JP2018002580A - Method for producing calcium phosphate sintered compact particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means of providing a calcium phosphate sintered compact particle group not causing an event of bubble generation in spite of using forms and also having a smaller particle dimeter.SOLUTION: Provided is a ceramic particle group made of almost spherical ceramic particles, in which the particle diameter of the ceramic particles lies in the range of 10 to 700 nm, also the variation coefficient of the particle diameter of the ceramic particle group is 20% or lower, the ceramic particles being calcium phosphate sintered compact particles, and also, the ceramic particle group substantially includes alkali metal elements and calcium carbonate. In the ceramic particle group, the ceramic particles preferably being hydroxyapatite sintered particles. Also provided is a medical material for introducing into the living body obtained by using the ceramic particle group. Also provided is medical equipment using the medical material for introducing into the living body as one material.SELECTED DRAWING: None

Description

  The present invention relates to a novel calcium phosphate sintered body particle, a production method thereof, and an application thereof.

  Hydroxyapatite (HAp) is known to exhibit high biocompatibility and is used in various fields including biomaterials.

  By the way, when using the HAp as a biomaterial, it has been proposed to produce hydroxyapatite particles (ceramic particles) with high crystallinity by firing at a high temperature in order to reduce in vivo solubility and degradability. Yes. However, during this sintering, bonding occurs due to fusion between hydroxyapatite (HAp) particles (primary particles), resulting in irregular secondary particles in which the primary particles are bonded, resulting in a decrease in dispersibility and specific surface area. There was a problem.

  Therefore, in prior patent document 1, mixed particles are mixed so that the anti-fusing agent is interposed between the primary particles made of the ceramic raw material before sintering, and the mixed particles are sintered, and after the firing A method for washing away the anti-fusing agent has been proposed. According to the production method, it is possible to obtain a group of ceramic particles having a high crystallinity and a small particle size.

Japanese Patent No. 5043436

  However, the present inventors have obtained knowledge that the ceramic particle group obtained by the production method contains a crystal phase of calcium carbonate. When calcium phosphate is used for an application, particularly a medical device introduced into a living body, an undesirable event may occur from the viewpoint of biocompatibility and change in solubility. Furthermore, calcium phosphate has a property of actively adsorbing endotoxin, and it is difficult to avoid adsorption of endotoxin contained in water, containers, and the like used for washing in the production process. On the other hand, endotoxin is known as a pyrogen, and when the calcium phosphate is used as a medical material for in vivo introduction (for example, DDS), it is necessary to remove the adsorbed endotoxin. Therefore, heat treatment is performed at about 300 ° C. to decompose the adsorbed endotoxin. However, at this time, the present inventors have found that the original particle size is doubled or more when the heat treatment is performed. In particular, such an increase in particle size is an undesirable phenomenon when the calcium phosphate fired body is used for applications where a finer particle size is required. Therefore, the present invention provides a calcium phosphate sintered body particle group having a smaller particle diameter, in which a decrease in biocompatibility and a change in solubility due to calcium carbonate are suppressed as much as possible regardless of the mode of use. It is an issue.

  Furthermore, the present inventors have found that the ceramic particle group obtained by the production method may not have sufficient cell adhesion when used for living organisms. Therefore, this invention makes it a 2nd subject to provide the calcium-phosphate sintered compact particle group which show | plays the outstanding cell adhesiveness, when it uses it for biological bodies.

  By the way, in Patent Document 1, a polymer compound having any of a carboxyl group, a sulfate group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, or an amino group in the side chain can be used as an anti-fusing agent. Specific examples of the compound include polyacrylic acid, polymethacrylic acid, polyglutamic acid, ethylenesulfonic acid, polymethacrylic acid alkylsulfonic acid ester, polyacryloylaminomethylphosphonic acid, and polypeptide. Based on this, the present inventors estimated that the cause of calcium carbonate generation was carbonic acid, which is a decomposition product of the polymer compound used as an anti-fusing agent. In addition, the present inventors have intensively studied, and when a specific component is included as an anti-fusing agent as described in Patent Document 1, cell adhesion in the obtained calcium phosphate sintered particles can be reduced. Estimated to be. Therefore, the present inventor examined a technique for obtaining a calcium phosphate sintered body particle group having a high crystallinity and a small particle diameter, and further exhibiting excellent cell adhesion without using an anti-fusing agent, The present invention has been completed.

That is, the present invention is as follows.
The present invention (1)
A group of ceramic particles composed of ceramic particles,
The particle diameter of the ceramic particles is within a range of 10 nm to 700 nm, and the coefficient of variation of the particle diameter of the ceramic particle group is 20% or less,
The ceramic particles are calcium phosphate sintered particles,
The ceramic particle group is substantially free of alkali metal elements and calcium carbonate.
The present invention (2)
The ceramic particle group of the invention (1), wherein the ceramic particles are substantially spherical.
The present invention (3)
A group of ceramic particles composed of ceramic particles,
The ceramic particles have a minor axis maximum diameter of 30 nm to 5 μm and a major axis of 75 nm to 10 μm, grow in the c-axis direction, and have an aspect ratio (c-axis length / a-axis length) of 1 ˜30, and the ceramic particles having a truncated columnar structure having a beveled front end angle,
The ceramic particles are a group of ceramic particles characterized by substantially not containing an alkali metal element and calcium carbonate.
The present invention (4)
The ceramic particle group according to any one of the inventions (1) to (3), wherein the ceramic particles are hydroxyapatite sintered particles.
The present invention (5)
The ceramic particles are washed with water,
Based on the particle diameter of the ceramic particle group after the water washing, the change rate of the particle diameter when heated at 300 ° C. under atmospheric pressure after the water washing is ± 20%. ) To (4).
The present invention (6)
A medical material for introduction into a living body, which is obtained using the ceramic particle group according to any one of the inventions (1) to (5).
The present invention (7)
A medical device obtained by using the medical material for introduction into a living body according to the invention (6) as one material.
The present invention (8)
In the method for producing a ceramic particle group consisting of ceramic particles,
A pre-process for obtaining a thawed body by thawing the frozen body after freezing the aqueous medium containing the primary particles that are the ceramic raw material before sintering and obtaining the frozen body;
A firing step of firing the primary particles obtained by removing the aqueous medium from the thawed body,
And crushing the fired body obtained by the firing step to obtain the ceramic particles,
The ceramic particles are calcium phosphate sintered particles,
The method for producing a ceramic particle group, wherein the ceramic particle group contains substantially no alkali metal element and calcium carbonate.
The present invention (9)
In the production method of the invention (8), the ceramic particles are substantially spherical.
The present invention (10)
It is the manufacturing method of the said invention (8) or (9) whose particle diameter of the said ceramic particle exists in the range of 10 nm-700 nm.
The present invention (11)
The ceramic particles are
The maximum diameter of the minor axis is 30 nm to 5 μm, the major axis is 75 nm to 10 μm, the particle diameter grows in the c-axis direction, and the crystal aspect ratio (c-axis length / a-axis length) is 1 to 30 The method according to the invention (8), wherein the tip angle is a truncated columnar structure having a beveled surface.
The present invention (12)
The method according to any one of the inventions (8) to (11), wherein the ceramic particles are hydroxyapatite sintered body particles.
The present invention (13)
The method according to any one of the inventions (8) to (12), wherein the ceramic particle group is a medical material for introduction into a living body.
The present invention (14)
It is a manufacturing method of the said invention (13) whose said ceramic particle group is an object for medical devices.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the ceramic particle group with a high crystallinity and a small particle diameter which the calcium carbonate crystal phase does not mix. Moreover, according to this invention, it becomes possible to provide the ceramic particle group which shows the outstanding cell adhesiveness. Furthermore, although a reason is not certain, the particle diameter of the calcined calcium phosphate according to the present invention hardly changes even when heated at 300 ° C. to remove endotoxin. Therefore, according to the present invention, it is possible to obtain a calcium phosphate sintered body particle group having a smaller particle diameter.

FIG. 1 is a chart showing the results of X-ray diffraction measurement of hydroxyapatite (HAp) sintered particles obtained in Example 1. FIG. 2 is a chart showing the results of X-ray diffraction measurement of hydroxyapatite (HAp) sintered body particles obtained in Example 2. FIG. 3 is a chart showing the results of X-ray diffraction measurement of the hydroxyapatite (HAp) sintered body particles obtained in the comparative example. 4 is a SEM photograph of the hydroxyapatite (HAp) sintered body particle group obtained in Example 1. FIG. FIG. 5 is an SEM photograph of the hydroxyapatite (HAp) sintered body particle group obtained in Example 1. 6 is an SEM photograph of the hydroxyapatite (HAp) sintered body particle group obtained in Example 2. FIG. FIG. 7 is a SEM photograph of the hydroxyapatite (HAp) sintered body particle group obtained in Example 2. FIG. 8 is an SEM photograph of a test material manufactured using the hydroxyapatite sintered body particle group according to Example 1. FIG. 9 is an SEM photograph of a test material manufactured using the hydroxyapatite sintered body particle group according to Example 2. FIG. 10 is an SEM photograph of a test material manufactured using a hydroxyapatite sintered body particle group according to a comparative example. FIG. 11 is a dyed image of a test material produced using the hydroxyapatite sintered body particle group according to Example 1. FIG. 12 is a dyed image of a test material produced using the hydroxyapatite sintered body particle group according to Example 2. FIG. 13 is an FTIR spectrum of hydroxyapatite sintered particles produced using a conventional manufacturing method. FIG. 14 is an FTIR spectrum of hydroxyapatite sintered particles produced using a conventional manufacturing method.

  Hereinafter, the manufacturing method of the calcium phosphate particle group according to the present invention will be described, then the ceramic particle group obtained by the manufacturing method will be described, and then the application of the ceramic particle group will be described.

<< 1. Manufacturing method of ceramic particles >>
<1-1. Raw material>
Specific examples of calcium phosphate (CaP) that 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, or a hydrothermal method. Naturally derived ones obtained from the above may be used.

<1-2. Process>
(1-1. Primary particle generation step)
Hereinafter, in the method for producing a calcium phosphate particle group according to the present invention, the case where the primary particles are substantially spherical and substantially rod-shaped is exemplified, but the present invention is not limited to this, and primary particles of any shape that can be produced can be applied. is there.

-Primary particle generation process of substantially spherical ceramic particles First, "primary particles" were formed of ceramic raw materials {calcium phosphate (CaP), hydroxyapatite (HAp), etc.} before sintering in the manufacturing process of the ceramic particles. Means particles. That is, it means a particle formed for the first time in the production process of ceramic particles. In a narrow sense, it means single crystal particles. In the claims and specification, “primary particles” include those in an amorphous state, a low crystalline state, and those in a sintered body state after sintering. Meaning. On the other hand, “secondary particles” are a combination of multiple “primary particles” by physical bonds such as fusion, chemical bonds such as Van der Waals force, electrostatic interaction, or covalent bonds. It means a particle in a state of being formed. In particular, the number of bonds between primary particles, the shape after bonding, and the like are not limited, and mean all bonded two or more primary particles. In particular, the “single crystal primary particle” means a primary particle made of a single crystal of a ceramic raw material or a particle lump in which the primary particles made of the single crystal are assembled by ionic interaction. The “particle aggregate assembled by ionic interaction” is a particle aggregate that self-assembles by ionic interaction when dispersed in a medium containing water or an organic solvent. It does not contain secondary particles that are melted and polycrystallized.

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

  The method for producing a ceramic particle group according to the present invention is a method for producing a ceramic particle group by sintering the primary particle group composed of the primary particles produced by the primary particle production step while preventing fusion or the like. . Therefore, the state (particle size, particle size distribution) of the primary particles generated by the primary particle generation step is directly reflected in the state (particle size, 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 a uniform particle size (narrow particle size distribution) must be generated.

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

  The “variation coefficient” is a value indicating the variation in particle diameter between particles that can be calculated by standard deviation ÷ average particle diameter × 100 (%).

  There is no particular limitation on the method of generating the primary particle group that is fine (nanometer size) and has a uniform particle size (narrow particle size distribution) as described above. For example, JP 2002-137910 A No. 5044336, Journal of Nanoscience and Nanotechnology Vol. 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 collecting the primary particles by centrifugation, filtration, or the like.

Primary rod generation process of substantially rod-shaped ceramic particles {Roughly rod-shaped ceramic particles {particles having a maximum minor axis diameter of 30 nm to 5 μm, a major axis of 75 nm to 10 μm, and growing in the c-axis direction to form crystals The primary particle production process of the aspect ratio (c-axis length / a-axis length) of 1 to 30 and the tip angle of the ceramic particles having a truncated columnar structure having an oblique surface is known per se, For example, the method described in Unexamined-Japanese-Patent No. 2002-137910, Journal of Nanoparticle Research (2007) 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 collecting the primary particles by centrifugation, filtration, or the like.

(Freezing process)
The freezing step is a step of freezing the aqueous medium containing calcium phosphate (CaP) before sintering. Here, the aqueous medium is a liquid medium containing water as a main component (preferably 90% by mass or more based on the total mass of the liquid medium). Preferably, only water is used, and a liquid miscible with water (alcohol or the like) may be appropriately added. In addition, a solution for producing calcium phosphate (CaP) as primary particles, that is, a solution obtained by dissolving a phosphate source and a 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. The freezing time is not particularly limited, but is preferably 1 minute to 24 hours.

(Thawing process)
The thawing step is a step in which the frozen body obtained in the freezing step is placed at a temperature exceeding the melting point of the aqueous medium of the frozen body to melt the aqueous medium.

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

(Baking process)
The said sintering process is a process which exposes the calcium-phosphate primary particle composition obtained by the said isolation | separation process to sintering temperature, and makes the primary particle contained in the said composition a ceramic particle (sintered body particle). Although the reason is not clear, even when the anti-fusing agent as in Patent Document 1 is not used, when the above freeze / thaw process is obtained and fired, it is exposed to high temperature conditions in the sintering process. Also, it is possible to prevent fusion between primary particles. 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 suitable. Moreover, 1-20 degrees C / min is suitable for the temperature increase rate in the said sintering process, for example. 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 is preferable. In addition, the apparatus etc. which are used for the said sintering process are not specifically limited, What is necessary is just to employ | adopt after selecting a commercially available baking furnace suitably according to a manufacturing scale, manufacturing conditions, etc.

(Crushing process)
The pulverization step is a step of pulverizing the aggregate after the sintering step to obtain a calcined calcium phosphate particle group having a desired size. Here, normally, the fired body that has been made into secondary particles can hardly be reduced to the size of the primary particles even if a considerable pulverization step is performed. On the other hand, according to the method of the present invention, it is possible to easily pulverize to the primary particle size level even with a simple pulverization process. Here, the pulverization method is not particularly limited, and examples thereof include ultrasonic treatment and pulverization using a pulverized sphere. After the pulverization treatment, particles having a smaller diameter may be collected by removing unpulverized ones.

(Washing process)
The washing step is a component other than the calcined calcium phosphate particles, for example, impurities derived from raw materials used in producing the primary particles of calcium phosphate (for example, calcium or phosphate-derived impurities not involved in calcium phosphate formation, nitric acid or ammonium-derived This is a step of cleaning the impurities. It is preferable to wash with water. In the case of hydroxyapatite (HAp) sintered body particles, the hydroxyapatite (HAp) sintered body particles are easily dissolved under the condition of pH less than 4.0, and therefore the removal step is performed at pH 4.0 to pH 12.0. Is preferred. Moreover, this order does not ask | require, such as performing a grinding | pulverization process and a washing | cleaning process alternately.

(Drying process)
A drying process is a process of removing a solvent by heating etc. after the said grinding | pulverization process or the said washing | cleaning process, and obtaining a calcium-phosphate particle group. The drying method is not particularly limited.

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

  An endotoxin removal process is a process performed by heating at 300 degreeC by the normal pressure under air, for example. The calcined calcium phosphate is preferably subjected to water washing in the washing step described above after firing. At this time, the calcined calcium phosphate adsorbs endotoxin that is slightly present in the environment such as in water, in a storage container or in a handling atmosphere. Here, this endotoxin is a harmful substance, and remaining in the calcined calcium phosphate is unsuitable particularly when the calcined calcium phosphate is used as a biomaterial. Therefore, this processing is performed. In addition, you may perform the said endotoxin removal process combining the drying process mentioned above.

  Here, according to this production method, unlike the prior art, fine calcium phosphate sintered particles can be produced without using an anti-fusing agent such as a polymer compound or a metal salt. More specifically, according to this production method, an anti-fusing agent 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 is provided. Compared with the conventional manufacturing method used for a manufacturing raw material, it becomes possible to obtain the calcium-phosphate sintered compact particle | grains which improved crystallinity more (thus, it is hard to melt | dissolve).

≪2. Calcium phosphate sintered particles obtained by this production method >>
<2-1. Spherical Calcium Phosphate Sintered Particle Group>
The substantially spherical calcium phosphate sintered body particle group obtained by the present production method is a ceramic particle group composed of substantially spherical ceramic particles, and the ceramic particle has a particle diameter in the range of 10 nm to 700 nm. The particle diameter variation coefficient of the group is 20% or less, the ceramic particle is a calcium phosphate sintered body particle, and the ceramic particle group does not substantially contain calcium carbonate. A group. The ceramic particle group is a fine particle and a uniform particle size (narrow particle size distribution). Therefore, there is an effect that it can be more uniformly adsorbed to the medical polymer material without performing an additional operation such as particularly high classification. Moreover, since calcium carbonate is not substantially contained, it is possible to prevent a situation in which the biocompatibility and solubility of the material change when used as a biomaterial.

  Further, from another aspect, the substantially spherical calcium phosphate sintered body particle group obtained by the present production method is a ceramic particle group composed of substantially spherical ceramic particles, and is composed of primary particles composed of a single crystal or the single crystal. When the particle aggregate in which the primary particles are aggregated by ionic interaction is a single crystal primary particle, the ratio of the single crystal primary particles contained in the ceramic particle group occupies a majority, and the ceramic particles are sintered with calcium phosphate. The ceramic particle group is a particle group, and the ceramic particle group substantially does not contain calcium carbonate. The ceramic particle group is a primary particle composed of a single crystal having a superior dispersibility in a solvent, or a particle lump in which the primary particles composed of the single crystal are assembled by ionic interaction (single crystal primary particles). Exist as. Therefore, there is an effect that the adsorption to the medical polymer base material described above is facilitated. Moreover, since there is no coupling | bonding of primary particles, a specific surface area is high. Furthermore, since it is highly stable in vivo and excellent in dispersibility, it has an effect that it can be used as a medical material capable of carrying and sustained release of 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 this way, a particle lump in which primary particles made of a single crystal are aggregated by ionic interaction may be used as ceramic particles.

  Further, in the ceramic particle group, the ratio of the single crystal primary particles contained in the ceramic particle group may be 70% or more. By adopting such a configuration, there is an effect that the adsorption to the medical polymer base material is facilitated.

  Moreover, the ceramic particle group may have a particle diameter of the ceramic particles in a range of 10 nm to 700 nm. According to the said structure, there exists an effect that it can adsorb | suck more uniformly with respect to medical polymeric material.

  The ceramic particle group may have a coefficient of variation of the particle diameter of the ceramic particle group of 20% or less. By adopting this configuration, there is an effect that it can be more uniformly adsorbed to the medical polymer material without performing an additional operation such as particularly high classification.

  The ceramic particles may be hydroxyapatite sintered particles. The particles are composed of a hydroxyapatite sintered body that has higher biocompatibility and can be used for a wide range of applications. Therefore, it is particularly preferable as a medical material.

  Further, the ceramic particle group was washed with water, and heated at 300 ° C. under atmospheric pressure in the air after the water washing, based on the particle diameter of the ceramic particle group after the water washing. The change rate of the particle diameter at the time may be ± 20%.

<2-2. Substantially rod-shaped calcium phosphate sintered body particle group>
The substantially rod-shaped calcium phosphate sintered body particle group obtained by this production method is a ceramic particle group composed of rod-shaped ceramic particles, and the ceramic particle has a short axis maximum diameter of 30 nm to 5 μm, long It has a truncated columnar structure having an axis of 75 nm to 10 μm, grown in the c-axis direction, a crystal aspect ratio (c-axis length / a-axis length) of 1 to 30, and a tip angle having an oblique surface. Ceramic particles, wherein the ceramic particles are calcium phosphate sintered particles, and the ceramic particles are substantially free of calcium carbonate. Since the rod-shaped calcium phosphate baked particle group has a much wider area for adhesion than conventional fine particles, it can improve the adhesion to the polymer substrate, so it can be applied to the polymer surface such as biocompatible medical materials such as catheters. Suitable for modification. And since it does not contain calcium carbonate substantially, when using as a biomaterial, it becomes possible to prevent the situation where carbon dioxide gas is generated from a material. As a method for modifying the polymer surface, an active group of calcium phosphate (for example, hydroxyapatite nanoparticles) and a polymer substrate, for example, a silicone rubber obtained by graft polymerization of a vinyl polymerizable monomer having a carboxyl group on the surface , A method of chemically reacting with an active group, a method of using a curable adhesive, a method of heating a polymer substrate to near the melting point and embedding it in the substrate, etc. The same applies to the substantially spherical calcium phosphate sintered body particle group).

  The ceramic particles may be hydroxyapatite sintered particles. The particles are composed of a hydroxyapatite sintered body that has higher biocompatibility and can be used for a wide range of applications. Therefore, it is particularly preferable as a medical material.

  Further, the ceramic particle group was washed with water, and heated at 300 ° C. under atmospheric pressure in the air after the water washing, based on the particle diameter of the ceramic particle group after the water washing. The change rate of the particle diameter at the time may be ± 20%.

<2-3. Properties>
Next, the properties of the calcium phosphate sintered particles obtained by the production method according to the present invention will be described.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to set it as the fine calcium-phosphate sintered compact particle group which does not contain an alkali metal element and calcium carbonate substantially.

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

  Further, by substantially not containing an alkali metal element, it becomes possible to obtain calcium phosphate sintered particles that are further improved in crystallinity, hardly dissolve, and are excellent in cell adhesion.

  Here, “substantially does not contain calcium carbonate” means that calcium carbonate is below the detection limit from the measurement result of X-ray diffraction {specifically, calcium carbonate (formula amount: 100.09) / hydroxyapatite (formula Amount: 1004.62) = 0.1 / 99.9 (formula conversion ratio) or less}, and the amount of bubbles generated was 0 when tested according to Quasi-drug raw material standard 2006 (hydroxyapatite). .Less than 25 mL.

  Further, “substantially free of alkali metal elements” means that the weight of alkali metal elements with respect to the total weight of the calcium phosphate sintered particles is 10 ppm or less (preferably 1 ppm or less). Indicates. In addition, a conventionally well-known method can be applied as an analysis method, for example, it may analyze by ICP-MS.

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

  Thus, by containing substantially no alkaline earth metal elements other than calcium and transition metal elements, the crystallinity is further enhanced, the dissolution is difficult, and the calcium phosphate sintered body particles are excellent in cell adhesion. It becomes possible to do. Moreover, it becomes possible to improve the safety | security to a biological body substantially by not containing a transition metal element.

  Note that “substantially free of alkaline earth metal elements” (“substantially free of transition metal elements”) means that calcium alkaline phosphate is used for each alkaline earth metal element (each transition metal element) as described above. It indicates that the weight of the alkaline earth metal element (transition metal element) is 10 ppm or less (preferably 1 ppm or less) with respect to the weight of the entire sintered body particle. In addition, a conventionally well-known method can be applied as an analysis method, for example, it may analyze by ICP-MS.

≪Usage≫
Since the calcium phosphate calcined particles according to the present invention have extremely high biocompatibility and bioactivity, in the medical field, for example, dental materials such as bone fillers, dental fillers, sustained drug release agents, and external preparations. Alternatively, it can be widely used as a medical material, and can be applied to all other uses such as food additives. In addition, since the calcium phosphate calcined particle group according to the present invention according to the present invention is excellent in cell adhesiveness, a medical material for introduction into a living body (for example, an in-vivo indwelling material) and a medical device using the material as one material, etc. It can also be suitably used. In addition, as a more specific use of the calcium phosphate calcined particles according to the present invention, for example, cosmetics (breast augmentation promoter, basic cosmetics, cosmetics, dentifrices, etc.), subcutaneous injections (dermal fillers, etc.), medical Devices {wound dressings (especially hernia mesh), etc.}, surgical implantable medical devices {artificial bones, artificial joints (orthopedic implants), dental implants, etc.}, cardiovascular medical devices {catheter (to cuff) Support / inner surface treatment), artificial blood vessel, stent, coil for cerebral embolism} and the like.

≪Production example≫
(Example 1: Spherical hydroxyapatite fired body particle group)
In a reaction vessel containing deionized water, calcium nitrate tetrahydrate, diammonium hydrogen phosphate aqueous solution and aqueous ammonia were added with stirring {calcium: phosphoric acid (molar ratio) = 5: 3}, and hydroxy Apatite primary particles were obtained. Thereafter, the operation of transferring the supernatant in the reaction vessel to a waste water vessel, adding deionized water, stirring with a stirrer, and transferring the supernatant to a waste vessel was repeated twice. Thereafter, the reaction vessel containing the precipitate was frozen overnight at −10 ° C. to −25 ° C. Thereafter, the mixture was thawed at room temperature and the thawed precipitate was collected by filtration. Thereafter, about 400 g of precipitate was put in a baking dish, put in a baking furnace, heated to 600 ° C. over 1 hour, kept at 600 ° C. for 1 hour, and then cooled for 1 hour or more to carry out baking. Then, deionized water was added to the fired body and irradiated with ultrasonic waves for 30 minutes or more. And it moved to the pod mill, put the pulverization ball | bowl, and grind | pulverized for 1 hour. After the pulverization was completed, the powder was transferred to a hand-held beaker, and an unground baked product was removed using a 150 μm sieve. After that, deionized water washing was repeated 6 times. Then, it dried at 60-80 degreeC, and obtained the hydroxyapatite baking body which concerns on Example 1 which does not contain an alkali metal element substantially.

(Example 2: Rod-like hydroxyapatite fired particles)
In a reaction vessel containing deionized water, while stirring the calcium nitrate tetrahydrate aqueous solution, diammonium hydrogen phosphate aqueous solution and aqueous ammonia were dropped into the calcium nitrate tetrahydrate aqueous solution {calcium: phosphoric acid (mol). Ratio) = 5: 3} to obtain primary particles of hydroxyapatite. Thereafter, the operation of transferring the supernatant in the reaction vessel to a waste water vessel, adding deionized water, stirring with a stirrer, and transferring the supernatant to a waste vessel was repeated 5 times. Thereafter, the reaction vessel containing the precipitate was frozen overnight at −10 ° C. to −25 ° C. Thereafter, the mixture was thawed at room temperature and the thawed precipitate was collected by filtration. Thereafter, about 400 g of precipitate was put in a baking dish, put in a baking furnace, heated to 600 ° C. over 1 hour, kept at 600 ° C. for 1 hour, and then cooled for 1 hour or more to carry out baking. Then, deionized water was added to the fired body and irradiated with ultrasonic waves for 30 minutes or more. And it moved to the pod mill, put the pulverization ball | bowl, and grind | pulverized for 1 hour. After the pulverization was completed, the powder was transferred to a hand-held beaker, and an unground baked product was removed using a 150 μm sieve. Thereafter, deionized water washing was repeated 7 times. Then, it dried at 60-80 degreeC, and obtained the hydroxyapatite baking body which concerns on Example 2 which does not contain an alkali metal element substantially. The ceramic particles have a minor axis average maximum diameter of 47 nm and a major axis of 157 nm, grow in the c-axis direction, and the crystal aspect ratio (c-axis length / a-axis length) is 3. The ceramic particles had a truncated columnar structure with a tip angle of 1 and a beveled surface.

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

≪X-ray diffraction test≫
1, FIG. 2 and FIG. 3 show the results of X-ray diffraction measurement of the fired hydroxyapatite bodies according to Example 1, Example 2 and Comparative Example, respectively. As can be seen from the figure, no peak of calcium carbonate was observed in FIG. 1, whereas a clear peak of calcium carbonate was observed in FIG. Moreover, also about Example 2, it can confirm that the peak of a calcium carbonate is not observed similarly to Example 1. FIG. More specifically, in FIG. 1, only a pattern corresponding to hydroxyapatite (PDF 74-0565) can be confirmed, while in FIG. 3, a peak that does not exist in hydroxyapatite is observed at 29.4 ° and calcium carbonate ( calcite: PDF 72-1937). The X-ray diffractometer and measurement conditions are as follows. Crystal structure analysis was performed using a powder X-ray diffraction device {MiniFlex} manufactured by Rigaku Corporation. The X-ray source used in XRD was a CuKα source {λ = 1.541841 Å (angstrom)}, output was 30 kV / 15 mA, scan speed was 1.0 ° / min, sampling width was 0.01 °, measurement The mode was a continuous condition.

≪Appearance observation test≫
4 and 5 are SEM photographs of the hydroxyapatite fired particles according to Example 1 (difference in scale). From these photographs, the hydroxyapatite fired body particle group according to Example 1 is a hydroxyapatite fired body particle group composed of substantially spherical hydroxyapatite fired body particles, and is composed of primary particles composed of a single crystal or the single crystal. When the particle lump in which the primary particles are aggregated by ionic interaction is defined as the single crystal primary particle, it can be seen that the majority of the single crystal primary particles contained in the hydroxyapatite fired body particle group occupy the majority. 6 and 7 are SEM photographs of the hydroxyapatite fired particles according to Example 2 (difference in scale).

≪Particle size measurement test≫
Regarding hydroxyapatite fired bodies (endotoxin non-inactivated) according to Example 1 and Comparative Example, average particle diameter and standard deviation were calculated (9 images were acquired by SEM, and 12 particles in 1 image) The total particle size of 108 particles was confirmed, and an average value and a standard deviation were calculated). In addition, regarding the inactivated hydroxyapatite fired bodies (endotoxin non-inactivated) according to Example 1 and Comparative Example, the average particle diameter and standard deviation were similarly calculated (9 images were obtained by SEM). The particle size of 12 particles in one point of the image was measured, the particle size of a total of 108 particles was confirmed, and the average value and standard deviation were calculated). The inactivation procedure is as follows: (1) dry heat sterilization (300 ° C., 2 hours), measure HAp powder into ampoule; (2) leave the ampoule with HAp open in a dry heat sterilizer. Dry heat sterilization (300 ° C., 2 hours), (3) sealing the ampule cooled to room temperature (sealed tube), and (4) sterilizing the sealed ampule again with a dry heat sterilizer (300 ° C, 2 hours). Table 1 shows the hydroxyapatite fired body according to Example 1, and Table 2 shows the hydroxyapatite fired body according to the comparative example.

≪Foaming confirmation test≫
The hydroxyapatite fired bodies according to Examples 1 and 2 and Comparative Example were tested according to the procedure shown in Purity Test (4) Carbonate Listed in Quasi-drug Raw Material Standard 2006 “Hydroxyapatite”. Specifically, 1.0 g of a sample was weighed at room temperature (20 ° C.), 5 mL of water was added and shaken, and the mixture was deaerated by reducing pressure for 1 hour using an aspirator. After deaeration, 2 mL of concentrated hydrochloric acid (35.0% by mass) was added to check for foaming. As a result, foaming could not be confirmed in the HAp obtained in the example (gas generation amount was less than 0.25 ml). On the other hand, in the HAp obtained by the comparative example, the resulting foam was so foamed that the supernatant became cloudy.

≪Cell adhesion test≫
<Manufacture of test materials>
(Cleaning process)
A circular polyethylene terephthalate (PET) sheet (diameter 9 mm, thickness 0.1 mm) was subjected to alcohol treatment (irradiation with ultrasonic waves in alcohol (ethanol, 2-propanol, etc.) for 5 minutes).

(Linker introduction process)
Corona discharge treatment (100 V, 15 seconds on one side) was applied to both surfaces of the PET sheet that had been subjected to the cleaning treatment. A 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 with a vacuum pump to perform a deaeration operation. The test tube was sealed under reduced pressure, and graft polymerization was performed in a water bath at a temperature of 60 ° C. for 60 minutes. After the treatment, in order to remove the acrylic acid homopolymer adhering to the substrate surface, the substrate was stirred in water at room temperature for 60 minutes, and then the substrate surface was washed with water and subsequently with ethanol.

(Sintered hydroxyapatite fine particle immobilization treatment)
After the above treatment, the base material was dispersed in 1% sintered hydroxyapatite fine particles (sintered hydroxyapatite fine particles according to Example 1, Example 2 or Comparative Example) (dispersion medium: ethanol) at 20 ° C. for 30 minutes. Left to stand. Then, ultrasonic irradiation was performed for 5 minutes, the base material was taken out and dried at 20 ° C. and normal pressure to obtain a test material.

<Evaluation of cell adhesion and cell morphology>
Cell adhesion and cell morphology evaluation tests were performed using the prepared test materials. Prior to the test, the test material was washed with ethanol, washed with phosphate buffered saline (PBS) and medium (MEM α (serum-free)) (3 times), then immersed in the medium and incubated until use. (37 ° C., 5% CO ₂ ). And cell culture was implemented in the following procedures. First, a culture medium (200 μL / hole) and a test material (test sheet) 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)
8 to 10 are SEM photographs of specimens manufactured using the hydroxyapatite sintered particles according to Example 1, Example 2, and Comparative Example, respectively. Based on the photograph, the state of cell aggregation, cell morphology, and cell density were determined. The results are shown in Table 3. As for the cell density, the greater the number of “+”, the higher the cell density. Moreover, the average coverage of each test material is calculated from SEM photographs.

(Dyeing observation)
Subsequently, staining observation was performed according to the following procedure. First, the test sheet was washed with PBS to remove non-adherent cells, fixed (4% PFA) and surfactant (0.5% Triton X-100 / PBS), and then AlexaFluor ( After adding 488-labeled Acti-stain, it was encapsulated with an encapsulating agent (with nuclear dye DAPI). With a fluorescence microscope (Nikon, ECLIPSE Ti-S), a laser beam of 488 nm was irradiated, and the cell morphology was confirmed by staining of actin fibers. A laser beam of 405 nm was irradiated to confirm staining of cell nuclei. The results are shown in FIGS. FIGS. 11 and 12 are images obtained by synthesizing actin-derived fluorescence images and cell-nucleus-derived fluorescence images in the test materials produced using the hydroxyapatite sintered particles according to Example 1 and Example 2. FIG. It is. As can be seen from the figure, when using the hydroxyapatite sintered particles according to Example 1 and Example 2, it can be confirmed that the actin fibers are sufficiently developed to exhibit excellent cell adhesion, It was also confirmed that most cells showed a greatly elongated cell morphology.

  From the above results, it was confirmed that the calcium phosphate fine particles according to the present invention had a polygonal cell shape and significantly promoted cell-cell adhesion. From this, it became clear that the calcium phosphate fine particles obtained by the present invention promote morphological differentiation of cells.

≪Crystallinity≫
Crystal structure analysis was performed using a powder X-ray diffraction apparatus {MiniFlex 600, manufactured by Rigaku Corporation). The X-ray source used in XRD is a CuKα source {λ = 1.541841 Å (angstrom)}, 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 crystallite size τ (nm) is given by the following Scherrer equation.
(Scherrer's formula) τ = Kλ / βcosθ
K is a shape factor of 0.9. 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 crystallite size τ (nm) 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 fine particles according to Example 1 was 11.6 nm, and the average size τ of the crystallites of the calcium phosphate fine particles according to the comparative example was 12.1 nm. Therefore, it is understood that the calcium phosphate fine particles obtained by the present invention have higher crystallinity.

(reference)
In the above-described examples, as a comparative example, a hydroxyapatite fired body obtained according to Example 1 of Japanese Patent No. 5043436, that is, obtained using calcium carbonate as an anti-fusing agent was used.

  Here, in the manufacturing method of Japanese Patent No. 5043436, a case where “a component containing a carbonic acid source” is not used as an anti-fusing agent will be examined.

  FIG. 13 and FIG. 14 are FTIR spectra (charts) in calcined hydroxyapatite particles when calcium nitrate and sodium nitrate are used as anti-fusing agents in the production method of Japanese Patent No. 5044336, respectively. As can be seen from the figure, a peak of calcium carbonate could be confirmed.

  Thus, when following the production method of Japanese Patent No. 5044336, a calcium carbonate film is formed on the surface of the calcined hydroxyapatite particles even when the “component containing a carbonic acid source” is not used as an anti-fusing 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 an air atmosphere. Here, when a component containing an alkali metal (sodium, potassium) or an alkaline earth metal (calcium) is used as an anti-fusing agent, an alkali metal oxide or an alkaline earth metal oxide is used in the firing process. It is formed. Here, since alkali metal oxides and alkaline earth metal oxides are basic, they neutralize with carbon dioxide in the atmosphere. Furthermore, when an alkali metal salt is used as an anti-fusing agent, calcium and alkali metal constituting the hydroxyapatite crystals are ion-exchanged near the surface of the particles. As a result, calcium carbonate is generated on the surface of hydroxyapatite by a reaction between carbonic acid radicals derived from carbon dioxide in the atmosphere and calcium derived from the anti-fusing agent or calcium generated on the surface by ion exchange.

Claims (14)

A group of ceramic particles composed of ceramic particles,
The particle diameter of the ceramic particles is within a range of 10 nm to 700 nm, and the coefficient of variation of the particle diameter of the ceramic particle group is 20% or less,
The ceramic particles are calcium phosphate sintered particles,
The ceramic particle group, wherein the ceramic particle group contains substantially no alkali metal element and calcium carbonate.   The ceramic particle group according to claim 1, wherein the ceramic particles are substantially spherical. A group of ceramic particles composed of ceramic particles,
The ceramic particles have a minor axis maximum diameter of 30 nm to 5 μm and a major axis of 75 nm to 10 μm, grow in the c-axis direction, and have an aspect ratio (c-axis length / a-axis length) of 1 ˜30, and the ceramic particles having a truncated columnar structure having a beveled front end angle,
The ceramic particles are substantially free of alkali metal elements and calcium carbonate.   The ceramic particle group according to any one of claims 1 to 3, wherein the ceramic particles are hydroxyapatite sintered body particles. The ceramic particles are washed with water,
The change rate of the particle diameter when heated at 300 ° C. under atmospheric pressure after the water cleaning is ± 20%, based on the particle diameter of the ceramic particle group after the water cleaning. The ceramic particle group according to any one of 4.   A medical material for introduction into a living body, wherein the medical material is obtained using the ceramic particle group according to any one of claims 1 to 5.   A medical device obtained using the medical material for introduction into a living body according to claim 6 as one material. In the method for producing a ceramic particle group consisting of ceramic particles,
A pre-process for obtaining a thawed body by thawing the frozen body after freezing the aqueous medium containing the primary particles that are the ceramic raw material before sintering and obtaining the frozen body;
A firing step of firing the primary particles obtained by removing the aqueous medium from the thawed body,
And crushing the fired body obtained by the firing step to obtain the ceramic particles,
The ceramic particles are calcium phosphate sintered particles,
The method for producing a ceramic particle group, wherein the ceramic particle group contains substantially no alkali metal element and calcium carbonate.   The manufacturing method according to claim 8, wherein the ceramic particles are substantially spherical.   The manufacturing method according to claim 8 or 9, wherein a particle diameter of the ceramic particles is in a range of 10 nm to 700 nm. The ceramic particles are
The maximum diameter of the minor axis is 30 nm to 5 μm, the major axis is 75 nm to 10 μm, the particle diameter grows in the c-axis direction, and the crystal aspect ratio (c-axis length / a-axis length) is 1 to 30 The manufacturing method according to claim 8, wherein the tip angle is a truncated columnar structure having a beveled surface.   The manufacturing method according to claim 8, wherein the ceramic particles are hydroxyapatite sintered particles.   The manufacturing method according to any one of claims 8 to 12, wherein the ceramic particle group is a medical material for introduction into a living body.   The manufacturing method according to claim 13, wherein the ceramic particle group is for a medical device.