JP6820537B2 - Hydroxyapatite derivative particle group - Google Patents

Description

The present invention relates to a group of hydroxyapatite derivative particles composed of antibacterial hydroxyapatite derivative particles in which a part of the crystal structure of hydroxyapatite is replaced with other ions.

Conventionally, it exists as single crystal primary particles in a solvent, and is useful for highly dispersible ceramic particles, especially medical materials having biocompatibility, adhesion or adhesion to biological tissues, and low biodegradation and absorption. A group of calcined calcium phosphate particles (ceramic particle group) including a single crystal hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ , hereinafter referred to as “HAp”) has been known (for example, a group of ceramic particles). See Patent Document 1). Applications of such single crystal HAp include dental materials such as bone fillers, dental fillers, and sustained-release agents, medical materials, immobilized carriers such as bacterial cells and yeast, and column chromatography. Various uses such as adsorbents such as fillers and deodorants, and nanometer-sized drug delivery systems (nano DDS) have been known.

Japanese Patent No. 5043436

Here, the calcium phosphate calcined particle group such as HAp is used as a dental material such as a bone filler, a dental filler, a drug sustained-release agent, a medical material, a protein, an amino acid, a polysaccharide, a physiologically active substance, or the like. When used for immobilization carriers, fillers for column chromatography, adsorbents such as deodorants, nanometer-sized drug delivery systems (nano DDS), etc., antibacterial action is often required. In such cases, until now, it has been common to simply combine and use an antibacterial metal such as Ag, ions of the metal, and other antibacterial components by mixing with HAp and the like. ..

However, as described above, when an antibacterial metal such as Ag or other antibacterial component is simply used in combination with HAp, the antibacterial property is too strong, so that not only the tissue infected with bacteria or the like but also a healthy tissue There was also a problem that it had an adverse effect on.

Therefore, the present invention has been made in view of the above circumstances, and is a healthy tissue while maintaining HAp functions such as excellent biocompatibility, adhesion or adhesiveness to living tissue, and low biodegradation and absorption. It is an object of the present invention to provide a group of hydroxyapatite derivative particles composed of hydroxyapatite derivative particles having mild antibacterial properties that have almost no adverse effect on.

As a result of intensive studies to solve the above problems, the present inventors have incorporated silver ion (Ag ⁺ ) or fluoride ion (F ⁻ ) into the crystal structure of HAp to obtain the crystal structure of HAp. It has been found that HAp derivatives such as Ag-HAp and F-HAp, which are partially substituted with silver ions or fluoride ions, have mild antibacterial properties. In addition, the present inventors have excellent biocompatibility, adhesion to biological tissues, or adhesiveness by using a group of HAp derivative particles composed of particles of HAp derivatives having such mild antibacterial properties as an antibacterial component. It has been found that a material having mild antibacterial properties that has almost no adverse effect on healthy tissues can be obtained while maintaining the HAp function such as low biocompatibility and absorption. The present invention has been completed based on the above findings.

That is, the present invention is characterized by comprising antibacterial hydroxyapatite derivative particles in which at least a part of calcium ions in the crystal structure of hydroxyapatite is replaced with silver ions (first hydroxyapatite derivative particle group). Apatite derivative particle group).
In the first hydroxyapatite derivative particle group, a predetermined microorganism of 5.0 × 10 ⁴ cells / μL and the antibacterial hydroxyapatite derivative particles coexist at a concentration of 2.47 mmol / L or more in terms of silver ions. It is preferable that the sterilization rate after preparing the prepared dispersion, spotting the dispersion on the medium, and culturing at 37 ° C. for 1 day is 90% or more.
In the first hydroxyapatite derivative particle group, the hydroxyapatite crystal phase purity quantified by XRD of the antibacterial hydroxyapatite derivative particles is preferably 90% or more.
The present invention also comprises a group of hydroxyapatite derivative particles (the first), which comprises antibacterial hydroxyapatite derivative particles in which at least a part of hydroxide ions in the crystal structure of hydroxyapatite is substituted with fluoride ions. 2) Hydroxyapatite derivative particle group).
In the second hydroxyapatite derivative particle group, 5.0 × 10 ⁴ cells / μL of a predetermined microorganism and the antibacterial hydroxyapatite derivative particles at a concentration of 89.3 mmol / L or more in terms of fluoride ions were used. It is preferable that the sterilization rate after preparing the coexisting dispersion, spotting the dispersion on the medium, and culturing at 37 ° C. for 1 day is 50% or more.
In the second hydroxyapatite derivative particle group, 5.0 × 10 ⁴ cells / μL of a predetermined microorganism and the antibacterial hydroxyapatite derivative particles at a concentration of 178.6 mmol / L or more in terms of fluoride ions were added. It is preferable that the sterilization rate after preparing a coexisting dispersion, spotting the dispersion on a medium, and culturing at 37 ° C. for 1 day is 95% or more.
In the first and second hydroxyapatite derivative particle groups, the antibacterial hydroxyapatite derivative particles are preferably fired hydroxyapatite derivative particles.
In the first and second hydroxyapatite derivative particle groups, the particle size of the antibacterial hydroxyapatite derivative particles is preferably in the range of 10 nm to 1,000 nm.

According to the present invention, by using a HAp derivative particle group consisting of HAp derivative particles such as Ag-HAp and F-HAp in which a part of the crystal structure of HAp is replaced with silver ion or fluoride ion as an antibacterial component, HAp is used. It is possible to obtain a material having mild antibacterial properties while maintaining the function of. In addition, such mild antibacterial properties make it possible to exert a bactericidal action and a bacteriostatic action with almost no adverse effect on healthy tissues that are not infected with bacteria or the like.

It is a figure which shows the FTIR spectrum of Ag-HAp prepared in an Example. It is a figure which shows the XRD diffraction pattern of Ag-HAp produced in an Example. It is a photograph which shows the SEM image (magnification 20,000 times) of Ag-HAp produced in an Example. It is a schematic diagram which shows the dilution method of the sample in the antibacterial property test using Ag-HAP or F-HAp in an Example. It is a photograph which shows the result of the antibacterial property test of Ag-HAp prepared in the Example. It is a figure which shows the XRD diffraction pattern of F-HAp produced in an Example. It is a photograph which shows the SEM image (magnification 20,000 times) of the commercially available F-HAp. It is a photograph which shows the SEM image (magnification 20,000 times) of F-HAp produced in an Example. It is a graph showing the acid resistance of F-HAp prepared in an Example and commercially available HAp (three kinds). It is a schematic diagram which shows the method of the antibacterial property test using NaF in an Example. It is a figure which shows the result of the antibacterial property test using NaF in an Example.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The HAp derivative particle group according to the present invention will be described in the following order.
1 Composition of HAp derivative particle group 2 Principle of antibacterial action 3 Use of HAp derivative particle group

<< Composition of HAp derivative particle group >>
First, the composition of the HAp derivative particle group according to the present invention will be described. The HAp derivative particle group according to the present invention is composed of HAp derivative particles containing silver ions or fluoride ions in the crystal structure of HAp. According to the findings of the present inventors, the HAp derivative particle group containing silver ion or fluoride ion has mild antibacterial properties. Here, the term "mild antibacterial property" as used herein means that it exerts a bactericidal action and a bacteriostatic action against bacteria and the like with almost no adverse effect on healthy tissues not infected with the bacteria and the like. Means antibacterial properties to the extent possible. In other words, "mild antibacterial property" means that, for HAp derivatives containing silver ions, the amount of silver ions required for the HAp derivative particle group to exert antibacterial properties is because silver ions alone exert antibacterial properties. It means that the antibacterial activity is suppressed, which is very large compared to the amount of silver ions required for. Regarding HAp derivatives containing fluoride ions, the amount of fluoride ions required for the HAp derivative particle group to exhibit antibacterial properties is the concentration of silver ions whose antibacterial properties are widely recognized, and the above silver ions. The amount of silver ions contained in the HAp derivative containing the above is very large, which means that the antibacterial activity is suppressed.

Examples of microorganisms in which the HAp derivative particle group according to the present invention can exhibit antibacterial properties (here, the concept includes not only "microorganisms" in a narrow sense but also viruses and fungi) include Escherichia. coli), Shigella (Shigella dysenteriae), Klebsiella pneumoniae (Klebsiella pneumoniae), Salmonella typhi (Salmonella typhimurium), salmonella (Salmonella enteritidis), Yersinia pestis (Yersinia. pestis), Yersinia enterocolitica (Yersinia enterocolitica), Serratia bacteria (Serratia marcescens ), Proteus, Providencia, Morganella, Citrobacter fluendii, Vibrio cholerae, Vibrio parahaemolysia, Pythropopterum, Vibrio parahaemolyticus, Green cepasia), Legionella pneumophila, Pertussis, Bordetella pertussis, Niserra gonorroea, Nesleria meningitidies, Catharbacillus (Mollia) Bacteria such as bacteria (Bacteroides fragilis), yellow staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), bacillus bacillus (Bacillus) Bacillus antibiotics, Clostridium tetani, Clostridium botulinum, Clostridi um perfringens), Listeria monocytogenes, Corynebacterium diphthriae, Norcadia (Nocardia spp.), Enterococcus Enterococcus, Enterococcus cerocococcus, Enterococcus enterococcus, Enterococcus , Acinetobacter calcoaceticus, Enterococcus (Enterococcus spp.), Vancomycin-resistant Enterococcus, VRE, Epidermoid bacillus (Staphylococcus) bacillus Staphylococcus. (Mycobacterium tuberculosis), Propionibacterium acnes (Propionibacterium acnes), Helicobacter pylori (Helicobacter pylori), Campylobacter (Campylobacter jejuni, Campylobacter coli), Chlamydia pneumoniae (Chlamydia pneumonia), sexually transmitted diseases chlamydia (Chlamydia trachomatis), periodontal bacteria (Porphyromonas gingivalis , Treponema denticola, Tannerella forsythensis) such as bacteria; Trichophyton (Trichophyton rubrum, Trichophyton mentagrophytes), Aspergillus (Aspergillus fumigatus), Candida (Candida albicans), fungi such as Cryptococcus bacteria (Cryptococcus neoformans); influenza virus (Influenzavirus ), Hepatitisvirus, Herpesviridae, Herpesvirus, adenovirus, AIDS virus (Human Immunodeficien) Examples include viruses such as cy Virus: HIV).

When the HAp derivative particle group according to the present invention is used as an antibacterial material, the HAp derivative particles may be a liquid antibacterial material dissolved or dispersed in water, an organic solvent or the like, and the HAp derivative particles are granulated. It may be a solid antibacterial material processed into a shape, a lump, a sheet, or the like, and has antibacterial properties such as mixing HAp derivative particles with another material or unevenly distributing HAp derivative particles on the surface of the other material. It may be a composite material having.

Examples of the antibacterial HAp derivative in the present invention include Ag-HAp in which a part of the crystal structure of HAp is replaced with silver ions and F-HAp in which a part of the crystal structure of HAp is replaced with fluoride ions. .. Hereinafter, Ag-HAp and F-HAp will be described in detail.

Here, as Ag-HAp and F-HAp used in the present invention, for example, when a group of HAp derivative particles composed of particles of these HAp derivatives is bound to a medical material, a polymer medical material, or for chromatography. When used as a filler, it is preferably a calcined HAp derivative particle group (calcined body) obtained by calcining the HAp derivative particle group in order to improve stability in a living body and ensure moldability. Further, the fired HAp derivative particle group (fired body) is crystalline, and by using such a crystalline fired body as an antibacterial material, milder antibacterial properties can be exhibited. As described above, the reason why the fired body has milder antibacterial properties than the unfired body is not clear, but the present inventors have a high ion elution rate because the unfired body is amorphous. Therefore, the antibacterial action is concentrated in a short period of time, and the ion concentration required for the antibacterial action is temporarily increased, so that the risk of damage to the living tissue is increased, and further, the antibacterial action is expressed. It is speculated that this is because the period during which the ion concentration required for sinter can be maintained is shortened. The fired body is a ceramic, and the ceramic here means not only a ceramic in a narrow sense but also a ceramic in a broad sense including a so-called "new ceramic" or "fine ceramic". The details of the firing method will be described later.

Further, in the calcined HAp derivative particle group composed of a set of calcined HAp derivative particles, the fusion of the primary particles is prevented by the action of the fusion inhibitor described later, so that the majority of the primary particles maintain the state of the primary particles. doing. Therefore, when the fired HAp derivative particle group is suspended in a solvent, the majority of the fired HAp derivative particle group is a primary particle composed of a single crystal, or the primary particle composed of the single crystal undergoes ionic interaction. It can be dispersed in aggregated particle masses (single crystal primary particles).

Further, when the calcined HAp derivative particle group is adsorbed on a medical polymer base material, it is important that the dispersibility is high. Further, when used as a packing material for chromatography, it is important that the surface area is high. The fired HAp derivative particle group according to the present invention is a primary particle whose majority is a single crystal, or a particle mass (single crystal primary particle) in which the primary particles composed of the single crystal are assembled by ionic interaction. It has good dispersibility in a solvent and has a high surface area because it does not form secondary particles. Therefore, the calcined HAp derivative particle group according to the present invention can be particularly suitably used for the above-mentioned applications.

Here, as a method for evaluating whether or not the fired HAp derivative particles are present as primary particles, for example, the result of measuring the particle size by electron microscope observation and the state of being suspended in a solvent by a dynamic light scattering method. By comparing the results of measuring the particle size with the above, if the results of both are almost the same, it can be determined that most of the fired HAp derivative particle group is in the state of primary particles. On the other hand, if the result of the particle size measurement by the dynamic light scattering method is larger than the result of the particle size measurement by the electron microscope observation, it is judged that the primary particles are fused with each other to form the secondary particles. Can be done.

The solvent for dispersing the calcined HAp derivative particles is not particularly limited as long as it does not dissolve the calcined HAp derivative particles. For example, water, alcohols such as methanol and ethanol, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone, amides such as N, N-dimethylformamide, sulfoxides such as dimethyl sulfoxide, toluene, xylene and hexane. , Hydrocarbons such as dodecane and cyclohexane, halogenated hydrocarbons such as chlorobenzene and chloroform, ethers such as diethyl ether and dioxane, etc., and one or two of these solvents may be used depending on the purpose of use. You can select and use it.

Based on the particle size distribution map obtained by the dynamic light scattering method, the ratio of particles having a particle size that is almost the same as the particle size of the primary particles obtained from an electron microscope is obtained to obtain primary particles composed of a single crystal. Alternatively, the ratio of the particle mass (single crystal primary particles) in which the primary particles composed of the single crystals are aggregated by ionic interaction can be calculated.

It may differ depending on the raw material of the calcined HAp derivative, the type of the fusion inhibitor, the calcining conditions, etc., but according to the method for producing the calcined HAp derivative particle group according to the present invention described later, at least 50% or more is single crystal primary. It exists as particles, and more preferably 60% or more exists as single crystal primary particles, and under the most preferable conditions, 70% or more can exist as single crystal primary particles.

Further, when the fired HAp derivative particles are adsorbed on a medical polymer base material, or when they are used as a packing material for chromatography, a medical material, or the like, the particles are preferably fine (nanometer size). In order to produce such a fine (nanometer-sized) calcined HAp derivative particle group, a fine (nanometer-sized) primary is used in the primary particle generation step of the method for producing a calcined HAp derivative particle group according to the present invention, which will be described later. Particles may be prepared. For example, in the primary particle generation step, by producing primary particles having a particle diameter in the range of 10 nm to 1,000 nm, more preferably 15 nm to 700 nm, and most preferably 20 nm to 500 nm, 10 nm to 1,000 nm. , More preferably, a fired HAp derivative particle group having a primary particle diameter in the range of 20 nm to 700 nm, most preferably 25 nm to 750 nm can be produced.

Further, it is preferable that the fired HAp derivative particle group has a uniform particle size (narrow particle size distribution). In order to produce a fired HAp derivative particle group having a uniform particle size (narrow particle size distribution), a primary particle group having a uniform particle size (narrow particle size distribution) should be prepared in the primary particle generation step. Just do it. The fired HAp derivative particle group having a uniform particle size (narrow particle size distribution) can be suitably used, for example, when adsorbing on a medical polymer base material, a packing material for chromatography, a medical material, or the like.

[Ag-HAp: structure]
Ag-HAp contains silver ions in the crystal structure, and the calcium ion (Ca ²⁺ ) portion (at least a part) in the crystal structure of HAp is replaced with silver ions (Ag ⁺ ). In other words, Ag-HAp is a HAp derivative in which silver ions are doped at the positions of calcium ions in HAp. This Ag-HAp has antibacterial properties unlike HAp which does not contain silver ions, and has mild antibacterial properties as compared with a simple mixture of silver ions and HAp. In Ag-HAp, when at least a part of calcium ions in the HAp crystal structure is replaced with silver ions, at least a part of hydroxide ions in the HAp crystal structure is fluoride ions or other shadows. It may be replaced with an ion.

[Ag-HAp: Synthesis method]
Ag-HAp having the above-mentioned characteristics can be produced by a known production method such as a wet method, a dry method, a hydrolysis method, or a hydrothermal method. In the following description, a method of synthesizing Ag-HAp using the coprecipitation method, which is one of the wet methods, will be taken as an example, and a method of synthesizing Ag-HAp will be described. By using a chemical reaction method including a wet method, low-temperature synthesis becomes possible, so that the chemical composition can be easily controlled, and it is also suitable for application in medical treatment.

The method for producing Ag-HAp according to the present invention may include at least a "primary particle generation step", but may also include a "mixing step", a "baking step", and a "removal step". .. In the following description, a manufacturing method including all the above four steps will be described.

In the method for producing Ag-HAp according to the present invention, the above four steps are, for example, in the order of "1. primary particle generation step"-> "2. mixing step"-> "3. firing step"-> "4. removal step". Will be done.

(1. Primary particle generation process)
Here, the "primary particles" mean particles formed by a HAp derivative raw material (fluorine, HAp, etc.) before firing in the manufacturing process of the HAp derivative particles. That is, it means the particles formed for the first time in the manufacturing process of HAp derivative particles. In a narrow sense, it means single crystal particles. In the description of the present invention, the term "primary particles" includes those in an amorphous state and those in a fired body that has been fired thereafter.

On the other hand, a "secondary particle" is a state in which a plurality of "primary particles" are bonded to each other by a physical bond such as fusion or a chemical bond such as an ionic bond or a covalent bond. Means particles. In particular, the number of bonds between the primary particles, the shape after bonding, and the like are not limited, and mean all of the two or more primary particles bonded together.

In particular, the "single crystal primary particle" means a primary particle made of a single crystal of a HAp derivative raw material, or a particle mass in which the primary particles made of the single crystal are assembled by ionic interaction. The "particle mass assembled by ionic interaction" is a particle mass that self-assembles by ionic interaction when dispersed in a medium containing water or an organic solvent, and is between particles by firing. Does not contain secondary particles that have been melted and polycrystallized.

The primary particle generation step is not particularly limited as long as it is a step capable of producing the above-mentioned primary particles, and may be appropriately selected and adopted depending on the raw material of the HAp derivative to be produced. For example, Ag-HAp primary particles can be produced by the following method. First, a silver (Ag) source (silver compound) and a calcium (Ca) source (calcium compound) are dissolved in a predetermined solvent and stirred for a predetermined time to prepare a silver compound solution and a calcium compound solution. Similarly, a phosphorus (P) source (phosphorus compound) is dissolved in a predetermined solvent and stirred for a predetermined time to prepare a phosphorus compound solution. Next, after mixing the silver compound solution and the calcium compound solution, the phosphorus compound solution is further added dropwise, mixed, heated to a predetermined temperature, and stirred. As a result, Ag-HAp particles precipitate.

Regarding the raw material of Ag-HAp, the Ag source is silver nitrate, silver perchlorate, silver ammonium complex, silver thiosulfate complex, silver cyano complex, etc., and the Ca source is calcium nitrate (including hydrate). Use phosphoric acid, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, etc. as the P source for calcium hydroxide, calcium nitrate, calcium chloride, etc. Can be done. The solvent for dissolving the raw material is not particularly limited as long as the raw material can be dissolved, and examples thereof include water, ethanol, dimethylformamide, dimethyl sulfoxide, acetonitrile, and toluene. As the Ag source, Ca source, P source, and solvent, the above-mentioned compounds may be used alone, or a plurality of types may be mixed and used.

In the method for producing HAp derivative particles according to the present invention, a group of primary particles composed of a collection of primary particles produced by the above primary particle generation step is fired while preventing fusion and the like, and HAp composed of a collection of HAp derivative particles is fired. It produces a group of derivative particles. Therefore, the state of the primary particles (particle size, particle size distribution) produced by the primary particle generation step is directly reflected in the state (particle size, particle size distribution) of the HAp derivative particles, which is the final product.

Further, this step may include a step of washing the generated primary particles with water or the like, a step of recovering the primary particles by centrifugation, filtration or the like.

(2. Mixing process)
This mixing step is a step of mixing the primary particles generated in the above step with a fusion inhibitor that prevents the primary particles from fusing to each other during firing. By interposing a fusion inhibitor in advance between the particles of the primary particle group obtained in the primary particle generation step, it is possible to prevent the primary particles from fusing to each other in the subsequent firing step. The mixture of the primary particles and the anti-fusion agent obtained in this mixing step is referred to as "mixed particles".

Here, the "fusion inhibitor" is not particularly limited as long as it can prevent fusion between the primary particles, but it is preferably non-volatile at the firing temperature in the subsequent firing step. .. This is because it is non-volatile under the firing temperature condition, so that it does not disappear from the primary particles during the firing step, and fusion of the primary particles can be reliably prevented. However, it is not necessary to have 100% non-volatility at the firing temperature, and it is sufficient that the non-volatileity is such that 10% or more remains between the primary particles after the firing step is completed. Further, the anti-fusion agent may be one that is chemically decomposed by heat after the completion of the firing step. That is, if it remains after the firing step is completed, it is not necessary that the substances (compounds) are the same before and after the firing step is started.

Further, it is preferable that the anti-fusion agent is a substance that dissolves in a solvent, particularly an aqueous solvent. In this way, by using a fusion inhibitor that dissolves in a solvent as the fusion inhibitor, the HAp derivative particles in which the fusion inhibitor is mixed are simply suspended in an aqueous solvent such as pure water for fusion. Inhibitors (eg, calcium nitrate, calcium carbonate, etc.) can be removed. In particular, if the anti-fusion agent is soluble in an aqueous solvent, it is not necessary to use an organic solvent when removing the anti-fusion agent. Therefore, equipment corresponding to the use of the organic solvent and organic solvent waste liquid treatment are required in the removal process. It becomes unnecessary. Therefore, the anti-fusion agent can be more easily removed from the HAp derivative particle group. The solvent is not particularly limited, and examples thereof include water, ethanol, methanol and the like as the aqueous solvent, and acetone, toluene and the like as the organic solvent.

In addition, the aqueous solvent may contain a chelate compound such as oxalate, ethylenediamine, bipyridine, or ethylenediaminetetraacetate in order to increase the solubility of the anti-fusion agent in water. Further, the aqueous solvent may contain electrolyte ions such as sodium chloride, ammonium nitrate and potassium carbonate in order to increase the solubility of the anti-fusion agent in water.

Specific examples of the anti-fusion agent include calcium salts (or complexes) such as calcium chloride, calcium oxide, calcium sulfate, calcium nitrate, calcium carbonate, calcium hydroxide, calcium acetate, and calcium citrate, potassium chloride, and potassium oxide. , Potassium salts such as potassium sulfate, potassium nitrate, potassium carbonate, potassium hydroxide, potassium phosphate, sodium chloride, sodium oxide, sodium sulfate, sodium nitrate, sodium carbonate, sodium hydroxide, sodium phosphate, etc. Be done.

The method of mixing the primary particles and the anti-fusion agent in this mixing step is not particularly limited, and the solid primary particles are mixed with the solid anti-fusion agent and then mixed using a blender. It may be a method, or it may be a method of dispersing the primary particles in a solution of the anti-fusion agent. However, since it is difficult to uniformly mix the solid and the solid, it can be said that the latter is a preferable method in order to uniformly and surely interpose the anti-fusion agent between the primary particles. When the latter method is adopted, it is preferable to dry the anti-fusion agent solution in which the primary particles are dispersed. This is because the state in which the primary particles and the anti-fusion agent are uniformly mixed can be maintained for a long period of time.

Further, in this mixing step, the primary particles are mixed with a solution containing a polymer compound having any one of a carboxyl group, a sulfate group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group or an amino group in the side chain. , Metal salts (alkali metal salts and / or alkaline earth metal salts and / or transition metal salts) may be further added. By adopting this step, the polymer compound is adsorbed on the surface of the HAp derivative to surely prevent the HAp derivatives from coming into contact with each other in the process of mixing the anti-fusion agent, and then the calcium salt is added to prevent the HAp derivatives from coming into contact with each other. It is possible to reliably deposit the anti-fusion agent on the surface of the derivative. In the following description, a polymer compound having any of a carboxyl group, a sulfate group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group or an amino group in the side chain is simply referred to as a "polymer compound". ..

The polymer compound is not particularly limited as long as it is a compound having any one of a carboxyl group, a sulfate group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group or an amino group in the side chain. For example, examples of the polymer compound having a carboxyl group in the side chain include polyacrylic acid, polymethacrylic acid, carboxymethyl cellulose, and a styrene-maleic anhydride copolymer, and examples of the polymer compound having a sulfate group in the side chain include. Examples of the polymer compound having a sulfonic acid group in the side chain include polyacrylic acid alkyl sulfonic acid ester, polymethacrylic acid alkyl sulfate ester, and polystyrene sulfuric acid. Examples of the polymer compound have polyacrylic acid alkyl sulfonic acid ester and polymethacrylate alkyl sulfone. Examples of the polymer compound having a phosphoric acid group in the side chain include acid ester and polystyrene sulfonic acid, and examples thereof include polyacrylic acid alkyl phosphoric acid ester, polymethacrylic acid alkyl phosphoric acid ester, polystyrene phosphoric acid, and polyacryloyl aminomethylphosphonic acid. Examples of the polymer compound having a phosphonic acid group in the side chain include polyacrylic acid alkylphosphonic acid ester, polymethacrylic acid alkylphosphonic acid ester, polystyrene phosphonic acid, polyacryloylaminomethylphosphonic acid, polyvinylalkylphosphonic acid and the like. Examples of the polymer compound having an amino group in the side chain include polyacrylamide, polyvinylamine, polymethacrylic acid aminoalkyl ester, polyaminostyrene, polypeptide, protein and the like. In the mixing step, any one of the above polymer compounds may be used, but a plurality of types of polymer compounds may be mixed and used.

The solution containing the polymer compound is preferably an aqueous solution. This is because the calcined particles of the HAp derivative dissolve under strongly acidic conditions. At this time, the pH of the aqueous solution containing the polymer compound is not particularly limited as long as it is 5 or more and 14 or less and the HAp derivative particles are insoluble. The pH of the aqueous solution containing the polymer compound may be adjusted by dissolving the polymer compound in distilled water, ion-exchanged water or the like, and adjusting the pH with an aqueous solution of ammonia, sodium hydroxide, potassium hydroxide or the like.

The concentration of the polymer compound contained in the aqueous solution is preferably 0.001% w / v or more and 50% w / v or less, more preferably 0.005% w / v or more and 30% w / v or less, and 0. Most preferably, it is 0.01% w / v or more and 10% w / v or less. If it is less than the above preferable range, the amount of penetration between the primary particles is small, and the ratio of blocking contact between the primary particles is low. Further, if it exceeds the above preferable range, it is not preferable because it becomes difficult to dissolve the polymer compound and the operability deteriorates such as the viscosity of the solution containing the polymer compound becomes high.

In the mixing step in the present invention, the solution containing the above polymer compound and the primary particles are mixed. For such mixing, for example, the primary particles may be put into the solution and the primary particles may be dispersed by a stirring operation or the like. By such an operation, in the method for producing a HAp derivative particle group according to the present invention, the polymer compound is adsorbed on the surface of the primary particles, and a carboxyl group, a sulfate group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group or an amino. Any of the groups can be added to the surface of the primary particle. At this time, the carboxyl group, sulfate group, sulfonic acid group, phosphoric acid group, phosphonic acid group or amino group exist in the ionic state in the solution.

Next, if a metal salt (alkali metal salt and / or alkaline earth metal salt and / or transition metal salt) is further added to the solution obtained by mixing the solution containing the polymer compound and the primary particles, the above primary particles can be obtained. Carborate ions, sulfate ions, sulfonate ions, phosphate ions, phosphonate ions, amino ions present on the surface and metal ions (alkali metal ions and / or alkaline earth metal ions and / or transition metal ions) It binds to form carboxylates, sulfates, sulfonates, phosphates, phosphonates, and amino acid salts on the surface of the primary particles. The carboxylates, sulfates, sulfonates, phosphates, phosphonates, and amino acid salts of such metals (alkali metals and / or alkaline earth metals and / or transition metals) function as the anti-fusion agents. .. Therefore, primary particles of metals (alkali metals and / or alkaline earth metals and / or transition metals) with carboxylates, sulfates, sulfonates, phosphates, phosphonates, amino acid salts on their surface , So-called "mixed particles". Since the carboxylates, sulfates, sulfonates, phosphates, phosphonates, and amino acid salts of such metals (alkali metals and / or alkaline earth metals and / or transition metals) precipitate, the precipitates Is collected, dried, and subjected to a baking step described later. Examples of the drying method include heating under reduced pressure conditions. The drying is preferably performed under reduced pressure conditions because the drying temperature can be lowered, but it may be performed under atmospheric pressure conditions.

The alkali metal salt is not particularly limited, but for example, sodium chloride, sodium hypochlorite, sodium chlorite, sodium bromide, sodium iodide, sodium iodide, sodium oxide, sodium peroxide. , Sodium sulfate, sodium thiosulfate, sodium selenate, sodium nitrite, sodium nitrate, sodium phosphate, sodium carbonate, sodium hydroxide, potassium chloride, potassium hypochlorite, potassium chlorate, potassium bromide, iodide Potassium, potassium iodide, potassium oxide, potassium peroxide, potassium sulfate, potassium thiosulfate, potassium selenate, potassium nitrite, potassium nitrate, potassium phosphate, potassium carbonate, potassium hydroxide and the like can be used.

Examples of the alkaline earth metal salt include magnesium chloride, magnesium hypochlorite, magnesium chlorite, magnesium bromide, magnesium iodide, magnesium iodide, magnesium oxide, magnesium peroxide, magnesium sulfate, and thio. Magnesium sulfate, magnesium selenate, magnesium nitrite, magnesium nitrate, magnesium phosphate, magnesium carbonate, magnesium hydroxide, calcium chloride, calcium hypochlorite, calcium chlorite, calcium bromide, calcium iodide, calcium iodide , Calcium oxide, calcium peroxide, calcium sulfate, calcium thiosulfate, calcium selenate, calcium nitrite, calcium nitrate, calcium phosphate, calcium carbonate, calcium hydroxide and the like can be used.

Examples of the transition metal salt include zinc chloride, zinc hypochlorate, zinc chlorate, zinc bromide, zinc iodide, zinc iodide, zinc oxide, zinc peroxide, zinc sulfate, and zinc thiosulfate. Zinc selenate, zinc nitrite, zinc nitrate, zinc phosphate, zinc carbonate, zinc hydroxide, iron chloride, iron hypochlorate, iron chlorate, iron bromide, iron iodide, iron iodide, iron oxide , Iron peroxide, iron sulfate, iron thiosulfate, iron selenate, iron nitrite, iron nitrate, iron phosphate, iron carbonate, iron hydroxide and the like can be used. Moreover, it may be a nickel compound.

Even if the metal salt (alkali metal salt, alkaline earth metal salt, transition metal salt) added to the solution in which the solution containing the polymer compound and the primary particles are mixed is one kind, it is a mixture of two or more kinds. It may be. Further, the metal salt (alkali metal salt, alkaline earth metal salt, transition metal) may be in a solid state, but can be added uniformly, and the concentration to be added can be controlled. For this reason, it is preferable to add it as an aqueous solution. The amount (concentration) of the metal salt (alkali metal salt and / or alkaline earth metal salt and / or transition metal salt) to be added depends on the carboxylic acid ion, sulfate ion, sulfonate ion, and phosphorus present on the surface of the primary particle. Couplates, sulfates, sulfonates, phosphates, phosphonates of metals (alkali metals and / or alkaline earth metals and / or transition metals) by combining with acid ions, phosphonate ions, amino ions. The conditions under which amino acid salts are generated are not particularly limited, and may be determined after appropriate consideration.

Here, carboxylates, sulfates, sulfonates, phosphates, phosphonates, of metals (alkali metals and / or alkaline earth metals and / or transition metals) produced on the surface of the primary particles by the above steps. Amino acid salts undergo thermal decomposition in the firing steps described below to become oxides of metals (alkali metals and / or alkaline earth metals and / or transition metals). For example, when calcium polyacrylate is generated on the surface of the primary particles, it becomes calcium oxide by the firing step. Since the metal oxide (alkali metal oxide and / or alkaline earth metal oxide (for example, calcium oxide) and / or transition metal oxide) is water-soluble, it can be easily removed by a removal step described later. Is possible.

In addition, since sodium polyacrylate is soluble in water, it can be used as it is as a fusion inhibitor in this mixing step, but since calcium polyacrylate is insoluble in water, only polyacrylate is once applied to the surface of the primary particles. It is preferable that calcium polyacrylate is precipitated on the surface of the primary particles by adding a calcium salt or the like after adsorbing the sodium polyacrylate.

(3. Baking process)
This firing step is a step of exposing the mixed particles obtained by the above mixing step to a firing temperature to turn the primary particles contained in the mixed particles into ceramic HAp derivative particles (fired body particles). Since the anti-fusion agent is interposed between the primary particles, it is possible to prevent the primary particles from fusing to each other even when exposed to high temperature conditions in the firing step.

The firing temperature in the firing step may be appropriately set so that the hardness of the HAp derivative particles becomes a desired hardness. For example, the firing temperature is more preferably in the range of 100 ° C. to 1800 ° C., further preferably 150 ° C. to 1500 ° C. Most preferably, 200 ° C to 1200 ° C. Further, from the viewpoint of exhibiting mild antibacterial properties, the firing temperature is preferably 500 ° C. or higher, and more preferably 700 ° C. or higher. Further, the firing time may be appropriately set based on the desired hardness of the HAp derivative particles and the like. For example, in the examples described later, firing is performed at 700 ° C. for 2 hours.

The apparatus or the like used in this firing step is not particularly limited, and a commercially available firing furnace (electric furnace or the like) may be appropriately selected and adopted according to the production scale, production conditions, and the like.

(4. Removal process)
This removal step is a step of removing the anti-fusion agent mixed between the particles of the HAp derivative particle group obtained by the above-mentioned firing step.

The means and method of removal may be appropriately adopted depending on the anti-fusion agent adopted in the above mixing step. For example, when a solvent-soluble anti-fusion agent is used, it is fused by using a solvent that does not dissolve the HAp derivative particles (insoluble) and a solvent that dissolves the anti-fusion agent (soluble). Only the inhibitor can be dissolved and removed. The solvent to be used is not particularly limited as long as it satisfies the above requirements, and may be an aqueous solvent or an organic solvent. For example, examples of the aqueous solvent include water, ethanol, methanol and the like, and examples of the organic solvent include acetone, toluene and the like.

In addition, the aqueous solvent may contain a chelate compound such as oxalate, ethylenediamine, bipyridine, or ethylenediaminetetraacetate in order to increase the solubility of the anti-fusion agent in water. Further, the aqueous solvent may contain electrolyte ions such as sodium chloride, ammonium nitrate and potassium carbonate in order to increase the solubility of the anti-fusion agent in water.

However, due to reasons such as the need for equipment that supports the use of organic solvents in the removal process, the need for organic solvent waste liquid treatment, high safety in manufacturing operations, and low environmental risk. The solvent used is preferably an aqueous solvent.

By the way, when the anti-fusion agent is removed using a solvent, the HAp derivative (calcined product) particles containing the anti-fusion agent obtained in the firing step are suspended in the solvent and then filtered or centrifuged. Only the HAp derivative (fired body) particles need to be recovered. In the method for producing the HAp derivative particle group according to the present invention, the above operation is not limited to one time, and may be performed twice or more. By performing the above operation a plurality of times, the removal rate of the anti-fusion agent between the HAp derivative particles is further improved. However, it is not preferable to perform the above operation more than necessary because the manufacturing process is complicated, the manufacturing cost is high, and the recovery rate of HAp derivative particles is lowered. Therefore, it is preferable that the number of operations is appropriately determined based on the target removal rate of the anti-fusion agent.

In addition, this step may further include a step of classifying in order to make the particle diameter uniform.

In addition to the method of removing the anti-fusion agent using the above solvent, the anti-fusion agent can be removed by using a magnet by using a magnetic material as the anti-fusion agent. More specifically, a group of HAp derivative particles (crude HAp derivative particles) containing an anti-fusion agent obtained in the firing step is suspended in an appropriate solvent (water, etc.) and dispersed, and then the suspension is used. A magnetic force is applied to attract only the anti-fusion agent to the magnet, and only the HAp derivative particles that have not been attracted are recovered. Further, a method may be used in which the crude HAp derivative particles are ground into powder without being suspended in a solvent, and then the anti-fusion agent is separated by a magnet. However, the HAp derivative particles and the anti-fusion agent are more easily peeled off from each other in a suspension, and the removal rate of the anti-fusion agent is higher. The HAp derivative particles to which this method can be applied are preferably non-magnetic materials or weak magnetic materials.

[Ag-HAp: Identification method]
Ag-HAp synthesized as described above can be identified by, for example, various structural analysis methods. For example, a diffraction pattern measured by using an X-ray diffractometer (XRD) is compared with a known diffraction pattern of Ag-HAp, and if these diffraction patterns are substantially the same, Ag- It can be identified that HAp is obtained. The axial length of the crystal lattice can be examined from the peak value in the diffraction pattern, and it can be confirmed from this axial length that calcium ions are replaced with silver ions.

Further, according to Fourier transform infrared spectroscopy (FTIR), the functional group contained in the obtained Ag-HAp can be identified.

Further, by observing the synthesized Ag-HAp with a scanning electron microscope (SEM), the surface shape and particle size of the Ag-HAp particles can be measured.

Further, by analyzing the synthesized Ag-HAp by inductively coupled plasma-atomic emission spectrometry (ICP-AES), the composition ratio of the elements and the amount of silver in the Ag-HAp nanocrystal can be determined.

If too many silver ions are contained in the crystal structure of Ag-HAp, metallic silver may be precipitated from the crystal structure of HAp, and the crystal structure of Ag-HAp may not be maintained. Then, the characteristics of HAp itself (for example, stability in a living body, biocompatibility, adhesion to a living tissue, adhesiveness, etc.) may not be exhibited, and biotoxicity may occur. On the other hand, if there are too many silver ions, the ratio of ionic species constituting the crystal structure of Ag-HAp collapses with the precipitation of metallic silver, and other crystal layers such as tricalcium phosphate (β-TCP) are formed. , Ag-HAp particles are water-soluble and the rate of metabolism in the living body is too high, so that they are not suitable for use as a HAp substitute material. Furthermore, since the crystal structure cannot be maintained, the release rate of silver ions increases, and mild antibacterial properties cannot be exhibited. From such a viewpoint, it is preferable that the substitution ratio of Ag in the crystal structure of Ag-HAp (= Ag / (Ca + Ag) × 100 [mol%]) is less than 0.75 mol%. In the case of such a substitution ratio of Ag, the purity of the hydroxyapatite crystal phase (determined from the diffraction pattern of XRD) quantified by XRD of Ag-HAp particles is 90% or more. Here, as a quantitative analysis method by XRD, a Rietveld method, a calibration curve method, a method using a diffraction integral intensity ratio and the like are generally known, but in the present invention, any of these methods is used. The crystal phase purity can be determined.

Furthermore, the composition ratio of the elements can be determined by analyzing the synthesized Ag-HAp with an inductively coupled plasma emission spectrophotometer (ICP-AES).

[Ag-HAp: antibacterial activity]
The antibacterial activity of Ag-HAp obtained as described above preferably satisfies, for example, the conditions described below. That is, a bacteria solution containing a predetermined cell value of absorbance measured by a microplate reader was adjusted to a concentration of 0.1 (10 ⁵ cells / [mu] L), the Ag-HAp particles NaCl solution fungus solution and the same volume It is preferable that the sterilization rate after spotting a mixed solution of a solution dispersed at a concentration of 2.47 mmol / L or more in terms of silver ions on a medium and culturing at 37 ° C. for 1 day is 90% or more. .. As a result, the HAp derivative particle group of the present invention can more reliably exhibit mild antibacterial properties capable of bacteriostatic or sterilizing bacteria and the like without adversely affecting healthy tissues. .. The "silver ion-equivalent concentration" here is the formula amount from the composition of Ag-HAp (Ca _10-x · Ag _x (PO ₄ ) ₆ (OH) ₂ ) used for the evaluation of antibacterial activity. It means the molar concentration (mol / L) of silver ions contained in the NaCl solution, which was calculated from this formula amount and the molar concentration (mol / L) of Ag-HAp in the NaCl solution.

[F-HAp: structure]
F-HAp contains fluoride ions in the crystal structure, and the hydroxide ion (OH ⁻ ) portion (at least a part) in the crystal structure of HAp is replaced with fluoride ions (F ⁻ ). Is. In other words, F-HAp is a HAp derivative in which fluoride ions are doped at the positions of the hydroxide ions of HAp. This F-HAp has antibacterial properties unlike HAp which does not contain fluoride ions, and has mild antibacterial properties as compared with a simple mixture of fluoride ions and HAp. In addition, F-HAp also has high acid resistance. In F-HAp, when at least a part of the hydroxide ion in the HAp crystal structure is replaced with a fluoride ion, at least a part of the calcium ion in the HAp crystal structure is a silver ion or other positive. It may be replaced with an ion.

Here, for example, a medical device such as a catheter is used for medical activities such as blood transfusion and drainage by connecting the outside and the inside of the body, but as a material for a catheter or the like, generally, a high elasticity such as silicone is excellent. Although molecular materials are used, when this device is actually worn for a long period of time, there is a concern that a gap will be created between the catheter and the living body, causing bacterial infection. Therefore, in recent years, a catheter has been developed in which a HAp nanosingle crystal having high dispersibility and crystallinity, which is useful for preventing bacterial infection, is produced and coated on a polymer base material. As a result, the living body and the device are in close contact with each other, and bacterial invasion from the insertion site can be significantly suppressed as compared with the conventional catheter. However, when extreme inflammation occurs around the transdermal device, phagocytic cells such as neutrophils and macrophages phagocytose it, releasing reactive oxygen species and lysosomal enzymes to dissolve the foreign substances they eat. Since active oxygen such as hydrogen peroxide releases protons and becomes weakly acidic in the inflamed surroundings, there is a concern that HAp, which is weak against acid, may be dissolved.

In such a case, by coating the surface of the polymer base material with F-HAp having high acid resistance, it is possible to suppress dissolution by active oxygen or the like. In addition, F-HAp has improved acid resistance compared to normal HAp and exhibits mild antibacterial properties. Therefore, by using F-HAp of the present invention as a dental material, caries can be prevented. Can be done.

[F-HAp: Synthesis method]
F-HAp having the above-mentioned characteristics can be produced by a known production method such as a wet method, a dry method, a hydrolysis method, and a hydrothermal method. In the following description, a method of synthesizing F-HAp using the coprecipitation method, which is one of the wet methods, will be taken as an example, and a method of synthesizing F-HAp will be described.

The method for producing F-HAp according to the present invention may include at least a "primary particle generation step", but may also include a "mixing step", a "baking step", and a "removal step". .. In the following description, a manufacturing method including all the above four steps will be described.

In the method for producing F-HAp according to the present invention, the above four steps are, for example, in the order of "1. primary particle generation step"-> "2. mixing step"-> "3. firing step"-> "4. removal step". Will be done.

(1. Primary particle generation process)
Here, the definitions of "primary particles", "secondary particles", and "single crystal primary particles" are the same as those of Ag-HAp.

The primary particle generation step is not particularly limited as long as it is a step capable of producing the above-mentioned primary particles, and may be appropriately selected and adopted depending on the raw material of the HAp derivative to be produced. For example, the primary particles of F-HAp can be produced by the following method. First, a fluorine (F) source (fluorine compound) and a calcium (Ca) source (calcium compound) are dissolved in a predetermined solvent and stirred for a predetermined time to prepare a fluorine compound solution and a calcium compound solution. Similarly, a phosphorus (P) source (phosphorus compound) is dissolved in a predetermined solvent and stirred for a predetermined time to prepare a phosphorus compound solution. Next, after mixing the fluorine compound solution and the calcium compound solution, the phosphorus compound solution is further mixed, heated to a predetermined temperature, and stirred. Then, the mixture is further stirred at room temperature for a predetermined time. As a result, F-HAp particles precipitate.

Regarding the raw material of F-HAp, the F source is sodium fluoride, potassium fluoride, etc., and the Ca source is calcium nitrate (including hydrate), calcium hydroxide, calcium nitrate, calcium chloride, etc. As the source, phosphoric acid, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate and the like can be used. The solvent for dissolving the raw material is not particularly limited as long as the raw material can be dissolved, and examples thereof include water, ethanol, dimethylformamide, dimethyl sulfoxide, acetonitrile, and toluene. As the Ag source, Ca source, P source, and solvent, the above-mentioned compounds may be used alone, or a plurality of types may be mixed and used.

In the method for producing HAp derivative particles according to the present invention, a group of primary particles composed of a collection of primary particles produced by the above primary particle generation step is fired while preventing fusion and the like, and HAp composed of a collection of HAp derivative particles is fired. It produces a group of derivative particles. Therefore, the state of the primary particles (particle size, particle size distribution) produced by the primary particle generation step is directly reflected in the state (particle size, particle size distribution) of the HAp derivative particles, which is the final product.

Further, this step may include a step of washing the generated primary particles with water or the like, a step of recovering the primary particles by centrifugation, filtration or the like.

(2. Mixing process)
This mixing step is a step of mixing the primary particles generated in the above step with a fusion inhibitor that prevents the primary particles from fusing to each other during firing. Since this mixing step is the same as the case of Ag-HAp described above, detailed description thereof will be omitted.

(3. Baking process)
This firing step is a step of exposing the mixed particles obtained by the above mixing step to a firing temperature to turn the primary particles contained in the mixed particles into ceramic HAp derivative particles (fired body particles). Since the main firing step is the same as the case of Ag-HAp described above, detailed description thereof will be omitted. However, the firing conditions in the examples described later are different from the case of Ag-HAp, and firing is performed at 800 ° C. for 1 hour.

(4. Removal process)
This removal step is a step of removing the anti-fusion agent mixed between the particles of the HAp derivative particle group obtained by the above-mentioned firing step. Since this removal step is the same as the case of Ag-HAp described above, detailed description thereof will be omitted.

[F-HAp: Identification method]
The F-HAp synthesized as described above can be identified by, for example, various structural analysis methods. For example, a diffraction pattern measured by using an X-ray diffractometer (XRD) is compared with a known diffraction pattern of F-HAp, and if these diffraction patterns are substantially the same, F- It can be identified that HAp is obtained. The axial length of the crystal lattice can be examined from the peak value in the diffraction pattern, and it can be confirmed from this axial length that the hydroxide ion is replaced with the fluoride ion.

Further, according to Fourier transform infrared spectroscopy (FTIR), the functional groups contained in the obtained F-HAp can be identified, and further, the synthesized F-HAp and the commercially available F-HAp can be identified. If the spectra of FTIR are compared and the absorption peaks belonging to each functional group are substantially the same, it can be inferred that the synthesized product is F-HAp.

Further, by observing the synthesized F-HAp with a scanning electron microscope (SEM), the surface shape and particle size of the F-HAp particles can be measured.

In addition, by using a fluorine ion densitometer, the concentration of fluoride ions in the synthesized F-HAp can be measured, and the measured value of the fluoride ion concentration and the theoretical concentration (F obtained from both chemical theories). -The fluoride ion substitution rate of hydroxide ions in HAp can also be determined from the ratio with (fluoride ion concentration in HAp).

[F-HAp: antibacterial activity]
The antibacterial activity of F-HAp obtained as described above preferably satisfies the conditions described below, for example. That is, a bacteria solution containing a predetermined cell value of absorbance measured by a microplate reader was adjusted to a concentration of 0.1 (10 ⁵ cells / [mu] L), the F-HAp particles NaCl solution fungus solution and the same volume A mixture of a solution dispersed at a concentration of 89.3 mmol / L or more in terms of fluoride ions and a mixed solution is spotted on the medium, and the sterilization rate after culturing at 37 ° C. for 1 day is 50% or more. preferable. As a result, the antibacterial agent of the present invention can more reliably exhibit mild antibacterial properties, which can sterilize bacteria and the like without adversely affecting healthy tissues. Furthermore, a bacterial solution containing a predetermined cell value of absorbance measured by a microplate reader was adjusted to a concentration of 0.1 (10 ⁵ cells / [mu] L), the F-HAp particles NaCl solution fungus solution and the same volume A mixture of a solution dispersed at a concentration of 178.6 mmol / L or more in terms of fluoride ions and a mixed solution are spotted on the medium, and the sterilization rate after culturing at 37 ° C. for 1 day is 95% or more. preferable. As a result, the antibacterial agent of the present invention can more reliably exhibit mild antibacterial properties capable of killing bacteria and the like without adversely affecting healthy tissues. The "concentration in terms of fluoride ion" here is the formula amount from the composition of F-HAp (Ca ₁₀ (PO ₄ ) ₆ F _2-y (OH) _y ) used for the evaluation of antibacterial activity. It means the molar concentration (mol / L) of fluoride ions contained in the NaCl solution, which was calculated from this formula amount and the molar concentration (mol / L) of F-HAp in the NaCl solution. ..

≪Principle of antibacterial action≫
The mechanism of action of the antibacterial agent according to the present invention is unknown, but as a result of converting the HAp derivative into nanoparticles as described above, the present inventors are likely to be taken into the inside of a bacterium having a size of about several μm. It is presumed that the mild antibacterial HAp derivative incorporated into the bacterium in this way exhibits an action of bacteriostatic or bactericidal the bacterium from the inside of the bacterium.

≪Use of antibacterial agent≫
Since the HAp derivative according to the present invention, particularly the calcined HAp derivative, has very high bioactivity, in the medical field, for example, a dental material such as a bone filler, a dental filler, a drug sustained-release agent, or a medical material. Can be widely used as. Moreover, since the HAp derivative has high bioactivity, it can be particularly suitably used as a medical material. Further, the calcined HAp derivative particle group can be suitably used as an immobilized carrier such as bacterial cells and yeast, a filler for column chromatography, an adsorbent such as a deodorant, and the like. Further, the HAp derivative in the present invention is expected to be used in a nanometer-sized drug delivery system (nano DDS). In particular, the HAp derivative and the antibacterial agent containing the HAp derivative according to the present invention are used among these applications where antibacterial properties are required, for example, applications such as medical devices such as catheters and artificial joints by connecting the outside and the inside of the body. It is preferably used for.

Next, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these examples.

≪Ag-HAp≫
First, Ag-HAp is synthesized, the obtained Ag-HAp is identified, and the result of evaluating the antibacterial property of the Ag-HAp will be described.

<Synthesis of Ag-HAp>
Using the coprecipitation method, which is one of the wet methods, Ag-HAp {Ca _10-x Ag _x (PO ₄ ) ₆ (OH) ₂ (in the formula, x = 0, 0.1, 0.2)} A nano-single crystal was prepared. For starting material, calcium nitrate tetrahydrate as Ca source _{{Ca (NO 3) 2 ·} 4H 2 O}, ammonium dihydrogen phosphate as the P source _{{(NH 4) 2 HPO 4} }, nitrate as Ag source { It was synthesized at room temperature using AgNO ₃ } while mixing in deionized water. Specifically, it was carried out as follows.

(Primary particle generation process)
As calcium nitrate tetrahydrate, silver nitrate, and ammonium dihydrogen phosphate, those purchased from Wako Pure Chemical Industries, Ltd. were used without purification. Moreover, in order to confirm the silver content in HAp, Ag-containing HAp of Ca: Ag = 80:20 (mol%) was prepared. Carried out at temperatures of all the reaction ^{100 ℃, (Ca 2+ + Ag} +): PO 4 3- molar ratio of 10: 6 and so as to calculate and defining the amount of reactants.

First, 80 mmol of calcium nitrate tetrahydrate and 20 mmol of silver nitrate were mixed with 300 mL of deionized water to prepare a 0.3 M solution (a nitrate solution of Ca and Ag). The solution was stirred at 100 ° C. for 30 minutes in an oil bath. Similarly, a predetermined amount of ammonium dihydrogen phosphate was mixed with 300 mL of deionized water to prepare a 0.2 M solution (phosphorus solution). The pH was adjusted to 10.0 by adding 28% concentration of NH ₄ OH to this solution, and the mixture was stirred at room temperature for 30 minutes. Then, the phosphorus solution was added drop by drop to the nitride solution of Ca and Ag maintained at a temperature of 100 ° C., and these solutions were mixed. After the addition of the phosphorus solution was completed, the mixture was stirred while being maintained at 100 ° C. for 2 hours. During stirring (reaction), the pH was constantly adjusted so that the pH of the solution was maintained at 10.0. Before mixing the phosphorus solution with the nitride solution of Ca and Ag, both solutions were transparent, but as the amount of the phosphorus solution added increased, the mixed solution gradually turned white. The resulting mixture was allowed to stand 12 hours, impurity ions ^{_{(NH 4 +, NO 3 -}} ) and washed 3 times with deionized water using a centrifugal separation method to remove. After washing, the mixture was filtered to give a moist cake (Ag-HAp cake). The damp cake was thoroughly dried by a low pressure pump and then left in the oven at 60 ° C. overnight. Further, the Ag-HAp cake was pulverized using a mortar and pestle to obtain Ag-HAp powder.

(Mixing process and firing process)
Next, a part of the Ag-HAp powder obtained above was fired without adding the anti-fusion agent, and the other part was mixed with the anti-fusion agent and then fired. The firing was carried out in a crucible at 700 ° C. for 2 hours.

(Removal process)
Finally, the pH was lowered to about 7 by adding an NH ₄ NO ₃ solution to the calcined product of Ag-HAp, and then washed with deionized water using centrifugation. After washing, the calcined product of Ag-HAp was filtered and sufficiently dried in an oven at 60 ° C. to obtain a sample of the calcined product of Ag-HAp.

<Identification of Ag-HAp>
Regarding the Ag-HAp sample obtained as described above, as identification of Ag-HAp, functional group analysis by Fourier transform infrared spectrophotometer (FTIR), crystal structure analysis by X-ray diffractometer (XRD), Surface shape and particle size were measured by a scanning electron microscope (SEM), and element composition analysis was performed by inductively coupled plasma-atomic emission spectrometry (ICP-AES).

(Functional group analysis by FTIR)
The functional group was measured by infrared spectroscopic measurement using a Fourier transform infrared spectrophotometer (Spectrum 50, manufactured by PerkinElmer Co., Ltd.). The KBr method was adopted as the measurement method. Potassium bromide (KBr) powder was mixed at a ratio of 99% by mass with respect to 1% by mass of a sample of Ag-HAp, thoroughly ground using a mortar, and the uniformly mixed powder was measured in the diffuse reflection mode. The measurement was performed with a resolution of 4000 cm ^-1 to 400 cm ^-1 and a resolution of 4, and the number of integrations was 16 times. The samples of F-HAp include (a) Ag-HAp prepared in the primary particle generation step and (b) a sample obtained by firing Ag-HAp of (a) without adding an anti-fusion agent. , (C) Ag-HAp of (a) was calcined by adding an anti-fusion agent {Ca (NO ₃ ) ₂ }. FIG. 1 shows the IR spectra of the samples (a) to (c) above.

As can be seen from FIG. 1, absorption peaks attributed to PO binding were observed near 1095 cm ^-1 , 1035 cm ^-1 , and 960 cm ^-1 . Further, the absorption peak is thought to be attributed to O-P-O ^bonds, 606 cm -1, were observed around 561cm ^-1 and 473cm ^-1. These absorption peaks suggested that all samples contained phosphate groups.

Further, the absorption peak attributable to a hydroxyl group (hydroxyl group) is, since it was observed in the vicinity of 3571cm ^-1 and 629cm ^-1, all of the samples also suggested to contain a hydroxyl group.

Further, in any of the samples, two broad absorption peaks attributed in _{H 2} O molecules, it was observed in the vicinity of 1630 cm ^-1 and 3400 cm ^-1, greatly peaks by baking for 2 hours at 700 ° C. It turned out that the strength was reduced.

In addition, as shown in FIG. 1, than the sample (b) and it is a sample of (c) (a), the peak intensity assigned to the hydroxyl group of 632 and 3571cm ^-1, phosphate groups of 962cm ^-1 The increase in peak intensity attributed to is suggesting that the crystallinity is increased in the calcined sample. This result is in agreement with the result by XRD described later.

Further, in the sample (a), the peak attributed to the hydroxyl group was not sharp, whereas in the calcined samples (b) and (c), the peak attributed to the hydroxyl group became sharp. From this, it was suggested that the calcined sample produced completely crystalline Ag-HAp. This was also observed at the diffraction peak of XRD, which will be described later.

(Crystal structure analysis by XRD)
The crystal structure of the produced Ag-HAp was analyzed using a powder X-ray diffractometer (RAD-X, manufactured by Rigaku Denki Co., Ltd.). A CuKα source (λ = 1.541841 Å (Angstrom)) was used as the X-ray source used in the XRD, the output was 30 kV / 15 mA, the scan speed was 1.0 ° / min, the sampling width was 0.01 °, and the measurement was performed. The mode was a continuous condition. FIG. 2 shows the X-ray diffraction peak of the prepared Ag-HAp. For the identification of the peak, the standard data of HAp of the ICCD-PDF card (00-009-0432) and the standard data of metallic silver of the ICCD-PDF card (04-0783) were used.

The prepared Ag-HAp shown in FIG. 2 was in good agreement with the HAp data (standard HAp data) of the ICCD-PDF card (00-009-0432) for the major peaks. Further, as shown in FIG. 2, the peak of metallic silver at 2θ = 38.1 ° was hardly observed in any of the above three types of samples. Therefore, it was found that the Ag-HAp synthesized by the above method was doped with silver ions in the HAp crystals both immediately after the formation of the primary particles and after firing (with or without the anti-fusion agent). ..

Further, based on the X-ray diffraction peak measured above, the full width at half maximum (FWHM: Full Width Half Maximum) in the (211) plane of the crystal lattices of the samples (a) to (c) is shown in Table 1 below. The results of samples (a), (b), and (c) are shown in order from the top of Table 1. The peak on the (211) plane in the standard HAp data was 2θ = 31.773 °.

It is generally known that the FWHM at the X-ray diffraction peak decreases as the firing temperature increases, and the particles become more complete crystalline. In this example, since the FWHMs of the samples (b) and (c) that were fired at 700 ° C. were smaller than the FWHM of the sample (a) that had not been fired, they were fired in this example. It can be said that Ag-HAp is more completely crystalline.

In addition, the axial length was examined from the X-ray diffraction peak value measured above. The shaft length can be calculated by using "unit cell software" based on the value of 2θ obtained from the XRD peak measured above. The results are shown in Table 2. The HAp standard data and the results of the samples (a), (b), and (c) are shown in order from the upper part of Table 2.

From Table 2, it was confirmed that the a-axis of the prepared Ag-HAp was longer than the axis length of the HAp standard data. Since the radius of silver ions (= 1.28 Å) is larger than the radius of calcium ions (= 0.99 Å), it was suggested that the calcium ions of HAp were replaced by silver ions.

(Measurement of surface shape and particle size by SEM)
The morphology of the prepared Ag-HAp was observed using SEM. The SEM images of the prepared Ag-HAp samples (a) to (c) are shown in FIG. As shown in FIG. 3, in the sample (a) in the state of primary particles, it was observed that the particles were close to each other. That is, it was found that the sample (a) was insufficiently crystalline (amorphous). Further, in the sample (b) fired without adding the anti-fusion agent, it was observed that the primary particles were fused to each other to form a large lump. On the other hand, in the sample (c) baked with the addition of an anti-fusion agent, the primary particles are sintered with almost no fusion, and are a mixture of spherical or rod-shaped particles (each particle is separated from each other). ) Was observed. Moreover, the particle size of the primary particles was almost the same with and without the fusion inhibitor.

In addition, the average particle size of the prepared Ag-HAp samples (a) to (c) was measured by a dynamic light scattering method (ELS, "ELS-8000 manufactured by Otsuka Electronics Co., Ltd." was used).

As a result, the average particle size of the sample (a) was 502.0 nm, whereas the average particle size of the sample (b) fired without adding the anti-fusion agent was as large as 1671.8 nm. I understood. On the other hand, the average particle size of the sample (c) calcined with the addition of the anti-fusion agent was as small as 374.7 nm. From this, it was suggested that when the fusion inhibitor was not used, the primary particles aggregated and fused to form secondary particles, and the particle size became large.

From the above, it was considered that small F-HAp could be produced in both the primary particles and the secondary particles by synthesizing F-HAp using an anti-fusion agent.

(Elemental composition analysis by ICP-AES)
The composition ratio of silver in Ag-HAp was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). As a measuring device, Optima 2000DV (manufactured by PerkinElmer Japan Co., Ltd.) was used. A 100 ppm Ag-HAp solution was prepared as a sample for ICP measurement. The results are shown in Table 3. Ag in the sample (a) in the state of primary particles (corresponding to "sample-1" in the table) is 1.5%, but the sample (c) fired by adding an anti-fusion agent is added (in the table). Ag was 0.5% (corresponding to "sample-3"). It was speculated that this decrease in Ag was due to the washing of Ag-HAp in the removal step.

<Antibacterial evaluation>
Next, the antibacterial property evaluation against Ag-HAp prepared above will be described. In the following description, the evaluation method and the evaluation result will be described in this order.

(Evaluation method)
Using the prepared Ag-HAp, the antibacterial property of Ag-HAp was evaluated by the powder addition method. The antibacterial property of Ag-HAp is E.I. I went to E. coli. E. E. coli is a microorganism that can cause implant-related infections and is a type of Gram-negative bacterium.

[Washing of Ag-HAp]
First, Ag-HAp (Ca _9.95 Ag _0.05 (PO ₄ ) ₆ (OH) ₂ : formula amount = 1015.0) used in the antibacterial property test was washed. 1 mL of 70% ethanol was added to 10 mg of Ag-HAp that had been sufficiently pulverized in a mortar and placed in a tube for pipetting. Then, the mixture was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was discarded. This was repeated twice and then washed with 140 mM NaCl in the same manner.

[Preparation of bacteria]
Absorbance was measured with a microplate reader to adjust the concentration of the bacterial solution. The liquid-cultured bacteria were diluted 20-fold, 150 μL was spotted on a plate, and the absorbance at a wavelength of 600 nm was measured. 140 mM NaCl was used as the background. Absorbance values were diluted bacterial solution so that 0.1 (10 ⁵ cells / [mu] L).

[Assay]
Powder addition method Put a solution containing Ag-HAp dispersed in 100 μL of 140 mM NaCl solution and 100 μL of bacterial solution in a tube, pipette, and stir with a petit rotor for 1 hour to mix Ag-HAp and bacteria. Made contact. Next, prepare 5 tubes containing 180 μL of NaCl solution, number them (1) to (5) in order, and add 20 μL of a mixed solution of bacteria and Ag-HAp stirred for 1 hour (1). , Pipetting and stirring thoroughly. Next, 20 μL of the solution of (1) was put into (2), pipetting was performed, and the mixture was sufficiently stirred. This was repeated in the same procedure as (2) to (3), (3) to (4) ... as shown in FIG. 4, and the mixed solution of bacteria and Ag-HAp was diluted (20 μL was added to 180 μL). Therefore, it is diluted 10 times at a time). Further, 5 μL of the solution diluted 10-fold in 5 steps was spotted on a petri dish of agar medium in order from the lowest concentration (5), and cultured in an incubator set at 37 ° C. for 1 day. HAp manufactured by Company B was used as a control for the antibacterial test. Based on the above evaluation, No. 1 in which Ag-HAp dispersed in 100 μL of NaCl solution was used as the above samples (a) to (c), respectively. 1-No. It was done for the solution of 3. The Ag-HAp concentration in the NaCl solution was 49.3 mmol / L (5.0 mg / 100 μL).

(Evaluation results)
[Antibacterial properties of Ag-HAp]
The results of the antibacterial test of Ag-HAp by the above powder addition method are shown in FIGS. 5 and 4. The uppermost row of FIG. 5 shows the result of HAp as a control, and the lower row shows the result of Ag-HAp. The sterilization rate shown in Table 4 was determined from the ratio of the number of Ag-HAp colonies to the number of control colonies, that is, (the number of reductions in the number of F-HAp colonies) / (the number of control colonies). As a result, all the samples (a) to (c) showed a sterilization rate of 90% or more, that is, regardless of the presence or absence of firing. In this antibacterial test, the above No. 1-No. Each of the solutions of 3 was performed 3 times (n = 3). Further, FIG. 5 shows the result of one specific test of n = 3 as an example.

Here, since the composition of Ag-HAp used in this antibacterial property test is Ca _9.95 Ag _0.05 (PO ₄ ) ₆ (OH) ₂ , the concentration in terms of Ag ions (Ag ion concentration). Is 49.3 mmol × 0.05 = 2.47 mmol / L. Generally, as the antibacterial property of Ag ion alone, it is said that if the Ag ion concentration is 5 to 10 ppb (0.093 × 10 ^-3 mmol), the antibacterial property is exhibited against Escherichia coli. On the other hand, as shown in the above test, in order to develop the antibacterial property as Ag-HAp, 2.47 mmol / L is required in terms of Ag ion, and the antibacterial property is improved as compared with the case of Ag ion alone. It was found that more Ag ions were required for expression. From this, it can be said that the antibacterial property of Ag-HAp is milder than that of Ag ion alone.

≪F-HAp≫
Next, the results of synthesizing F-HAp, identifying the obtained F-HAp, and evaluating the acid resistance and antibacterial properties of the F-HAp will be described.

<Synthesis of F-HAp>
An F-HAp nano-single crystal was prepared by using the coprecipitation method, which is one of the wet methods. As the starting material, calcium nitrate tetrahydrate was used as the Ca source, phosphoric acid was used as the P source, and sodium fluoride was used as the F source. Specifically, it was carried out as follows.

(Primary particle generation process)
7.03 g of calcium nitrate tetrahydrate and 0.254 g of sodium fluoride were each dissolved in 200 ml of ethanol (99.5% aqueous solution), and the mixture was stirred under a nitrogen atmosphere for 30 minutes. Similarly, 2.04 g of phosphoric acid was dissolved in 50 ml of ethanol, and the mixture was stirred under a nitrogen atmosphere for 30 minutes. Next, sodium fluoride was mixed with calcium nitrate at once, and after stirring for 5 minutes, phosphoric acid was added, and the mixture was stirred and reacted for 1 hour while being kept at 80 ° C. in a water bath. Then, the mixture was stirred at room temperature for 15 hours. After stirring, the mixture was centrifuged at 6,500 rpm for 3 minutes using a centrifuge, and the supernatant was discarded. Pure water was added and ultrasonic irradiation was performed to sufficiently disperse the precipitated reactant, and centrifugation was performed at 6,500 rpm for 3 minutes. This purification was repeated 3 times, and the obtained sample (F-HAp) was dispersed in water.

(Mixing process and firing process)
For 1.0 g of F-HAp prepared above, 1.0 g of polyacrylic acid is dissolved in 100 ml of pure water, and aqueous ammonia (28.0%) is used to adjust the pH of the polyacrylic acid to 5. (Solution A). Then, while stirring the F-HAp solution, solution A was added dropwise at a rate of 10 ml for 1 minute using a perista pump. Further, the mixture was stirred as it was for 5 minutes (solution B). Similarly, 3.7 g of calcium nitrate was dissolved in 370 ml of pure water with respect to 1.0 g of the prepared F-HAp (solution C). While stirring the solution B, the solution C was added dropwise at a rate of 10 ml for 1 minute using a perista pump. Then, the mixed solution was suction-filtered using an aspirator, and the taken-out sample was dried under reduced pressure for 1 hour. The dried sample was crushed in a mortar, placed in a crucible, and baked in an electric furnace at 800 ° C. for 1 hour. After cooling in the furnace, the calcined F-HAp was crushed in a mortar.

(Removal process)
8.0 g of ammonium nitrate was dissolved in 800 ml of pure water, and the mixture was stirred under a nitrogen atmosphere for 30 minutes. A solution was added to the calcined F-HAp, ultrasonic irradiation was performed to sufficiently disperse the precipitated reactant, and centrifugation was performed at 8,500 rpm for 3 minutes. The supernatant was discarded and an aqueous ammonium hydrogen nitrate solution was added. Hereinafter, this purification was repeated 4 times until the pH of the supernatant became neutral. Further, the taken-out sample was purified by the same method using pure water. Finally, the sample was dried under reduced pressure for 1 hour, pulverized in a mortar and collected.

<Identification of F-HAp>
Regarding the F-HAp sample obtained as described above, as identification of F-HAp, crystal structure analysis by an X-ray diffractometer (XRD), surface shape measurement and particle size measurement by a scanning electron microscope (SEM) ( Dispersibility evaluation by DLS) and concentration analysis by a fluorine ion densitometer were performed.

(Crystal structure analysis by XRD)
The crystal structure of the produced F-HAp was analyzed using a powder X-ray diffractometer (Mini Flex / HCM manufactured by Rigaku Denki Co., Ltd.). A CuKα source (λ = 1.541841 Å (Angstrom)) was used as the X-ray source used in the XRD, the output was 30 kV / 15 mA, the scan speed was 1.0 ° / min, the sampling width was 0.01 °, and the measurement was performed. The mode was a continuous condition. FIG. 6 shows the X-ray diffraction peak of the prepared F-HAp. The upper part of FIG. 6 is the peak of F-HAp, and the lower part is the F-HAp data of the JCPDS card.

As can be seen from FIG. 6, the prepared F-HAp substantially matched the peak value of the F-HAp data (standard data) of the JCPDS card. Since the diffraction pattern of F alone was not detected in the prepared F-HAp, it was presumed that fluoride ions were substituted in the crystal structure of HAp. From the above, it was presumed that F-HAp having a crystal structure similar to that of the F-HAp data of the JCPDS card could be produced.

In addition, the axial length was examined from the X-ray diffraction peak value measured above. The results are shown in Table 5.

From Table 5, it was confirmed that the a-axis of the prepared F-HAp was shorter than the shaft length of the HAp standard product. Since the radius of the fluoride ion (= 1.33 Å) is smaller than the radius of the hydroxide ion (= 1.37 Å), it was found that the hydroxide ion of HAp was replaced with the fluoride ion.

(Surface shape measurement by SEM and dispersibility evaluation by DLS)
The morphology of the prepared F-HAp was observed using SEM. A commercially available F-HAp is shown in FIG. 7, and an SEM image of the produced F-HAp is shown in FIG. From FIGS. 7 and 8, it was found that the F-HAp synthesized using the anti-fusion agent (prepared above) had smaller primary particles than the commercially available F-HAp and had a uniform spherical shape. It was.

Moreover, the dispersibility of the prepared F-HAp was evaluated from the result of the average particle size measurement by DLS. Samples (prepared F-HAp and commercially available F-HAp) were dispersed in ethanol to prepare a 1% by weight ethanol dispersion. After preparation, a sample / ethanol dispersion was added to the tetrahedral cell. As the analysis conditions, the analysis method: NNLS, the display items: autocorrelation function, scattering intensity histogram, weight conversion histogram, and number conversion histogram were used, and the measurement wavelength range was 8500 cm ^{-1 to} 100 cm ^-1, and the particle size distribution analysis was performed.

As a result, the average particle size of the commercially available F-HAp of FIG. 8 was 17507.7 ± 1161.0 nm, and the average particle size of the prepared F-HAp of FIG. 8 was 319.2 ± 51.3 nm. Therefore, it was found that the F-HAp synthesized using the anti-fusing agent had smaller secondary particles and a smaller particle size distribution.

From the above, it was considered that small F-HAp could be produced in both the primary particles and the secondary particles by synthesizing F-HAp using an anti-fusion agent.

(Concentration analysis by fluorine ion densitometer)
The fluoride ion concentration of commercially available F-HAp and the prepared F-HAp was measured using a fluorine ion concentration meter (F-10Z, manufactured by Kasa Principle Industry Co., Ltd.). To about 50 mg of a sample (commercially available F-HAp and prepared F-HAp), 4 ml of 0.5 M hydrochloric acid and 15 ml of pure water were mixed, and pure water was added until the volume became 25 ml. After shaking the solution for a while, the dissolution of the sample was confirmed, and if it was not dissolved, 0.5 M hydrochloric acid was added to dissolve it. 25 ml of the solution containing the sample and 2 ml of the masking agent were placed in a 100 ml volumetric flask, and pure water was further added to adjust the solution to 100 ml. After making the concentration in the solution uniform by ultrasonic irradiation, ammonia water was added to adjust the pH to 5.0. Table 6 shows the theoretical concentration (mmol / L) and the measured concentration (mmol / L) of each sample.

From Table 6, it was confirmed that the prepared F-HAp had a higher fluoride ion concentration. From the ratio of the theoretical concentration to the measured concentration, the substitution rate of hydroxide ions with fluoride ions was estimated to be about 90%.

<Acid resistance evaluation>
Next, the acid resistance evaluation for F-HAp prepared above will be described. In the following description, the evaluation method and the evaluation result will be described in this order.

(Evaluation method)
An acid resistance test was conducted to investigate the dissolution point of the prepared F-HAp. For comparison, the acid resistance of a total of four samples including F-HAp manufactured by A company, HAp manufactured by B company, and HAp manufactured by C company was evaluated.

First, 0.02 mM hydrochloric acid solution is added to pure water, and solvents with different pH (pH = 1.6, 2.0, 2.5, 2.8, 3.2, 3.6, 4.0, 4) .6) was prepared. 2 mg of the sample was added to 5 mL of the pH-adjusted solution, stirred for 1 minute with a vortex mixer, transferred to a quartz glass cell, and the absorbance was measured with an ultraviolet / visible spectrophotometer (V550 manufactured by JASCO Corporation). Fixed wavelength measurement was selected, and measurement was performed for each sample and each concentration under the conditions of metering mode: Abs, response: Quick, bandwidth: 2.0 nm, measurement wavelength: 595 nm, number of repetitions: 3, and repetition interval: 0. .. The transmittance was obtained from the measured absorbance, a graph was created with the vertical axis representing the transmittance and the horizontal axis representing the pH, and the dissolution points of the four samples were compared. Two asymptote lines were drawn on the graph, and the pH at the intersection was defined as the dissolution point. The created graph is shown in FIG.

(Evaluation results)
From FIG. 9, the dissolution points of HAp manufactured by Chemical Company C and HAp manufactured by B Company are pH = 4.0, the dissolution points of F-HAp manufactured by A Company are pH = 3.2, and the dissolution points of the prepared F-HAp are prepared. Was pH = 2.9. Therefore, it was confirmed that the prepared F-HAp had higher acid resistance than HAp. The difference in acid resistance between the F-HAp synthesized this time and the F-HAp manufactured by Company A was considered to be due to the difference in the fluorine content.

<Antibacterial evaluation>
Next, the antibacterial property evaluation against F-HAp prepared above will be described. In the following description, the evaluation method and the evaluation result will be described in this order.

(Evaluation method)
Using the prepared F-HAp, the antibacterial property of F-HAp was evaluated by the powder addition method. The antibacterial property of F-HAp was performed against Escherichia coli. The antibacterial property of fluoride ions was also evaluated using sodium fluoride. The antibacterial property evaluation of fluoride ions was performed on the 7 bacterial species listed in Table 7.

[Preparation of bacteria]
Absorbance was measured with a microplate reader to adjust the concentration of the bacterial solution. The liquid-cultured bacteria were diluted 20-fold, 150 μL was spotted on a plate, and the absorbance at a wavelength of 600 nm was measured. 140 mM NaCl was used as the background. Absorbance values were diluted bacterial solution so that 0.1 (10 ⁵ cells / [mu] L).

[Assay]
Powder addition method A solution containing F-HAp (Ca ₁₀ (PO ₄ ) ₆ F _1.8 (OH) _0.2 : formula amount = 1008.2) dispersed in 100 μL of 140 mM NaCl solution and 100 μL of bacterial solution were added. It was placed in a tube, pipetted, and stirred with a petit rotor for 1 hour to bring F-HAp into contact with bacteria. Next, prepare 5 tubes containing 180 μL of NaCl solution, number them (1) to (5) in order, and add 20 μL of a mixed solution of bacteria and F-HAp stirred for 1 hour (1). , Pipetting and stirring thoroughly. Next, 20 μL of the solution of (1) was put into (2), pipetting was performed, and the mixture was sufficiently stirred. This was repeated in the same procedure as (2) to (3), (3) to (4), etc. as shown in FIG. 4, and the mixed solution of bacteria and F-HAp was diluted (20 μL was added to 180 μL). Therefore, it is diluted 10 times at a time). Further, 5 μL of the solution diluted 10-fold in 5 steps was spotted on a petri dish of agar medium in order from the lowest concentration (5), and cultured in an incubator set at 37 ° C. for 1 day. HAp manufactured by Company B was used as a control for the antibacterial test. The above evaluation was performed on the solutions of Examples 1 to 7 in which the content of F-HAp dispersed in 100 μL of NaCl solution was changed. The composition formula of F-HAp is based on the theoretical concentration of fluoride ions in the F-HAp sample of 18.8 mg / L (when substituted with 100% fluoride ions), and the actual measurement of the prepared sample is performed. The ratio to the value (16.9 mg / L) was calculated and replaced with the original HAp; Ca ₁₀ (PO ₄ ) ₆ (OH ₂ ) and 100% fluoride ion F-HAp; Ca ₁₀ (PO ₄ ). from ₆ F _2, ionic species lacking the theoretical amount, all determined in terms of a hydroxide ion.

Method of antibacterial test using sodium fluoride A 50 mM NaF solution having a concentration harmless to the human body was prepared, and the bacteria were eradicated by filtration. It was mixed with an autoclaved LB solution to prepare a mixed solution having five different concentrations of 5 mM, 0.5 mM, 0.05 mM, 0.005 mM, and 0 mM, and poured into a plate to prepare an agar medium containing NaF. Bacteria were liquid cultured and spread with a Conradi rod as 5μL spot diagram 10 into five plates made that the concentration values of OD ₆₀₀ as a 1 ⁽¹⁰⁶ / [mu] L). Then, the cells were cultured in an incubator set at 37 ° C. for 1 day.

(Evaluation results)
[Antibacterial properties of F-HAp]
As a result of conducting an antibacterial test of F-HAp by the above powder addition method, when the concentration of the F-HAp solution added to the bacterial solution is 49.6 mmol / L or more, 53.7 to 99.9% antibacterial property is confirmed. I was able to. The results are shown in Table 8. The upper part of Table 8 shows the result of HAp as a control, and the lower part shows the result of F-HAp. The sterilization rate shown in Table 8 was determined from the ratio of the number of F-HAp colonies to the number of control colonies, that is, (the number of reductions in the number of F-HAp colonies) / (the number of control colonies).

When the antibacterial property test was conducted by changing the concentration of F-HAp as shown in Table 8, the antibacterial property could be confirmed in all the examples. The sterilization rate was 98.0% to 99.9% when the F-HAp concentration was 99.2 mmol / L or more.

When the F-HAp concentration was 49.6 mmol / L, a bactericidal rate of 53.7% was observed. Therefore, when the F-HAp concentration is 49.6 mmol / L or more, it can be said that the bactericidal property is obtained. Further, when F-HAp is 99.2 mmol / L or more, it can be said that it has bactericidal properties.

Here, since the composition of F-HAp used in this antibacterial test is Ca ₁₀ (PO ₄ ) ₆ F _1.8 (OH) _0.2 , F-HAp is antibacterial (bacteriostatic). The concentration in terms of fluoride ions (fluoride ion concentration) for expressing the above is 49.6 mmol × 1.8 = 89.3 mmol / L or more (furthermore, in order to exhibit antibacterial properties having a bactericidal rate of 95% or more). 178.6 mmol / L or more). The fluoride ion concentration is, for example, 5 to 10 ppb (0.093 × 10 ^-3 mmol), which is a concentration at which silver ions, which are widely recognized for antibacterial properties, can exhibit antibacterial properties against Escherichia coli, and Ag-HAp. It was found that a higher (fluoride) ion concentration was required to develop the antibacterial property, as compared with 2.47 mmol / L, which is the silver ion concentration capable of expressing the antibacterial property of. From this, it can be said that the antibacterial property of F-HAp is milder than the antibacterial property of silver ion alone and further, the antibacterial property of Ag-HAp.

[Antibacterial properties of fluoride ions]
The result of the antibacterial property test using sodium fluoride performed by the above method is shown in FIG. (1) is Bacillus cereus, (2) is Salmonella, (3) is Staphylococcus aureus, (4) is Escherichia coli, (5) is Pseudomonas aeruginosa, (6) is Klebsiella pneumoniae, and (7) is Streptococcus pyogenes. is there.

As shown in FIG. 11, when five plates having different concentrations were compared, it was confirmed that the Streptococcus pyogenes (7) became thinner as the concentration increased. Therefore, it was found that the fluoride ion itself has antibacterial activity against Streptococcus pyogenes. Here, although it is not clear why F-HAp has antibacterial activity against Escherichia coli even though fluoride ion itself does not have antibacterial activity against Escherichia coli, it is not clear why F-HAp has antibacterial activity against Escherichia coli. It was speculated that the sex was not simply due to the antibacterial properties of the fluoride ion itself. As described above, from the results of this test and the antibacterial property test of F-HAp, there are bacterial species that can express antibacterial activity and bacterial species that cannot express antibacterial activity by fluoride ion alone, but F-HAp is fluoride ion It was found that antibacterial activity can be expressed even against bacterial species that cannot express antibacterial activity by themselves (for example, Streptococcus pyogenes).

Although preferred embodiments of the present invention have been described above with reference to the drawings, the present invention is not limited to the above-described embodiments. That is, it is understood that other forms or various modifications that can be conceived by those skilled in the art within the scope of the invention described in the claims also belong to the technical scope of the present invention.

Claims (3)

A group of hydroxyapatite derivative particles composed of antibacterial hydroxyapatite derivative particles in which at least a part of hydroxide ions in the crystal structure of hydroxyapatite is replaced with fluoride ions.
The particle size of the antibacterial hydroxyapatite derivative particles is in the range of 10 nm to 1000 nm.
The hydroxyapatite derivative particle group, wherein the majority of the antibacterial hydroxyapatite derivative particles contained in the hydroxyapatite derivative particle group are primary particles composed of a single crystal. A dispersion in which a predetermined microorganism of 5.0 × 10 ⁴ cells / μL and the antibacterial hydroxyapatite derivative particles coexist at a concentration of 89.3 mmol / L or more in terms of fluoride ions was prepared, and the dispersion was prepared. The sterilization rate after spotting the solution on the medium and culturing at 37 ° C. for 1 day is 50% or more.
The hydroxyapatite derivative particle group according to claim 1, wherein the predetermined microorganism is Escherichia coli. A dispersion in which a predetermined microorganism of 5.0 × 10 ⁴ cells / μL and the antibacterial hydroxyapatite derivative particles at a concentration of 178.6 mmol / L or more in terms of fluoride ions coexist was prepared, and the dispersion was prepared. The sterilization rate after spotting the solution on the medium and culturing at 37 ° C. for 1 day was 95% or more.
The hydroxyapatite derivative particle group according to claim 1, wherein the predetermined microorganism is Escherichia coli.