibacterial bone graft material with excellent antibacterial properties and bone formation ability.

[0026] Furthermore, the low-crystallinity calcium phosphate-based antibacterial bone graft material according to another aspect of this specification exhibits excellent antibacterial properties, reducing the risk of infection due to bacterial growth, while simultaneously possessing absorbability and osteoconductivity, and demonstrating bone formation capabilities equivalent to those of allogeneic bone.

[0027] The effects of one aspect of this specification should be understood to include all effects that can be inferred from the detailed description or claims herein, not limited to those described above. [Brief explanation of the drawing]

[0028] [Figure 1] These are the results of the antibacterial evaluation of bone graft materials based on the examples and comparative examples described herein. [Figure 2] These are the results of the cytotoxicity evaluation of bone graft materials based on the examples and comparative examples described herein. [Figure 3] These are the XRD analysis results of bone graft materials according to the examples and comparative examples described herein. [Figure 4] This is the degree of crystallinity based on the XRD analysis results of bone graft materials according to the examples and comparative examples described herein. [Figure 5] This is a variance graph of EDS measurements for bone graft materials according to the examples described herein. [Figure 6] This is a SEM image of a bone graft material according to one embodiment described herein. [Figure 7] This shows the BET specific surface area analysis results of bone graft materials according to the examples and comparative examples described herein. [Modes for carrying out the invention]

[0029] The following describes one aspect of this specification with reference to the attached drawings. However, the provisions of this specification can be embodied in various different forms and are not limited to the embodiments described herein. In order to clearly illustrate one aspect of this specification, parts unrelated to the description have been omitted in the drawings, and similar parts throughout the specification are denoted by similar reference numerals.

[0030] Throughout the specification, when a part is described as being "connected" to another part, this includes not only cases where they are "directly connected," but also cases where they are "indirectly connected" through other components in between. Furthermore, when a part is described as "containing" a certain component, this means, unless otherwise stated, that it may further contain other components, rather than excluding them.

[0031] When a range of numerical values ​​is described herein, unless otherwise specified, the value shall have the precision of significant figures provided in accordance with the standard rules of significant figures in chemistry. For example, 10 includes the range of 5.0 to 14.9, and the figure 10.0 includes the range of 9.50 to 10.49.

[0032] An embodiment of this specification will be described in detail below with reference to the attached drawings.

[0033] Method for manufacturing low-crystallinity calcium phosphate-based antibacterial bone graft material

[0034] A method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to one aspect of this specification includes: (a) a step of synthesizing calcium phosphate by adding a calcium precursor and a phosphate precursor together; (b) a step of adding the calcium phosphate to an antibacterial substance precursor solution and allowing it to react; (c) a step of freeze-drying the product of step (b); (d) a step of mixing the product of step (c) with a shape control agent and press-molding to produce a molded body; and (e) a step of removing the shape control agent from the molded body with a solvent at a temperature above its boiling point and then drying it.

[0035] As used herein, the term "low crystallinity" refers to a lower proportion of the apatite structure, which is a crystalline phase, among the phases of the bone graft material's components. For example, low crystallinity may mean, but is not limited to, a crystallinity of 50% or less or 25% or less, and may refer to low crystallinity components recognized within the normal technical range in this industry. The "crystallinity" can be determined by X-ray diffraction (XRD) analysis in accordance with ISO 13779-3.

[0036] The aforementioned step (a) is a step of preparing the raw materials for a low-crystallinity calcium phosphate bone graft material.

[0037] The 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.

[0038] The phosphate precursor may include, but is not limited to, one or more selected from the group consisting of phosphorus oxide, monoammonium phosphate, diammonium phosphate, triammonium phosphate, monosodium phosphate, disodium phosphate, trisodium phosphate, and potassium phosphate.

[0039] In step (a) above, 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 two of these values. If the ratio is outside this range, the reaction may proceed inefficiently, or it may be difficult to produce a bone graft material with the desired composition.

[0040] The above step (a) may be, but is not limited to, a wet precipitation method in which each precursor is dissolved in distilled water and mixed.

[0041] The aforementioned wet precipitation method can be carried out by dropping the precursor dropwise to maintain vacuum conditions, but since the above manufacturing method does not require vacuum conditions as a necessary condition, calcium phosphate can be easily synthesized by adding the precursor all at once.

[0042] The calcium phosphate can be used after being aged for 1 to 24 hours before step (b), followed by filtration and washing.

[0043] The aforementioned step (b) is a step of imparting antibacterial properties to a low-crystallinity calcium phosphate-based bone graft material.

[0044] The aforementioned antimicrobial substance precursor solution may, but is not limited to, contain an antimicrobial substance precursor and distilled water.

[0045] The aforementioned antimicrobial substance may include, but is not limited to, one or more substances selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

[0046] The reaction in step (b) above may be an ion exchange reaction. For example, the antimicrobial substance may exist substituted within the calcium phosphate crystals.

[0047] The reaction in step (b) above may be carried out at a temperature of 15 to 25°C for 10 to 20 hours. For example, it may 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 at a temperature between 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 at a temperature between two of these times.

[0048] The product of step (b) can be washed with ammonia before step (c), filtered, and then used.

[0049] The (c) step is a step of producing the product of the (b) step as a powder.

[0050] The aforementioned freeze-drying method may involve reducing atmospheric pressure at temperatures below room temperature to sublimate and remove moisture. Such freeze-drying conditions are not limited as long as they are at a temperature and pressure at which ice can sublimate.

[0051] The freeze-drying can be carried out for 36 to 48 hours. Freeze-drying can minimize damage or degeneration of the bone graft material. If high-temperature drying is performed in step (c), the proportion of the apatite structure, which is the crystalline phase, increases, which may make it difficult to achieve the desired properties.

[0052] The aforementioned step (d) is a step of shaping the bone graft material.

[0053] The shape-controlling agent may be, but is not limited to, sodium chloride (NaCl). The shape-controlling agent can function as a template for forming the skeleton of the bone graft material during molding and hydrothermal treatment.

[0054] In step (d), the weight ratio of the product of step (c) to the shape-controlling agent may be 1:1 to 1.5. For example, it 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 two of these weight ratios. If the weight ratio of the product of step (c) to the shape-controlling agent falls outside this range, it may be difficult to remove the shape-controlling agent, or the pores or grooves of the bone graft material may not be properly formed, ultimately resulting in a decrease in bone regeneration and osteoconduction ability.

[0055] The average particle size of the shape-controlling agent may be between 100 and 500 μm. For example, it 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 two of these values. The area from which the shape-controlling agent has been removed can form grooves or pores in the bone graft material. Therefore, if the average particle size of the shape-controlling agent satisfies the above range, a low-crystallinity calcium phosphate-based antibacterial bone graft material containing at least one groove or pore with an average diameter of 100 to 500 μm can be manufactured.

[0056] The aforementioned step (e) is a step of removing the shape-controlling agent that acted as a mold and imparting the desired properties to the bone graft material.

[0057] The (e) step may include, for example, a hydrothermal treatment step of removing shape-controlling agents present inside and outside the molded body with water at a temperature of 100°C, which is above its boiling point. The hydrothermal treated molded body can then be dried to remove moisture at a temperature of 250°C or lower, 225°C or lower, 200°C or lower, 175°C or lower, 150°C or lower, 125°C or lower, or 100°C or lower.

[0058] The process may further include (f) heat-treating the molded body at a temperature of 250°C or less, after step (e). The heat treatment may be carried out at a temperature in the range of 250°C, 225°C, 200°C, 175°C, 150°C, 125°C, 100°C, 75°C, 50°C, or between two of these values. If the heat treatment temperature exceeds the above range, the crystallinity may increase while the surface area decreases due to densification by grain growth. As a result, the absorbability and bone formation ability of the bone graft material may decrease.

[0059] In other words, by adjusting the temperature during the heat treatment process, the surface area of ​​the bone graft material is increased, facilitating the binding and preservation of proteins and growth factors, enhancing blood wettability and clot formation, and improving the adhesion of cells related to bone formation. Ultimately, this can promote bone fusion between the graft material and bone tissue.

[0060] Furthermore, the absorption rate of the bone graft material can be controlled by adjusting the temperature during the heat treatment process. Relatively high-temperature heat treatment increases the degree of crystallinity, which slows down the absorption rate.

[0061] The antimicrobial substance content of the low-crystallinity calcium phosphate-based antimicrobial bone graft material may be 0.2 to 1.8% by weight. For example, it may be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, or a value between two of these values. If the antimicrobial substance content is below the above range, the antimicrobial properties of the bone graft material may decrease, and the risk of infection due to bacterial growth may increase. If the antimicrobial substance content exceeds the above range, a secondary phase may be formed in addition to low-crystallinity calcium phosphate, and the physical properties may deteriorate.

[0062] In one example, the antimicrobial substance content of the low-crystallinity calcium phosphate antimicrobial bone graft material may be 0.5 to 0.9% by weight, but is not limited thereto. If the antimicrobial substance content is below the above range, the antimicrobial properties of the bone graft material may decrease. If the antimicrobial substance content exceeds the above range, the antimicrobial substance may crystallize and exist in particulate form, and an additional washing step may be performed to remove the antimicrobial substance particles that were not replaced within the lattice.

[0063] Low-crystallinity calcium phosphate antibacterial bone graft material

[0064] Another aspect of this specification, the low-crystallinity calcium phosphate antimicrobial bone graft material contains an antimicrobial substance and comprises at least one of grooves and pores with an average diameter of 100 to 500 μm, and has a BET specific surface area of ​​100 m². 2 It is greater than or equal to / g, contains both crystalline apatite and amorphous calcium phosphate, and may have a crystallinity of 25% or less.

[0065] The aforementioned antimicrobial substance may include, but is not limited to, one or more substances selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

[0066] The content of the antimicrobial substance may be 0.2 to 1.8% by weight relative to the total elements constituting the bone graft material. For example, it may be 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, or a value between two of these values. If the content of the antimicrobial substance is below the above range, the antimicrobial properties of the bone graft material may decrease, and the risk of infection due to bacterial growth may increase. If it exceeds the above range, a secondary phase may be formed in addition to low-crystallinity calcium phosphate, and the physical properties may deteriorate.

[0067] In one example, the content of the antimicrobial substance may be 0.5 to 0.9% by weight relative to the total elements constituting the bone graft material, but is not limited thereto. If the content of the antimicrobial substance is below the above range, the antimicrobial properties of the bone graft material may decrease, and if it exceeds the above range, the antimicrobial substance may crystallize and exist in particulate form.

[0068] The aforementioned low-crystallinity calcium phosphate antibacterial bone graft material may have antibacterial activity against oral single bacteria such as Streptococcus oralis, Porphyromonas gingivalis, and Fusobacterium nucleatum, or complex bacteria including combinations thereof.

[0069] The aforementioned low-crystallinity calcium phosphate antibacterial bone graft material can exhibit a bacterial growth inhibitory effect of 90% or more compared to low-crystallinity calcium phosphate bone graft materials that do not contain antibacterial substances.

[0070] The aforementioned low-crystallinity calcium phosphate antibacterial bone graft material may contain at least one of the following: a groove or pore recessed in the center of the particle. Such grooves or pores can promote neovascularization and the growth of new bone by allowing blood cells, osteoblasts, osteoclasts, proteins, or growth factors to be carried on the bone graft material particles.

[0071] The grooves or pores of the low-crystallinity calcium phosphate 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 grooves or pores falls outside this range, the bone-forming ability of the bone graft material may decrease, or the specific surface area or crystallinity may fall outside the desired range.

[0072] As used herein, the term "specific surface area" means the specific surface area determined by the BET (Brunauer, Emmett and Teller theory) method, which measures the amount of gas (e.g., nitrogen) physically adsorbed on the surface of a sample and calculates and estimates the area required for the gas to be adsorbed on a smooth surface.

[0073] The low-crystalline calcium phosphate-based bone graft material may have a BET specific surface area of 100 m 2 / g or more. For example, 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 m 2 / 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 may be in the range between two of these values, but is not limited thereto. The BET specific surface area is 100 m 2If the BET specific surface area is less than / g, blood cells, osteoblasts, osteoclasts, proteins, or various growth factors may not bind to and preserve well. On the other hand, if the BET specific surface area meets the above range, blood clot formation is easy, blood wettability is excellent, adhesion of bone formation-related cells is excellent, and bone fusion between the graft material and bone tissue can be promoted.

[0074] Conventional synthetic bone has the disadvantage of having a small surface area because the particles grow and become dense during the high-temperature sintering process. On the other hand, natural bone is known to have a large surface area due to its microstructure characteristics, and thus excellent bone healing properties. The bone graft material according to one aspect of this specification, while being synthetic bone, has a high surface area and can therefore have excellent bone healing properties.

[0075] The term "crystalline apatite" may refer to a hexagonal inorganic calcium phosphate. For example, the inorganic calcium phosphate may be one or more selected from the group consisting of hydroxyapatite (HA), fluoroapatite, chloroapatite, and carbonate apatite, but is not limited to these.

[0076] As an example, the crystalline apatite may contain additional inorganic ions.

[0077] The aforementioned inorganic ion is Mg 2+ Sr 2+ Ba 2+ Mn 2+ Zn 2+ na + , K + Ag + , Ga 3+ VO4 3- SO4 2- CO3 2- SiO4 4- F - Cl -The inorganic ions may be one or more selected from the group consisting of the following, but are not limited to these. The inorganic ions may be added in the process of synthesizing calcium phosphate from calcium precursors and phosphate precursors, but are not limited to this.

[0078] The bone graft material may have a crystallinity of 25% or less. For example, it 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 two of these values. If the crystallinity of the bone graft material falls outside this range, the absorption rate may become excessively fast, or it may not be absorbable at all.

[0079] The examples of this specification will be described in more detail below. However, the experimental results described below represent only representative results from the aforementioned examples, and the scope and content of this specification should not be narrowed or limited by these examples. The effects of various examples of this specification that are not explicitly presented below will be described specifically in the relevant sections. [Examples]

[0080] Example 1

[0081] A calcium ion solution was prepared by dissolving 0.3 M of 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 of diammonium hydrogen phosphate ((NH4)2HPO4), a phosphate precursor, in 500 ml of distilled water whose pH was adjusted to 9.5-11.5 with ammonia water. A hydroxyapatite precipitate was prepared by mixing the calcium ion solution and the phosphate ion solution.

[0082] After aging at 22°C for 6 hours, the hydroxyapatite precipitate was filtered and washed using a suction filter, vacuum pump, and filter paper.

[0083] 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 bone graft material was 0.2% by weight. The filtered hydroxyapatite precipitate was then added to the silver precursor solution, and an ion exchange reaction was carried out at 19°C for 16 hours.

[0084] After washing the ion exchange reaction products with ammonia, they were filtered using a suction filter, vacuum pump, and filter paper.

[0085] After freezing the filtered material, silver-hydroxyapatite powder was produced by freeze-drying it for 36-48 hours.

[0086] Sodium chloride (NaCl), a shape-controlling agent, was sieved to a size of 100-500 μm, and the sieved sodium chloride was dry-mixed with the silver-hydroxyapatite powder in a weight ratio of 1:1-1.5. The mixed powder was placed in a mold and pressurized to produce a molded body. The produced molded body was placed in distilled water and heated for 2 to 4 hours to remove the sodium chloride inside the molded body.

[0087] The hydrothermally treated molded body was dried at 100°C for 12 to 24 hours, and the dried molded body was pulverized to 0.25 to 2 mm to produce particles. The aforementioned particles were placed in a sieve and immersed in distilled water, and washed for 4 to 8 hours by running the distilled water over them to remove impurities.

[0088] The washed particles were dried at 110°C for 12 to 24 hours to produce bone graft material.

[0089] Example 2

[0090] Bone graft material was manufactured in the same manner as in Example 1, except that silver nitrate was dissolved to produce a silver precursor solution so that the silver ion content of the final bone graft material was 0.3% by weight.

[0091] Example 3

[0092] Bone graft material was manufactured in the same manner as in Example 1, except that silver nitrate was dissolved to produce a silver precursor solution so that the silver ion content of the final bone graft material was 0.4% by weight.

[0093] Example 4

[0094] Bone graft material was manufactured in the same manner as in Example 1, except that silver nitrate was dissolved to produce a silver precursor solution so that the silver ion content of the final bone graft material was 0.6% by weight.

[0095] Example 5

[0096] Bone graft material was prepared in the same manner as in Example 1, except that silver nitrate was dissolved to produce a silver precursor solution so that the silver ion content of the final bone graft material was 0.7% by weight.

[0097] Example 6

[0098] Bone graft material was prepared in the same manner as in Example 1, except that silver nitrate was dissolved to produce a silver precursor solution so that the silver ion content of the final bone graft material was 1.0% by weight.

[0099] Example 7

[0100] Bone graft material was manufactured in the same manner as in Example 1, except that silver nitrate was dissolved to produce a silver precursor solution so that the silver ion content of the final bone graft material was 1.5% by weight.

[0101] Comparative Example 1

[0102] A calcium ion solution was prepared by dissolving 0.3 M of 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 of diammonium hydrogen phosphate ((NH4)2HPO4), a phosphate precursor, in 500 ml of distilled water whose pH was adjusted to 9.5-11.5 with ammonia water. A hydroxyapatite precipitate was prepared by mixing the calcium ion solution and the phosphate ion solution.

[0103] After aging at 22°C for 6 hours, the hydroxyapatite precipitate was filtered and washed using a suction filter, vacuum pump, and filter paper.

[0104] After freezing the filtered hydroxyapatite precipitate, hydroxyapatite powder was produced by freeze-drying for 36-48 hours.

[0105] Sodium chloride (NaCl), a shape-controlling agent, was sieved to a size of 100-500 μm, and the sieved sodium chloride was dry-mixed with the hydroxyapatite powder in a weight ratio of 1:1-1.5.

[0106] The mixed powder was placed in a mold and pressurized to produce a molded body. The produced molded body was placed in distilled water and heated for 2 to 4 hours to remove the sodium chloride inside the molded body.

[0107] The hydrothermally treated molded body was dried at 100°C for 12 to 24 hours, and the dried molded body was pulverized to 0.25 to 2 mm to produce particles.

[0108] The aforementioned particles were placed in a sieve and immersed in distilled water, and washed for 4 to 8 hours by running the distilled water over them to remove impurities.

[0109] The washed particles were dried at 110°C for 12 to 24 hours to produce bone graft material.

[0110] Comparative Example 2

[0111] Bone graft material was prepared in the same manner as in Example 1, except that silver nitrate was dissolved to produce a silver precursor solution so that the silver ion content of the final bone graft material was 1.9% by weight.

[0112] Experimental Example 1: Antibacterial Evaluation

[0113] Streptococcus oralis (5%) and 10% respectively were applied to the bone graft materials prepared in Examples 1-5 and Comparative Example 1 for 3 minutes, and then removed. The bone graft materials contaminated with bacteria were then loaded onto a culture medium and incubated at 37°C for 7 hours. Subsequently, the optical density (OD) at 595 nm was measured. 595 The bacterial growth inhibition rate was calculated by measuring the bacterial growth inhibition rate of the bone graft material of Comparative Example 1. The results are shown in Figure 1.

[0114] Referring to Figure 1, it was confirmed that the bone graft materials of Examples 1 to 5, which contained silver, showed a greater inhibitory effect on bacterial growth compared to the bone graft material of Comparative Example 1, which did not contain silver. In particular, the bone graft materials of Examples 4 and 5, which contained 0.6% by weight and 0.7% by weight of silver ions relative to calcium ions, showed a superior bacterial growth inhibition rate of over 90% compared to the bone graft material of Comparative Example 1, which did not contain silver.

[0115] Experimental Example 2: Cytotoxicity Evaluation

[0116] The eluate of the bone graft material prepared in Examples 2-7 and Comparative Example 1 was dispensed into fibroblast L929 cells and cultured for 24 hours. Cell viability was then confirmed using the CCK-8 analytical method. The results are shown in Figure 2.

[0117] Referring to Figure 2, the bone graft materials used in Examples 2-7 and Comparative Example 1 all showed high cell viability rates of over 90%, confirming that they were non-cytotoxic.

[0118] Experimental Example 3: XRD Analysis

[0119] To confirm the crystalline structure of the bone graft materials produced in Examples 1-7 and Comparative Examples 1 and 2, X-ray diffraction (XRD) analysis was performed according to ISO 13779-3, and the results are shown in Figure 3. Furthermore, the degree of crystallinity was calculated by substituting the values ​​obtained from the XRD analysis into the following formula 1, and the results are shown in Figure 4.

[0120] <Formula 1>

[0121] Crystallinity = (Sum of 10 peak intensities of the sample / Sum of 10 peak intensities of the HA standard) × 100

[0122] Referring to Figure 3, it was confirmed that the bone graft materials of Examples 1 to 7 did not differ significantly in crystalline structure from the bone graft material of Comparative Example 1, which did not contain silver. On the other hand, it was confirmed that the bone graft material of Comparative Example 2, which had a silver ion content of 1.9% by weight, generated a secondary phase in addition to low-crystallinity calcium phosphate.

[0123] Referring to Figure 4, it was confirmed that the bone graft materials of Examples 1-7 and Comparative Example 1 all had a low degree of crystallinity of 25% or less.

[0124] Experimental Example 4: SEM / EDS Analysis

[0125] To determine whether the silver component exists as particles or is substituted within the apatite crystal, the bone graft materials prepared in Examples 1 to 6 were imaged using a scanning electron microscope (SEM) and subjected to energy-dispersive X-ray spectroscopy (EDS). The EDS analysis results are shown in Figure 5 and Table 1 below. Figure 6 is an SEM image of the bone graft material prepared in Example 6. [Table 1]

[0126] Referring to Figure 5, the bone graft materials of Examples 1-4 did not contain silver particles, while the bone graft material of Example 5 contained a small amount of silver particles.

[0127] As can be seen in Table 1 below, no significant concentrations of silver particles were observed in Examples 1-4, but in Example 5, some silver particles crystallized outside the bone graft material, resulting in a high Ag content. This trend was confirmed to be even more pronounced in Example 6.

[0128] In particular, as can be seen in Figures 5 and 6, the bone graft material of Example 6, which had a high silver ion content of 1.0% by weight, contained crystallized silver particles.

[0129] Through this, it was confirmed that the silver components added in Examples 1 to 6 were substituted into the apatite crystals, however, it was confirmed that when the amount of silver added increased, saturated silver particles did not substituted into the lattice but instead crystallized.

[0130] Experimental Example 5: ICP Analysis

[0131] To confirm the silver ion content in the bone graft material, inductively coupled plasma (ICP) analysis was performed on the bone graft materials prepared in Examples 1-7 and Comparative Examples 1 and 2. The results are shown in Table 2 below. The silver ion content (weight %) below is calculated as the weight ratio of silver ions to all elements constituting the bone graft material. [Table 2]

[0132] Referring to Table 2 above, it was confirmed that the bone graft materials of Examples 1-7 and Comparative Example 2 all exhibited the target silver ion content. The silver ion content in the bone graft materials of Examples 1-7 increased in proportion to the amount of silver added.

[0133] Experimental Example 6: Specific Surface Area Analysis

[0134] The specific surface area of ​​the bone graft materials produced in Example 4 and Comparative Example 1 was analyzed using the BET method with nitrogen gas, and the results are shown in Figure 7.

[0135] Referring to Figure 7, the bone graft material in both Example 4 and Comparative Example 1 is 100 m 2 We confirmed that it exhibits a high BET specific surface area of ​​1 / g or more.

[0136] The descriptions herein, as set forth above, are illustrative, and those with ordinary skill in the art to which one aspect of this specification belongs will understand that it is possible to easily modify the descriptions herein into other specific forms without altering the technical ideas or essential features. Therefore, the embodiments described above should be understood in all respects as illustrative and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

[0137] The scope of this specification is defined by the claims set forth below, and all modifications or alterations derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included within the scope of this specification.

Claims

1. (a) A step of synthesizing calcium phosphate by simultaneously adding a calcium precursor and a phosphate precursor; (b) A step of adding the calcium phosphate to the antimicrobial precursor solution and allowing it to react; (c) A step of freeze-drying the product of step (b); (d) A step of mixing the product of step (c) with a shape control agent and press-molding to produce a molded body; and (e) A step of removing the shape-controlling agent from the molded body with a solvent at a temperature above its boiling point, and then drying it; A method for producing a low-crystalline calcium phosphate-based antibacterial bone graft material containing [the specified ingredient].

2. The method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 1, wherein the calcium precursor comprises one or more selected from the group consisting of calcium nitrate, calcium chloride, calcium fluoride, calcium iodide, calcium acetate, ammonium carbonate, and ammonium bicarbonate.

3. The method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 1, wherein the phosphate precursor comprises one or more selected from the group consisting of phosphorus oxide, monoammonium phosphate, diammonium phosphate, triammonium phosphate, monosodium phosphate, disodium phosphate, trisodium phosphate, and potassium phosphate.

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

5. The method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 1, wherein step (a) is a wet precipitation method in which each precursor is dissolved in distilled water and mixed.

6. The method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 1, wherein the antibacterial substance comprises one or more selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

7. The method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 1, wherein the reaction in step (b) is carried out at a temperature of 15 to 25°C for 10 to 20 hours.

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

5.

9. A method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 1, wherein the average particle size of the shape-controlling agent is 100 to 500 μm.

10. A method for producing a low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 1, further comprising the step of (f) heat-treating the molded body at a temperature of 250°C or less, after the step of (e) above.

11. The method for producing a low-crystallinity calcium phosphate antibacterial bone graft according to claim 1, wherein the antibacterial substance content of the low-crystallinity calcium phosphate antibacterial bone graft is 0.2 to 1.8% by weight.

12. The method for producing a low-crystallinity calcium phosphate antibacterial bone graft according to claim 1, wherein the antibacterial substance content of the low-crystallinity calcium phosphate antibacterial bone graft material is 0.5 to 0.9% by weight.

13. It contains an antimicrobial substance and includes at least one of the grooves and pores with an average diameter of 100 to 500 μm, and has a BET specific surface area of ​​100 m². 2 A low-crystallinity calcium phosphate-based antibacterial bone graft material containing both crystalline apatite and amorphous calcium phosphate, with a crystallinity of 25% or less, and having a concentration of 1 / g or more.

14. The low-crystalline calcium phosphate antibacterial bone graft material according to claim 13, wherein the antibacterial substance comprises one or more selected from the group consisting of silver (Ag), magnesium (Mg), gallium (Ga), zinc (Zn), and copper (Cu).

15. The low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 13, wherein the content of the antibacterial substance is 0.2 to 1.8% by weight relative to the total elements constituting the bone graft material.

16. The low-crystallinity calcium phosphate-based antibacterial bone graft material according to claim 13, wherein the content of the antibacterial substance is 0.5 to 0.9% by weight relative to the total elements constituting the bone graft material.

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