JP6548617B2 - Collagen production promoter - Google Patents

Description

  The present invention relates to a collagen production promoter, and to a collagen production promoter that stimulates fibroblasts present in the dermal layer of skin.

  Collagen is present in all organs throughout the body, and in particular in the skin, it is known that about 70% of the dry weight of the dermis is collagen, and in the function retention of the skin's contractility, flexibility, moisture retention, etc. As well as being useful, it is known to maintain the morphology of various cells normally and to affect metabolism, adhesion and the like.

  In addition, the skin is always exposed to the outside world, and loses its elasticity and tension with age, causing aging phenomena such as wrinkles and tarmi. These phenomena are considered to occur because the amount of collagen present in the skin decreases with aging. In order to eliminate such wrinkles and tears, a large number of collagen production promoting agents using substances having a collagen production promoting action have been reported (Patent Document 1). Conventionally, fibroblasts present in the dermis are known to produce collagen, and the promoter is said to act on fibroblasts to promote collagen production.

  In addition, hydroxyapatite (HAp) is known to exhibit high biocompatibility, and is used in various fields including biomaterials.

  By the way, when using the said HAp for a biomaterial, in order to reduce the solubility and degradability in in vivo, producing at high temperature and producing hydroxyapatite particle (ceramic particle) of high crystallinity is proposed. There is. However, during this sintering, bonding occurs due to fusion between hydroxyapatite (HAp) particles (primary particles), resulting in irregular secondary particles in which primary particles are bonded, and the dispersibility and specific surface area decrease. There was a problem of that.

  Therefore, in Patent Document 2, a mixture of primary particles made of ceramic raw materials before sintering is mixed such that an anti-fusion agent is interposed to form mixed particles, the mixed particles are sintered, and after the sintering A process has been proposed for washing away the anti-fusion agent. According to the manufacturing method, it is possible to obtain a ceramic particle group having a high degree of crystallinity and a small particle size.

JP 2008-260747 A Patent No. 5043436 gazette

  When the ceramic particle group described in Patent Document 2 was used, it was also possible to be a novel collagen production promoter that increases the amount of collagen contained in skin tissue. However, in recent years, agents having higher collagen production ability are required.

  The present inventors also found that the ceramic particle group obtained by the process of Patent Document 2 is an undesirable event from the viewpoint of expression of biocompatibility and change in solubility when introduced into applications, particularly in vivo. It has been found that it can occur. Therefore, this invention makes it a subject to provide the calcium phosphate sintered compact particle group with a small particle size in which the bioaffinity fall and solubility change are suppressed as much as possible.

  As a result of intensive research conducted by the inventor to improve the collagen-producing ability of functional particles, when calcium carbonate is contained in particles, carbonic acid is generated in the use stage etc., and the carbonic acid adversely affects. It came to complete this invention, acquiring the knowledge that it is.

That is, the present invention is as follows.
The present invention (1) is
A collagen production promoter comprising ceramic particles as an active ingredient,
The particle diameter of the ceramic particles is in the range of 10 nm to 700 nm,
The ceramic particles are calcium phosphate sintered particles,
The collagen production promoter, wherein the ceramic particles do not contain calcium carbonate.
The present invention (2) is
The collagen production promoter of said invention (1) whose said ceramic particle is spherical.
The present invention (3) is
A collagen production promoter comprising ceramic particles as an active ingredient,
The particle diameter of the ceramic particles is 30 nm to 5 μm in the short axis and 75 nm to 10 μm in the long axis, and grows in the c axis direction, and the aspect ratio (c axis length / a axis length) of the crystal is 1 ~ 30,
The ceramic particles are calcium phosphate sintered particles,
The collagen production promoter, wherein the ceramic particles do not contain calcium carbonate.
The present invention (4) is
The collagen production promoter according to any one of the inventions (1) to (3), wherein the ceramic particles are sintered hydroxyapatite particles.
The present invention (5) is
The collagen production promoter according to any one of the inventions (1) to (4), wherein the ceramic particles do not contain an alkali metal element.
The present invention (6) is
The collagen production promoter according to any one of the inventions (1) to (5), wherein the ceramic particles contain carbonate apatite on at least the surface thereof.
The present invention (7) is
The collagen production promoter according to any one of the inventions (1) to (6), wherein the ceramic particles satisfy the following property (A).
(A) After sufficient drying (for example, after drying at normal pressure, temperature 60 ° C., humidity 45 to 85% for 18 hours or more), normal pressure, temperature 25 ° C., humidity 50% After leaving to stand for 3 days or more, under a nitrogen stream, a thermal gravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under a nitrogen stream at 10 ° C./min. The weight decreased in the temperature range of 25 to 200 ° C. is 2% or less.
The present invention (8) is
The ceramic particle according to any one of the inventions (1) to (7), wherein the half width at d = 2.814 measured by the X-ray diffraction method is in the range of 0.2 to 0.8. Collagen production promoter.
The present invention (9) is
Invention (1) to (1), which contain at least one substance selected from the group consisting of alcohols, saccharides, proteins, amino acids, water-soluble vitamins, fat-soluble vitamins, lipids, mucopolysaccharides, and surfactants. The collagen production promoter according to any one of (8).

Here, as described later, "does not contain calcium carbonate" in the present invention means that substantially no calcium carbonate is contained, and more specifically, the criteria of (1) to (3) below: To satisfy all
(1) From the measurement results of X-ray diffraction, calcium carbonate is calcium carbonate (formula weight: 100.09) / hydroxyapatite (formula weight: 1004.62) = 0.1 / 99.9 (formula conversion ratio) or less It is.
(2) In thermogravimetric differential thermal analysis (TG-DTA) measurement, a weight loss of 2% or more at a temperature of 650 ° C. to 800 ° C. with a clear endotherm is not observed.
(3) In the FT-IR spectra obtained in the measurement Kubelka-Munk (KM) chart showing the calculated absorbance by the equation, separated peaks wavenumber appears between ^{860cm -1 ~890cm} -1, is attributed to calcium carbonate The peak around 877 cm ^-1 is not observed. The peak separation is performed, for example, by using a software of fityk 0.9.4, under the condition of Function Type: Gaussian, Fitting Method: Levenberg-Marquardt.

Further, in the present invention, when “does not contain calcium carbonate”, it is preferable to further satisfy the following criteria (4).
(4) When tested according to the quasi-drug raw material standard 2006 (hydroxyapatite), the air bubble generation amount is 0.25 mL or less.

  In the present invention, "spherical" indicates that the aspect ratio of the particles is 1.35 or less (more preferably 1.25 or less, and even more preferably 1.2 or less). In addition, the aspect ratio of particle | grains here shows the numerical value obtained by the following method. In the SEM image obtained by photographing the particle, two line segments whose both ends are located on the outer periphery of the particle are drawn on the particle. At this time, one of the line segments has its maximum length. Furthermore, at the midpoint of the line segment, the other line segment is drawn so as to be orthogonal to each other. Of the two line segments drawn in this manner, the length of the shorter line segment is taken as the minor axis, and the length of the longer line segment as the major axis, and the ratio of the major axis / minor axis is determined. However, particles whose outlines appear blurry, particles that are too close to another particle and whose boundary is ambiguous, and particles in which some of the particles are behind other particles are excluded from the measurement target.

  In the present invention, “ceramic particle group” including “ceramic particles having certain properties” means that “ceramic particles having certain properties” is 50% by mass or more with respect to the entire “ceramic particle groups”. The content is preferably 70% by mass or more, more preferably 90% by mass or more.

  According to the present invention, as a result of the particles not containing calcium carbonate, the generation of carbonic acid is suppressed during use, etc., whereby it is possible to provide a collagen production promoter that exhibits higher collagen production ability.

1-1 is a chart showing the results of X-ray diffraction measurement of the hydroxyapatite (HAp) sintered particles obtained in Example 1-1. 1-2 is a chart showing the results of X-ray diffraction measurement of the hydroxyapatite (HAp) sintered particles obtained in Example 1-2. FIG. 1-3 is a chart showing the results of X-ray diffraction measurement of the hydroxyapatite (HAp) sintered particles obtained in Comparative Example 1-1. 1-4 is a SEM photograph of the hydroxyapatite (HAp) sintered body particle group obtained in Example 1-1. 1-5 is a SEM photograph of the hydroxyapatite (HAp) sintered compact particle group obtained in Example 1-1. 1-6 is a SEM photograph of the hydroxyapatite (HAp) sintered body particle group obtained in Example 1-2. FIGS. 1-7 is a SEM photograph of the hydroxyapatite (HAp) sintered particle group obtained in Example 1-2. 1-8 is a SEM photograph of a test material manufactured using the hydroxyapatite sintered body particle group according to Example 1-1. FIG. 1-9 is a SEM photograph of a test material manufactured using the hydroxyapatite sintered body particle group according to Example 1-2. FIGS. 1-10 is a SEM photograph of a test material manufactured using the hydroxyapatite sintered body particle group according to Comparative Example 1-1. FIG. 1-11 is a dyed image of a test material manufactured using the hydroxyapatite sintered body particle group according to Example 1-1. 1-12 is a dyed image of a test material manufactured using the hydroxyapatite sintered body particle group according to Example 1-2. 2-1 is the result of the thermogravimetric differential thermal analysis (TG-DTA) measurement of the hydroxyapatite (HAp) baking body particle group which concerns on Example 2-1. 2-2 is the result of the thermogravimetric differential thermal analysis (TG-DTA) measurement of the hydroxyapatite (HAp) baked body particle group which concerns on Comparative Example 2-1. FIG. 2-3 is a chart showing the absorbance calculated by the Kubelkamkck (KM) equation, the spectrum obtained in the FT-IR measurement. When manufacturing the hydroxyapatite (HAp) particle | grains of Example 2-1, it was investigated how the quantity of calcium carbonate changes by an acid-washing process. The left chart in FIG. 2-3 is hydroxyapatite (HAp) particles in which the pickling process has not been performed, and the right chart in FIG. 2-3 is hydroxyapatite particles in which the pickling process is performed. 2-4 is a chart which shows the FT-IR spectrum of the hydroxyapatite (HAp) sintered compact particle obtained in Example 2-1. 2-5 is a chart which shows the FT-IR spectrum of the hydroxyapatite (HAp) sintered compact particle obtained in Example 2-2. FIG. 2-6 is a SEM photograph of hydroxyapatite (HAp) sintered particle group obtained in Example 2-1. FIGS. 2-7 is a SEM photograph of the hydroxyapatite (HAp) sintered particle group obtained in Example 2-1. FIG. 2-8 is a SEM photograph of the hydroxyapatite (HAp) sintered particle group obtained in Example 2-2. FIG. 2-9 is a SEM photograph of the hydroxyapatite (HAp) sintered particle group obtained in Example 2-2. FIG. 2-10 is a SEM photograph of a test material manufactured using the hydroxyapatite (HAp) sintered particle group according to Example 2-1. Cell adhesion was assessed. FIG. 2-11 is a SEM photograph of a test material manufactured using the hydroxyapatite (HAp) sintered particle group according to Example 2-2. Cell adhesion was assessed. FIG. 2-12 is a SEM photograph of a test material manufactured using the hydroxyapatite (HAp) sintered particle group according to Comparative Example 2-1. Cell adhesion was assessed.

  The collagen production promoter according to the present invention contains, as ceramic particles, specific calcium phosphate sintered particles. In particular, in the collagen production promoter containing ceramic particles, the present invention is mainly characterized in that the ceramic particles have a minute size and do not contain calcium carbonate. So, below, after demonstrating the basic composition of a collagen production promoter, the ceramic particle characteristic of this invention is demonstrated. Moreover, the present inventors have found two types of production methods as methods for obtaining the characteristic ceramic particles of the present invention. Such a manufacturing method will be described roughly divided into a first manufacturing method and a second manufacturing method below. Among these, the first production method is capable of achieving more stringent standards in terms of "does not contain calcium carbonate". The “ceramic particle group” may be read as “ceramic particle”.

<<<< collagen production promoter >>
The collagen production promoter according to the present best mode contains calcium phosphate sintered particles. In addition, alcohols, saccharides, proteins, amino acids, water-soluble vitamins, fat-soluble vitamins, lipids, mucopolysaccharides, surfactants are preferably included as optional constituent requirements.

  As calcium phosphate sinter particles according to the present best mode, calcium phosphate particles obtained by a first manufacturing method and a second manufacturing method described later can be used. About basic collagen production ability at the time of using a calcium-phosphate sintered compact particle, it is detailed to WO2010 / 137122 grade | etc.,. The collagen production promoter according to the present invention can further improve the collagen production ability by not containing calcium carbonate, in addition to the basic collagen production ability of the calcium phosphate sintered particles.

  The collagen production promoter according to the best mode preferably contains alcohols, saccharides, proteins, amino acids, water-soluble vitamins, fat-soluble vitamins, lipids, mucopolysaccharides, and surfactants. It is.

  Examples of the alcohol include ethanol and glycerin. By including the alcohol, the dispersion stability of the calcium phosphate fine particles is improved, and the rapid permeation into the dermal layer is promoted. It also exerts cleansing, astringent and moisturizing effects on the skin. Examples of sugars include hydrolyzed hydrogenated starch and the like. The inclusion of the sugar improves the moisturizing effect and the anti-inflammatory effect. Proteins include, for example, collagen. By including proteins, it becomes a carrier of calcium phosphate and can promote delivery to the dermal layer. Examples of amino acids include glutamic acid, arginine acid, and sodium salts thereof. By including amino acids, collagen production can be promoted. Examples of water-soluble vitamins include magnesium ascorbyl phosphate and the like. By including water-soluble vitamins, collagen production is promoted, and a synergistic effect with calcium phosphate can be expected. Examples of fat-soluble vitamins include vitamin A. By containing fat-soluble vitamins, it is possible to promote mucopolysaccharide production and moisturize the skin. As a lipid, ceramide and phospholipids are mentioned, for example. By containing a lipid, it exerts a moisturizing effect. Examples of mucopolysaccharides include hyaluronic acid. By containing mucopolysaccharides, it exerts a moisturizing effect. Examples of surfactants include polyethylene glycol and stearic acid derivatives. By including the surfactant, the dispersibility of the particulates and fat-soluble components such as vitamin A is improved.

  The calcium phosphate fine particles according to the present best mode are not particularly limited, but are suitable for being formulated into cosmetics, orthopedic preparations and dental preparations. The type of the cosmetic is not particularly limited, and specific examples thereof include a face wash, a lotion, a cosmetic solution, an ointment, a cream, a milk, a lotion, a pack, a lipstick, a bath agent and the like. The blending amount of calcium phosphate fine particles in the cosmetic according to the present best mode can be appropriately adjusted depending on the type of cosmetic and the physiological activity of the extract, etc., but 0.01 to 30% by mass is suitable and 0.1 is preferable. -15 mass% is more preferable, and 0.1-10 mass% is still more preferable. Although the compounding agent of alcohol is not specifically limited, 0.01-70 mass% is suitable, 0.01-50 mass% is more suitable, 0.01-30 mass% is still more suitable. Although the compounding agent of saccharides is not specifically limited, 0.01-70 mass% is suitable, 0.01-50 mass% is more suitable, and 0.01-30 mass% is still more preferable. Although the compounding agent of proteins is not specifically limited, 0.01-30 mass% is suitable, 0.01-20 mass% is more suitable, and 0.01-10 mass% is still more preferable. The blending amount of the amino acids is not particularly limited, but is preferably 0.01 to 30% by mass, more preferably 0.01 to 20% by mass, and still more preferably 0.01 to 10% by mass. The compounding agent of the water-soluble vitamins is not particularly limited, but preferably 0.001 to 30% by mass, more preferably 0.001 to 20% by mass, and still more preferably 0.001 to 10% by mass. is there. The blending amount of the fat-soluble vitamins is not particularly limited, but preferably 0.001 to 30% by mass, more preferably 0.001 to 20% by mass, and still more preferably 0.001 to 10% by mass. is there. The lipid composition is not particularly limited, but preferably 0.001 to 30% by mass, more preferably 0.001 to 20% by mass, and still more preferably 0.001 to 10% by mass. There are no particular limitations on the mucopolysaccharide blending agent, but it is preferably 0.01 to 70% by mass, more preferably 0.01 to 50% by mass, and still more preferably 0.01 to 30% by mass. . The composition of the surfactant is not particularly limited, but is preferably 0.001 to 30% by mass, more preferably 0.001 to 20% by mass, and still more preferably 0.001 to 10% by mass. .

  In the cosmetic according to the present best mode, various main and auxiliary agents usually used in the preparation of cosmetics can be used, as long as they do not hinder the collagen production promoting action of the calcium phosphate fine particles. In the cosmetic according to the present best mode, known additives used in cosmetics can be used as the cosmetic component together with calcium phosphate fine particles, for example, astringents, bactericidal / antibacterial agents, whitening agents Agents, ultraviolet light absorbers, moisturizers, cell activators, anti-inflammatory and anti-allergic agents, anti-oxidation and active oxygen scavengers and the like. In addition, when manufacturing cosmetics, selection of other manufacturing materials is hardly restricted, such as fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, perfumes, etc. Common substrates and assistants can all be used.

  In addition, examples of orthopedic preparations include soft tissue fillers and the like, which regenerate the defect site due to the self tissue by injecting into a part where a dent is made in the skin due to an injury or the like. As a dental preparation, after performing implant treatment etc., it applies to the site | part to which the clearance gap was made, and the gum fixative etc. which make it easy to fix a base material, a tooth, etc. are mentioned.

<<<< calcium phosphate sintered particles >>
<<< First manufacturing method >>>
Hereinafter, the first production method of the calcium phosphate particles according to the present invention will be described, and then, the ceramic particles obtained by the first production method will be described.

<< 1. First production method of ceramic particles >>
<1-1. Raw material>
Specific examples of calcium phosphate (CaP), which is a ceramic raw material before firing, include hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ) and calcium metaphosphate _{(Ca (PO 3) 2)} , Ca 10 (PO 4) 6 F 2, Ca 10 (PO 4) 6 Cl 2 , and the like. Here, the calcium phosphate (CaP) may be artificially produced by a known production method such as a wet method, a dry method, a hydrolysis method, a hydrothermal method, etc., and bones and teeth It may be of natural origin obtained from E. coli.

<1-2. Process>
(1-1. Primary particle generation process)
Hereinafter, although the case where primary particles are spherical and rod-like is illustrated in the manufacturing method of the calcium phosphate particles concerning this form, it is not limited to this, The primary particles of all the shapes which can be manufactured are applicable.

· Primary particle formation step of spherical ceramic particles First, "primary particles" refers to particles formed of ceramic raw materials {calcium phosphate (CaP), hydroxyapatite (HAp), etc.} before sintering in the production step of ceramic particles. It means that. That is, it means the particles formed for the first time in the process of producing ceramic particles. In a narrow sense, it means single crystal particles. In the claims and the specification, the term "primary particles" includes those in an amorphous (amorphous) state, in a low crystalline state, and in the state of a sintered body after sintering. It is a meaning. On the other hand, with "secondary particles", a plurality of "primary particles" are bonded by physical bonding such as fusion, or by chemical bonding such as Van der Waals force, electrostatic interaction, or covalent bonding. Mean particles in the formed state. 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. Further, in particular, "single-crystal primary particles" mean primary particles consisting of single crystals of ceramic raw material, or particle agglomerates 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 is obtained by sintering. It is a thing which does not contain the secondary particle which the particle | grains fuse | melted and polycrystallized.

  The primary particle generation step is not particularly limited as long as it is a step known per se and a step capable of generating the above primary particles, and may be appropriately selected depending on the raw material of the ceramic to be produced. For example, when phosphoric acid is dropped to a calcium hydroxide slurry at normal temperature, particles of calcium phosphate (CaP) are precipitated.

  The method for producing a ceramic particle group according to the present embodiment is to produce a ceramic particle group by sintering a primary particle group consisting of primary particles produced by the above primary particle producing step while preventing fusion or the like. . Therefore, the state (particle diameter, particle size distribution) of the primary particles generated in the primary particle generation step is directly reflected in the state (particle diameter, particle size distribution) of the ceramic particles as the final product. Therefore, in the case of producing a ceramic particle group having a fine particle size (nanometer size) and a uniform particle size (narrow particle size distribution), the particle size is fine (nanometer size) in the primary particle generation step. And primary particles having a uniform particle diameter (narrow particle size distribution).

  The particle diameter (average particle diameter and particle size distribution) of the preferred primary particles in this case is preferably 10 nm to 700 nm, more preferably 15 nm to 450 nm, and most preferably 20 nm to 400 nm. The variation coefficient of the particle diameter of the primary particle group consisting of primary particles is preferably 20% or less, more preferably 18% or less, and most preferably 15% or less. The particle size and coefficient of variation of primary particles may be calculated by measuring the particle size of at least 100 or more primary particles using a dynamic light scattering method or an electron microscope.

  The “variation coefficient” is a value representing the variation in particle diameter among particles which can be calculated by standard deviation / average particle diameter × 100 (%).

  There is no particular limitation on the method of producing the above fine (nanometer size) primary particle group having uniform particle diameter (narrow particle size distribution), for example, JP-A-2002-137910. Patent No. 5043436, Journal of Nanoscience and Nanotechnology Vol. 7, 848-851, 2007 can be mentioned.

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

· Primary particle generation process of rod-like ceramic particles {Rod-like ceramic particles {The particle diameter of the particles is 30 nm to 5 μm in the maximum diameter of the short axis and 75 nm to 10 μm in the long axis. The primary particle generation process of ceramic particles having a ratio (c axis length / a axis length) of 1 to 30 is known per se, for example, JP-A-2002-137910, Journal of Nanoparticle Research (2007) The method described in 9: 807-815 can be mentioned.

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

(Freezing process)
The freezing step is a step of freezing an aqueous medium containing calcium phosphate (CaP) before sintering. Here, the aqueous medium is a liquid medium containing water as a main component (preferably 90 mass% or more based on the total mass of the liquid medium). Preferably, a liquid (such as alcohol) which is water only and is otherwise miscible with water may be added as appropriate. In addition, the liquid from which calcium phosphate (CaP) which is primary particles is produced, that is, the liquid obtained by dissolving the phosphate source and the calcium source in water may be frozen as it is. Here, the freezing temperature is not particularly limited, but is preferably −1 to −269 ° C. More preferably, it is -5 to -196 ° C. Moreover, freezing time is although it does not specifically limit, Preferably, they are 1 minute-24 hours.

(Decompression process)
The thawing step is a step of disposing the frozen body obtained in the freezing step at a temperature above the melting point of the aqueous medium of the frozen body, and thawing the aqueous medium.

(Separation process)
The separation step is a step of separating calcium phosphate from the calcium phosphate-containing aqueous medium melted in the thawing step. The separation method may be a method of filtering the precipitate after thawing, or may be a method of centrifugal collection.

(Firing process)
The said sintering process is a process of exposing the calcium-phosphate primary particle composition obtained by the said isolation | separation process to sintering temperature, and making the primary particle contained in the said composition into a ceramic particle (sintered particle). Although the reason is not clear, it is the case of being exposed to the high temperature condition in the sintering process when the above-mentioned freezing and thawing process is obtained and fired without using the anti-fusion agent as disclosed in Patent Document 1 Also, the fusion between primary particles can be prevented. Here, the sintering temperature in the sintering step may be appropriately set so that the crystallinity of the ceramic particles has a desired hardness, and for example, 300 to 1000 ° C. is preferable. Moreover, as for the temperature rising rate in the said sintering process, for example, 1 to 20 ° C./min is suitable. Furthermore, the sintering time in the sintering step may be appropriately set based on the hardness of the ceramic particles and the like, and for example, 0.5 to 3 hours are suitable. In addition, the apparatus etc. which are used for the said sintering process are not specifically limited, According to a manufacturing scale, manufacturing conditions, etc., after selecting a commercially available calcination furnace suitably, it may be adopted.

(Crushing process)
The pulverizing step is a step of pulverizing the aggregate after the sintering step to obtain calcined calcium phosphate particles having a desired size. Here, in general, it is almost impossible to reduce the size of the secondary particles to the primary particle size even if a considerable degree of pulverizing process is performed. On the other hand, according to the method of the present embodiment, it is possible to easily grind to the primary particle size level even with a simple grinding step. Here, the pulverizing method is not particularly limited, and for example, ultrasonication and pulverizing treatment using a pulverizing ball. In addition, after the pulverizing treatment, particles having a smaller diameter may be collected by removing the uncrushed ones or the like.

(Washing process)
In the washing step, components other than calcined calcium phosphate particles, for example, impurities derived from raw materials used when producing primary particles of calcium phosphate (for example, impurities derived from calcium or phosphoric acid not involved in calcium phosphate formation, nitric acid or ammonium derived Of impurities) of the Preferably, washing with water is preferred. In the case of sintered hydroxyapatite (HAp) particles, the sintered particles of hydroxyapatite (HAp) are easily dissolved under conditions of pH less than 4.0, so the removal process is performed at pH 4.0 to pH 12.0. Is preferred. Moreover, this order does not matter, such as alternately performing the grinding process and the washing process.

(Drying process)
The drying step is a step of removing the solvent by heating or the like after the pulverizing step and the washing step to obtain calcium phosphate particles. The drying method is not particularly limited.

(Endotoxin removal process)
Here, in the present step, an endotoxin removal step may be provided as necessary.

  The endotoxin removal step is, for example, a step performed by heating at 300 ° C. under normal pressure under air. The calcium phosphate fired body is preferably subjected to water washing in the washing step described above after firing. At this time, the calcium phosphate fired body adsorbs endotoxin which is slightly present in the environment, such as water, a storage container and a handling atmosphere. Here, the endotoxin is a harmful substance, and remaining in the calcium phosphate fired body is not suitable particularly when the calcium phosphate fired body is used as a biomaterial. Thus, the treatment is carried out to degrade the adsorbed endotoxin. In addition, the said endotoxin removal process may perform the drying process mentioned above collectively.

  Here, the present inventors obtained the knowledge that when the calcium phosphate fired body obtained by the conventional production method is subjected to the heat treatment, the original particle diameter may be double or more. In particular, when using a calcium phosphate fired body for applications where finer particle sizes are required, such increase in particle size is an undesirable event. On the other hand, although the reason is not clear, the calcium phosphate calcined body obtained according to the first production method hardly changes in particle size even when heated at 300 ° C. to remove endotoxin. Therefore, according to the present production method, it is also possible to obtain calcium phosphate sintered particles having a smaller particle size from which endotoxin has been removed.

  Here, according to the present production method, unlike the prior art, minute calcium phosphate sintered particles can be produced without using a fusion inhibitor such as a polymer compound or a metal salt. More specifically, according to the present production method, a fusion inhibitor containing a polymer compound or a different metal element (for example, an alkali metal element, an alkaline earth metal element other than calcium, a transition metal element, etc.) in the production process It becomes possible to obtain calcium phosphate sinter particles having higher crystallinity (as a result, it is more difficult to dissolve) as compared with the conventional manufacturing method used for manufacturing raw materials.

<< 2. Calcium phosphate sintered particles obtained by the first production method >>
<2-1. Spherical calcium phosphate sintered particles>
The spherical calcium phosphate sintered body particle group obtained by the present production method is a ceramic particle group including spherical ceramic particles, and the particle diameter of the ceramic particle is in the range of 10 nm to 700 nm, and the ceramic particle is It is a calcium phosphate sintered particle, and the ceramic particle group is a ceramic particle group characterized by containing substantially no calcium carbonate.

  Furthermore, in the spherical calcium phosphate sintered body particles obtained by the present production method, the variation coefficient of the particle diameter of the ceramic particles is preferably 30% or less, and more preferably 25% or less. It is particularly preferable that the content is 20% or less. The ceramic particle group is a fine particle having a uniform particle size (narrow particle size distribution). Therefore, there is an effect that it can be adsorbed more uniformly to the medical polymer material without performing additional operations such as particularly high classification. And since it does not contain calcium carbonate, when using as a biomaterial, it becomes possible to prevent the situation where biocompatibility and solubility of material change.

  From another aspect, the spherical calcium phosphate sintered body particle group obtained by the present manufacturing method is a ceramic particle group including spherical ceramic particles, and is a primary particle consisting of a single crystal, or a primary consisting of the single crystal When a particle aggregate in which particles are aggregated by ionic interaction is a single crystal primary particle, the proportion of single crystal primary particles contained in the ceramic particle group accounts for a majority, and the ceramic particles are calcium phosphate sintered particles. And the ceramic particle group does not contain calcium carbonate. The ceramic particle group is a primary particle consisting of a single crystal, the majority of which is excellent in dispersibility in a solvent, or a particle aggregate (single crystal primary particle) in which the primary particles consisting of the single crystal are aggregated by ionic interaction. As exists. Therefore, there is an effect that the adsorption onto the aforementioned medical polymer base material is facilitated. In addition, since there is no bonding between primary particles, the specific surface area is high. Furthermore, since the stability in a living body is high and the dispersibility is excellent, there is an effect that it can be used as a medical material which can carry and release a drug. And since calcium carbonate is not contained, when it uses as a biomaterial, the biocompatibility of material and the original solubility of calcium phosphate are maintained. In addition, it is good also as particle | grains which the particle | grains which the primary particle which consists of single crystals gathered by ionic interaction in this way are ceramic particles.

  In the ceramic particle group, the ratio of single crystal primary particles contained in the ceramic particle group may be 70% or more. By adopting such a configuration, an effect of facilitating adsorption to the medical polymer substrate is exhibited.

  In the ceramic particle group, the particle diameter of the ceramic particles may be in the range of 10 nm to 700 nm. According to the said structure, it is effective in the ability to make it adsorb | suck more uniformly with respect to a medical polymeric material.

  The ceramic particle group may have a coefficient of variation in particle diameter of the ceramic particle group of 20% or less. By adopting the configuration, it is possible to more uniformly adsorb the medical polymer material without performing additional operation such as particularly high classification.

  In addition, the ceramic particles may be sintered hydroxyapatite particles. The particles are further composed of a hydroxyapatite sintered body that is more biocompatible and can be used in a wide range of applications. Therefore, it is particularly preferable as a medical material.

  In addition, the ceramic particle group is water-washed, and when the particle diameter of the ceramic particle group after the water-washing is used as a reference, the ceramic particle group is heated at 300 ° C. in air under normal pressure after the water-washing. The rate of change of the particle size at that time may be ± 20%.

2-2. Rod-like sintered calcium phosphate particles>
The rod-like sintered body of calcium phosphate particles obtained by this production method is a ceramic particle group containing ceramic particles, and the particle diameter of the ceramic particles is 30 nm to 5 μm in the maximum diameter of the short axis and 75 nm to 75 nm in the long axis. Ceramic particles which are 10 μm, grow in the c-axis direction, and have a crystal aspect ratio (c-axis length / a-axis length) of 1 to 30, and the ceramic particles are calcium phosphate sintered particles, And, the ceramic particle group is a ceramic particle group characterized by not containing calcium carbonate.

  Since the area to be provided for adhesion is much larger than that of the conventional fine particles, the rod-like calcium phosphate calcined particle group can improve the adhesion to the polymer substrate, and therefore, the polymer surface such as a biocompatible medical material such as a catheter can be improved. Suitable for modification. And since it does not contain calcium carbonate, when using it as a biomaterial, it becomes possible to prevent the situation which a carbon dioxide gas generate | occur | produces from a material. In addition, as a method of modifying on the polymer surface, silicone rubber obtained by graft polymerizing an active group of calcium phosphate (for example, hydroxyapatite nanoparticles) and a polymer substrate, for example, a vinyl-based polymerizable monomer having a carboxyl group on the surface A method of chemically reacting with an active group of to make a complex, a method of using a curable adhesive, a method of heating a polymer substrate to near the melting point and embedding it in a substrate can be used (this is The same applies to the above-mentioned spherical calcium phosphate sintered particle group). In addition, as a rod shape, it may be a truncated columnar structure in which the tip angle has an oblique surface.

  In addition, the ceramic particles may be sintered hydroxyapatite particles. The particles are further composed of a hydroxyapatite sintered body that is more biocompatible and can be used in a wide range of applications. Therefore, it is particularly preferable as a medical material.

  In addition, the ceramic particle group is water-washed, and when the particle diameter of the ceramic particle group after the water-washing is used as a reference, the ceramic particle group is heated at 300 ° C. in air under normal pressure after the water-washing. The rate of change of the particle size at that time may be ± 20%.

<2-3. Nature>
Next, the properties of the calcium phosphate sintered body particles obtained by the manufacturing method according to the present embodiment will be described.

  According to the present embodiment, it is possible to obtain a minute calcium phosphate sintered particle group which does not contain calcium carbonate.

  By not containing calcium carbonate, it becomes possible to obtain calcium phosphate sintered particles in which the decrease in biocompatibility and the change in solubility are suppressed.

  Furthermore, according to this embodiment, it is also possible to make a minute calcium phosphate sintered body particle group not containing an alkali metal element.

  Further, by not containing an alkali metal element, it is possible to obtain calcium phosphate sintered particles which are further improved in crystallinity, difficult to be dissolved, and are excellent in cell adhesion.

Here, "does not contain calcium carbonate" means that it does not substantially contain calcium carbonate, and does not necessarily exclude a small amount of inclusion, but satisfies the criteria of (1) to (3) below And, preferably, further fulfilling the criterion of (4).
(1) From the measurement results of X-ray diffraction, calcium carbonate is calcium carbonate (formula weight: 100.09) / hydroxyapatite (formula weight: 1004.62) = 0.1 / 99.9 (formula conversion ratio) or less It is.
(2) In thermogravimetric differential thermal analysis (TG-DTA) measurement, a weight loss of 2% or more at a temperature of 650 ° C. to 800 ° C. with a clear endotherm is not observed.
(3) In the FT-IR spectra obtained in the measurement Kubelka-Munk (KM) chart showing the calculated absorbance by the equation, separated peaks wavenumber appears between ^{860cm -1 ~890cm} -1, is attributed to calcium carbonate The peak around 877 cm ^-1 is not observed. The peak separation is performed, for example, by using a software of fityk 0.9.4, under the condition of Function Type: Gaussian, Fitting Method: Levenberg-Marquardt.
(4) When tested according to the quasi-drug raw material standard 2006 (hydroxyapatite), the air bubble generation amount is 0.25 mL or less.

  Moreover, "does not contain an alkali metal element" means that the alkali metal element is not substantially contained, and with respect to each alkali metal element, the weight of the alkali metal element with respect to the weight of the entire calcium phosphate sintered body particles is 10 ppm or less ( Preferably, it is 1 ppm or less). In addition, a conventionally well-known method is applicable as an analysis method, For example, what is necessary is just to analyze by ICP-MS.

  Furthermore, as described above, according to the present embodiment, it is also possible to use a calcium phosphate sintered particle group that does not contain an alkaline earth metal element other than calcium or a transition metal element.

  As described above, by containing substantially no alkaline earth metal element other than calcium or a transition metal element, the crystallinity is further enhanced, and it is difficult to dissolve, and further, calcium phosphate sintered particles having excellent cell adhesion property and It is possible to In addition, the safety to the living body can be further enhanced by substantially not containing the transition metal element.

  In addition, "does not contain an alkaline earth metal element" ("does not contain a transition metal element") refers to all the sintered calcium phosphate particles with respect to each alkaline earth metal element (each transition metal element) as described above. The weight of the alkaline earth metal element (transition metal element) relative to the weight is 10 ppm or less (preferably, 1 ppm or less). In addition, a conventionally well-known method is applicable as an analysis method, For example, what is necessary is just to analyze by ICP-MS.

Here, the calcium phosphate particle group obtained by the present production method further has the following property (A) as moisture adsorption.
(A) After sufficient drying (for example, after drying at normal pressure, temperature 60 ° C., humidity 45 to 85% for 18 hours or more), normal pressure, temperature 25 ° C., humidity 50% After leaving to stand for 3 days or more, under a nitrogen stream, a thermal gravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under a nitrogen stream at 10 ° C./min. The weight decreased in the temperature range of 25 to 200 ° C. is 2% or less.

  It has been found that the cell adhesion can be further improved by the calcium phosphate particles satisfying such properties.

  The calcined calcium phosphate particles in this embodiment preferably have a half width at d = 2.814 of 0.2 to 0.8 as measured by X-ray diffraction (XRD), and 0 It is more preferable to satisfy .3 to 0.7. By setting the half value width of the calcined calcium phosphate particles in this range, it is possible to further reduce the solubility and to further improve the cell adhesion.

  In the adjustment of the half width, the firing temperature and the firing time may be appropriately adjusted. When the above half width is desired, for example, the firing temperature (maximum achieved temperature) is set to 600 to 800 ° C. The holding time may be 0 to 1 h or the like.

<<< Second manufacturing method >>>
Hereinafter, the second production method of the calcium phosphate particles according to the present invention will be described, and then, the ceramic particles obtained by the second production method will be described.

<< 1. Second Production Method of Ceramic Particles >>
<1-1. Raw material>
Specific examples of calcium phosphate (CaP), which is a ceramic raw material before firing, include hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ) and calcium metaphosphate _{(Ca (PO 3) 2)} , Ca 10 (PO 4) 6 F 2, Ca 10 (PO 4) 6 Cl 2 , and the like. Here, the calcium phosphate (CaP) may be artificially produced by a known production method such as a wet method, a dry method, a hydrolysis method, a hydrothermal method, etc., and bones and teeth It may be of natural origin obtained from E. coli.

<1-2. Process>
The method for producing a ceramic particle group according to the present embodiment only needs to include at least a "mixing step", a "sintering step" and a "pickling step", but other steps (eg, primary particle generation step) , “Removal process”, “grinding process”, “drying process”, etc.) may be included.

  For example, the method for producing a ceramic particle group according to the present embodiment is performed in the order of “1. primary particle generation process” → “2. Mixing process” → “3. Sintering process” → “4. pickling process” .

(Primary particle generation process)
Hereinafter, in the method for producing a calcium phosphate particle group according to the present embodiment, the case is exemplified where the primary particles are spherical and rod-like, but the production method of the present embodiment is not limited thereto, and primary particles can be produced as primary particles. It may be of any shape.

· Primary particle formation step of spherical ceramic particles First, "primary particles" refers to particles formed of ceramic raw materials {calcium phosphate (CaP), hydroxyapatite (HAp), etc.} before sintering in the production step of ceramic particles. It means that. That is, it means the particles formed for the first time in the process of producing ceramic particles. In a narrow sense, it means single crystal particles. In the claims and the specification, the term "primary particles" includes those in an amorphous (amorphous) state, in a low crystalline state, and in the state of a sintered body after sintering. It is a meaning. On the other hand, with "secondary particles", a plurality of "primary particles" are bonded by physical bonding such as fusion, or by chemical bonding such as Van der Waals force, electrostatic interaction, or covalent bonding. Mean particles in the formed state. 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. Further, in particular, "single-crystal primary particles" mean primary particles consisting of single crystals of ceramic raw material, or particle agglomerates 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 is obtained by sintering. It is a thing which does not contain the secondary particle which the particle | grains fuse | melted and polycrystallized.

  The primary particle generation step is not particularly limited as long as it is a step known per se and a step capable of generating the above primary particles, and may be appropriately selected depending on the raw material of the ceramic to be produced. For example, when phosphoric acid is dropped to a calcium hydroxide slurry at normal temperature, particles of calcium phosphate (CaP) are precipitated.

  The method for producing a ceramic particle group according to the present embodiment is to produce a ceramic particle group by sintering a primary particle group consisting of primary particles produced by the above primary particle producing step while preventing fusion or the like. . Therefore, the state (particle diameter, particle size distribution) of the primary particles generated in the primary particle generation step is directly reflected in the state (particle diameter, particle size distribution) of the ceramic particles as the final product. Therefore, in the case of producing a ceramic particle group having a fine particle size (nanometer size) and a uniform particle size (narrow particle size distribution), the particle size is fine (nanometer size) in the primary particle generation step. And primary particles having a uniform particle diameter (narrow particle size distribution).

  The particle diameter (average particle diameter and particle size distribution) of the preferred primary particles in this case is preferably 10 nm to 700 nm, more preferably 15 nm to 450 nm, and most preferably 20 nm to 400 nm. The variation coefficient of the particle diameter of the primary particle group consisting of primary particles is preferably 20% or less, more preferably 18% or less, and most preferably 15% or less. The particle size and coefficient of variation of the primary particles may be calculated by measuring the particle size of at least 100 or more primary particles using dynamic light scattering or an electron microscope.

  The “variation coefficient” is a value representing the variation in particle diameter among particles which can be calculated by standard deviation / average particle diameter × 100 (%).

  There is no particular limitation on the method of producing the above fine (nanometer size) primary particle group having uniform particle diameter (narrow particle size distribution), for example, JP-A-2002-137910. No. 5,043,436, Journal of Nanoscience and Nanotechnology 7, 848-851, 2007.

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

· Primary particle generation process of rod-like ceramic particles {Rod-like ceramic particles {The particle diameter of the particles is 30 nm to 5 μm in the maximum diameter of the short axis and 75 nm to 10 μm in the long axis. The primary particle generation process of ceramic particles having a ratio (c axis length / a axis length) of 1 to 30 is known per se, for example, JP-A-2002-137910, Journal of Nanoparticle Research 9, 8077 -815, 2007 can be mentioned.

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

(Mixing process)
The mixing step is a step of mixing the primary particles and the anti-fusion agent. By interposing an anti-fusion agent in advance between the particles of the primary particle group obtained in the above primary particle forming step, it is possible to prevent the fusion of primary particles in the subsequent sintering step. It is. The mixture of the primary particles and the anti-fusion agent obtained by the mixing step is called "mixed particles".

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

  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, the ceramic particle group in which the anti-fusion agent is mixed is suspended only in an aqueous solvent such as pure water. The inhibitor (eg 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, and equipment and organic solvent waste treatment corresponding to the use of the organic solvent are unnecessary in the removal step. It becomes. Therefore, it can be said that the anti-fusion agent can be more easily removed from the ceramic 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.

  The aqueous solvent may contain a chelate compound such as oxalate, ethylenediamine, bipyridine and ethylenediaminetetraacetate 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.

  Here, the solubility of the anti-fusion agent in the solvent is preferably higher because the higher the removal efficiency is, the better. The preferable solubility is preferably 0.01 g or more, more preferably 1 g or more, and most preferably 10 g or more, when the amount (g) of the solute in 100 g of the solvent is the solubility.

  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 the mixing step is not particularly limited, and a solid anti-fusion agent is mixed with the solid primary particles and then mixed using a blender. A method may be used, or a method of dispersing primary particles in a solution of anti-fusion agent may be performed. 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. Also in the examples described later, 0.5 g of primary particles of hydroxyapatite (HAp) are dispersed in a calcium carbonate saturated aqueous solution and dried at 80 ° C. to obtain mixed particles.

  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, It may be a step of further adding a metal salt (alkali metal salt and / or alkaline earth metal salt and / or transition metal salt). By employing the above steps, the adsorption of the polymer compound on the surface of hydroxyapatite (HAp) makes it possible to reliably prevent contact between hydroxyapatite (HAp) in the process of mixing the anti-fusion agent, and thereafter calcium By adding a salt, it becomes possible to reliably deposit the anti-fusion agent on the surface of hydroxyapatite (HAp). 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 molecular weight of the polymer compound is not particularly limited, but is preferably 100 g / mol or more and 1,000,000 g / mol or less, more preferably 500 g / mol or more and 500,000 g / mol or less, and 1,000 g / mol. Most preferably, it is mol or more and 300,000 g / mol or less. If it is less than the above-mentioned preferable range, the ratio of entering between primary particles decreases, and the ratio of preventing contact between primary particles decreases. Moreover, when the said preferable range is exceeded, it is unpreferable in order that operativity, such as that the solubility of a high molecular compound becomes low, and the viscosity of the solution containing the said high molecular compound, worsen will worsen.

  The solution containing the polymer compound is preferably an aqueous solution. This is because the hydroxyapatite (HAp) sintered particles dissolve under strongly acidic conditions. 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 particles are insoluble. The aqueous solution containing the polymer compound may be prepared by dissolving the polymer compound in distilled water, ion-exchanged water or the like, and adjusting the pH with an aqueous solution of ammonia, sodium hydroxide, potassium hydroxide or the like.

  In addition, 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, and 0. The range of 01% w / v to 10% w / v 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 embodiment, a solution containing the above-described polymer compound and primary particles are mixed. 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 a ceramic particle group according to the present embodiment, the polymer compound is adsorbed on the primary particle surface, and a carboxyl group, a sulfuric acid group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group or an amino group Either can be added to the surface of the primary particles. 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 surface of the above primary particles is obtained. A combination of a carboxylate ion, a sulfate ion, a sulfonate ion, a phosphate ion, a phosphonate ion, an amino ion, and a metal ion (alkali metal ion and / or alkaline earth metal ion and / or transition metal ion) On the surface of primary particles, carboxylates, sulfates, sulfonates, phosphates, phosphonates and amino acid salts are formed. 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 Is collected, dried, and subjected to the sintering step described later. The drying is performed, for example, under reduced pressure (1 × 10 ⁵ Pa or more and 1 × 10 ⁵ Pa or less is preferable, 1 × 10 ³ Pa or more and 1 × 10 ³ Pa or less is more preferable, 1 × 10 ² Pa or more and 1 × 10 ^-2 Pa or less is most preferable.), heated (0 preferably 200 ° C. or less over ° C., further preferably 20 ° C. or higher 0.99 ° C. or less, and a method in which the 40 ° C. or higher 120 ° C. or less performed most preferred.) to Be 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.

  Examples of the alkali metal salt include, but are not limited to, 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, potassium iodide 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.

  Further, 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, magnesium thiosulfate 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, calcium peroxide, calcium sulfate, calcium thiosulfate, calcium selenate, calcium nitrite, calcium nitrate, calcium phosphate, calcium carbonate, calcium hydroxide and the like can be used.

  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, selenium Acid, zinc nitrite, zinc nitrate, zinc phosphate, zinc carbonate, zinc hydroxide, iron chloride, 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. It may also be a nickel compound.

  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. Also, metal salts (alkali metal salts, alkaline earth metal salts, transition metals) may be in the solid state, but can be added uniformly, and because the concentration to be added can be controlled, etc. It is preferable to add as an aqueous solution. 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, phosphoric acid present on the primary particle surface. Ion, phosphonate ion, amino ion, metal carboxylate (alkali metal and / or alkaline earth metal and / or transition metal), 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.

  In addition, although sodium polyacrylate is soluble in water, it can be used as an antifusing agent as it is in the present mixing step, 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. In addition, since the polymer compound is decomposed when the primary particles are calcined at a high temperature (about 300 ° C. or more), the metal salt of the polymer compound is used as the primary particle so as to function as an anti-fusion agent even after calcination. It is preferable to deposit on the surface. However, when the primary particles are calcined (heat treatment) at a temperature at which the polymer compound does not decompose (do not soften), it is not particularly necessary to precipitate the metal salt of the polymer compound on the surface of the primary particles.

(Sintering process)
The said sintering process is a process of exposing the mixed particle obtained by the said mixing process to sintering temperature, and making the primary particle contained in the said mixed particle into a ceramic particle (sintered particle). Since the antifusing agent intervenes 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 sintering step.

  The sintering temperature in the sintering step may be appropriately set so that the hardness of the ceramic particles becomes a desired hardness, for example, more preferably in the range of 100 ° C. to 1800 ° C., still more preferably 150 ° C. to 1500 ° C. And 200 ° C. to 1200 ° C. are most preferable. The sintering time may be appropriately set based on the desired hardness of the ceramic particles and the like. In the examples described below, sintering is performed at 800 ° C. for 1 hour.

  In addition, the apparatus etc. which are used for the said sintering process are not specifically limited, According to a manufacturing scale, manufacturing conditions, etc., after selecting a commercially available calcination furnace suitably, it may be adopted.

(Removal process)
The said removal process is a process of removing the anti-fusion agent mixed between the particle | grains of the ceramic particle group obtained by the sintering process.

  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 prevented by using a solvent which does not dissolve the ceramic particles (non-dissolving) and a solvent which dissolves the fusion inhibitor (dissolution). Only the agent 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.

  The aqueous solvent may contain a chelate compound such as oxalate, ethylenediamine, bipyridine and ethylenediaminetetraacetate 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.

  In the case of sintered hydroxyapatite (HAp) particles, the sintered hydroxyapatite (HAp) particles are dissolved under conditions of pH 4.0 or less, so the removal process should be performed at pH 4.0 to pH 12.0. preferable.

  By the way, when removing the anti-fusion agent using a solvent, after suspending the ceramic particle group containing the anti-fusion agent obtained by the sintering step in the solvent, only the ceramic particles are filtered or centrifuged. It may be recovered. In the method for producing a ceramic particle group according to the present embodiment, the above operation is not limited to once, and may be performed twice or more. It can be said that the removal rate of the anti-sticking agent between the ceramic particles is further improved by performing the above operation a plurality of times. 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 ceramic particles decreases. Therefore, the number of times of the above operation may be appropriately determined based on the target removal rate of the anti-fusion agent.

  The process may further include a process of classifying in order 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, the group of ceramic particles (rough ceramic particles) containing the anti-fusion agent obtained by the sintering step is suspended and dispersed in a suitable solvent (such as water), and then the magnetic force is applied to the suspension. The magnet is made to adsorb only the anti-fusion agent, and only the non-adsorbed ceramic particles are recovered. In addition, a method may be used in which the anti-sticking agent is separated by a magnet after the crude ceramic particles are ground into powder without being suspended in a solvent. However, when the suspension is used, the ceramic particles and the anti-fusion agent are easily separated, and it can be said that the removal rate of the anti-fusion agent is high. The ceramic particle to which this method can be applied is preferably a nonmagnetic material or a weak magnetic material.

(Acid wash process)
The pickling step is a step of washing the ceramic particles (sintered particles) obtained by the sintering step with an acid. The purpose of the cleaning step is to remove calcium carbonate and the like contained in the sintered particles.

  As the acid, a weak acid which does not damage the ceramic particles is used. For example, hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, or ammonium salts thereof can be mentioned.

  The pickling step is performed by suspending a calcium phosphate sintered body in an acid solution or the like. For example, the calcium phosphate sintered body is suspended in an acid solution, centrifuged, and then the supernatant is discarded. The pH of the supernatant is 6.0 to 9 (preferably 6.5 to 8.5, and further, The pickling step is carried out preferably by repeating the process to 7.0 to 7.5). More specifically, for example, HAp is suspended in 0.2 wt% ammonium nitrate aqueous solution, ultrasonicated for 5 minutes, solid-liquid separation is performed by centrifugation, and the supernatant is discarded. This operation may be repeated until the pH of the supernatant reaches 8, and then suspended in deionized water and centrifuged, and the operation of discarding the supernatant may be repeated three or more times to perform the acid washing step. .

(Crushing process)
The pulverizing step is a step of pulverizing the aggregate after the sintering step to obtain calcined calcium phosphate particles having a desired size. Here, in general, it is almost impossible to reduce the size of the secondary particles to the primary particle size even if a considerable degree of pulverizing process is performed. On the other hand, according to the method of the present embodiment, it is possible to easily grind to the primary particle size level even with a simple grinding step. Here, the pulverizing method is not particularly limited, and for example, ultrasonication and pulverizing treatment using a pulverizing ball. In addition, after the pulverizing treatment, particles having a smaller diameter may be collected by removing the uncrushed ones or the like.

(Drying process)
The drying step is a step of removing the solvent by heating or the like after the pulverizing step and the washing step to obtain calcium phosphate particles. The drying method is not particularly limited.

<< 2. Calcium phosphate sintered particles obtained by the second production method >>
<2-1. Spherical calcium phosphate sintered particles>
The spherical calcium phosphate sintered body particle group obtained by the present manufacturing method is a ceramic particle group containing ceramic particles, and the ceramic particle is sintered with calcium phosphate in a particle diameter range of 10 nm to 700 nm. It is a body particle, and the ceramic particle is a ceramic particle group characterized by not containing calcium carbonate substantially. Furthermore, the ceramic particles contain carbonated apatite on at least the surface thereof.

  Furthermore, in the spherical calcium phosphate sintered body particles obtained by the present production method, the variation coefficient of the particle diameter of the ceramic particles is preferably 30% or less, and more preferably 25% or less. It is particularly preferable that the content is 20% or less. The ceramic particle group is a fine particle having a uniform particle size (narrow particle size distribution). Therefore, there is an effect that it can be adsorbed more uniformly to the medical polymer material without performing additional operations such as particularly high classification. And since it does not contain calcium carbonate, when using as a biomaterial, it becomes possible to prevent the situation where biocompatibility and solubility of material change. Moreover, the said ceramic particle group shows high dispersibility.

  From another aspect, the spherical calcium phosphate sintered body particle group obtained by the present manufacturing method is a ceramic particle group including spherical ceramic particles, and is a primary particle consisting of a single crystal, or a primary consisting of the single crystal When a particle aggregate in which particles are aggregated by ionic interaction is a single crystal primary particle, the proportion of single crystal primary particles contained in the ceramic particle group accounts for a majority, and the ceramic particles are calcium phosphate sintered particles. And the ceramic particle group is substantially free of calcium carbonate. The ceramic particle group is a primary particle consisting of a single crystal, the majority of which is excellent in dispersibility in a solvent, or a particle aggregate (single crystal primary particle) in which the primary particles consisting of the single crystal are aggregated by ionic interaction. As exists. Therefore, there is an effect that the adsorption onto the aforementioned medical polymer base material is facilitated. In addition, since there is no bonding between primary particles, the specific surface area is high. Furthermore, since the stability in a living body is high and the dispersibility is excellent, there is an effect that it can be used as a medical material which can carry and release a drug. And since it does not contain calcium carbonate substantially, when it uses as a biomaterial, the biocompatibility of material and the original solubility of calcium phosphate are maintained. In addition, it is good also as particle | grains which the particle | grains which the primary particle which consists of, or the said single crystal gathered by ionic interaction in this way are ceramic particles.

  In the ceramic particle group, the ratio of single crystal primary particles contained in the ceramic particle group may be 70% or more. By adopting such a configuration, an effect of facilitating adsorption to the medical polymer substrate is exhibited.

  In the ceramic particle group, the particle diameter of the ceramic particles may be in the range of 10 nm to 700 nm. According to the said structure, it is effective in the ability to make it adsorb | suck more uniformly with respect to a medical polymeric material.

  The ceramic particle group may have a coefficient of variation in particle diameter of the ceramic particle group of 20% or less. By taking the configuration, it is possible to more uniformly adsorb the medical polymer material without performing additional operations such as particularly high classification.

  In addition, the ceramic particles may be hydroxyapatite (HAp) sintered particles. The particles are further composed of a hydroxyapatite (HAp) sintered body that is more biocompatible and can be used in a wide range of applications. Therefore, it is particularly preferable as a medical material.

2-2. Rod-like sintered calcium phosphate particles>
The rod-like sintered body of calcium phosphate particles obtained by this production method is a ceramic particle group containing ceramic particles, and the particle diameter of the ceramic particles is 30 nm to 5 μm in the maximum diameter of the short axis and 75 nm to 75 nm in the long axis. Ceramic particles which are 10 μm, grow in the c-axis direction, and have a crystal aspect ratio (c-axis length / a-axis length) of 1 to 30, and the ceramic particles are calcium phosphate sintered particles, And, the ceramic particle group is substantially free of calcium carbonate. Furthermore, the ceramic particles contain carbonated apatite on at least the surface thereof.

  Since the area to be provided for adhesion is much larger than that of the conventional fine particles, the rod-like calcium phosphate calcined particle group can improve the adhesion to the polymer substrate, and therefore, the polymer surface such as a biocompatible medical material such as a catheter can be improved. Suitable for modification. And since it does not contain calcium carbonate substantially, when using as a biomaterial, it becomes possible to prevent the situation which a carbon dioxide gas generate | occur | produces from a material. In addition, as a method of modifying the polymer surface, graft polymerization of an active group of calcium phosphate (for example, hydroxyapatite (HAp) nanoparticles) and a polymer substrate, for example, a vinyl-based polymerizable monomer having a carboxyl group on the surface is performed. A method of chemically reacting with the active group of the silicone rubber to form a complex, a method of using a curable adhesive, a method of heating the polymer substrate to near the melting point and embedding it in the substrate can be used. (This also applies to the above-mentioned spherical calcium phosphate sintered particle group). In addition, as a rod shape, it may be a truncated columnar structure in which the tip angle has an oblique surface.

  In addition, the ceramic particles may be sintered hydroxyapatite particles. The particles are further composed of a hydroxyapatite sintered body that is more biocompatible and can be used in a wide range of applications. Therefore, it is particularly preferable as a medical material.

<2-3. Calcium phosphate sintered particles expressed by general formula>
The above-mentioned 2-1 and 2-2 calcium phosphate sintered particles obtained by the present production method have the following general formula Ca _x (PO ₄ ) _6-y (CO ₃ ) _y (OH) ₂
(Wherein x is a number of 8 or more and 12 or less (preferably a number of 9 or more and 11 or less, more preferably a number of 9.5 or more and 10.5 or less), y is greater than 0 and 3 or less A number (preferably a number greater than 0 and less than or equal to 2 and more preferably a number greater than 0 and less than or equal to 1)
It may be represented by

The measuring method of Ca, (PO ₄ ) and CO ₃ is as follows. Ca and PO ₄ dissolve the measurement sample in 1 N nitric acid, adjust the measurement solution to 100 ppm in terms of formula weight, and use ICP emission analysis (Thermo Fisher Scientific, iCAP 7600 Duo) to measure the above solution Measure Further, CO ₃ was measured by a thermogravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under nitrogen stream at 10 ° C./min. The decarbonated amount is evaluated from the decreased weight in the temperature range of 700 ° C to 950 ° C.

<2-3. Structure>
Next, the structure of the calcium phosphate sintered body particle group obtained by the manufacturing method according to the present embodiment will be described.

  The present embodiment is a calcium phosphate sintered particle group characterized mainly by containing substantially no calcium carbonate. In addition, at least the surface thereof contains carbonated apatite.

  The inventors of the present invention found that when the invention of Patent Document 1 was carried out, calcium carbonate was generated on the surface of calcium phosphate sintered particles. It is surprising that calcium phosphate sintered bodies are used not only when using an anti-fusion agent containing carbonic acid such as calcium carbonate but also when using an anti-fusion agent containing no carbonic acid such as calcium nitrate and sodium nitrate. Calcium carbonate was produced on the surface of the particles (see Comparative Examples 2-2 and 2-3 below). The reason is that calcium nitrate and sodium nitrate are converted to calcium oxide and sodium oxide by heating. These substances are strong bases and react with carbon dioxide present in the electric furnace to produce calcium carbonate. In addition, since hydroxyapatite (HAp) easily causes ion exchange, ions forming the salt used as the anti-fusion agent and ions forming hydroxyapatite (HAp) are exchanged to change the crystal structure. At that time, it is presumed that the hydroxyapatite surface is rich in calcium ions, and reacts with carbon dioxide in the electric furnace to form calcium carbonate. The present inventors have found that carbonate apatite is produced on the surface or the like of sintered calcium phosphate particles (particle group) by removing this calcium carbonate by an acid washing step or the like.

  In this embodiment, by containing carbonated apatite on the surface of at least the surface of sintered calcium phosphate particles (particles) without containing calcium carbonate, the decrease in biocompatibility and the change in solubility are suppressed, and high dispersibility is achieved. It becomes possible to obtain the calcium phosphate sintered particles shown.

  Furthermore, in the present embodiment, by not containing an alkali metal element, crystallinity can be further improved, dissolution is difficult, and calcium phosphate sintered particles excellent in cell adhesion can be obtained.

Here, "does not contain calcium carbonate" means that it does not substantially contain calcium carbonate and does not necessarily exclude a slight amount of inclusion, but more specifically, the following (1) to (1) It is to meet the criteria of 3).
(1) From the measurement results of X-ray diffraction, calcium carbonate is calcium carbonate (formula weight: 100.09) / hydroxyapatite (formula weight: 1004.62) = 0.1 / 99.9 (formula conversion ratio) or less It is.
(2) In thermogravimetric differential thermal analysis (TG-DTA) measurement, a weight loss of 2% or more at a temperature of 650 ° C. to 800 ° C. with a clear endotherm is not observed.
(3) In the FT-IR spectra obtained in the measurement Kubelka-Munk (KM) chart showing the calculated absorbance by the equation, separated peaks wavenumber appears between ^{860cm -1 ~890cm} -1, is attributed to calcium carbonate The peak around 877 cm ^-1 is not observed. The peak separation is performed, for example, by using a software of fityk 0.9.4, under the condition of Function Type: Gaussian, Fitting Method: Levenberg-Marquardt.

"Containing carbonated apatite" refers to the presence of carbonated apatite as a result of measurement according to a known method of measuring carbonated apatite. For example, in the FT-IR spectrum, it means that absorption is observed at 1350 cm ^{−1 to} 1500 cm ⁻¹ .

The FT-IR analyzer and the measurement conditions are as follows. The particles of hydroxyapatite (HAp) fired particles are taken to be 10% by weight with respect to potassium bromide [KBr], and the measurement sample sufficiently ground in an agate mortar is diffusely reflected using FT-IR Spectrum 100 made by Perkin Elmer. by measuring the range of ^{450cm -1 ~4000cm} -1 at 8 cumulative.

  Moreover, the thermogravimetric differential thermal analysis (TG-DTA) measurement conditions are as follows. A thermogravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under nitrogen stream at 10 ° C./min. The decarbonated amount is evaluated from the decreased weight in the temperature range of 700 ° C to 950 ° C.

  Moreover, "does not contain an alkali metal element" shows that the weight of the alkali metal element with respect to the weight of the whole calcium phosphate sintered body particle is 10 ppm or less (preferably 1 ppm or less) with respect to each alkali metal element. In addition, a conventionally well-known method is applicable as an analysis method, For example, what is necessary is just to analyze by ICP-MS.

  Furthermore, as described above, according to the present embodiment, it is also possible to use a calcium phosphate sintered particle group that does not contain an alkaline earth metal element other than calcium or a transition metal element.

  As described above, by containing substantially no alkaline earth metal element other than calcium or a transition metal element, the crystallinity is further enhanced, and it is difficult to dissolve, and further, calcium phosphate sintered particles having excellent cell adhesion property and It is possible to In addition, the safety to the living body can be further enhanced by substantially not containing the transition metal element.

  The phrase “does not contain any alkaline earth metal element other than calcium” (“does not contain a transition metal element”) means, as in the above, calcium phosphate sintered bodies for each alkaline earth metal element (each transition metal element) The weight of the alkaline earth metal element (transition metal element) other than calcium relative to the weight of the whole particle is 10 ppm or less (preferably, 1 ppm or less). In addition, a conventionally well-known method is applicable as an analysis method, For example, what is necessary is just to analyze by ICP-MS.

  Here, when it is desired to manufacture calcium phosphate sintered particles that do not contain an alkali metal element, do not contain an alkaline earth metal element other than calcium, and / or do not contain a transition metal element As the selection of the anti-fusion agent, it is conceivable to use one not containing the element.

Here, the calcium phosphate particle group obtained by the present production method further has the following property (A) as moisture adsorption.
(A) After sufficient drying (for example, after drying at normal pressure, temperature 60 ° C., humidity 45 to 85% for 18 hours or more), normal pressure, temperature 25 ° C., humidity 50% After leaving to stand for 3 days or more, under a nitrogen stream, a thermal gravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under a nitrogen stream at 10 ° C./min. The weight decreased in the temperature range of 25 to 200 ° C. is 2% or less.

  It has been found that the cell adhesion can be further improved by the calcium phosphate particles satisfying such properties.

  The calcined calcium phosphate particles in this embodiment preferably have a half width at d = 2.814 of 0.2 to 0.8 as measured by X-ray diffraction (XRD), and 0 It is more preferable to satisfy .3 to 0.7. By setting the half value width of the calcined calcium phosphate particles in this range, it is possible to further reduce the solubility and to further improve the cell adhesion.

  In the adjustment of the half width, the firing temperature and the firing time may be appropriately adjusted. When the above half width is desired, for example, the firing temperature (maximum achieved temperature) is set to 600 to 800 ° C. The holding time may be 0 to 1 h or the like.

  Here, the calcium phosphate obtained by the present production method further contains B-type carbonated apatite. It has been found that, by satisfying such properties, calcium phosphate can further improve cell adhesion.

In addition, in the analysis by the FT-IR spectrum mentioned above, when two absorption peaks are observed at 1350 cm ^{−1 to} 1500 cm ⁻¹ , it is considered to be “containing carbonate apatite” and “containing B-type carbonate apatite”. {for example, in Figure 2-4 to be described ^later, among the peaks of the two absorption observed in the range of 1350cm ^-1 ~1500cm -1, a peak of the absorption peak type B carbonated apatite right (1350 cm ^-1 side) Is considered equivalent to

<<<< Dose of Usage >>>
The means of delivery to the application site of the collagen production promoter according to the present best mode includes, for example, direct application to the indication site, percutaneous absorption, subcutaneous injection, and surgical procedure.
The collagen production promoter according to the best mode exhibits its effect by being used as a cosmetic twice a day, once at 150 mg (however, 1.5 mg of calcium phosphate is contained therein).

<<< First Embodiment >>>
<< Production example >>
(Example 1-1: spherical hydroxyapatite fired body particles)
In a reaction vessel containing deionized water, while stirring, calcium nitrate tetrahydrate, an aqueous solution of diammonium hydrogen phosphate and aqueous ammonia are added {calcium: phosphoric acid (molar ratio) = 5: 3}, hydroxy Primary particles of apatite were obtained. Thereafter, the supernatant in the reaction vessel was transferred to a waste water vessel, deionized water was added, the mixture was stirred with a stirrer, and the supernatant was transferred to a waste vessel twice. Thereafter, the reaction vessel containing the precipitate was frozen overnight at -10 ° C to -25 ° C. Thereafter, it was thawed at room temperature, and the precipitate after thawing was collected by filtration. Thereafter, about 400 g of the precipitate was put in a baking dish, put in a baking furnace, brought to 600 ° C. for 1 hour, kept at 600 ° C. for 1 hour, and then cooled for 1 hour or more to carry out baking. Thereafter, deionized water was added to the sintered body, and ultrasonication was performed for 30 minutes or more. Then, it was transferred to a pod mill, put in a grinding ball, and ground for 1 hour. After completion of the pulverization, it was transferred to a hand-made beaker, and the uncrushed sintered body was removed using a 150 μm mesh sieve. After this, deionized water washing was repeated six times. Then, it dried at 60-80 degreeC and obtained the hydroxyapatite baked body which concerns on Example 1-1 which does not contain an alkali metal element.

(Example 1-2: rod-like hydroxyapatite sintered body particle group)
In a reaction vessel containing deionized water, while stirring the aqueous solution of calcium nitrate tetrahydrate, an aqueous solution of diammonium hydrogen phosphate and aqueous ammonia are added dropwise to the aqueous solution of calcium nitrate tetrahydrate {{calcium: phosphoric acid (molar Ratio: 5: 3}, primary particles of hydroxyapatite were obtained. Thereafter, the supernatant in the reaction vessel was transferred to a waste water vessel, deionized water was added, and the operation of stirring with a stirrer and transferring the supernatant to a waste vessel was repeated five times. Thereafter, the reaction vessel containing the precipitate was frozen overnight at -10 ° C to -25 ° C. Thereafter, it was thawed at room temperature, and the precipitate after thawing was collected by filtration. Thereafter, about 400 g of the precipitate was put in a baking dish, put in a baking furnace, brought to 600 ° C. for 1 hour, kept at 600 ° C. for 1 hour, and then cooled for 1 hour or more to carry out baking. Thereafter, deionized water was added to the sintered body, and ultrasonication was performed for 30 minutes or more. Then, it was transferred to a pod mill, put in a grinding ball, and ground for 1 hour. After completion of the pulverization, it was transferred to a hand-made beaker, and the uncrushed sintered body was removed using a 150 μm mesh sieve. After this, deionized water washing was repeated seven times. Then, it dried at 60-80 degreeC and obtained the hydroxyapatite baked body which does not contain an alkali metal element which concerns on Example 1-2. The particle diameter of the ceramic particles is 47 nm in the average maximum diameter in the minor axis and 157 nm in the major axis, and grows in the c axis direction, and the aspect ratio of the crystal (c axis length / a axis length) is 3.3. It is 3 and the tip angle was ceramic particles of a truncated columnar structure having an oblique surface.

(Comparative Example 1-1)
According to Example 1 of Japanese Patent No. 5043436, a hydroxyapatite fired body according to Comparative Example 1-1 was obtained.

<< X-ray diffraction test >>
FIGS. 1-1, 1-2, and 1-3 are the results of X-ray diffraction measurement of the hydroxyapatite fired body according to Example 1-1, Example 1-2, and Comparative Example 1-1, respectively. As can be seen from the figure, the peak of calcium carbonate was not observed in FIG. 1-1, while the clear peak of calcium carbonate was observed in FIG. Moreover, it can confirm that the peak of a calcium carbonate is not observed similarly to Example 1-1 also about Example 1-2. More specifically, in FIG. 1-1, only a pattern corresponding to hydroxyapatite (PDF 74-0565) can be confirmed, while in FIG. 1-3, a peak not existing in hydroxyapatite is observed at 29.4 °. And calcium carbonate (calcite: PDF 72-1937). The X-ray diffraction apparatus and the measurement conditions are as follows. Crystal structure analysis was performed using a powder X-ray diffraction apparatus {Mini Flex 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 °, the measurement The mode was a continuous condition.

  In addition, when the half value width in d = 2.814 was measured on the same conditions as the said X-ray-diffraction test, Example 1-1 is about 0.4, and Example 1-2 is also about 0.5. there were.

<< Appearance observation test >>
1-4 and 1-5 are SEM photographs of hydroxyapatite fired body particle groups according to Example 1-1 (scale difference). From these photographs, the hydroxyapatite fired body particle group according to Example 1-1 is a hydroxyapatite fired body particle group including spherical hydroxyapatite fired body particles, and is made of a primary particle consisting of a single crystal or the single crystal. It can be seen that, when a particle mass in which primary particles are aggregated by ionic interaction is a single crystal primary particle, the ratio of single crystal primary particles contained in the hydroxyapatite fired body particle group occupies a majority. 1-6 and 1-7 are SEM photographs of hydroxyapatite fired body particle groups according to Example 1-2 (scale difference).

<< Particle size measurement test >>
With respect to the hydroxyapatite fired body (endotoxin not inactivated) according to Example 1-1 and Comparative Example 1-1, the average particle diameter and the standard deviation are calculated (9 images are obtained by SEM, and within 1 image) The particle size of 12 particles was measured, the particle size of a total of 108 particles was confirmed, and the average value and the standard deviation were calculated). In addition, with respect to those obtained by inactivating the hydroxyapatite fired body (endotoxin not inactivated) according to Example 1-1 and Comparative example 1-1, the average particle diameter and the standard deviation are similarly calculated (9 points by SEM The image of (1) was obtained, and the particle diameter of 12 particles was measured in one image point, the particle diameter of a total of 108 particles was confirmed, and the average value and the standard deviation were calculated). The inactivation procedure is as follows: (1) Measure the HAp powder into an ampoule in advance with dry heat sterilization (300 ° C, 2 hours), (2), put the HAp loaded ampoule into the dry heat sterilizer as it is opened. Dry heat sterilization (300 ° C, 2 hours), (3) heat sealed ampoules sealed to room temperature (sealed tube), (4) sealed heat sealed ampoules again in dry heat sterilizer, dry heat sterilized (300 ° C, 2 hours). Table 1-1 is a hydroxyapatite fired body according to Example 1-1, and Table 1-2 is a hydroxyapatite fired body according to Comparative Example 1-1.

<< Thermogravimetric differential thermal analysis >>
The thermogravimetric differential thermal analysis (TG-DTA) measurement of the hydroxyapatite fired body particle group according to Example 1-1, Example 1-2 and Comparative Example 1-1 is performed, and in the example, it is at 650 ° C. to 800 ° C. It was confirmed that no weight loss of 2% or more was observed with a clear endotherm. On the other hand, in Comparative Example 1-1, a weight loss of 2% or more with a clear endotherm at 650 ° C. to 800 ° C. was observed.

  Moreover, the thermogravimetric differential thermal analysis (TG-DTA) measurement conditions are as follows. A thermogravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under nitrogen stream at 10 ° C./min. The decarbonated amount was evaluated from the decreased weight in the temperature range of 700 ° C. to 950 ° C.

  Furthermore, as a moisture adsorption test, after drying the hydroxyapatite fired body particle group according to Example 1-1 under the conditions of a temperature of 60 ° C. and a humidity of 45 to 85% for 18 hours or more, normal pressure and a temperature of 25 ° C. And left at a humidity of 50% for 3 days or more, a thermal gravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under nitrogen stream at 10 ° C./min. The weight was measured in the temperature range of 25 to 200 ° C. to obtain 1.43%. Moreover, when the same test was performed also about Example 2-1, the result of the same level was obtained.

<< FT-IR Analysis >>
FT-IR analysis was performed on the hydroxyapatite fired body particle groups according to Example 1-1, Example 1-2, and Comparative Example 1-1. In particular, in chart showing the absorbance of calculating the spectrum obtained in the FT-IR measurement in Kubelka-Munk (KM) equation, to separate the peak wavenumber appears between ^{860cm -1 ~890cm} -1, it was evaluated. As a result, in the hydroxyapatite fired body particle group according to Example 1-1 and Example 1-2, it was confirmed that a peak near 877 cm ⁻¹ attributed to calcium carbonate was not observed. On the other hand, in the hydroxyapatite fired body particle group according to Comparative Example 1-1, a peak around 877 cm ⁻¹ attributed to calcium carbonate was observed.

The FT-IR analyzer and the measurement conditions are as follows. The particles of hydroxyapatite (HAp) fired particles are taken to be 10% by weight with respect to potassium bromide [KBr], and the measurement sample sufficiently ground in an agate mortar is diffusely reflected using FT-IR Spectrum 100 made by Perkin Elmer. the range of ^{450cm -1 ~4000cm} -1 at was measured at 8 cumulative. In addition, peak separation was performed using a software of fityk 0.9.4 under the condition of Function Type: Gaussian, Fitting Method: Levenberg-Marquardt.

From the results of the X-ray diffraction test, the thermogravimetric differential thermal analysis and the FT-IR analysis, Example 1-1 and Example 1-2 were substantially free of calcium carbonate.
On the other hand, Comparative Example 1-1 and Comparative Example 1-2 contained calcium carbonate according to the results of thermogravimetric differential thermal analysis.

<< Foaming Confirmation Test >>
Furthermore, for the hydroxyapatite fired bodies according to Examples 1-1 and 1-2 and Comparative Example 1-1, the purity test (4) carbonate listed in the quasi-drug raw material standard 2006 “hydroxyapatite” is shown. The test was conducted according to the procedure. Specifically, 1.0 g of a sample was weighed at normal temperature (20 ° C.), 5 mL of water was added and shaken, and pressure reduction was performed using an aspirator for 1 hour for degassing. After degassing, 2 mL of concentrated hydrochloric acid (35.0% by mass) was added to confirm the presence or absence of foaming. As a result, in the HAp obtained according to the example, no foaming could be confirmed (gas generation amount is less than 0.25 ml). On the other hand, in the HAp obtained by the comparative example, the foam generated so much that the supernatant became cloudy (gas generation amount was 0.25 ml or more).

<< Cell adhesion test >>
<Manufacturing of test material>
(Washing process)
Alcohol treatment (ultrasonic irradiation in alcohol (ethanol, 2-propanol, etc.) for 5 minutes) was performed on a circular polyethylene terephthalate (PET) sheet (diameter 9 mm, thickness 0.1 mm).

(Linker introduction process)
Corona discharge treatment (100 V, 15 seconds on one side) was applied to both sides of the cleaned PET sheet. The PET sheet after corona discharge treatment and 10 ml of acrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were placed in a 20 ml test tube, and the inside of the test tube was depressurized by a vacuum pump to perform a degassing operation. The test tube was melted under reduced pressure, and graft polymerization was performed for 60 minutes in a water bath at a temperature of 60 ° C. After the treatment, in order to remove the acrylic acid homopolymer adhering on the surface of the substrate, after stirring for 60 minutes at room temperature in water, the surface of the substrate was washed with water and then with ethanol.

(Sintered hydroxyapatite fine particle immobilization treatment)
After the above treatment, the base material is in a dispersion of 1% of sintered hydroxyapatite fine particles (sintered hydroxyapatite fine particles according to Example 1-1, Example 1-2 or Comparative Example 1-1) (dispersion medium: ethanol ), Left at 20 ° C. for 30 minutes. Thereafter, the substrate was irradiated with ultrasonic waves for 5 minutes, the base material was taken out, and dried at 20 ° C. under normal pressure to obtain a test material.

<Cell adhesion / Cell morphology evaluation>
A cell adhesion / cell morphology evaluation test was performed using the prepared test material. Prior to the test, the test material is washed with ethanol, washed with phosphate buffered saline (PBS) and medium (MEM α (serum free)) (3 times), immersed in medium, and incubated until use (37 ° C., 5% CO ₂ ). And cell culture was implemented in the following procedures. First, culture medium (200 μL / hole) and a test material (sheet for test) were placed in a 48-well plate. Thereafter, a suspension of osteoblast-like cells (MC3T3-E1 (subclone 14)) was added (about 1 × 10 ⁵ cells / 200 μL / well) and cultured for 3 hours.

(Cell observation)
1-8 to 1-10 are SEM photographs of test materials manufactured using the hydroxyapatite sintered body particle group according to Example 1-1, Example 1-2 and Comparative Example 1-1, respectively. It is. Based on the photograph, the state of cell aggregation, cell morphology, and cell density were determined. The results are shown in Table 1-3. In addition, regarding cell density, it shows that cell density is so high that there are many "+" numbers. In addition, the average coverage of each of the test materials is calculated from the SEM photograph.

(Staining observation)
Subsequently, staining observation was performed according to the following procedure. First, the test sheet is washed with PBS to remove non-adherent cells, fixed (4% PFA) and treated with surfactant (0.5% Triton X-100 / PBS), and then treated with AlexaFluor ( After the addition of 488-labeled Acti-stain (trademark), it was encapsulated with a mounting agent (with nuclear staining dye DAPI). It was irradiated with a 488 nm laser beam with a fluorescence microscope (Nikon, ECLIPSE Ti-S), and cell morphology was confirmed by staining of actin fibers. Irradiation with a laser beam of 405 nm confirmed staining of cell nuclei. The results are shown in FIGS. 1-11 and 1-12. FIGS. 1-11 and 1-12 show fluorescence images derived from actin and cell nuclei derived from the test materials manufactured using the hydroxyapatite sintered body particle groups according to Example 1-1 and Example 1-2. And the fluorescence image of As can be seen from the figure, when sintered hydroxyapatite particles according to Example 1-1 and Example 1-2 are used, actin fibers are sufficiently developed to exhibit excellent cell adhesion. It was also confirmed that most of the cells exhibited greatly elongated cell morphology.

  From the above results, it was confirmed that the calcium phosphate fine particles according to this example had a polygonal cell shape and significantly promoted intercellular adhesion. From this, it was clarified that the calcium phosphate fine particles according to this example promote morphological differentiation of cells.

<< Crystallinity >>
Crystal structure analysis was performed using a powder X-ray diffraction apparatus {Mini Flex 600, 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, output is 30 kV / 15 mA, scan speed is 10.0 ° / min, sampling width is 0.02 °, measurement The mode was a continuous condition. The average size τ (nm) of crystallites is given by the Scheller equation shown below.
(Scheller's formula) τ = Kλ / β cos θ
In addition, K was 0.9 in the form factor. λ is the X-ray wavelength, β is the half width of the top peak, and θ is the Bragg angle of the top peak. The average size τ (nm) of crystallites was calculated from the measured value of X-ray diffraction using the above equation.

  The average size τ of the crystallites of the calcium phosphate microparticles according to Example 1-1 was 11.6 nm, and the average size τ of the crystallites of the calcium phosphate microparticles according to Comparative Example 1-1 was 12.1 nm. Accordingly, it is understood that the calcium phosphate fine particles according to the present example have higher crystallinity.

<<< Second Embodiment >>>
<< Production example >>
(Example 2-1: Spherical hydroxyapatite (HAp) fired body particle group containing carbonate apatite)
(Primary particle generation process)
In a reaction vessel containing deionized water, while stirring, calcium nitrate tetrahydrate, an aqueous solution of diammonium hydrogen phosphate and aqueous ammonia are added {calcium: phosphoric acid (molar ratio) = 5: 3}, 24 The reaction was allowed to stir for an hour at room temperature. Primary particles of hydroxyapatite (HAp) were obtained.
(Mixing process)
By dispersing 1.0 g of hydroxyapatite (HAp) primary particles in 100 ml of an aqueous solution of pH 12.0 containing 1.0 g of sodium polyacrylate (manufactured by ALDRICH, weight average molecular weight 15,000 g / mol), Sodium polyacrylate was adsorbed on the surface of the particles. The pH of this aqueous solution was measured using EUTECH CyberScan pH 1100 EUTECH CyberScan pH 1100.
Next, 100 ml of a 0.12 mol / l calcium nitrate [Ca (NO ₃ ) ₂ ] aqueous solution was added to the dispersion prepared above to precipitate calcium polyacrylate on the surface of the same primary particles. Such calcium polyacrylate is an anti-fusion agent. The resulting precipitate was collected and dried at 80 ° C. under reduced pressure (about 0.1 Pa) to obtain mixed particles.
(Sintering process)
The above mixed particles were placed in a crucible, and the temperature was raised to a sintering temperature of 600 ° C. at a temperature rising rate of 200 ° C./hour, and sintering was performed for 1 hour. At this time, calcium polyacrylate was thermally decomposed to be calcium oxide [CaO], and partly calcium carbonate [CaCO ₃ ].
(Acid wash process)
In order to increase the solubility of the anti-fusion agent and calcium carbonate in water, an aqueous 50 mmol / l ammonium nitrate [NH ₄ NO ₃ ] aqueous solution was prepared. Next, the sinter obtained in the above step is suspended in 500 ml of the aqueous solution prepared above, irradiated with ultrasonic waves for 5 minutes, and solid-liquid separated by centrifugation, and the obtained precipitate is again made into an aqueous ammonium nitrate solution. The above steps were repeated until the pH of the supernatant became 7.5 or less. Furthermore, by suspending in distilled water and performing separation and washing similarly by centrifugation, the anti-fusion agent and ammonium nitrate were removed, and highly crystalline hydroxyapatite (HAp) fine particles were recovered.

(Example 2-2: rod-like hydroxyapatite (HAp) fired body particles group containing carbonated apatite)
(Primary particle generation process)
In a reaction vessel containing deionized water, while stirring, calcium nitrate tetrahydrate, an aqueous solution of diammonium hydrogen phosphate and aqueous ammonia are added {calcium: phosphoric acid (molar ratio) = 5: 3}, 24 The reaction was carried out with stirring at 80 ° C. for a time. Primary particles of hydroxyapatite (HAp) were obtained.
(Mixing process) · (Sintering process) · (Acid washing process)
It carried out similarly to Example 2-1.

(Comparative Example 2-1: hydroxyapatite-baked (HAp) adult particle group manufactured as a polyacrylate with a fusion inhibitor based on Japanese Patent No. 5043436)
(Primary particle generation process)
In a reaction vessel containing deionized water, while stirring, calcium nitrate tetrahydrate, an aqueous solution of diammonium hydrogen phosphate and aqueous ammonia are added {calcium: phosphoric acid (molar ratio) = 5: 3}, 24 The reaction was allowed to stir for an hour at room temperature. Primary particles of hydroxyapatite (HAp) were obtained.
(Mixing process)
By dispersing 1.0 g of hydroxyapatite (HAp) primary particles in 100 ml of an aqueous solution of pH 12.0 containing 1.0 g of sodium polyacrylate (manufactured by ALDRICH, weight average molecular weight 15,000 g / mol), Sodium polyacrylate was adsorbed on the surface of the particles. The pH of this aqueous solution was measured using EUTECH CyberScan pH 1100 EUTECH CyberScan pH 1100.
Next, 100 ml of a 0.12 mol / l calcium nitrate [Ca (NO ₃ ) ₂ ] aqueous solution was added to the dispersion prepared above to precipitate calcium polyacrylate on the surface of the same primary particles. Such calcium polyacrylate is an anti-fusion agent. The resulting precipitate was collected and dried at 80 ° C. under reduced pressure (about 0.1 Pa) to obtain mixed particles.
(Sintering process)
The mixed particles were placed in a crucible, and the temperature was raised to a sintering temperature of 600 ° C. at a temperature rising rate of 200 ° C./hour for sintering for 1 hour. At this time, calcium polyacrylate was thermally decomposed to be calcium oxide [CaO], and partly calcium carbonate [CaCO ₃ ].
(Removal process)
In order to increase the solubility of the anti-fusion agent in water, an aqueous 50 mmol / l ammonium nitrate [NH ₄ NO ₃ ] aqueous solution was prepared. Next, the sintered body obtained in the above step is suspended in 500 ml of the aqueous solution prepared above, separated and washed by centrifugation, suspended in distilled water, and similarly separated and washed by centrifugation. The antifusing agent and ammonium nitrate were removed, and highly crystalline hydroxyapatite (HAp) fine particles were recovered.

(Comparative Example 2-2: Hydroxyapatite fired body particles manufactured by using a fusion preventing agent as calcium nitrate based on Japanese Patent No. 5043436)
(Primary particle generation process)
The same as in Comparative Example 2-1.
(Mixing process)
1.0 g of hydroxyapatite (HAp) primary particles were suspended in 100 ml of a 0.12 mol / l aqueous solution of calcium nitrate. The resulting precipitate was collected and dried at 80 ° C. under reduced pressure (about 0.1 Pa) to obtain mixed particles.
(Sintering process) / (Removal process)
The same as in Comparative Example 2-1.

(Comparative example 2-3: hydroxyapatite fired body particles manufactured by using the fusion preventing agent as sodium nitrate based on Japanese Patent No. 5043436)
(Primary particle generation process)
The same as in Comparative Example 2-1.
(Mixing process)
1.0 g of hydroxyapatite (HAp) primary particles were suspended in 100 ml of a 0.12 mol / l aqueous solution of sodium nitrate. The resulting precipitate was collected and dried at 80 ° C. under reduced pressure (about 0.1 Pa) to obtain mixed particles.
(Sintering process) / (Removal process)
The same as in Comparative Example 2-1.

  In Examples 2-1 to 2-2, the weight of the alkali metal element or the like was 10 ppm or less.

<< X-ray diffraction test >>
The X-ray-diffraction measurement of the hydroxyapatite baked body which concerns on Example 2-1, Example 2-2, and Comparative example 2-1-Comparative example 2-3 was performed. As a result, a peak of calcium carbonate was not observed in the hydroxyapatite fired body according to the example, whereas a peak of calcium carbonate was observed in the hydroxyapatite fired body according to the comparative example. More specifically, in the hydroxyapatite fired body according to the example, only the pattern matching the hydroxyapatite (PDF 74-0565) can be confirmed, while in the hydroxyapatite fired body according to the comparative example, it does not exist in the hydroxyapatite A peak was observed at 29.4 °, which was in agreement with calcium carbonate (calcite: PDF 72-1937). The X-ray diffraction apparatus and the measurement conditions are as follows. Crystal structure analysis was performed using a powder X-ray diffraction apparatus {Mini Flex 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 °, the measurement The mode was a continuous condition.

  In addition, when the half value width in d = 2.814 was measured on the same conditions as the said X-ray-diffraction test, Example 2-1 is about 0.5, and Example 2-2 is also about 0.5. there were.

<< Thermogravimetric differential thermal analysis >>
FIGS. 2-1-2-2 are the results of the thermogravimetric differential thermal analysis (TG-DTA) measurement of the hydroxyapatite baked body particle group which concerns on Example 2-1 and Comparative Example 2-1. As can be seen from the figure, in Example 2-1 (FIG. 2-1), no weight loss of 2% or more was observed at 650 ° C. to 800 ° C. with a clear endotherm. On the other hand, in Comparative Example 2-1 (FIG. 2-2), a weight loss of 2% or more was observed at 650 ° C. to 800 ° C. with a clear endotherm.

  Similarly, Example 2-2 did not observe a weight loss of 2% or more with a definite endotherm at 650 ° C. to 800 ° C., while Comparative Example 2-2 to 2-3 showed 650%. A weight loss of more than 2% with a clear endotherm between 0 C and 800 0 C was observed.

  Furthermore, as a moisture adsorption test, the hydroxyapatite fired body particles according to Example 2-1 are dried at a temperature of 60 ° C. and a humidity of 45 to 85% for 18 hours or longer, under normal pressure, at a temperature of 25 ° C. And left at a humidity of 50% for 3 days or more, a thermal gravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under nitrogen stream at 10 ° C./min. The weight was measured in the temperature range of 25 to 200 ° C. to obtain 1.34%. Moreover, when the same test was performed also about Example 2-1, the result of the same level was obtained.

<< FT-IR Analysis >>
(About calcium carbonate)
The present inventors examined how the amount of calcium carbonate is changed by the pickling step when producing the hydroxyapatite (HAp) particles of Example 2-1. It is shown in Figure 2-3. The left chart in FIG. 2-3 is the hydroxyapatite particles for which the pickling process has not been performed, and the right chart in FIG. 2-3 is the hydroxyapatite (HAp) particles for which the pickling process has been performed. In particular, in chart showing the absorbance of calculating the spectrum obtained in the FT-IR measurement in Kubelka-Munk (KM) equation, to separate the peak wavenumber appears between ^{860cm -1 ~890cm} -1, it was evaluated. As a result, it was confirmed that in the hydroxyapatite particles (right figure in FIG. 2-3) subjected to the acid washing step, a peak near 877 cm ⁻¹ attributed to calcium carbonate was not observed. On the other hand, in the hydroxyapatite particle (left figure of FIG. 2-3) in which the acid-washing process was not performed, the peak around 877 cm < ^-1 > attributed to calcium carbonate was observed.

(About carbonated apatite)
FIGS. 2-4 to 2-5 are FT-IR spectra of hydroxyapatite (HAp) fired body particles according to Example 2-1 and Example 2-2. As can be seen from the figure, in Example 2-1 and Example 2-2, absorption was observed at 1350 cm ^{−1 to} 1500 cm ⁻¹ . Therefore, it was confirmed that Example 2-1 and Example 2-2 contain carbonated apatite.

<< Consideration based on results of X-ray diffraction test, thermogravimetric differential thermal analysis and FT-IR analysis >>
From the results of X-ray diffraction test, thermogravimetric differential thermal analysis and FT-IR analysis, Example 2-1 and Example 2-2 are substantially free of calcium carbonate and contain carbonate apatite. The
On the other hand, Comparative Examples 2-1 to 2-3 contained calcium carbonate according to the results of the thermogravimetric differential thermal analysis.
When producing hydroxyapatite particles, it is considered that calcium carbonate was removed by an acid washing step or the like.
Moreover, Example 2-1 and Example 2-2 are confirmed to contain B-type carbonated apatite.

The FT-IR analyzer and the measurement conditions are as follows. The particles of hydroxyapatite (HAp) fired particles are taken to be 10% by weight with respect to potassium bromide [KBr], and the measurement sample sufficiently ground in an agate mortar is diffusely reflected using FT-IR Spectrum 100 made by Perkin Elmer. the range of ^{450cm -1 ~4000cm} -1 at was measured at 8 cumulative. In addition, peak separation was performed using a software of fityk 0.9.4 under the condition of Function Type: Gaussian, Fitting Method: Levenberg-Marquardt.

  Moreover, the thermogravimetric differential thermal analysis (TG-DTA) measurement conditions are as follows. A thermogravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., EXSTAR 6000) under nitrogen stream at 10 ° C./min. The decarbonated amount was evaluated from the decreased weight in the temperature range of 700 ° C. to 950 ° C.

  In addition, when according to the manufacturing method of patent 5043436, the high molecular 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 is used as a fusion prevention agent. It is described that it is possible, and specific compounds include polyacrylic acid, polymethacrylic acid, polyglutamic acid, ethylenesulfonic acid, polymethacrylic acid alkylsulfonic acid ester, polyacryloylaminomethylphosphonic acid, and polypeptides. ing. Referring to Comparative Example 2-1, it can be confirmed that calcium carbonate is generated when such a compound is used. Based on this, the present inventors estimated that the cause of calcium carbonate formation is carbonic acid, which is a decomposition product of the polymer compound used as the anti-fusion agent.

  In the case of the method of Patent No. 5043436, a calcium carbonate film is formed on the surface of the fired hydroxyapatite particles even when the "component containing a carbonic acid source" is not used as the anti-fusion agent. The present inventors considered the reason as follows.

  According to the description of Japanese Patent No. 5043436, the firing is considered to be performed in the atmosphere. Here, when a component containing an alkali metal (sodium, potassium) or an alkaline earth metal (calcium) is used as the anti-fusion agent, the alkali metal oxide or the alkaline earth metal oxide is It is formed. Here, since alkali metal oxides and alkaline earth metal oxides are basic, they react with carbon dioxide in the atmosphere. Furthermore, when an alkali metal salt is used as the anti-fusion agent, calcium and alkali metal which constitute crystals of hydroxyapatite ion-exchange in the vicinity of the surface of the particles. As a result, on the surface of hydroxyapatite, calcium carbonate is generated by the reaction between a carbonate root derived from carbon dioxide in the air and calcium derived from the anti-fusion agent or calcium produced on the surface by ion exchange.

<< Appearance observation test >>
FIGS. 2-6 and 2-7 are SEM photographs of hydroxyapatite (HAp) fired body particles according to Example 2-1 (scale difference). From these photographs, it is understood that the hydroxyapatite (HAp) fired body particle group according to Example 2-1 is a hydroxyapatite fired body particle group including spherical hydroxyapatite (HAp) fired body particles. Moreover, FIGS. 2-8 and 2-9 are SEM photographs of hydroxyapatite fired body particle groups according to Example 2-2 (scale difference). From these photographs, it is understood that the hydroxyapatite (HAp) fired body particle group according to Example 2-1 is a hydroxyapatite (HAp) fired body particle group including rod-like hydroxyapatite fired body particles.

<< Composition Analysis >>
As a result of composition analysis of the ceramic particles according to Examples 2-1 and 2-2,
Ca _x (PO ₄ ) _6-y (CO ₃ ) _y (OH) ₂
It was found that it is in the range of what is represented by (wherein x is a number of 8 or more and 12 or less, y is a number of more than 0 and 3 or less).
The measurement method is as described above.

<< Dispersibility test >>
60 mg of sintered hydroxyapatite (HAp) (sintered hydroxyapatite (HAp) according to Example 2-1 and Comparative Example 2-1) and 3 ml of ethanol are mixed and irradiated with ultrasonic waves for 30 minutes to disperse the sintered hydroxyapatite The solution was prepared. In a water circulation flow cell of a laser scattering particle size distribution analyzer (SALD-7500 nano, manufactured by Shimadzu Corporation), 200 ml of water was added, and a sintered hydroxyapatite dispersion was dropped so that the laser scattered light intensity became a measurement region of the device. The circulation tank was irradiated with ultrasonic waves for 5 minutes, and the particle size distribution was measured. The dispersibility was evaluated by examining the abundance ratio (%) of 500 nm or less in terms of volume from the obtained particle size distribution. The higher the abundance ratio (%) of 500 nm or less, the higher the dispersibility. The results are shown in Table 2-1 below.

  From the above table, it is clear that Example 2-1 exhibits significantly higher dispersibility than Comparative Example 2-1.

  In addition, the variation coefficient of the particle diameter of Example 2-1 was 17%. In Example 2-2, the maximum diameter of the minor axis is 51 nm, the major axis is 170 nm, and the crystal is grown in the c-axis direction and the aspect ratio of the crystal (c-axis length / a-axis length) is 3.3. It was 33.

<< Cell adhesion test >>
<Manufacturing of test material>
(Washing process)
Alcohol treatment (ultrasonic irradiation in ethanol, 2-propanol or the like for 5 minutes) was performed on a circular polyethylene terephthalate (PET) sheet (diameter 9 mm, thickness 0.1 mm).

(Linker introduction process)
Corona discharge treatment (100 V, 15 seconds on one side) was applied to both sides of the cleaned PET sheet. The PET sheet after corona discharge treatment and 10 ml of acrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were placed in a 20 ml test tube, and the inside of the test tube was depressurized by a vacuum pump to perform a degassing operation. The test tube was melted under reduced pressure, and graft polymerization was performed for 60 minutes in a water bath at a temperature of 60 ° C. After the treatment, in order to remove the acrylic acid homopolymer adhering on the substrate surface, after stirring for 60 minutes at room temperature in water, the substrate surface was washed with water followed by ethanol.

(Sintered hydroxyapatite (HAp) fine particle immobilization treatment)
After the above treatment, 1% of sintered hydroxyapatite (HAp) fine particles (sintered hydroxyapatite (HAp) fine particles according to Example 2-1, Example 2-2, and Comparative Example 2-1) of 1% of the base material is dispersed In the liquid (dispersion medium: ethanol), it was allowed to stand at 20 ° C. for 30 minutes. Thereafter, the substrate was irradiated with ultrasonic waves for 5 minutes, the base material was taken out, and dried at 20 ° C. under normal pressure to obtain a test material.

<Cell adhesion / Cell morphology evaluation>
A cell adhesion / cell morphology evaluation test was performed using the prepared test material. Prior to the test, the test material is washed with ethanol, washed with phosphate buffered saline (PBS) and medium (MEM α (serum free)) (3 times), immersed in medium, and incubated until use (37 ° C., 5% CO ₂ ). And cell culture was implemented in the following procedures. First, culture medium (200 μL / hole) and a test material (sheet for test) were placed in a 48-well plate. Thereafter, a suspension of osteoblast-like cells (MC3T3-E1 (subclone 14)) was added (about 1 × 10 ⁵ cells / 200 μL / well) and cultured for 3 hours.

(Cell observation)
FIGS. 2-10 to 2-12 show samples produced using the hydroxyapatite (HAp) sintered particle group according to Example 2-1, Example 2-2, and Comparative Example 2-1, respectively. It is a SEM photograph of material. Based on the photograph, the state of cell aggregation, cell morphology, and cell density were determined. The results are shown in Table 2. In addition, regarding cell density, it shows that cell density is so high that there are many "+" numbers. In addition, the average coverage of each of the test materials is calculated from the SEM photograph.

As can be seen from FIGS. 2-10 to 2-12 and Table 2-2, when using the hydroxyapatite sintered body particles according to Example 2-1 and Example 2-2, excellent cell adhesion is exhibited. It was also confirmed that most of the cells exhibited greatly elongated cell morphology. On the other hand, when the hydroxyapatite sintered compact particle group which concerns on Comparative Example 2-1 was used, it has confirmed that cell adhesiveness was inferior.

Claims (9)

A collagen production promoter comprising ceramic particles as an active ingredient,
The particle diameter of the ceramic particles is in the range of 10 nm to 700 nm,
The ceramic particles are calcium phosphate sintered particles,
The collagen production promoter, wherein the ceramic particles do not contain calcium carbonate.   The collagen production promoter according to claim 1, wherein the ceramic particles are spherical. A collagen production promoter comprising ceramic particles as an active ingredient,
The particle diameter of the ceramic particles is 30 nm to 5 μm in the short axis and 75 nm to 10 μm in the long axis, and grows in the c axis direction, and the aspect ratio (c axis length / a axis length) of the crystal is 1 ~ 30,
The ceramic particles are calcium phosphate sintered particles,
The collagen production promoter, wherein the ceramic particles do not contain calcium carbonate.   The collagen production promoter according to any one of claims 1 to 3, wherein the ceramic particles are sintered hydroxyapatite particles.   The collagen production promoter according to any one of claims 1 to 4, wherein the ceramic particles do not contain an alkali metal element.   The collagen production promoter according to any one of claims 1 to 5, wherein the ceramic particles contain carbonate apatite on at least the surface thereof. The collagen production promoter according to any one of claims 1 to 6, wherein the ceramic particles satisfy the following property (A).
(A) After sufficient drying, after standing for 3 days or more under conditions of temperature 25 ° C. and humidity 50% under normal pressure, a thermogravimetric differential thermal analyzer (TG-DTA) (manufactured by Seiko Instruments Inc., In EXSTAR 6000), under nitrogen stream, 10 ° C./min. The weight decreased in the temperature range of 25 to 200 ° C. is 2% or less.   The collagen according to any one of claims 1 to 7, wherein the ceramic particles have a half width at d = 2.814 measured by X-ray diffractometry within a range of 0.2 to 0.8. Production promoter.   9. The method according to claim 1, comprising at least one substance selected from the group consisting of alcohols, saccharides, proteins, amino acids, water-soluble vitamins, fat-soluble vitamins, lipids, mucopolysaccharides, and surfactants. The collagen production promoter according to any one of the above.