JP2009213723A - Apatite coated biological implant having biomolecule fixed on its surface, and cell culture carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological implant, such as an artificial joint and an artificial dental root, having basic protein, such as bone morphogenetic protein, fixed on the surface of hydroxyapatite, and to provide a cell culture carrier. <P>SOLUTION: A biomaterial consists of ceramic primarily composed of hydroxyapatite, wherein a (001) face (c face) of a crystal of the hydroxyapatite HA in the ceramic has a face preferentially exposed to the surface, an orientation by a thin film orientation analysis of XRD of HA (004) surface shows a high crystalline orientation of 13.5° or more and the basic protein is selectively fixed to a part at least near the surface of the applicable face. The biomaterial provides the hydroxyapatite membrane which has a high orientation observed in a dental enamel of a person, and scarcely includes calcium oxide which is supposed to cause inflammation especially in a living body, at least near the surface of the membrane, the biological implant having the membrane, and the cell culture carrier. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a biomaterial in which a basic protein is immobilized on the surface of a hydroxyapatite film. More specifically, the (001) plane (c-plane) of a hydroxyapatite (HA) crystal is preferential to the surface. The present invention relates to a biomaterial having a surface exposed to the surface, a crystal orientation with a high degree of orientation of 13.5 ° or more, and a basic protein selectively immobilized at least near the surface of the surface. The present invention provides biological implant materials and cell culture carriers such as artificial joints and artificial roots.

  Bone morphogenetic protein (BMP) promotes the differentiation of undifferentiated mesenchymal cells into osteoblasts and chondroblasts and suppresses differentiation into cells of other strains, and so on around the carrier on which BMP is fixed The bone tissue is induced. In order to effectively express the medicinal effects of a specific substance in the living body of BMP, a carrier that holds the substance locally and releases it slowly at an appropriate rate is essential.

  In general, it is known that proteins are not strongly fixed on the surface of hydroxyapatite (HA) coated on the surface of an artificial joint or an artificial tooth root. As a method of fixing a basic protein such as BMP on the surface of an artificial joint, a method of depositing a calcium phosphate film containing protein from a precursor solution of calcium phosphate containing protein has been proposed (see Patent Documents 1 and 2). .

  However, in these methods, it takes about 1 to 7 days to form a film, and since the amount of protein to be immobilized is small relative to the amount of protein used, a large amount of expensive protein such as BMP is used. There is a problem of having to. Moreover, in these methods, there exists a problem that the adhesiveness of the protein containing calcium phosphate membrane | film | coat formed is low. Thus, conventionally, a simple immobilization method that simply adsorbs a basic protein such as BMP to the surface of the HA material and an HA material in which the protein is immobilized on the surface have not been known so far.

  In the hydroxyapatite (HA) crystal, (100) or (010) plane (a plane) and (001 plane) (c plane) mainly appear. In these aspects, it is known that there is a difference in adsorption of molecules such as proteins (Non-Patent Document 1), and there is a difference in solubility in body fluids such as saliva (Non-Patent Document 2). The living body makes good use of the difference in the crystal surface properties of hydroxyapatite (HA). For example, human tooth enamel has a (001) -oriented structure of HA crystals, In contrast, an inactive c-plane appears preferentially on the surface.

  If an HA material with arbitrarily controlled crystal orientation can be realized, it can be expected that the material can be used as a new biomaterial that can bring out the anisotropy of the physical properties of the HA crystal and the properties of the crystal plane. Conventionally, it has been reported that a c-plane oriented HA film is produced using a plasma spraying method (Non-Patent Documents 3 and 4). And although it was known that basic protein mainly adsorb | sucks to the c surface of HA, in HO-HAC which has high crystal orientation, basic protein is selective with an orientation degree of 13.5 degreeC or more. It was not known to be fixed to.

Japanese Patent Laid-Open No. 2005-21208 Japanese Patent Laid-Open No. 2005-112848 T.A. Kawasaki, J. et al. chromatogr. 554, 147 (1991) Hideki Aoki, Surface Science, 10.96-104 (1989) M.M. Inagaki et al. , Journal of Materials Science; Materials in Medicine. 14.919-22 (2003) M.M. Inagaki et al. , BIOMATERIALS, 28, 2923-31 (2007)

  Under such circumstances, in view of the above-mentioned conventional technology, the present invention provides a simple fixing method in which a basic protein such as BMP is adsorbed on the HA surface, and an HA material having the protein fixed on the surface. As a result of intensive research aimed at developing biomaterials as the main component, (001) plane (c-plane) of hydroxyapatite (HA) crystals having a high crystal orientation with an orientation degree of 13.5 ° or more ) Has been found to be able to achieve the intended purpose by using HA having a surface preferentially exposed on the surface, and further research has been made to complete the present invention. And in this invention, when orientation degree was 13.5 degrees or more, More preferably, it was confirmed that selective carrying | support of a basic protein arises when it is 18.0 degrees or more.

  The present invention solves the above problems, and provides biological implants such as artificial joints and artificial tooth roots and cell culture carriers, in which basic proteins such as bone morphogenetic proteins are fixed on the surface of hydroxyapatite. It is for the purpose. In addition, the present invention is to provide a hydroxyapatite having a basic protein immobilized on its surface, a biological implant having a film of the hydroxyapatite, and a cell culture carrier in a short time using a smaller amount of protein, and providing It is the purpose.

The present invention for solving the above-described problems comprises the following technical means.
(1) A biomaterial composed of ceramics mainly composed of hydroxyapatite, wherein the (001) plane (c-plane) of hydroxyapatite (HA) crystals in the ceramic is preferentially exposed on the surface. And has a high crystal orientation of at least 13.5 ° by XRD thin film orientation analysis of the HA (004) plane, and a basic protein is selectively fixed at least near the surface of the plane. A biomaterial characterized by that.
(2) The biomaterial has an apatite film mainly composed of hydroxyapatite formed on a base material made of ceramics or metal, and the film is composed of particles deposited in a lamellar shape. (1) The (001) plane (c plane) has a plane preferentially exposed on the surface by preferentially orienting the c-axis direction of the hydroxyapatite crystal in the direction perpendicular to the substrate. The biomaterial described in 1.
(3) The biomaterial according to (2), comprising calcium triphosphate and calcium tetraphosphate in which the a-axis direction of the crystal is preferentially oriented in a direction perpendicular to the substrate.
(4) A cell culture carrier comprising the biomaterial according to any one of (1) to (3) as a constituent element.
(5) The cell culture carrier according to (4), which has an apatite film having a thickness of 5 to 1000 μm.
(6) A living body implant material comprising the living body material according to any one of (1) to (3) as a constituent element.
(7) Having an apatite film on a substrate made of titanium or a titanium alloy, a layer containing a mixture of titanium or a titanium alloy and a nitride thereof is formed in the film, and the adhesion of the film to the substrate The living body implant material according to (6), wherein
(8) The living body implant material according to (6) or (7), which has an apatite film having a thickness of 5 to 1000 μm.
(9) The biological implant material according to any one of (6) to (7), wherein unevenness is formed in a limited range of the surface of the biological implant material.

Next, the present invention will be described in more detail.
The present invention is a biomaterial made of ceramics mainly composed of hydroxyapatite, and the (001) plane (c-plane) of hydroxyapatite (HA) crystals in the ceramic is preferentially exposed on the surface. It has a crystal orientation with a high degree of orientation of at least 13.5 ° by XRD thin film orientation analysis of the HA (004) plane, and a basic protein is selectively fixed at least near the surface of the surface. It is characterized by that.

  In the present invention, the biomaterial has an apatite film mainly composed of hydroxyapatite formed on a base material made of ceramics or metal, and the film is composed of particles deposited in a lamellar shape. The c-axis direction of the hydroxyapatite crystal is preferentially oriented in the direction perpendicular to the base material, so that the (001) plane (c-plane) has a plane preferentially exposed on the surface, calcium triphosphate In addition, it is preferable to include calcium tetraphosphate in which the a-axis direction of the crystal is preferentially oriented in a direction perpendicular to the base material.

  In addition, the present invention is characterized by the point of a cell culture carrier having the biomaterial as a constituent element and the point of a bioimplant material having the biomaterial as a constituent element. Further, in the present invention, the base material of the film has an apatite film on a base material made of titanium or a titanium alloy, and a layer containing a mixture of titanium or a titanium alloy and a nitride thereof is formed in the film. It is a preferred embodiment that the adhesion to the surface of the living body is enhanced and that the irregularities are formed in a limited range of the surface of the biological implant material.

  The present invention has the greatest feature in that the c-axis direction of a hydroxyapatite crystal is preferentially oriented in the direction perpendicular to the surface, and a basic protein is fixed at least near the surface of the hydroxyapatite. A hydroxyapatite, a biological implant material having the hydroxyapatite film, and a cell culture carrier. In the present invention, it is possible to appropriately use the hydroxyapatite on which the basic protein is immobilized by coating it on a substrate made of ceramic, polymer or metal.

  In the present invention, a hydroxyapatite or a coating thereof, in which the c-axis direction of hydroxyapatite crystals is preferentially oriented in the direction perpendicular to the surface, and a basic protein is fixed at least near the surface of the hydroxyapatite film An article is coated on a substrate made of ceramic, polymer or metal to produce a biological implant. The base material made of ceramics, polymer or metal in the present invention is not particularly limited as long as it has the necessary properties for the purpose of use, and the composition, shape and form of use of the ceramics or metal.

  The biological implant material referred to in the present invention means that the c-axis direction of a hydroxyapatite crystal is preferentially oriented in the direction perpendicular to the surface at least near the surface, and is basic at least near the surface of the hydroxyapatite film. It means a molded body for use in a living body in which a coating composition immobilizing proteins is formed. If a biological implant material has the characteristic and safety | security required in order to use it in_vivo | within_body, a shape, a usage form, etc. will not be specifically limited.

  For example, the biological implant material of the present invention can have any shape such as a columnar shape, a plate shape, a sheet shape, a block shape, a wire shape, a fiber shape, and a powder shape. Moreover, as a usage form, product forms such as an artificial hip joint stem, an artificial knee joint, an artificial vertebral body, an artificial intervertebral disc, a bone filling material, a bone plate, a bone screw, and an artificial tooth root can be appropriately employed.

  The cell culture carrier referred to in the present invention means that the c-axis direction of hydroxyapatite crystals is preferentially oriented in the direction perpendicular to the surface in the vicinity of the surface, and the basic protein is at least in the vicinity of the surface of the hydroxyapatite film. It means a molded body for culturing the cells on which the coating composition to which is fixed is formed. As long as it has the characteristics necessary for using the cells for culturing, the shape and form of use are not particularly limited.

  As the cell culture carrier of the present invention, for example, any shape such as a plate shape, a sheet shape, a block shape, a column shape, a wire shape, a fiber shape, and a powder shape can be used. Moreover, as a use form, it is possible to employ | adopt suitably product forms, such as a petri dish for cell cultures, a sheet | seat for cell cultures.

  As a method for producing the biomaterial of the present invention, specifically, for example, a hydroxyapatite powder having an average particle size of 80 μm is introduced into a thermal plasma and deposited on a substrate directly under the plasma to form a coating composition. A method of immersing the heat-treated one after the formation in a basic protein solution is exemplified. However, the present invention is not limited to these methods, and the composition of the plasma gas, the type of powder and the particle size, or the type of substrate may be appropriately selected, designed and changed according to the target product. It is possible to carry out by an appropriate method.

  The c-axis direction of hydroxyapatite crystals referred to in the present invention is preferentially oriented in the direction perpendicular to the surface. The average of the surface irregularities formed by the hydroxyapatite molded product is neglected to be 1000 μm or less. This means that the c-axis direction of the hydroxyapatite crystal is oriented in the normal direction of the surface. In the present invention, the degree of orientation refers to the diffraction intensity plotted against the angle ψ of the normal surface of the sample surface to the normal surface of the lattice plane of the HA (004) plane under the Bragg condition, and the integration when ψ = 0. It means the angle of ψ at which the integrated intensity is 50% of the intensity.

  Conventionally, as a method for fixing a basic protein such as BMP on the surface of an artificial joint, for example, a method of depositing a calcium phosphate film containing a protein from a precursor solution of calcium phosphate containing a protein has been proposed. In this method, it takes about 1 to 7 days to form a film, and since the amount of protein to be immobilized is small relative to the amount of protein to be used, a large amount of expensive protein such as BMP must be used. In addition, there are problems such as that the adhesion of the formed protein-containing calcium film is low.

  In contrast, in the present invention, the (001) plane (c-plane) of the hydroxyapatite (HA) crystal has a plane that is preferentially exposed on the surface, and the degree of orientation is 13.5 ° or more, preferably Use a large amount of protein in a short time by using an HA surface with a crystal orientation of 18.0 ° or more and selectively fixing a basic protein at least near the surface of the surface. In addition, it is possible to provide a biomaterial having an apatite film-protein complex having high adhesion to a substrate.

The present invention has the following effects.
(1) A biomaterial in which a basic protein such as BMP is selectively adsorbed and immobilized on the surface of the HA material can be provided.
(2) A simple immobilization technique for selectively immobilizing a basic protein on the surface of the HA material can be provided.
(3) It is possible to provide a cell culture carrier and a biological implant material in which a specific basic protein is selectively immobilized near the surface.
(4) A biological implant material having an apatite film on a substrate made of titanium or a titanium alloy and having improved adhesion of the film to the substrate can be provided.
(5) It is possible to provide an implant material in which the ability to form bone tissue or blood vessels is enhanced by a bone morphogenetic protein immobilized on the surface, a fibroblast growth factor that promotes the formation of new blood vessels, or the like.

  EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.

  A c-plane oriented HA coating was prepared using plasma spraying. A HA film with high orientation (HO-HAC) and a HA film with low orientation (LO-HAC) were produced according to the thermal spraying conditions. As a reference, a sintered body HA (S-HA) (1200 ° C., 2 h sintering) was produced. The degree of crystal orientation of each sample was evaluated by XRD thin film orientation analysis using the (004) diffraction peak of HA. The diffraction intensity was plotted against the angle ψ of the sample surface normal to the specific lattice plane normal under Bragg conditions.

  FIG. 1 shows a ψ-diffraction intensity curve. The angle of ψ at which the integrated intensity is 50% with respect to the integrated intensity when ψ = 0 of the sample was defined as the degree of orientation (OD). In HO-HAC, films with OD = 13.5 ° and 18.0 ° having different orientation degrees were obtained. The above S-HA, LO-HAC, HO-HAC (OD = 13.5 °) and HO-HAC (OD = 18.0 °) were used as samples.

  In this example, lactoferrin was used as a basic protein to be adsorbed on the sample. A solution of FITC-labeled lactoferrin (FITC-LAC) dissolved in phosphate buffer (PBS) at 22 μg / 100 μl was adsorbed on the surface of each sample at room temperature for 30 minutes. This was washed with a phosphate buffer with NaCl concentrations of 154, 250, 500, and 1000 mM, then observed with a fluorescence microscope, and the fluorescence intensity was measured from the obtained image.

  FIG. 2 shows a plot of the fluorescence intensity after washing the protein-adsorbed sample surface with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. In any case, the control S-HA had low fluorescence intensity and low protein adsorption.

  In the case of lactoferrin, which is a basic protein with an isoelectric point of 8.5, the fluorescence intensity of HO-HAC with high orientation is higher than that of LO-HAC with low orientation when the NaCl concentration is washed at 1000 mM. It was found that the HA film having a higher orientation of 75% or more and higher orientation has a higher protein retention. In HO-HAC with high orientation, the fluorescence intensity was stronger when OD = 13.5 ° with higher orientation, and about 65% of protein was adsorbed.

  In this example, cytochrome C was used as the basic protein to be adsorbed on the sample. A solution of FITC-labeled cytochrome C (FITC-CCC) dissolved in phosphate buffer (PBS) at 22 μg / 100 μl was adsorbed on the surface of each sample at room temperature for 30 minutes. After washing with a phosphate buffer with a NaCl concentration of 154, 250, 500, or 1000 mM, the fluorescence intensity was measured from the obtained image by observing with a fluorescence microscope.

  FIG. 3 shows a plot of the fluorescence intensity after washing each sample surface adsorbed with protein with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. In any case, the control S-HA had low fluorescence intensity and low protein adsorption.

  In the case of cytochrome C, which is a basic protein having an isoelectric point of about 10.9, the fluorescence intensity of HO-HAC (OD = 13.5 °) having high orientation is lower than that of LO-HAC having low orientation. When the NaCl concentration was washed at 1000 mM, it was found that the HA coating having a higher orientation was higher by about 83% and the protein retention was higher. In a highly oriented HO-HAC, when the degree of orientation is about 18 °, there is no significant difference compared to LO-HAC with a low degree of orientation, and in the case of a highly basic protein, the degree of orientation is affected. Strongly expressed.

  In this example, lysozyme was used as the basic protein to be adsorbed on the sample. A solution of FITC-labeled lysozyme (FITC-Lyz) dissolved in phosphate buffer (PBS) at 22 μg / 100 μl was adsorbed on the surface of each sample at room temperature for 30 minutes. After washing with a phosphate buffer with a NaCl concentration of 154, 250, 500, or 1000 mM, the fluorescence intensity was measured from the obtained image by observing with a fluorescence microscope.

  FIG. 4 shows a plot of the fluorescence intensity after washing each sample surface adsorbed with protein with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. In any case, the control S-HA had low fluorescence intensity and low protein adsorption.

  In the case of lysozyme, which is a basic protein having an isoelectric point of about 11.2, the fluorescence intensity of HO-HAC (OD = 13.5 °) with high orientation is higher than that of LO-HAC with low orientation. When washed at a concentration of 1000 mM, it was found that the HA film having a higher orientation was higher by about 2.2 times and the protein retention was higher. In a highly oriented HO-HAC, when the degree of orientation is about 18 °, there is no significant difference compared to LO-HAC with a low degree of orientation, and in the case of a highly basic protein, the degree of orientation is affected. Strongly expressed.

Comparative Example 1
A solution of FITC-labeled bovine serum albumin (FITC-BSA) dissolved in phosphate buffer (PBS) at 22 μg / 100 μl was adsorbed on the surface of each sample at room temperature for 30 minutes. After washing with a phosphate buffer with a NaCl concentration of 154, 250, 500, or 1000 mM, the fluorescence intensity was measured from the obtained image by observing with a fluorescence microscope.

  FIG. 5 shows a plot of the fluorescence intensity after washing each sample surface adsorbed with protein with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. In any case, the control S-HA had low fluorescence intensity and low protein adsorption.

  In the case of albumin, which is an acidic protein having an isoelectric point of about 4.5, the fluorescence intensity of LO-HAC with low orientation is higher than that of HO-HAC with high orientation when the NaCl concentration is washed at 1000 mM. It was found that the amount of protein retained was higher in the HA film having a higher orientation of about 32% and lower orientation. In the highly oriented HO-HAC, there was almost no difference in fluorescence intensity due to the difference in orientation degree, and no difference was observed in the amount of protein adsorbed.

Comparative Example 2
A solution in which FITC-labeled immunoglobulin G (FITC-IgG) was dissolved in phosphate buffer (PBS) at 22 μg / 100 μl was adsorbed on the surface of each sample at room temperature for 30 minutes. After washing with a phosphate buffer with a NaCl concentration of 154, 250, 500, or 1000 mM, the fluorescence intensity was measured from the obtained image by observing with a fluorescence microscope.

  FIG. 6 shows a plot of the fluorescence intensity after washing each sample surface adsorbing each protein with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. In any case, the control S-HA had low fluorescence intensity and low protein adsorption.

  In the case of immunoglobulin G, which is a neutral protein with an isoelectric point of 6.6, the fluorescence intensity of LO-HAC with low orientation is higher than that of HO-HAC with high orientation, when the NaCl concentration is washed at 1000 mM. It was found that the HA film having a higher orientation of 55% and lower orientation has a higher protein retention. In the highly oriented HO-HAC, there was almost no difference in fluorescence intensity due to the difference in orientation degree, and no difference was observed in the amount of protein adsorbed.

  As described above in detail, the present invention relates to an apatite-coated biological implant and a cell culture carrier in which a biomolecule is immobilized on the surface, and according to the present invention, a biomaterial in which a basic protein is selectively immobilized in the vicinity of the surface. Can be provided. According to the present invention, it is possible to produce and provide a living body implant material in which a basic protein is immobilized on the surface of a hydroxyapatite film and a cell culture carrier by a simple method of immersing the protein in a protein solution and adsorbing the protein. it can. INDUSTRIAL APPLICABILITY The present invention is useful for providing biological implant materials such as artificial joints and artificial dental roots and cell culture carriers in which basic proteins such as BMP are selectively fixed on the surface.

FIG. 1 shows the ψ-diffraction intensity curve of Example 1. FIG. 2 plots the fluorescence intensity after washing each sample surface adsorbing FITC-labeled lactoferrin (FITC-LAC) of Example 2 with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. It shows things. FIG. 3 is a plot of fluorescence intensity after washing each sample surface adsorbing FITC-labeled cytochrome C (FITC-CCC) of Example 3 with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. It shows what was done. FIG. 4 is a plot of the fluorescence intensity after washing each sample surface adsorbing FITC-labeled lysozyme (FITC-Lyz) of Example 4 with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. It shows things. FIG. 5 shows the fluorescence intensity after washing each sample surface adsorbing FITC-labeled bovine serum albumin (FITC-BSA) of Comparative Example 1 with a phosphate buffer having a different NaCl concentration with respect to the NaCl concentration of the washing solution. It shows what was plotted. FIG. 6 is a plot of fluorescence intensity after washing each sample surface adsorbing FITC-labeled immunoglobulin G (FITC-IgG) of Comparative Example 2 with a phosphate buffer having a different NaCl concentration against the NaCl concentration of the washing solution. It shows what was done.

Claims (9)

  A biomaterial composed of ceramics mainly composed of hydroxyapatite, wherein the (001) plane (c-plane) of hydroxyapatite (HA) crystals in the ceramics is preferentially exposed on the surface. Characterized by high crystal orientation of at least 13.5 ° orientation by XRD thin film orientation analysis of the HA (004) plane, and basic protein selectively immobilized at least near the surface of the surface A biomaterial.   The biomaterial has an apatite film mainly composed of hydroxyapatite formed on a substrate made of ceramics or metal, and the film is composed of particles deposited in a lamellar shape, and the hydroxyapatite in the film 2. The living body according to claim 1, wherein the (001) plane (c plane) has a plane preferentially exposed on the surface by preferentially orienting the c-axis direction of the crystal of the crystal in a direction perpendicular to the substrate. material.   The biomaterial according to claim 2, comprising calcium triphosphate and calcium tetraphosphate in which the a-axis direction of the crystal is preferentially oriented in a direction perpendicular to the substrate.   A cell culture carrier comprising the biomaterial according to any one of claims 1 to 3 as a constituent element.   The cell culture carrier according to claim 4, which has an apatite film having a thickness of 5 to 1000 μm.   A biological implant material comprising the biomaterial according to any one of claims 1 to 3 as a constituent element.   It has an apatite film on a substrate made of titanium or a titanium alloy, and a layer containing a mixture of titanium or a titanium alloy and a nitride thereof is formed in the film, thereby improving the adhesion of the film to the substrate. The biological implant material according to claim 6.   The biological implant material according to claim 6 or 7, which has an apatite film having a thickness of 5 to 1000 µm.   The living body implant material according to any one of claims 6 to 7, wherein irregularities are formed in a limited range of the surface of the living body implant material.