JP2019077720A - Hydroxyapatite derivative aggregate - Google Patents

Abstract

To provide a hydroxyapatite derivative aggregate comprising hydroxyapatite derivative particles, which has mild antibacterial activity that has little adverse effect on healthy tissue while maintaining functions of HAp such as excellent biocompatibility, stickiness or adhesion to biological tissues, and low biodegradable and absorptive property.SOLUTION: As an antibacterial material, there is used the hydroxyapatite aggregate, comprising: antibacterial hydroxyapatite derivative particles where at least a part of calcium ions in the crystalline structure of hydroxyapatite are substituted with silver ions; or antibacterial hydroxyapatite derivative particles where at least a part of hydroxide ions in the crystalline structure of hydroxyapatite are substituted with fluoride ions.SELECTED DRAWING: None

Description

  The present invention relates to a hydroxyapatite derivative particle group consisting of antibacterial hydroxyapatite derivative particles in which a part of the crystal structure of hydroxyapatite is substituted by another ion.

Conventionally, it is useful as a medical material which exists as single crystal primary particles in a solvent and has high dispersibility, in particular, biocompatibility, adhesiveness to a living tissue, or adhesiveness, and low bioabsorbability. Calcium phosphate calcined particles (ceramic particles) including single crystal hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ , hereinafter referred to as “HAp”) that is See Patent Document 1). As applications of such single crystal HAp, dental materials such as bone fillers, dental fillers, drug sustained release agents, medical materials, immobilized carriers such as bacteria, yeast, etc., for column chromatography Various applications such as fillers, adsorbents such as deodorants, etc., and nanometer-sized drug delivery systems (nano DDS) have been known.

Patent No. 5043436 gazette

  Here, calcium phosphate calcined particles such as HAp are used as dental fillers such as bone fillers, dental fillers, drug sustained release agents, medical materials, proteins, amino acids, polysaccharides, physiologically active substances, etc. When used for applications such as immobilized carriers, packing agents for column chromatography, adsorbents such as deodorants, and nanometer-sized drug delivery systems (nano DDS), in many cases, an antibacterial action is required. In such a case, until now, it has been common to use an antibacterial metal such as Ag, an ion of the metal and other antibacterial components simply by combining it with HAp or the like. .

  However, as described above, when an antimicrobial metal such as Ag or other antimicrobial component is simply used in combination with HAp, since the antimicrobial activity is too strong, not only tissues infected with bacteria etc., but also healthy tissues Also had an adverse effect.

  Therefore, the present invention has been made in view of the above circumstances, and it is a sound tissue while maintaining the functions of HAp such as excellent biocompatibility, adhesiveness or adhesion to a living tissue, and low bioresorbability. It is an object of the present invention to provide a hydroxyapatite derivative particle group consisting of hydroxyapatite derivative particles having mild antibacterial properties which hardly adversely affect the

As a result of intensive studies to solve the above problems, the present inventors have found that the crystal structure of HAp can be achieved by incorporating silver ion (Ag ⁺ ) or fluoride ion (F ⁻ ) into the crystal structure of HAp. It was found that HAp derivatives such as Ag-HAp and F-HAp in which a part was replaced with silver ion or fluoride ion had mild antibacterial activity. In addition, the inventors of the present invention have excellent biocompatibility, adhesiveness to living tissue, or adhesiveness by using HAp derivative particles consisting of particles of HAp derivatives having such mild antibacterial properties as an antibacterial component. It has been found that while maintaining the function of HAp such as low bioresorbability, it is possible to obtain a material having mild antibacterial properties that hardly adversely affect healthy tissue. The present invention has been completed based on the above findings.

That is, according to the present invention, hydroxyapatite derivative particles (the first hydroxyapatite derivative particle group) are characterized in that they consist of antibacterial hydroxyapatite derivative particles in which at least a part of calcium ions in the crystal structure of hydroxyapatite is substituted by silver ions. Apatite derivative particles).
In the first hydroxyapatite derivative particle group, a coexistence of a predetermined microorganism of 5.0 × 10 ⁴ / μL and the antimicrobial hydroxyapatite derivative particles at a concentration of 2.47 mmol / L or more in terms of silver ion It is preferable that the dispersion after preparation is spotted, the dispersion is spotted on a culture medium, and the sterilization rate after culturing for 1 day at 37.degree. C. is 90% or more.
In the first hydroxyapatite derivative particle group, it is preferable that the hydroxyapatite crystal phase purity determined by the XRD of the antibacterial hydroxyapatite derivative particles is 90% or more.
Further, the present invention is characterized in that the hydroxyapatite derivative particles according to the present invention are composed of 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 particles).
In the second hydroxyapatite derivative particle group, a predetermined microorganism of 5.0 × 10 ⁴ / μL and the antimicrobial hydroxyapatite derivative particles at a concentration of 89.3 mmol / L or more in terms of fluoride ion, It is preferable that the dispersion which made the coexistence be prepared, the said dispersion is spotted on a culture medium, and the sterilization rate after culture | cultivating at 37 degreeC for 1 day is 50% or more.
In the second hydroxyapatite derivative particle group, a predetermined microorganism of 5.0 × 10 ⁴ / μL and the antimicrobial hydroxyapatite derivative particles at a concentration of 178.6 mmol / L or more in terms of fluoride ion, It is preferable that the dispersion which made the coexistence be prepared, the said dispersion is spotted on a culture medium, and the sterilization rate after culture | cultivating at 37 degreeC for 1 day is 95% or more.
In the first and second hydroxyapatite derivative particle groups, the antibacterial hydroxyapatite derivative particles are preferably calcined hydroxyapatite derivative particles.
In the first and second hydroxyapatite derivative particle groups, the particle diameter of the antibacterial hydroxyapatite derivative particles is preferably in the range of 10 nm to 1,000 nm.

  According to the present invention, HAp derivative particles composed of HAp derivative particles such as Ag-HAp and F-HAp in which a part of the crystal structure of HAp is substituted with silver ions or fluoride ions are used as antibacterial components. 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 bactericidal action and bacteriostatic action with little adverse effect on healthy tissues not infected with bacteria and the like.

It is a figure which shows the FTIR spectrum of Ag-HAp produced in the Example. It is a figure which shows the XRD diffraction pattern of Ag-HAp produced in the Example. It is a photograph which shows the SEM image (magnification 20,000 times) of Ag-HAp produced in the example. It is a schematic diagram which shows the dilution method of the sample in the antimicrobial property test using Ag-HAP or F-HAp in an Example. It is a photograph which shows the result of the antimicrobial property test of Ag-HAp produced in the Example. It is a figure which shows the XRD diffraction pattern of F-HAp produced in the Example. It is a photograph which shows the SEM image (magnification 20,000 times) of commercial F-HAp. It is a photograph which shows the SEM image (magnification 20,000 times) of F-HAp produced in the example. It is a graph showing F-HAp and commercial HAp (three kinds) acid resistance created in the example. It is a schematic diagram which shows the method of the antimicrobial property test using NaF in an Example. It is a figure which shows the result of the antimicrobial 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 Particles 2 Principle of Antibacterial Action 3 Application of HAp Derivative Particles

«Configuration of HAp Derivative Particles»
First, the configuration of the HAp derivative particle group according to the present invention will be described. The HAp derivative particles according to the present invention consist 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 particles containing silver ions or fluoride ions have mild antibacterial properties. Here, "mild antibacterial activity" as referred to in the present specification exerts a bactericidal action or bacteriostatic action on bacteria and the like with almost no adverse effect on healthy tissues not infected with bacteria and the like. It means the degree of antibacterial that is possible. In other words, "mild antibacterial activity" means that, with regard to HAp derivatives containing silver ions, the amount of silver ions necessary for the HAp derivative particles to exhibit antibacterial activity exerts antibacterial activity by silver ions alone. This is very large compared to the amount of silver ion required, meaning that the antibacterial activity is suppressed. With respect to HAp derivatives containing fluoride ion, the amount of fluoride ion necessary for the HAp derivative particle group to exhibit the antibacterial property is a concentration of silver ion whose antibacterial property is widely recognized, and the above silver ion This means that the antibacterial activity is suppressed much more than the amount of silver ion contained in the HAp derivative containing

  In addition, as microorganisms (here, not only the “microorganisms” in a narrow sense but also a concept including viruses and fungi) in which the HAp derivative particles according to the present invention can exhibit antibacterial properties, for example, Escherichia coli (Escherichia) E. coli), Shigella dysenteriae, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella enteritidis, Yersinia. ), Proteus (Proteus, Providencia, Morganel) a) Citrobacter (Citrobacter freundii), Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Burkholderia (Pseudomonas) cepacia, L. , Bordetella pertussis, Niserria gonorrhoeae, Niserria meningitides catarrhalis (Moraxella (Branhamella) catarrhalis), Haemophilus i bacteria (Bacteroides fragilis), Staphylococcus aureus (Staphylococcus aureus), methicillin-resistant Staphylococcus aureus (MRSA), Paleobacteria (Bacillus subtilis), Bacillus cereus (Bacereus cereus) ), Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Listeria monocytogenes, diphtheria bacteria (Corynebacterium diphthriae), Nocardia (Nocardia sp.), Enterobacteria enterobacteria (Enterobacter cloacae), Streptococcus pneumoniae, Streptococcus pyogenes, Acinetobacterium (Acinetobacter sp. Enterococcus), vancomycin-resistant Enterococci (VRE), Staphylococcus epidermidis (Staphylococcus epidermidis), Group B Streptococcus (Streptococcus agalactiae), Mycobacterium tuberculosis (My) Cacterium tuberculosis, Propionibacterium acnes, Helicobacter pylori, Campylobacter jejuni, Campylobacter coli, Pneumonia chlamydia (Chlamydia pneumonia), sexually transmitted disease Chlamydia traumatics Bacteria such as Treponema denticola, Tannerella forsythensis); Trichophyton rubrum, Trichophyton mentagrophytes, Aspergi Fungi such as Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, influenza virus (influenzavirus), hepatitis virus, herpes virus (Herpesviridae), herpes virus (herpesvirus), adenovirus Viruses (adenovirus), viruses such as AIDS virus (Human Immunodeficiency Virus (HIV)), etc. may be mentioned.

  When the HAp derivative particles according to the present invention are used as an antibacterial material, it may be a liquid antibacterial material in which the HAp derivative particles are dissolved or dispersed in water, an organic solvent or the like, and the HAp derivative particles are granulated. May be a solid antimicrobial material processed into a solid, a block, a sheet, etc. An antimicrobial property in which HAp derivative particles are mixed with another material, or HAp derivative particles are unevenly distributed on the surface of another material It may be a composite material having

  The antimicrobial HAp derivatives in the present invention include Ag-HAp in which part of the crystal structure of HAp is substituted by silver ions, and F-HAp in which part of the crystal structure of HAp is substituted by 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 HAp derivative particles consisting of particles of these HAp derivatives are bonded to medical materials, polymeric medical materials, or for chromatography In the case of using as a filler, in order to improve the stability in the living body and to ensure the formability, it is preferable to be a calcined HAp derivative particle group (sintered body) obtained by calcining the HAp derivative particle group. Further, the calcined HAp derivative particle group (sintered body) is crystalline, and by using such a crystalline sintered body as an antibacterial material, more mild antibacterial property can be exhibited. Thus, although it is not clear why the fired body is milder in antibacterial activity than the unfired body, the inventors of the present invention have a faster rate of ion elution because the unfired body is amorphous. As a result, the antibacterial action is concentrated in a short period of time, and the ion concentration necessary for the expression of the antibacterial action is temporarily increased, so that the risk of damage to the living tissue is increased, and the antibacterial action is further developed. It is estimated that the period of time to maintain the ion concentration required for In addition, this sintered body is a ceramic, and as the ceramic in this case, not only the ceramic in a narrow sense, but also a broad ceramic including so-called "new ceramic" or "fine ceramic" is meant. The details of the firing method will be described later.

  In addition, since the fusion of primary particles is prevented by the action of the anti-fusion agent described later, the majority of the calcined HAp derivative particle group consisting of an assembly of particles of the calcined HAp derivative maintains the state of primary particles. doing. Therefore, when the calcined HAp derivative particle group is suspended in a solvent, primary particles in which a majority of the calcined HAp derivative particle group consists of single crystals, or primary particles consisting of the single crystals form an ionic interaction. It can be dispersed in aggregated particle mass (single crystal primary particles).

  Furthermore, when the calcined HAp derivative particle group is adsorbed to a medical polymer base material, it is important that the dispersibility is high. In addition, when used as a chromatography filler, it is important that the surface area is high. The calcined HAp derivative particle group according to the present invention is a primary particle of which the majority is a single crystal, or a particle aggregate (single crystal primary particle) in which primary particles of the single crystal are aggregated by ionic interaction. It has good dispersibility in a solvent, and its surface area is high because secondary particles are not formed. Therefore, the calcined HAp derivative particle group according to the present invention can be suitably used particularly for the above application.

  Here, as a method for evaluating whether or not the calcined HAp derivative particles are present as primary particles, for example, the result of measuring the particle diameter by electron microscope observation and the state of being suspended in the solvent by dynamic light scattering method By comparing the results when the particle diameter is measured, it is possible to judge that most of the calcined HAp derivative particle group is in the state of primary particles, if the both results are almost the same. On the other hand, if the result of the particle diameter measurement by the dynamic light scattering method is large from the measurement result of the particle diameter by electron microscope observation, it is judged that the fusion between the primary particles occurs and forms the secondary particles. Can.

  The solvent for dispersing the calcined HAp derivative particle group 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 And hydrocarbons such as dodecane and cyclohexane, halogenated hydrocarbons such as chlorobenzene and chloroform, ethers such as diethyl ether and dioxane, etc. These solvents may be used alone or in combination depending on the purpose of use. It should be selected and used.

  Based on the particle size distribution map determined by the dynamic light scattering method, the primary particle consisting of a single crystal is determined by determining the proportion of particles having a particle size substantially matching the particle size of the primary particle determined by the electron microscope. Alternatively, it is possible to calculate the proportion of a particle mass (single crystal primary particles) in which primary particles consisting of the single crystals are aggregated by ionic interaction.

  Depending on the raw material of the calcined HAp derivative, the type of the anti-fusion agent, the condition of the calcination, etc., at least 50% or more of the single-crystal primary according to the method for producing the calcined HAp derivative particles according to the present invention described later. It is present as particles, more preferably 60% or more as single crystal primary particles if more preferred, and 70% or more as single crystal primary particles under most preferred conditions.

  When the calcined HAp derivative particles are adsorbed to a medical polymer base material, or when used as a chromatography filler, 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, in the step of producing primary particles of the calcined HAp derivative particle group according to the present invention described later, the primary (fine) The particles may be prepared. For example, in the primary particle generation step, primary particles having a particle diameter within the range of 10 nm to 1,000 nm, more preferably 15 nm to 700 nm, and most preferably 20 nm to 500 nm are prepared to 10 nm to 1,000 nm. A calcined HAp derivative particle group having a primary particle diameter within the range of 20 nm to 700 nm, most preferably 25 nm to 750 nm can be produced, more preferably.

  The calcined HAp derivative particles preferably have a uniform particle size (narrow particle size distribution). In order to produce such a fired HAp derivative particle group having a uniform particle size (narrow particle size distribution), it is possible to prepare a primary particle group having a uniform particle size (narrow particle size distribution) in the primary particle generation step. Just do it. Such a calcined HAp derivative particle group having a uniform particle diameter (narrow particle size distribution) can be suitably used, for example, when it is adsorbed on a medical polymer base, a filler for chromatography, a medical material and the like.

[Ag-HAp: Structure]
Ag-HAp contains silver ions in the crystal structure, and calcium ions (Ca ²⁺ ) in the crystal structure of HAp (at least partially) are replaced with silver ions (Ag ⁺ ). In other words, Ag-HAp is an HAp derivative in which silver ion is doped at the position of calcium ion of HAp. The Ag-HAp, unlike the HAp not containing silver ions, has an antibacterial property, and the antibacterial property is mild compared to a mere mixture of silver ions and HAp. In the 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 the hydroxide ions in the HAp crystal structure are fluoride ions or other negative ions. It may be substituted by ions.

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

  The method for producing Ag-HAp according to the present invention may include at least the "primary particle generation step", but may additionally 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 of producing Ag-HAp according to the present invention, the above four steps are carried out, for example, in the order of “1. primary particle generation process” → “2. Mixing process” → “3. Baking process” → “4. removal process” To be done.

(1. Primary particle generation process)
Here, “primary particles” mean particles formed of HAp derivative raw materials (fluorine, HAp, etc.) before firing in the production process of HAp derivative particles. That is, it means the particles formed for the first time in the production process of the HAp derivative particles. Also, in a narrow sense, it means single crystal particles. In the description of the present invention, the term "primary particles" is meant to include those in an amorphous (amorphous) state and those in a state of a fired body fired thereafter.

  On the other hand, the “secondary particle” is a state in which a plurality of “primary particles” are formed by physical bonds such as fusion, etc., or chemical bonds such as ionic bonds or covalent bonds. Means particles. In particular, the number of bonds between primary particles, the shape after bonding, and the like are not limited, and all those in which two or more primary particles are bonded are meant.

  In particular, “single-crystal primary particles” mean primary particles consisting of single crystals of HAp derivative raw material, or particle clusters in which primary particles consisting of the single crystals are aggregated by ionic interaction. The “particle aggregate aggregated by ionic interaction” is a particle aggregate that self-assembles by ionic interaction when dispersed in a medium containing water or an organic solvent, and it is an interparticle particle by firing. Is free of molten and polycrystallized secondary particles.

  The primary particle production step is not particularly limited as long as it is a step capable of producing the above-described primary particles, and may be appropriately selected depending on the raw material of the HAp derivative to be produced. For example, primary particles of Ag-HAp 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 the mixture is 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 dropped and mixed, and the mixture is heated to a predetermined temperature and stirred. As a result, particles of Ag-HAp precipitate.

  Regarding Ag-HAp raw materials, silver nitrate, silver perchlorate, silver ammine complex, silver thiosulfate complex, silver cyano complex etc. as Ag source, calcium nitrate (including hydrate) as Ca source, Use calcium hydroxide, calcium nitrate, calcium chloride etc. P source such as phosphoric acid, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate etc. Can. 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, toluene and the like. As said Ag source, Ca source, P source, and a solvent, the compound mentioned above may be used independently and may be used in mixture of multiple types.

  The method for producing HAp derivative particles according to the present invention comprises: HAp comprising a collection of HAp derivative particles by firing a primary particle group consisting of a collection of primary particles generated by the above primary particle generation step while preventing fusion or the like It is for producing a group of derivative particles. Therefore, the state (particle diameter and particle size distribution) of primary particles generated in the primary particle generation step is directly reflected on the state (particle diameter and particle size distribution) of HAp derivative particles as the final product.

  In addition, this step may include a step of washing the generated primary particles with water or the like, and a step of collecting the primary particles by centrifugation, filtration or the like.

(2. mixing process)
The present mixing step is a step of mixing the primary particles generated in the above step and the anti-fusion agent for preventing the fusion between the primary particles at the time of firing. By interposing an anti-fusion agent in advance between the particles of the primary particle group obtained in the above primary particle generation step, it is possible to prevent fusion between primary particles in the subsequent firing step. The mixture of the primary particles and the anti-fusion agent obtained by the mixing step is referred to as "mixed particles".

  Here, the “fusion inhibitor” is not particularly limited as long as it can prevent fusion between primary particles, but is preferably non-volatile at the firing temperature of the subsequent firing step. . Because it is non-volatile under the firing temperature conditions, it does not disappear from between primary particles during the firing step, and fusion between primary particles can be reliably prevented. However, it is not necessary to have 100% nonvolatility at the firing temperature, as long as it is 10% or more remaining between primary particles after completion of the firing step. In addition, the anti-fusion agent may be chemically decomposed by heat after completion of the firing step. That is, if it remains after the completion of the firing step, it is not necessary to be the same substance (compound) before and after the start of the firing step.

  The anti-fusion agent is preferably a substance that dissolves in a solvent, particularly an aqueous solvent. As described above, by using an anti-fusion agent that dissolves in a solvent as the anti-fusion agent, only by suspending the HAp derivative particles containing the anti-fusion agent in an aqueous solvent such as pure water, Inhibitors (eg, calcium nitrate, calcium carbonate, etc.) can be removed. In particular, if it is an anti-fusion agent that dissolves in an aqueous solvent, there is no need to use an organic solvent when removing the anti-fusion agent, so the equipment corresponding to the use of the organic solvent in the removal process, organic solvent waste treatment 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, but examples of the aqueous solvent include water, ethanol, methanol and the like, and examples of the organic solvent include acetone, toluene and the like.

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

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

  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 may be a method of dispersing primary particles in a solution of anti-fusion agent. However, since it is difficult to uniformly mix the solid and the solid, it can be said that the latter is the preferred method in order to interpose the anti-fusion agent uniformly and reliably 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 time.

  In the mixing step, the primary particles are mixed with a solution containing a polymer compound having any of a carboxyl group, a sulfuric acid group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group or an amino group in a side chain. And the step of further adding a metal salt (alkali metal salt and / or alkaline earth metal salt and / or transition metal salt). By adopting this process, the polymer compound can be adsorbed to the surface of the HAp derivative, and the contact between the HAp derivatives in the process of mixing the anti-fusion agent can be reliably prevented, and thereafter the calcium salt is added to the HAp. It becomes 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 sulfuric acid 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 said high molecular compound will not be specifically limited if it is a compound which has any of a carboxyl group, a sulfuric acid group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, or an amino group in a side chain. For example, as a polymer compound having a carboxyl group in a side chain, polyacrylic acid, polymethacrylic acid, carboxymethylcellulose, styrene-maleic anhydride copolymer, etc. may be mentioned, and as a polymer compound having a sulfate group in a side chain Examples include polyacrylic acid alkyl sulfuric acid ester, polymethacrylic acid alkyl sulfuric acid ester, polystyrene sulfuric acid and the like, and as a polymer compound having a sulfonic acid group in the side chain, polyacrylic acid alkyl sulfonic acid ester, polymethacrylic acid alkyl sulfone Acid ester, polystyrene sulfonic acid, etc., and as the polymer compound having a phosphoric acid group in the side chain, polyacrylic acid alkyl phosphoric acid ester, polymethacrylic acid alkyl phosphoric acid ester, polystyrene phosphoric acid, polyacryloyl aminomethyl phosphonic acid Etc Examples of polymer compounds having a phosphonic acid group in the side chain include polyacrylic acid alkyl phosphonic acid ester, polymethacrylic acid alkyl phosphoric acid ester, polystyrene phosphonic acid, polyacryloyl aminomethyl phosphonic acid, polyvinyl alkyl phosphonic 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 kind of the above-mentioned polymer compounds may be used, but a plurality of kinds 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 a condition of 5 or more and 14 or less and the HAp derivative particle is insoluble. The aqueous solution containing the polymer compound may be prepared by dissolving the polymer compound in distilled water, ion-exchanged water and the like, and adjusting the pH with an aqueous solution of ammonia aqueous solution, sodium hydroxide, potassium hydroxide and the like.

  Further, the concentration of the polymer compound contained in the aqueous solution is preferably 0.001% w / v to 50% w / v, more preferably 0.005% w / v to 30% w / v, 0 .01% w / v or more and 10% w / v or less is most preferable. If the amount is less than the above-mentioned preferred range, the amount of penetration between primary particles is small, and the ratio of preventing contact between primary particles is low. Moreover, when it exceeds the said preferable range, since the melt | dissolution of a high molecular compound becomes difficult, and the operativity of the viscosity of the solution containing the said high molecular compound becoming high worsens, it is unpreferable.

  In the mixing step in the present invention, the solution containing the above-described polymer compound is mixed with primary particles. For such mixing, for example, primary particles may be introduced into the solution, and the primary particles may be dispersed by stirring operation or the like. By the above operation, in the method for producing HAp derivative particles 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 sulfonate group, a phosphate group, a phosphate group, a phosphonate group or an amino Any of the groups can be attached to the surface of the primary particle. At this time, the carboxyl group, sulfuric acid group, sulfonic acid group, phosphoric acid group, phosphonic acid group or amino group is present in the form of ions 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 a solution in which a solution containing a polymer compound and primary particles are mixed, the primary particles Carboxylate ion, sulfate ion, sulfonate ion, phosphate ion, phosphonate ion, amino ion and metal ion (alkali metal ion and / or alkaline earth metal ion and / or transition metal ion) present on the surface It bonds to form carboxylate, sulfate, sulfonate, phosphate, phosphonate, amino acid salt on the surface of the primary particle. Such metal (alkali metal and / or alkaline earth metal and / or transition metal) carboxylates, sulfates, sulfonates, phosphates, phosphonates, amino acid salts function as the above anti-fusion agent . Therefore, primary particles having a carboxylate, sulfate, sulfonate, phosphate, phosphonate, amino acid salt of metal (alkali metal and / or alkaline earth metal and / or transition metal) formed on the surface thereof are , So-called "mixed particles". In addition, since such metal (alkali metal and / or alkaline earth metal and / or transition metal) carboxylate, sulfate, sulfonate, phosphate, phosphonate, amino acid salt precipitates, the precipitate After recovery, it may be dried and subjected to the firing step described later. The drying may be carried out, for example, by heating under reduced pressure. In addition, in the said drying, although drying temperature can be lowered, although pressure reduction conditions are preferable, you may carry out on atmospheric pressure conditions.

  The above-mentioned alkali metal salt is not particularly limited. For example, sodium chloride, sodium hypochlorite, sodium chlorite, sodium bromide, sodium bromide, sodium iodide, sodium iodide, sodium oxide, sodium peroxide , Sodium sulfate, sodium thiosulfate, sodium selenate, sodium nitrite, sodium nitrate, sodium nitrate, sodium phosphate, sodium carbonate, sodium hydroxide, potassium chloride, potassium hypochlorite, potassium chlorite, 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.

  Moreover, as the alkaline earth metal salt, for example, magnesium chloride, magnesium hypochlorite, magnesium chlorite, magnesium bromide, magnesium iodide, magnesium iodide, magnesium iodide, magnesium oxide, magnesium peroxide, magnesium sulfate, 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 phosphate, calcium carbonate, calcium hydroxide, etc.

  Moreover, as the transition metal salt, for example, zinc chloride, zinc hypochlorite, zinc chlorite, zinc bromide, zinc iodide, zinc iodide, zinc iodide, zinc oxide, zinc peroxide, zinc sulfate, zinc thiosulfate, Zinc selenate, zinc nitrite, zinc nitrate, zinc phosphate, zinc carbonate, zinc hydroxide, iron hydroxide, iron hypochlorite, iron chlorite, iron bromide, iron iodide, iron iodide, iron iodide, iron oxide Iron peroxide, iron sulfate, iron thiosulfate, iron selenate, iron nitrite, iron nitrate, iron phosphide, iron carbonate, iron hydroxide and the like can be used. Moreover, a nickel compound may be sufficient.

  The metal salt (alkali metal salt, alkaline earth metal salt, transition metal salt) to be added to the mixed solution of the solution containing the polymer compound and the primary particles is a mixture of two or more kinds even if it is one kind. It may be In addition, metal salts (alkali metal salts, alkaline earth metal salts, transition metals) may be in the solid state, but can be uniformly added, and the concentration to be added can be controlled, etc. It is preferable to add as an aqueous solution from the reason. Further, the amount (concentration) of the metal salt (alkali metal salt and / or alkaline earth metal salt and / or transition metal salt) to be added is the carboxylate ion, sulfate ion, sulfonate ion, phosphorus present on the primary particle surface Acid salt, phosphonate ion, amino ion in combination with metal (alkali metal and / or alkaline earth metal and / or transition metal) carboxylate, sulfate, sulfonate, phosphate, phosphonate The condition is not particularly limited as long as the amino acid salt is generated, and it may be determined after appropriate examination.

  Here, carboxylates, sulfates, sulfonates, phosphates, phosphonates of metals (alkali metals and / or alkaline earth metals and / or transition metals) generated on the surface of primary particles by the above-mentioned steps The amino acid salt is thermally decomposed in the calcination step described later to form an oxide of a metal (an alkali metal and / or an alkaline earth metal and / or a transition metal). For example, when calcium polyacrylate is produced on the surface of the primary particles, it becomes calcium oxide in the firing step. In addition, since the metal oxide (alkali metal oxide and / or alkaline earth metal oxide (eg, calcium oxide) and / or transition metal oxide) is water soluble, it can be easily removed by the removal step described later. Is possible.

  In addition, sodium polyacrylate is soluble in water, so it can be used as an antifusing agent in this mixing step as it is, but calcium polyacrylate is insoluble in water, so only polyacrylic acid is once primary particle surface. Preferably, calcium polyacrylate is precipitated on the surface of the primary particles by adding a calcium salt or the like after the adsorption.

(3. Baking process)
The firing step is a step of exposing the mixed particles obtained in the mixing step to a firing temperature to convert primary particles contained in the mixed particles into ceramic HAp derivative particles (fired particles). Since the antifusing agent is present between the particles of the primary particles, it is possible to prevent the fusion of the primary particles 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, and for example, the range of 100 ° C. to 1800 ° C. is more preferable, and 150 ° C. to 1500 ° C. is more preferable. 200 ° C. to 1200 ° C. are most preferred. Furthermore, from the viewpoint of expressing mild antibacterial properties, the firing temperature is preferably 500 ° C. or higher, and more preferably 700 ° C. or higher. The firing time may be appropriately set based on the desired hardness of the HAp derivative particles and the like. For example, in the Example mentioned later, it bakes at 700 degreeC for 2 hours.

  The apparatus and the like used in the main baking step are not particularly limited, and a commercially available baking furnace (electric furnace or the like) may be appropriately selected and adopted according to the manufacturing scale, manufacturing conditions, and the like.

(4. Removal process)
The main removing 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 removal means and method may be appropriately adopted in accordance with the anti-fusion agent employed in the mixing step. For example, in the case of using a fusion inhibitor having solvent solubility, fusion is achieved by using a solvent which does not dissolve HAp derivative particles (non-dissolving) and a solvent which dissolves the fusion inhibitor (dissolving) Only the inhibitor can be dissolved and removed. It will not specifically limit, if it is a solvent which satisfy | fills the said requirements as a solvent to be used, It may be a water-based solvent, and an organic solvent may be sufficient. For example, water, ethanol, methanol and the like can be mentioned as the aqueous solvent, and acetone, toluene and the like can be mentioned as the organic solvent.

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

  However, because there is no need for equipment corresponding to the use of the organic solvent in the removal step, no need for organic solvent waste liquid treatment, high safety of the manufacturing operation, and a low risk to the environment, etc. The solvent used is preferably an aqueous solvent.

  By the way, in the case of removing the anti-fusion agent using a solvent, the HAp derivative (calcined body) particles containing the anti-fusion agent obtained by the baking step are suspended in a solvent and then filtered or centrifuged. Only the HAp derivative (sintered body) particles may be recovered. In the method for producing the HAp derivative particle group according to the present invention, the above operation is not limited to once but 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 carry out the above-described operation more than necessary because the production process becomes complicated, the production cost becomes high, and the recovery rate of the HAp derivative particles decreases. Therefore, it is preferable to appropriately determine the number of times of the above operation based on the target removal rate of the anti-fusion agent.

  The process may further include a classification step to make the particle diameter uniform.

  In addition to the method of removing the anti-fusion agent using the solvent, the anti-fusion agent can be removed using a magnet by using a magnetic substance as the anti-fusion agent. More specifically, after suspending and dispersing the HAp derivative particles (crude HAp derivative particles) group containing the anti-fusion agent obtained by the baking step in a suitable solvent (such as water), A magnetic force is applied, and only the anti-fusion agent is adsorbed to the magnet, and only the non-adsorbed HAp derivative particles are recovered. Alternatively, the crude HAp derivative particles may be ground into powder without being suspended in a solvent, and then the anti-sticking agent may be separated by a magnet. However, when the suspension is used, the HAp derivative particles and the anti-fusion agent are easily peeled off, and the removal rate of the anti-fusion agent is high. The HAp derivative particle to which this method can be applied is preferably a nonmagnetic material or a weak magnetic material.

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

  Moreover, according to Fourier transform infrared spectroscopy (FTIR), it is possible to identify functional groups contained in the obtained Ag-HAp.

  Moreover, the surface shape and particle diameter of Ag-HAp particle | grains can be measured by observing synthetic | combination Ag-HAp with a scanning electron microscope (SEM).

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

  If too much silver ions are contained in the crystal structure of Ag-HAp, metallic silver may precipitate from the crystal structure of HAp, and the crystal structure of Ag-HAp may not be maintained. Then, the properties of HAp itself (for example, stability in a living body, biocompatibility, adhesion to a living tissue, adhesiveness, etc.) may not be exhibited, and there is a possibility that a biotoxin may occur. When the amount of silver ions is too large, the ratio of ion species constituting the crystal structure of Ag-HAp collapses with precipitation of metallic silver, and another crystalline layer such as tricalcium phosphate (β-TCP) is formed. Since the water solubility of Ag-HAp particles and the metabolic rate in the living body are too high, it is unsuitable when used as a HAp substitute material. Furthermore, since the crystal structure can not be maintained, the release rate of silver ions is increased, and the mild antibacterial property can not 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%. When it is such a substitution ratio of Ag, the hydroxyapatite crystal phase purity (determined from the diffraction pattern of XRD) determined by XRD of Ag-HAp particles is 90% or more. Here, as a method of quantitative analysis by XRD, a Rietveld method, a calibration curve method, a method using a diffraction integral intensity ratio, etc. are generally known, but in the present invention, any one 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 using an inductively coupled plasma emission spectrometer (ICP-AES).

[Ag-HAp: antibacterial activity]
The antimicrobial activity of Ag-HAp obtained as described above preferably satisfies, for example, the conditions described below. That is, a cell suspension containing predetermined cells adjusted to a concentration of 0.1 (10 ⁵ cells / μL) in absorbance value measured by a microplate reader, and Ag-HAp particles in the same volume of NaCl solution as the cell suspension It is preferable that a mixed solution obtained by mixing a solution in which a solution dispersed at a concentration of 2.47 mmol / L or more in terms of silver ion is mixed is spotted on a culture medium and cultured at 37 ° C. for 1 day is 90% or more . Thereby, the HAp derivative particle group of the present invention can more reliably exhibit mild antibacterial property capable of bacteriostatic or sterilizing bacteria etc. without adversely affecting healthy tissue. . Here, “the concentration in terms of silver ion” means the formula weight from the composition (Ca _10-x Ag _x (PO ₄ ) ₆ (OH) ₂ ) of Ag-HAp used for the evaluation of the antimicrobial activity. The molar concentration (mol / L) of the silver ion contained in the NaCl solution is calculated from the formula weight and the molar concentration (mol / L) of Ag-HAp in the NaCl solution.

[F-HAp: structure]
F-HAp includes a fluoride ion in the crystal structure, the hydroxide ions in the crystal structure of HAp (OH ^{-) -} those substituted in the portion (at least part) of the fluoride ion (F) It is. In other words, F-HAp is an HAp derivative in which fluoride ion is doped at the position of hydroxide ion of HAp. Unlike F-HAp which does not contain fluoride ion, this F-HAp has anti-bacterial activity, and the anti-bacterial activity is mild as compared with a mere mixture of fluoride ion and HAp. Furthermore, F-HAp also has high acid resistance. In F-HAp, when at least a part of hydroxide ion in HAp crystal structure is substituted with fluoride ion, at least a part of calcium ion in HAp crystal structure is a silver ion or other positive ions. It may be substituted by ions.

  Here, for example, a medical device such as a catheter is used for medical activities such as blood transfusion and drainage by connecting the inside and outside of the body with a body, but as a material such as a catheter, it is generally high in elasticity such as silicone. Although molecular materials are used, there is a concern that when the device is actually worn for a long period of time, a gap is formed in the adhesion part between the catheter and the living body to cause bacterial infection. Therefore, in recent years, a catheter having high dispersibility and crystallinity, which is useful for preventing bacterial infection, prepared and coated on a polymer base has been developed. 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 percutaneous device, phagocytes such as neutrophils and macrophages phagocytose them and release active oxygen and lysosomal enzymes to dissolve foreign substances eaten. Since the active oxygen such as hydrogen peroxide releases protons and becomes weakly acidic around the inflammation, it is feared that weak HAp dissolves in the acid.

  In such a case, by coating F-HAp having high acid resistance on the surface of the polymer substrate, dissolution by active oxygen or the like can be suppressed. In addition, since F-HAp is improved in acid resistance as compared to normal HAp, and exhibits mild antibacterial activity, using the F-HAp of the present invention as a dental material prevents caries. Can.

[F-HAp: synthesis method]
F-HAp having the characteristics as described above can be produced by known production methods 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 will be described as an example of a method of synthesizing F-HAp using a coprecipitation method which is one of wet methods.

  The method for producing F-HAp according to the present invention may include at least the "primary particle generation step", but may additionally include "mixing step", "baking step" and "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 carried out, for example, in the order of “1. primary particle generation process” → “2. Mixing process” → “3. Baking process” → “4. removal process” To be done.

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

  The primary particle production step is not particularly limited as long as it is a step capable of producing the above-described primary particles, and may be appropriately selected depending on the raw material of the HAp derivative to be produced. For example, primary particles of F-HAp can be produced by the following method. First, a fluorine (F) source (a fluorine compound) and a calcium (Ca) source (a 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 the mixture is 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. Thereafter, the mixture is further stirred at normal temperature for a predetermined time. As a result, particles of F-HAp precipitate.

  As for F-HAp raw materials, sodium fluoride, potassium fluoride and the like as the F source, calcium nitrate (including hydrate) as the Ca source, calcium hydroxide, calcium nitrate, calcium chloride and the like, P As a 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, toluene and the like. As said Ag source, Ca source, P source, and a solvent, the compound mentioned above may be used independently and may be used in mixture of multiple types.

  The method for producing HAp derivative particles according to the present invention comprises: HAp comprising a collection of HAp derivative particles by firing a primary particle group consisting of a collection of primary particles generated by the above primary particle generation step while preventing fusion or the like It is for producing a group of derivative particles. Therefore, the state (particle diameter and particle size distribution) of primary particles generated in the primary particle generation step is directly reflected on the state (particle diameter and particle size distribution) of HAp derivative particles as the final product.

  In addition, this step may include a step of washing the generated primary particles with water or the like, and a step of collecting the primary particles by centrifugation, filtration or the like.

(2. mixing process)
The present mixing step is a step of mixing the primary particles generated in the above step and the anti-fusion agent for preventing the fusion between the primary particles at the time of firing. About this mixing process, since it is the same as that of the case of Ag-HAp mentioned above, detailed explanation is omitted.

(3. Baking process)
The firing step is a step of exposing the mixed particles obtained in the mixing step to a firing temperature to convert primary particles contained in the mixed particles into ceramic HAp derivative particles (fired particles). The main firing step is also the same as in the case of Ag-HAp described above, and thus detailed description will be omitted. However, unlike the case of Ag-HAp, the firing conditions in the examples described later are firing at 800 ° C. for 1 hour.

(4. Removal process)
The main removing 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 main removing step is also the same as in the case of Ag-HAp described above, and thus detailed description will be omitted.

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

  Also, according to Fourier transform infrared spectroscopy (FTIR), it is possible to identify the functional group contained in the obtained F-HAp, and further, for the synthesized F-HAp and commercially available F-HAp, The spectra of FTIRs are compared, and if the absorption peaks attributable to the respective functional groups are almost identical, it can be inferred that the synthesized one is F-HAp.

  Further, by observing the synthesized F-HAp with a scanning electron microscope (SEM), it is possible to measure the surface shape and the particle diameter of the F-HAp particle.

  In addition, the concentration of fluoride ion in the synthesized F-HAp can be measured by using a fluorine ion concentration meter, and the measured value of the fluoride ion concentration and the theoretical concentration (the theoretically determined F It is also possible to determine the fluoride ion substitution rate of hydroxide ion in HAp 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, for example, the conditions described below. That is, a cell suspension containing predetermined cells whose absorbance value measured with a microplate reader was adjusted to 0.1 (10 ⁵ cells / μL), and F-HAp particles in the same volume of NaCl solution as the cell suspension A mixed solution obtained by mixing a solution dispersed at a concentration of 89.3 mmol / L or more in terms of fluoride ion is spotted on a culture medium, and the sterilization rate after culturing for 1 day at 37 ° C. is 50% or more preferable. Thus, the antibacterial agent of the present invention can more reliably exhibit mild antibacterial properties capable of bacteriostatic bacteria and the like without adversely affecting healthy tissue. Furthermore, a bacterial solution containing predetermined cells whose concentration is adjusted to 0.1 (10 ⁵ cells / μL) and the absorbance value measured with a microplate reader, and F-HAp particles in the same volume of NaCl solution as the bacterial solution. A mixed solution obtained by mixing a solution dispersed at a concentration of 178.6 mmol / L or more in terms of fluoride ion is spotted on a culture medium, and the bactericidal rate after culturing for 1 day at 37 ° C. is 95% or more preferable. Thus, the antibacterial agent of the present invention can more reliably exhibit mild antibacterial properties that can kill bacteria and the like without adversely affecting healthy tissue. Here, "the concentration of fluoride ion in terms" is the formula weight of the composition of F-HAp was used to evaluate the antibacterial activity _{(Ca 10 (PO 4) 6} F 2-y (OH) y) Calculated is the molar concentration (mol / L) of fluoride ion contained in the NaCl solution calculated from the formula weight and the molar concentration (mol / L) of F-HAp in the NaCl solution. .

«Principle of antibacterial action»
Although the mechanism of action of the antibacterial agent according to the present invention is unknown, as a result of using the HAp derivative as nanoparticles as described above, the present inventors are likely to be easily incorporated into bacteria of about several μm in size. Thus, it is presumed that the mildly antibacterial HAp derivative incorporated into the inside of the fungus exhibits an action of bacteriostatic or sterilizing the fungus from the inside of the fungus.

«Applications of antibacterial agents»
The HAp derivatives according to the present invention, in particular calcined HAp derivatives, have a very high biological activity, and therefore, in the medical field, for example, dental materials or medical materials such as bone fillers, dental fillers, drug sustained release agents, etc. It can be used widely as In addition, since HAp derivatives have high biological activity, they can be particularly suitably used as medical materials. In addition, 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. Furthermore, the HAp derivative according to the present invention is expected to be used for nanometer sized drug delivery systems (nano DDS). In particular, the HAp derivative according to the present invention and the antibacterial agent containing the same are, among these uses, applications requiring antibacterial properties, for example, medical devices such as catheters, artificial joints, etc. by connecting the outside and the body Are preferably used.

  Next, the present invention will be described more specifically by Examples and Comparative Examples, but the present invention is not limited by these examples.

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

<Synthesis of Ag-HAp>
Using a coprecipitation method, which is one of the wet _{method, Ag-HAp {Ca 10-} x Ag x (PO 4) 6 (OH) 2 ( wherein, 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 { AgNO ₃ } was synthesized at room temperature with mixing in deionized water. Specifically, it carried out as follows.

(Primary particle generation process)
As calcium nitrate tetrahydrate, silver nitrate and ammonium dihydrogen phosphate, any of those purchased from Wako Pure Chemical Industries, Ltd. was used without purification. Moreover, in order to confirm the silver content in HAp, Ag containing HAp of Ca: Ag = 80: 20 (mol%) was prepared. All reactions were carried out at a temperature of 100 ° C., and the amount of reactants was determined by calculation such that the molar ratio of (Ca ²⁺ + Ag ⁺ ): PO ₄ ^3- is 10: 6.

First, 80 mmol of calcium nitrate tetrahydrate and 20 mmol of silver nitrate were respectively 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 in an oil bath at 100 ° C. for 30 minutes. 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% strength NH ₄ OH to this solution and stirred at room temperature for 30 minutes. Next, the phosphorus solution was added dropwise to a 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 complete, the mixture was stirred for 2 hours, maintained at 100 ° C. During stirring (reaction), the pH was always adjusted so that the pH of the solution was kept at 10.0. Before the phosphorus solution was mixed with the Ca and Ag nitride solution, both solutions were clear, but as the addition of the phosphorus solution was increased, the mixed solution gradually became white. The resulting mixed solution was allowed to stand for 12 hours and then washed three times with deionized water using centrifugation to remove impurity ions (NH ₄ ⁺ , NO ₃ ⁻ ). After washing, the mixture was filtered to obtain a wet cake (Ag-HAp cake). The wet cake was fully dried by a low pressure pump and then left in an oven at 60 ° C. overnight. Further, the Ag-HAp cake was crushed using a mortar and pestle to obtain an 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 another part was mixed with the anti-fusion agent and then fired. Baking was performed at 700 ° C. for 2 hours in a crucible.

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

<Identification of Ag-HAp>
Regarding the sample of Ag-HAp 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), The surface shape and particle size measurement by a scanning electron microscope (SEM) and the elemental composition analysis by inductively coupled plasma-atomic emission spectrometry (ICP-AES) were performed.

(Functional group analysis by FTIR)
The measurement of functional groups by infrared spectroscopy was performed using a Fourier transform infrared spectrophotometer (Spectrum 50, manufactured by Perkin Elmer Co., Ltd.). The KBr method was adopted as the measurement method. Mix potassium bromide (KBr) powder in a ratio of 99% by weight to 1% by weight of Ag-HAp sample, fully grind using a mortar, and uniformly mix the powder in the diffuse reflection mode and measure the wavelength range The number of integrations was measured 16 times at 4000 cm ⁻¹ to 400 cm ^{−1 with} a resolution of 4. In addition, as a sample of F-HAp, (a) Ag-HAp prepared in the above-mentioned primary particle generation step, and (b) a sample obtained by firing Ag-HAp of (a) without adding an anti-fusion agent were used and samples fired by the addition of (c) anti-fusing agent Ag-HAp of _{(a) {Ca (NO 3} ) 2}. The IR spectrum of the sample of said (a)-(c) is shown in FIG.

As can be seen from FIG. 1, absorption peaks attributable to PO bonds were observed around 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.

In addition, absorption peaks attributed to hydroxyl groups (hydroxyl groups) were observed at around 3571 cm ⁻¹ and 629 cm ⁻¹ , which suggested that all samples contained hydroxyl groups.

Further, in any of the samples, two broad absorption peaks attributed in _{H 2} O molecules, was observed in the vicinity of 1630 cm ^-1 and 3400 cm ^-1, greatly peaks by baking for 2 hours at 700 ° C. It was found 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 the peak intensity attributable to <10> indicates that the crystallinity is increased in the fired sample. In addition, this result is in agreement with the result by XRD mentioned later.

  Also, in the sample (a), the peak attributed to the hydroxyl group was not sharp, while in the fired samples (b) and (c), the peak attributed to the hydroxyl group became sharp From these results, it was suggested that in the fired sample, completely crystalline Ag-HAp was produced. In addition, this was observed also in the diffraction peak of XRD mentioned later.

(Crystal structure analysis by XRD)
The crystal structure analysis of the produced Ag-HAp was performed using a powder X-ray diffraction apparatus (manufactured by Rigaku Corporation, RAD-X). As a X-ray source used in the XRD, a CuKα source (λ = 1.541841 Å (angstrom)) is used, the output is 30 kV / 15 mA, the scan speed is 1.0 ° / min, the sampling width is 0.01 °, and the measurement The mode was a continuous condition. FIG. 2 shows an X-ray diffraction peak of the produced Ag-HAp. In addition, the standard data of HAp of ICCD-PDF card (00-009-0432) and the standard data of metallic silver of ICCD-PDF card (04-0783) were used for the identification of a peak.

  The prepared Ag-HAp shown in FIG. 2 substantially matched the HAp data (standard HAp data) of the ICCD-PDF card (00-009-0432) for the main peak. 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 Ag-HAp synthesized by the above method was doped with silver ions in HAp crystals immediately after primary particle formation and after firing (regardless of presence or absence of anti-fusion agent). .

  Further, based on the X-ray diffraction peaks measured above, the full width at half maximum (FWHM: Full Width Half Maximum) in the (211) plane of the crystal lattice of the above 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 °.

  In general, it is known that the FWHM in the X-ray diffraction peak decreases as the firing temperature increases, and the particles become more completely crystalline. In the present example, since the FWHMs of the samples (b) and (c) having a firing temperature of 700 ° C. are lower than the FWHM of the sample (a) not subjected to the firing, the firing was performed in the present example. It can be said that Ag-HAp is more completely crystalline.

  Moreover, the axial length was investigated from the X-ray diffraction peak value measured above. The axial length can be calculated using “unit cell soft” 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 samples (a), (b) and (c) are shown in order from the top of Table 2.

  From Table 2, it was confirmed that the a-axis of the produced Ag-HAp was longer than the axis length of the HAp standard data. The larger radius of silver ion (= 1.28 Å) than the radius of calcium ion (= 0.99 Å) suggested that the calcium ion of HAp was replaced with silver ion.

(Measurement of surface shape and particle size by SEM)
The form of the produced Ag-HAp was observed using SEM. The SEM images of the prepared samples (a) to (c) of Ag-HAp 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 approaching each other. That is, it was found that sample (a) was insufficiently crystalline (amorphous). Moreover, in the sample (b) fired without adding the anti-fusion agent, it was observed that the primary particles were fused to form a large block. On the other hand, in the sample (c) fired by adding the anti-fusion agent, the primary particles are sintered with almost no fusion and are a mixture of spherical or rod-like particles (each particle is separated from each other) ) Was observed. In addition, the particle size of the primary particles was hardly changed by the presence or absence of the anti-fusion agent.

  Moreover, the average particle diameter of the prepared samples (a) to (c) of Ag-HAp was measured by a dynamic light scattering method (ELS, “ELS-8000 manufactured by Otsuka Electronics Co., Ltd.”).

  As a result, while the average particle size of the sample (a) was 502.0 nm, the average particle size of the sample (b) fired without the addition of the anti-fusion agent increased to 1671.8 nm. I understand. On the other hand, the average particle diameter of the sample (c) fired by adding the anti-fusion agent was as small as 374.7 nm. From this, it was suggested that when the anti-fusion agent is not used, primary particles aggregate and fuse to form secondary particles, and the particle size becomes large.

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

(Element composition analysis by ICP-AES)
The composition ratio of silver in Ag-HAp was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). As a measurement apparatus, Optima 2000DV (made by Perkin-Elmer Japan) 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 the anti-fusion agent (in the table) Ag of “sample-3” in the above was 0.5%. This decrease in Ag was presumed to be due to the washing of Ag-HAp in the removal step.

<Antimicrobial evaluation>
Next, the antimicrobial evaluation with respect to Ag-HAp created above is demonstrated. The following description will be made in the order of the evaluation method and the evaluation result.

(Evaluation method)
The antibacterial property of Ag-HAp was evaluated by the powder addition method using Ag-HAp produced. In addition, the antibacterial property of Ag-HAp is E. coli. I went to Coli. E. E. coli is a microorganism that can cause infections associated with implants, and is a type of gram-negative bacteria.

[Washing of Ag-HAp]
First, washing of Ag-HAp (Ca _9.95 Ag _0.05 (PO ₄ ) ₆ (OH) ₂ : formula weight = 1015.0) used in the antibacterial property test was performed. To 10 mg of Ag-HAp sufficiently ground in a mortar and placed in a tube, 1 mL of 70% ethanol was added and pipetted. Thereafter, it was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was discarded. This was repeated twice and then similarly washed with 140 mM NaCl.

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

[Assay]
Powder addition method A solution containing Ag-HAp dispersed in 100 μL of 140 mM NaCl solution and 100 μL of bacterial solution are placed in a tube, pipetted, stirred for 1 hour with a petit rotor, and Ag-HAp and bacteria I was in contact. Next, prepare 180 μL of NaCl solution in 5 tubes, numbered as (1) to (5) in order, and stir for 1 hour to make 20 μL of a mixed solution of bacteria and Ag-HAp (1) , Pipetting and stirring well. Next, 20 μL of the solution of (1) was added to (2), and the solution was pipetted and sufficiently stirred. This was repeated in the same procedure as (2) to (3), (3) to (4) ... as shown in FIG. 4 to dilute the mixed solution of bacteria and Ag-HAp (add 20 μL to 180 μL Will be diluted by 10 times). Furthermore, 5 μl of the solution diluted in 10 steps of 5 steps was spotted in order of 5 μl each from the lightest concentration (5) on the petri dish of agar medium, and cultured in an incubator set at 37 ° C. for 1 day. As a control of the antibacterial test, HAp manufactured by B company was used. In the above evaluations, the samples No. 1 and No. 2 in which Ag-HAp dispersed in 100 μL of NaCl solution was used as the above samples (a) to (c) respectively. 1 to No. Performed on 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 property of Ag-HAp]
The result of having carried out the antimicrobial test of Ag-HAp by the said powder addition method is shown in FIG. The top row in FIG. 5 shows the result of HAp as a control, and the lower row shows the result of Ag-HAp. The bactericidal rate described in Table 4 was determined from the ratio of the colony number of Ag-HAp to the colony number of the control, that is, (the number of colonies of F-HAp decreased) / (the colony number of control). As a result, in all the samples (a) to (c), that is, 90% or more of the sterilization rate was shown regardless of the presence or absence of baking. In this antimicrobial test, the above-mentioned No. 1 to No. Each of the solutions of 3 was carried out three 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 the present antibacterial test is Ca _9.95 Ag _0.05 (PO ₄ ) ₆ (OH) ₂ , the concentration in terms of Ag ion (Ag ion concentration) Is 49.3 mmol × 0.05 = 2.47 mmol / L. Generally, as an antibacterial property by Ag ion alone, it is said that when the Ag ion concentration is 5 to 10 ppb (0.093 × 10 ⁻³ mmol), the antibacterial property is expressed against E. coli. On the other hand, as shown in the above test, 2.47 mmol / L in terms of Ag ion is required to express the antimicrobial property as Ag-HAp, and the antimicrobial property is required compared to the case of Ag ion alone. It was found that more Ag ions were needed to express. From this, it can be said that the antibacterial activity of Ag-HAp is mild as compared to the antibacterial activity of Ag ion alone.

«F-HAp»
Next, F-HAp is synthesized, and the obtained F-HAp is identified, and then the results of evaluation of the acid resistance and antibacterial properties of the F-HAp will be described.

<Synthesis of F-HAp>
Preparation of F-HAp nano single crystal was performed using the coprecipitation method which is one of the wet methods. For the starting materials, calcium nitrate tetrahydrate as a Ca source, phosphoric acid as a P source, and sodium fluoride as an F source were used. Specifically, it carried out as follows.

(Primary particle generation process)
In 200 ml of ethanol (99.5% aqueous solution), 7.03 g of calcium nitrate tetrahydrate and 0.254 g of sodium fluoride were respectively dissolved, and stirred for 30 minutes under a nitrogen atmosphere. Similarly, 2.04 g of phosphoric acid was dissolved in 50 ml of ethanol and stirred for 30 minutes under a nitrogen atmosphere. Next, sodium fluoride was mixed with calcium nitrate in a stroke, and after stirring for 5 minutes, phosphoric acid was added, and the mixture was reacted by stirring for 1 hour while maintaining at 80 ° C. in a water bath. Then, it stirred at normal 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 to perform ultrasonic irradiation, the precipitated reaction product was sufficiently dispersed, and centrifugation was performed at 6,500 rpm for 3 minutes. This purification was repeated three times, and the obtained sample (F-HAp) was dispersed in water.

(Mixing process and firing process)
1.0 g of polyacrylic acid is dissolved in 100 ml of pure water with respect to 1.0 g of the above-prepared F-HAp, and the pH of the polyacrylic acid is adjusted to 5 using aqueous ammonia (28.0%) Prepared (solution A). After that, while stirring the F-HAp solution, solution A was dropped at a rate of 10 ml for 1 minute using a peristaltic pump. Further, it was allowed to stir 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 Solution B, Solution C was dropped at a rate of 10 ml for 1 minute using a peristaltic pump. Then, the mixture was suction filtered using an aspirator, and the removed sample was dried under reduced pressure for 1 hour. The dried sample was ground in a mortar, placed in a crucible and fired at 800 ° C. for 1 hour in an electric furnace. After furnace cooling, the fired F-HAp was crushed in a mortar.

(Removal process)
In 800 ml of pure water, 8.0 g of ammonium nitrate was dissolved and stirred for 30 minutes under a nitrogen atmosphere. The solution was added to the calcined F-HAp, the mixture was irradiated with ultrasonic waves, the precipitated reaction product was sufficiently dispersed, and centrifuged at 8,500 rpm for 3 minutes. The supernatant was discarded and an aqueous ammonium hydrogen nitrate solution was added, and this purification was repeated four times until the pH of the supernatant became neutral. Furthermore, the sample taken out was purified in the same manner using pure water. Finally, the sample was dried under vacuum for 1 hour, crushed and collected in a mortar.

<Identification of F-HAp>
With respect to the sample of F-HAp obtained as described above, as identification of F-HAp, crystal structure analysis by X-ray diffractometer (XRD), surface shape measurement by scanning electron microscope (SEM) and particle size measurement ( Evaluation of dispersibility by DLS) and concentration analysis by a fluorine ion concentration meter were performed.

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

  As can be seen from FIG. 6, the produced F-HAp substantially matches the peak value of the F-HAp data (standard data) of the JCPDS card. In the produced F-HAp, since the diffraction pattern of F alone was not detected, 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.

  Moreover, the axial length was investigated 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 produced F-HAp was shorter than the axial length of the HAp standard. Since the radius of the fluoride ion (= 1.33 Å) was 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 dispersion evaluation by DLS)
The form of the produced F-HAp was observed using SEM. The commercially available F-HAp is shown in FIG. 7, and the SEM image of the prepared F-HAp is shown in FIG. From FIGS. 7 and 8, it is understood that F-HAp synthesized (using the above-mentioned) using an anti-fusion agent has smaller primary particles and more uniform spherical shape than commercial F-HAp. The

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

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

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

(Concentration analysis by fluorine ion concentration meter)
The fluoride ion concentration of commercially available F-HAp and produced F-HAp was measured using a fluorine ion densitometer (F-10Z, manufactured by Sakai Principles, Ltd.). About 50 mg of a sample (commercially available F-HAp and prepared F-HAp) was mixed with 4 ml of 0.5 M hydrochloric acid and 15 ml of pure water, and pure water was added to a volume of 25 ml. After shaking the solution for a while, the sample was checked for dissolution, and if not dissolved, 0.5 M hydrochloric acid was added and dissolved. 25 ml of a solution containing a sample and 2 ml of a masking agent were placed in a 100 ml measuring flask, and pure water was further added to adjust the solution to 100 ml. After the concentration in the solution was made 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 could be confirmed that the produced F-HAp had a higher fluoride ion concentration. From the ratio of the theoretical concentration to the measured concentration, it was estimated that the substitution rate of hydroxide ion by fluoride ion was about 90%.

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

(Evaluation method)
An acid resistance test was conducted to determine the melting point of the produced F-HAp. For comparison, the acid resistance of a total of four samples to which three of A-made F-HAp, B- made HAp and C- made HAp were added were evaluated.

  First, 0.02 mM hydrochloric acid solution is added to pure water, and solvents having different pH (pH = 1.6, 2.0, 2.5, 2.8, 3.2, 3.6, 4.0, 4 I prepared .6). 2 mg of a 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 a UV-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 photometry mode: Abs, response: Quick, bandwidth: 2.0 nm, measurement wavelength: 595 nm, number of repetitions: 3, repetition interval: 0 . The transmittance was determined from the measured absorbance, and a graph in which the vertical axis represents the transmittance and the horizontal axis represents the pH was created, and the melting points of the four samples were compared. Two asymptotes were drawn on the graph, and the pH at the intersection was taken as the melting point. The created graph is shown in FIG.

(Evaluation results)
From FIG. 9, the dissolution point of HAP manufactured by Chemical Co., Ltd. and HAp manufactured by B Co. is pH = 4.0, the dissolution point of F-HAp manufactured by A Co. Was pH = 2.9. Therefore, it could be confirmed that the prepared F-HAp was higher in acid resistance as compared to 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 derived from the difference in the fluorine content.

<Antimicrobial evaluation>
Next, the antimicrobial evaluation with respect to F-HAp created above is demonstrated. The following description will be made in the order of the evaluation method and the evaluation result.

(Evaluation method)
The antibacterial property of F-HAp was evaluated by the powder addition method using the produced F-HAp. The antibacterial activity of F-HAp was carried out against E. coli. The antibacterial activity of fluoride ion was also evaluated using sodium fluoride. About the antimicrobial property evaluation of the fluoride ion, it did with respect to seven types of bacteria described in Table 7.

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

[Assay]
Powder Addition Method A solution containing F-HAp (Ca ₁₀ (PO ₄ ) ₆ F _1.8 (OH) _0.2 : formula weight = 1008.2) dispersed in 100 μL of 140 mM NaCl solution, and 100 μL of bacterial solution The mixture was placed in a tube, pipetted, and stirred for 1 hour in a peti-rotor to bring F-HAp into contact with bacteria. Next, prepare 180 μL of NaCl solution in 5 tubes, numbered as (1) to (5) in order, and mix for 1 hour with 20 μL of a mixed solution of bacteria and F-HAp (1) , Pipetting and stirring well. Next, 20 μL of the solution of (1) was added to (2), and the solution was pipetted and sufficiently stirred. Repeat this procedure as in (2) to (3), (3) to (4) ... as shown in Fig. 4 to dilute the mixture of bacteria and F-HAp (add 20 μL to 180 μL Will be diluted by 10 times). Further, 5 μl of the solution diluted in 10 steps of 5 steps was spotted in order of 5 μl each from the lightest concentration (5) on the petri dish of agar medium and cultured in an incubator set at 37 ° C. for 1 day. As a control of the antibacterial test, HAp manufactured by B company was used. 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 an actual measurement of a sample prepared based on the theoretical concentration of 18.8 mg / L of fluoride ion in the F-HAp sample (when substituted by 100% fluoride ion). The ratio to the value (16.9 mg / L) was calculated, and the original HAp; F-HAp substituted for 100% fluoride ion with Ca ₁₀ (PO ₄ ) ₆ (OH ₂ ) and Ca ₁₀ (PO ₄ ) _{From 6} F ₂ , all ion species lacking in the theoretical amount were determined in terms of hydroxide ion.

Method of Antibacterial Test Using Sodium Fluoride A 50 mM NaF solution at a concentration not harmful to human body was prepared, filtered and sterilized. It was mixed with an autoclaved LB solution to prepare mixed mixtures of 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 for 1 day in an incubator set at 37 ° C.

(Evaluation results)
[Antibacterial property of F-HAp]
As a result of conducting an antibacterial test of F-HAp by the above-mentioned powder addition method, when the concentration of F-HAp solution added to the bacterial solution is 49.6 mmol / L or more, 53.7 to 99.9% of the 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 bactericidal rate described in Table 8 was determined from the ratio of the number of colonies of F-HAp to the number of colonies of the control, that is, (the number of decreases in the number of colonies of F-HAp) / (the number of colonies of control).

  When the concentration of F-HAp was changed as described in Table 8 and the antibacterial test was conducted, the antibacterial properties 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 is 49.6 mmol / L or more, it can be said to be bacteriostatic. Moreover, 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 (fluoride ion concentration) in terms of fluoride ion for expressing the acid is 49.6 mmol × 1.8 = 89.3 mmol / L or more (in order to further express the antibacterial property of 95% or more of the sterilization rate , 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 as having antibacterial activity, can exhibit antibacterial activity against E. coli, or the above Ag-HAp It was found that a higher (fluoride) ion concentration is required to express the antibacterial property, as compared to 2.47 mmol / L, which is the silver ion concentration capable of expressing the antibacterial property. From this, it can be said that the antibacterial activity of F-HAp is mild also in comparison with the antibacterial activity of silver ion alone, and further, the antibacterial activity of Ag-HAp.

[Antibacterial properties of fluoride ion]
The results of the antibacterial test using sodium fluoride conducted by the above method are shown in FIG. (1) is Bacillus cereus, (2) is Salmonella, (3) is Staphylococcus aureus, (4) is E. coli, (5) is Pseudomonas aeruginosa, (6) is Klebsiella pneumoniae, (7) is Streptococcus pyogenes is there.

  As shown in FIG. 11, when the plates of five different concentrations were compared, it could be confirmed that (7) S. pylori streptococcus became thinner as the concentration increased. Thus, it was found that fluoride ion itself is antibacterial against Streptococcus pyogenes. Here, although the fluoride ion itself has no antibacterial activity against E. coli, the reason why F-HAp has antibacterial activity against E. coli is not clear, but the antibacterial effect of F-HAp It was speculated that the sex was not simply due to the antibacterial activity of the fluoride ion itself. Thus, according to the results of the present test and the antibacterial test of F-HAp, there are species of bacteria capable of expressing antibacterial activity and species of bacteria that can not express antibacterial activity with fluoride ion alone, but F-HAp is a fluoride ion. It has been found that antibacterial properties can be expressed even against bacterial species which can not express antibacterial properties alone (for example, Streptococcus pyogenes).

Although the 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 fall within the technical scope of the present invention.

Claims (4)

It consists of antibacterial hydroxyapatite derivative particles in which at least a part of calcium ions in the crystal structure of hydroxyapatite is replaced by silver ions,
90% or more of hydroxyapatite crystal phase purity determined by XRD of the antibacterial hydroxyapatite derivative particles,
The particle size of the antibacterial hydroxyapatite derivative particles is in the range of 10 nm to 1,000 nm,
The hydroxyapatite derivative particle group, wherein the length of the a axis of the antimicrobial hydroxyapatite derivative particles is 9.424 Å or more. A dispersion liquid in which 5.0 × 10 ⁴ cells / μL of a predetermined microorganism and the antibacterial hydroxyapatite derivative particles are allowed to coexist at a concentration of 2.47 mmol / L or more in terms of silver ion is prepared, and the dispersion liquid Is spotted on the medium, and the sterilization rate after culturing for 1 day at 37.degree. C. is 90% or more,
The hydroxyapatite derivative particle group according to claim 1, wherein the predetermined microorganism is Escherichia coli.   The hydroxyapatite derivative particles according to claim 1 or 2, wherein the antibacterial hydroxyapatite derivative particles are calcined hydroxyapatite derivative particles.   The hydroxyapatite derivative particles according to any one of claims 1 to 3, wherein the length of the a-axis of the antimicrobial hydroxyapatite derivative particles is 9.442 Å or less.