Bone regeneration material, method for manufacturing bone regeneration material, and bone regeneration material manufacturing device

Abstract

To provide a bone regeneration material, a method for manufacturing a bone regeneration material, and a bone regeneration material manufacturing device that control the adsorption amount of protein with respect to octacalcium phosphate.SOLUTION: A bone regeneration material is formed by a complex that includes: a base material including octacalcium phosphate; and functional molecules adsorbed to the octacalcium phosphate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a bone regeneration material, a method for producing a bone regeneration material, and an apparatus for producing a bone regeneration material. [Background technology]

[0002] Conventionally, a method has been known in which the precipitation of a new octacalcium phosphate is promoted on octacalcium phosphate seed crystals under supersaturated conditions, thereby increasing the amount of protein adsorbed onto octacalcium phosphate (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2021-16724 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the method of Patent Document 1 has a problem in that it is not possible to control the amount of protein adsorbed to octacalcium phosphate, and therefore it is not possible to adjust the amount of protein adsorbed depending on the application.

[0005] The present invention has been made in consideration of the above circumstances, and aims to provide a bone regeneration material in which the amount of protein adsorbed to octacalcium phosphate is controlled, a method for producing the bone regeneration material, and an apparatus for producing the bone regeneration material. [Means for solving the problem]

[0006] The present invention has the following aspects. [1] A bone regeneration material comprising a composite material including a base material containing octacalcium phosphate and a biofunctional molecule adsorbed to the octacalcium phosphate. [2] The bone regeneration material according to [1], wherein the biofunctional molecule is at least one selected from a growth factor, a serum-derived protein, an antibacterial molecule and an antibacterial compound. [3] The bone regeneration material according to [1] or [2], wherein the base material contains a lactic acid-glycolic acid copolymer. [4] Dissolving a biofunctional molecule in phosphate buffered saline to prepare a phosphate buffered saline solution; contacting the phosphate buffered saline solution with a substrate containing octacalcium phosphate to adsorb the biofunctional molecule onto the octacalcium phosphate; and freeze-drying the substrate containing the octacalcium phosphate having the biofunctional molecule adsorbed thereon. [5] preparing a buffer solution containing a biofunctional molecule by dissolving the biofunctional molecule in a buffer solution saturated or supersaturated with respect to octacalcium phosphate; a step of immersing a substrate containing octacalcium phosphate in the buffer solution containing the biofunctional molecule, thereby allowing the biofunctional molecule to be adsorbed onto the octacalcium phosphate; and freeze-drying the substrate containing the octacalcium phosphate having the biofunctional molecule adsorbed thereon. [6] A reaction vessel containing a slurry containing a base material including octacalcium phosphate; an acid / base supply unit for supplying an acid or a base to the slurry in the reaction vessel; a calcium ion supply unit that supplies calcium ions to the slurry in the reaction tank; an inorganic phosphate ion supply unit that supplies inorganic phosphate ions to the slurry in the reaction tank; a biofunctional molecule supplying unit for supplying a biofunctional molecule to the slurry in the reaction tank; a biofunctional molecule concentration unit that recovers and concentrates the biofunctional molecules remaining in the supernatant of the slurry after the biofunctional molecules are adsorbed onto the substrate in the reaction tank, and supplies the biofunctional molecules again to the reaction tank; A pH electrode for measuring the pH of the slurry in the reaction vessel; a calcium ion electrode for measuring a calcium ion concentration of the slurry in the reaction tank; a control unit that calculates a degree of supersaturation of the slurry with respect to octacalcium phosphate based on the pH of the slurry in the reaction tank measured by the pH electrode and the calcium ion concentration of the slurry in the reaction tank measured by the calcium ion electrode, and issues instructions to the acid / base supply unit, the calcium ion supply unit, and the inorganic phosphate ion supply unit to supply an acid or base, calcium ions, and inorganic phosphate ions to the slurry in the reaction tank based on the degree of supersaturation so that the degree of supersaturation of the slurry in the reaction tank becomes a set value. Effect of the Invention

[0007] According to the present invention, it is possible to provide a bone regenerating material in which the amount of protein adsorbed to octacalcium phosphate is controlled, a method for producing the bone regenerating material, and an apparatus for producing the bone regenerating material. [Brief description of the drawings]

[0008] [Figure 1] 1 is a schematic diagram showing an apparatus for producing a bone regenerating material according to one embodiment of the present invention. [Diagram 2] FIG. 1 is a diagram illustrating the mechanism by which adsorption of a biofunctional molecule to a substrate containing an OCP is promoted in a buffer solution that is supersaturated with respect to the OCP. [Diagram 3] FIG. 1 is a diagram illustrating the mechanism by which adsorption of a biofunctional molecule to a substrate containing an OCP is promoted in a buffer solution that is supersaturated with respect to the OCP. [Figure 4] FIG. 2 is a graph showing the ratio of new bone area to defect area in Example 1. [Diagram 5] FIG. 2 is a graph showing the ratio of the remaining OCP granule area to the defect area in Example 1. [Figure 6] FIG. 1 shows the DNA concentration of cells on the 14th or 21st day after the start of culture in Example 2. [Figure 7]FIG. 1 shows ALP activity on the 14th or 21st day after the start of culture in Example 2. [Figure 8] FIG. 13 is a graph showing the relationship between the initial concentration of cytochrome c in a buffer solution saturated with respect to OCP or a buffer solution supersaturated with respect to OCP, and the amount of cytochrome c adsorbed to OCP in Example 4. [Figure 9] FIG. 13 is a diagram showing the results of X-ray diffraction analysis of the crystal structure of OCP having cytochrome c adsorbed thereon in Example 4. [Figure 10] 13 is a transmission electron microscope image of OCP granules before cytochrome c is adsorbed in Example 5. [Figure 11] 13 is a transmission electron microscope image of OCP granules to which cytochrome c has been adsorbed using cytochrome c-containing buffer solution E in Example 5. [Figure 12] 13 is a transmission electron microscope image of OCP granules to which cytochrome c has been adsorbed using cytochrome c-containing buffer solution F in Example 5. [Figure 13] FIG. 13 shows adsorption isotherms of fetuin to OCP in a fetuin-containing 0.5Ca0.5Pi buffer solution and a fetuin-containing 3.0Ca1.0Pi buffer solution in Example 6. [Figure 14] FIG. 13 shows the results of measuring CD spectra of fetuin in 0.5Ca0.5Pi buffer and 3.0Ca1.0Pi buffer before and after immersion in OCP in Example 6. [Figure 15] FIG. 13 shows secondary structure elements of fetuin in 0.5Ca0.5Pi buffer and in 3.0Ca1.0Pi buffer in Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] BEST MODE FOR CARRYING OUT THE DISCLOSURE The bone regenerating material, the method for producing the bone regenerating material, and the apparatus for producing the bone regenerating material according to the present invention will be described below. It should be noted that the present embodiment is specifically described to allow a better understanding of the gist of the invention, and does not limit the present invention unless otherwise specified.

[0010] [Bone regeneration material] A bone regenerating material according to one embodiment of the present invention comprises a complex containing a base material containing octacalcium phosphate and a biofunctional molecule adsorbed to the octacalcium phosphate.

[0011] The shape of the complex can be set arbitrarily taking into consideration the shape and size of the affected area to be filled with it, etc. For example, the complex is preferably in the form of a rectangular parallelepiped (block), a cube, a cylinder, a tablet, or granules.

[0012] "Base material" The substrate contains octacalcium phosphate (Ca8H2(PO4)6·5H2O, hereinafter sometimes referred to as "OCP"). The substrate may consist of only OCP, or may contain OCP and other components.

[0013] When the base material contains an OCP and other components, the other components are preferably poly(lactic acid-co-glycolic acid) (hereinafter sometimes referred to as "PLGA").

[0014] When the base material contains OCP and PLGA, the ratio of OCP to PLGA is not particularly limited, but for example, based on the total amount of OCP and PLGA, the base material preferably contains 20% by mass or more and 65% by mass or less of OCP, more preferably 20% by mass or more and 40% by mass or less of OCP. Also, the base material preferably contains 35% by mass or more and 80% by mass or less of PLGA, more preferably 60% by mass or more and 80% by mass or less of PLGA. This allows the bone regeneration material to have a good balance between the osteoconductivity of OCP and the bioabsorbability of PLGA, and has excellent osteoconductivity.

[0015] OCP is a known substance and can be prepared, for example, by the LeGeros dropping method (LeGeros RZ, Calcif Tissue Int 37:194-197, 1985) or the method using a synthesis apparatus (three-flow tube) described in Japanese Patent No. 3115642.

[0016] "Biofunctional molecules" Biofunctional molecules are adsorbed to the substrate and promote cell migration and proliferation in addition to increasing bone regeneration by OCP. For example, as reported in Tohoku J Exp Med 164:37-50, 1991 and Bone Miner 20:151-166, 1993, it is believed that biofunctional molecules accumulate in the gaps between OCP crystals and function.

[0017] As the biofunctional molecule, a growth factor, a serum-derived protein, an antibacterial molecule, or an antibacterial compound is preferred. Examples of growth factors include stromal cell-derived factor 1 (SDF-1), bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), etc. For example, by adsorbing SDF-1 onto a substrate, it is expected to promote the migration of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts.

[0018] Examples of serum-derived proteins include fetuin (α2-HS glycoproteins), albumin, fibronectin, etc. For example, fetuin can be adsorbed onto a substrate to bind with surrounding calcium ions and phosphate ions and promote bone formation. Fibronectin can be adsorbed onto a substrate to enhance cell adhesion.

[0019] Examples of antibacterial molecules include nitroimidazole-based metronidazole, alcohols such as ethanol, phenolic compounds such as thymol, etc. For example, metronidazole can be adsorbed onto a substrate and used to obtain an antibacterial effect against anaerobic bacteria. Examples of antibacterial compounds include antibacterial iodine compounds, such as povidone-iodine.

[0020] The biofunctional molecules may be used alone or in combination of two or more. By using two or more biofunctional molecules in combination, two or more actions can be exerted on the living body.

[0021] The amount of biofunctional molecules adsorbed on the substrate is expressed as the unit area (cm 2 ), preferably 50 ng to 70 μg, more preferably 100 ng to 60 μg, and even more preferably 200 ng to 20 μg. When the amount of biofunctional molecule adsorbed is equal to or greater than the lower limit, bone regeneration is expected to be promoted. When the amount of biofunctional molecule adsorbed is less than the lower limit, the effect of promoting bone regeneration cannot be obtained. When the amount of biofunctional molecule adsorbed exceeds the upper limit, cytotoxicity, inflammation, etc. are induced, and sufficient bone regeneration effect cannot be obtained.

[0022] The bone regeneration material of this embodiment may contain components generally contained in bone regeneration materials within the range in which the effect of the present invention is not impaired. Examples of such components include collagen, gelatin, alginic acid, hyaluronic acid, chitosan, bioabsorbable polymers (polylactic acid, polylactic acid-polyethylene glycol copolymer, etc.), bioabsorbable calcium phosphates (β-tricalcium phosphate (β-TCP), α-tricalcium phosphate (α-TCP), tetracalcium phosphate (Ca4(PO4)2O; TTCP), calcium hydrogen phosphate (CaHPO4; DCP), calcium hydrogen phosphate dihydrate (CaHPO4·2H2O; DCPD), low-crystalline HA, nano HA, carbonate-containing HA, etc.), and non-bioabsorbable materials (HA ceramics, etc.).

[0023] According to the bone regeneration material of the present embodiment, since it is composed of a complex containing a base material containing an OCP and a biofunctional molecule adsorbed to the OCP, when applied to a living body, in addition to increasing bone regeneration by the OCP, it can bring about the effect of the biofunctional molecule. Moreover, the bone regeneration material of the present embodiment can be made to have a controlled amount of adsorption of the biofunctional molecule by the manufacturing method of the bone regeneration material described later.

[0024] [Method of manufacturing bone regenerative materials] (First embodiment) A method for producing a bone regenerating material according to one embodiment of the present invention comprises the steps of: dissolving a biofunctional molecule in phosphate buffered saline to prepare a phosphate buffered saline solution (hereinafter referred to as the "first step"); contacting the phosphate buffered saline solution with a substrate containing octacalcium phosphate to adsorb the biofunctional molecule to the octacalcium phosphate (hereinafter referred to as the "second step"); and freeze-drying the substrate containing the octacalcium phosphate to which the biofunctional molecule has been adsorbed (hereinafter referred to as the "third step").

[0025] "First step" The concentration of the phosphate buffered saline is preferably 1.0 mmol / L or more and 30.0 mmol / L or less, more preferably 5.0 mmol / L or more and 15.0 mmol / L or less. If the concentration of the phosphate buffered saline is less than the lower limit, the pH buffering ability decreases. If the concentration of the phosphate buffered saline is more than the upper limit, cytotoxicity may occur due to precipitated phosphate.

[0026] The content (concentration) of the biofunctional molecule in the phosphate buffered saline solution is preferably 3.0 μmol / L to 30.0 μmol / L, more preferably 4.0 μmol / L to 10.0 μmol / L, and even more preferably 4.5 μmol / L to 7.5 μmol / L. If the content of the biofunctional molecule is less than the lower limit, the amount of the biofunctional molecule carried is small, and sufficient bone regeneration effect may not be obtained. If the content of the biofunctional molecule exceeds the upper limit, cytotoxicity, inflammation, etc. are induced, and sufficient bone regeneration effect cannot be obtained.

[0027] "Second step" In the second step, contacting the substrate containing OCP with the phosphate buffered saline solution includes, for example, dropping the phosphate buffered saline solution onto the substrate containing OCP or immersing the substrate containing OCP in the phosphate buffered saline solution.

[0028] When the substrate containing an OCP is immersed in the phosphate buffered saline solution, the immersion time is not particularly limited, but is preferably, for example, from 1 second to 10 minutes.

[0029] When a phosphate buffered saline solution is dropped onto a substrate containing an OCP, the amount of the phosphate buffered saline solution dropped per unit mass (mg) of the substrate is preferably 2.0 μL to 8.0 μL, more preferably 3.0 μL to 7.0 μL, and even more preferably 4.0 μL to 6.0 μL. When the amount of the phosphate buffered saline solution dropped is equal to or more than the lower limit, the biofunctional molecule can be supported uniformly on the surface of the OCP. When the amount of the phosphate buffered saline solution dropped is equal to or less than the upper limit, dissolution of the OCP crystals can be suppressed.

[0030] When the substrate containing OCP is immersed in a phosphate buffered saline solution, the mass (mg) of the substrate containing OCP added per unit volume (μL) of the phosphate buffered saline solution is preferably 0.10 mg to 0.5 mg in terms of OCP, more preferably 0.15 mg to 0.30 mg in terms of OCP, and even more preferably 0.15 mg to 0.25 mg in terms of OCP. When the mass of the substrate containing OCP is equal to or greater than the lower limit, dissolution of OCP crystals can be suppressed. When the mass of the substrate containing OCP is equal to or less than the upper limit, biofunctional molecules can be supported uniformly on the OCP.

[0031] "The third step" In the third step, it is preferable to pre-freeze the substrate containing the OCP with the biofunctional molecule adsorbed thereon before freeze-drying the substrate containing the OCP with the biofunctional molecule adsorbed thereon. The pre-freezing temperature is preferably −196° C. or higher and −10° C. or lower.

[0032] The freeze-drying time is not particularly limited, but is preferably, for example, from 12 hours to 120 hours.

[0033] According to the bone regeneration material of this embodiment, the biofunctional molecules are adsorbed onto the OCP using a phosphate-buffered saline solution with an adjusted content of biofunctional molecules, so that the amount of biofunctional molecules adsorbed onto the OCP can be controlled.

[0034] Second Embodiment A method for producing a bone regenerating material according to one embodiment of the present invention comprises the steps of: dissolving a biofunctional molecule in a buffer solution that is saturated or supersaturated with octacalcium phosphate to prepare a buffer solution containing the biofunctional molecule (hereinafter referred to as the "first step"); immersing a substrate containing octacalcium phosphate in the buffer solution containing the biofunctional molecule to allow the biofunctional molecule to be adsorbed onto the octacalcium phosphate (hereinafter referred to as the "second step"); and freeze-drying the substrate containing the octacalcium phosphate to which the biofunctional molecule has been adsorbed (hereinafter referred to as the "third step").

[0035] "First step" Examples of buffer solutions that are saturated with OCP include those containing calcium ions (Ca 2+ ), inorganic phosphate ions, and a Tris-HCl buffer solution, and has a pH of 5.0 or more and 8.0 or less.

[0036] The calcium ion concentration in the buffer saturated with OCP is preferably 0.1 mmol / L to 1.0 mmol / L, more preferably 0.4 mmol / L to 0.6 mmol / L. If the calcium ion concentration is below the lower limit, the OCP substrate will dissolve. If the calcium ion concentration is above the upper limit, hydroxyapatite will precipitate.

[0037] The concentration of inorganic phosphate ions in the buffer saturated with OCP is preferably 0.1 mmol / L to 1.0 mmol / L, more preferably 0.4 mmol / L to 0.6 mmol / L. If the concentration of inorganic phosphate ions is less than the lower limit, the OCP substrate will dissolve. If the concentration of inorganic phosphate ions is more than the upper limit, hydroxyapatite will precipitate.

[0038] The concentration of trishydroxymethylaminomethane in the tris-HCl buffer in a buffer saturated with OCP is preferably 50 mmol / L to 300 mmol / L, more preferably 100 mmol / L to 200 mmol / L. If the concentration of the tris-HCl buffer is below the lower limit, the pH buffering ability is lost. If the concentration of the tris-HCl buffer exceeds the upper limit, there is a possibility that cytotoxicity will occur due to residual trishydroxymethylaminomethane.

[0039] The pH of the buffer saturated with OCP is preferably 5.0 to 8.0, more preferably 6.8 to 8.0, and even more preferably 7.2 to 7.6. If the pH is less than the lower limit, OCP dissolves. If the pH is greater than the upper limit, hydroxyapatite precipitates.

[0040] Examples of buffer solutions that are supersaturated with respect to OCP include calcium ions (Ca 2+ ), inorganic phosphate ions, and a Tris-HCl buffer solution, and has a pH of 5.0 or more and 8.0 or less.

[0041] The calcium ion concentration in the buffer solution supersaturated with respect to OCP is preferably 0.5 mmol / L to 5.0 mmol / L, more preferably 1.0 mmol / L to 4.0 mmol / L, and even more preferably 2.5 mmol / L to 3.5 mmol / L. When the calcium ion concentration is within the above range, new OCP crystals are precipitated on the OCP crystals. When the calcium ion concentration is below the lower limit, the OCP crystals are dissolved. When the calcium ion concentration exceeds the upper limit, hydroxyapatite is precipitated.

[0042] The concentration of inorganic phosphate ions in a buffer solution supersaturated with respect to OCP is preferably 0.5 mmol / L to 5.0 mmol / L, more preferably 0.75 mmol / L to 4.0 mmol / L, and even more preferably 1.0 mmol / L to 3.0 mmol / L. When the concentration of inorganic phosphate ions is within the above range, new OCP crystals are precipitated on the OCP crystals. When the concentration of inorganic phosphate ions is below the above lower limit, the OCP crystals are dissolved. When the concentration of inorganic phosphate ions exceeds the above upper limit, hydroxyapatite is precipitated.

[0043] The concentration of trishydroxymethylaminomethane in the tris-hydrochloric acid buffer in a buffer solution supersaturated with respect to OCP is preferably 50 mmol / L to 300 mmol / L, more preferably 100 mmol / L to 200 mmol / L. If the concentration of the tris-hydrochloric acid buffer is less than the lower limit, the pH buffering ability is lost. If the concentration of the tris-hydrochloric acid buffer exceeds the upper limit, there is a possibility that cytotoxicity will occur due to residual trishydroxymethylaminomethane.

[0044] The pH of the buffer solution supersaturated with respect to the OCP is preferably 5.0 to 8.0, more preferably 6.8 to 8.0, and even more preferably 7.2 to 7.6. If the pH is less than the lower limit, the OCP dissolves. If the pH is greater than the upper limit, hydroxyapatite precipitates.

[0045] "Second step" When the substrate containing OCP is immersed in a saturated buffer solution, the mass (mg) of the substrate containing OCP added per unit volume (mL) of the saturated buffer solution is preferably 0.5 mg to 10 mg in terms of OCP, more preferably 1.0 mg to 7.5 mg, and even more preferably 3.0 mg to 6.0 mg. If the mass of the substrate containing OCP is less than the lower limit, the surface area of ​​the substrate is reduced and the amount of biofunctional molecules adsorbed is reduced. If the mass of the substrate containing OCP exceeds the upper limit, the amount of adsorption per unit area of ​​the substrate is reduced.

[0046] When a substrate containing OCP is immersed in a supersaturated buffer solution, the mass (mg) of the substrate containing OCP added per unit volume (mL) of the supersaturated buffer solution is preferably 0.5 mg to 10 mg, more preferably 1.0 mg to 7.5 mg, and even more preferably 3.0 mg to 6 mg, calculated as OCP. If the mass of the substrate containing OCP is less than the lower limit, the surface area of ​​the substrate is reduced and the amount of biofunctional molecules adsorbed is reduced. If the mass of the substrate containing OCP exceeds the upper limit, the amount of new OCP crystals precipitated on the OCP crystals is reduced.

[0047] When the substrate containing OCP is immersed in a saturated buffer solution, the immersion time is not particularly limited, but is preferably, for example, from 10 hours to 120 hours. When the substrate containing OCP is immersed in a supersaturated buffer solution, the immersion time is not particularly limited, but is preferably, for example, from 10 hours to 120 hours.

[0048] "The third step" In the third step, it is preferable to wash the substrate containing the OCP having the biofunctional molecule adsorbed thereon with pure water before freeze-drying the substrate containing the OCP having the biofunctional molecule adsorbed thereon. After washing the substrate containing the OCP with the biofunctional molecule adsorbed thereon, it is preferable to pre-freeze the substrate containing the OCP with the biofunctional molecule adsorbed thereon before freeze-drying. The pre-freezing temperature is preferably −196° C. or higher and −10° C. or lower.

[0049] The freeze-drying time is not particularly limited, but is preferably, for example, from 12 hours to 120 hours.

[0050] According to the bone regeneration material of this embodiment, the biofunctional molecule is adsorbed onto the OCP by using a saturated or supersaturated buffer solution containing the biofunctional molecule for the OCP with the adjusted content of the biofunctional molecule, thereby making it possible to control the amount of the biofunctional molecule adsorbed onto the OCP.

[0051] [Bone regeneration material manufacturing equipment] FIG. 1 is a schematic diagram showing an apparatus for producing a bone regenerating material according to one embodiment of the present invention. As shown in Figure 1, the bone regenerative material manufacturing apparatus 1 of this embodiment includes a reaction tank 2, an acid / base supply unit 3, a calcium ion supply unit 4, an inorganic phosphate ion supply unit 5, a biofunctional molecule supply unit 6, a biofunctional molecule concentrator 7, a pH electrode 8, a calcium ion electrode 9, and a control unit 10.

[0052] The reaction vessel 2 is a vessel for reacting a base material containing an OCP with a biofunctional molecule to adsorb the biofunctional molecule to the OCP. Specifically, in the reaction vessel 2, a slurry containing a base material containing an OCP with a biofunctional molecule dissolved therein (hereinafter, abbreviated as "slurry") is reacted with a buffer solution supersaturated with respect to the OCP.

[0053] The reaction vessel 2 may be provided with a stirring means for stirring the slurry in which the biofunctional molecules are dissolved and the buffer solution that is supersaturated with respect to the OCP. Examples of the stirring means include a stirring blade and a magnetic stirrer.

[0054] The acid / base supply unit 3 is for supplying an acid or a base to the reaction tank 2. An acid or a base is supplied from the acid / base supply unit 3 to the reaction tank 2 to adjust the pH of the slurry in the reaction tank 2. A pump 11 is provided in the piping 21. The pump 11 is connected to the control unit 10 via wiring 31.

[0055] Examples of the acid include hydrochloric acid, nitric acid, acetic acid, and citric acid. Examples of the base include sodium hydroxide, potassium hydroxide, and ammonia.

[0056] The calcium ion supplying unit 4 is for supplying calcium ions to the reaction tank 2. Calcium ions are supplied from the calcium ion supplying unit 4 to the reaction tank 2 to adjust the calcium ion concentration of the slurry in the reaction tank 2. The calcium ion supplying unit 4 is connected to the reaction tank 2 via a pipe 22. The pipe 22 is provided with a pump 12. The pump 12 is connected to the control unit 10 via a wiring 32.

[0057] The inorganic phosphate ion supplying unit 5 is for supplying inorganic phosphate ions to the reaction tank 2. Inorganic phosphate ions are supplied from the inorganic phosphate ion supplying unit 5 to the reaction tank 2 to adjust the inorganic phosphate ion concentration of the slurry in the reaction tank 2. The inorganic phosphate ion supplying unit 5 is connected to the reaction tank 2 via a pipe 23. A pump 13 is provided in the pipe 23. The pump 13 is connected to the control unit 10 via a wiring 33.

[0058] The biofunctional molecules can be contained in the reaction vessel 2 in advance. The biofunctional molecule supplying section 6 is connected to the reaction vessel 2 via a pipe 24. The pipe 24 is provided with a pump 14. The pump 14 is connected to the control section 10 via a wiring 34. The biofunctional molecules may be supplied from the biofunctional molecule supplying section 6 to the reaction vessel 2 by the pump 14, and the biofunctional molecules may be dissolved in the slurry in the reaction vessel 2.

[0059] The biofunctional molecule concentrating section 7 recovers and concentrates the biofunctional molecules remaining in the supernatant of the slurry after the biofunctional molecules are adsorbed onto the substrate, and supplies (reuses) them again to the reaction vessel 2. The biofunctional molecule concentrating section 7 is connected to the reaction vessel 2 via a pipe 25. The pipe 25 is provided with a pump 15 and a pump 16.

[0060] The pH electrode 8 is installed in the reaction tank 2 and serves to measure the pH of the slurry in the reaction tank 2. The pH electrode 8 is connected to the control unit 10 via a wire 35.

[0061] The calcium ion electrode 9 is installed in the reaction tank 2 and serves to measure the concentration of calcium ions in the slurry in the reaction tank 2. The calcium ion electrode 9 is connected to the control unit 10 via a wiring .

[0062] In the bone regenerative material manufacturing apparatus 1, the pH of the slurry in the reaction tank 2 is measured by the pH electrode 8, and the calcium ion concentration of the slurry in the reaction tank 2 is measured by the calcium ion electrode 9. The control unit 10 calculates the degree of supersaturation of the slurry with respect to the OCP based on these measurement results (concentrations). Based on the calculated degree of supersaturation, the control unit 10 operates pumps 11, 12, and 13 as necessary to supply an acid or base, calcium ions, and inorganic phosphate ions to the slurry in the reaction tank 2 as necessary so that the degree of supersaturation of the slurry in the reaction tank 2 becomes a set value.

[0063] When the pH and calcium ion concentration of the slurry being stirred in the reaction vessel 2 reach predetermined values, the biofunctional molecules are adsorbed onto the OCP.

[0064] This results in a bone regeneration material.

[0065] According to the bone regeneration material manufacturing apparatus of this embodiment, based on the pH of the slurry in the reaction tank 2 measured by the pH electrode 8 and the calcium ion concentration of the slurry in the reaction tank 2 measured by the calcium ion electrode 9, the control unit 10 calculates the degree of supersaturation with respect to the OCP based on the pH of the slurry in the reaction tank 2 measured by the pH electrode 8 and the calcium ion concentration of the slurry in the reaction tank 2 measured by the calcium ion electrode 9, and supplies acid or base, calcium ions and inorganic phosphate ions as necessary to the slurry in the reaction tank 2 so that the degree of supersaturation of the slurry in the reaction tank 2 becomes a set value, thereby controlling the amount of biofunctional molecules adsorbed to the OCP.

[0066] Here, the mechanism of promotion of adsorption of a biofunctional molecule to a substrate containing an OCP in a buffer solution supersaturated with respect to the OCP in this embodiment will be described with reference to FIGS. In a buffer solution saturated with OCP, the biofunctional molecule is adsorbed to the substrate containing OCP due to the interaction between the surface of the substrate and the biofunctional molecule. On the other hand, in a buffer solution that is supersaturated with respect to OCP as shown in Figure 2, new OCP 110 is selectively precipitated on the surface of the substrate 100 as shown in Figure 3, and the biofunctional molecule 200 is adsorbed to the new OCP 110, thereby increasing the amount of adsorption of the biofunctional molecule 200. EXAMPLES

[0067] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0068] [Example 1] SDF-1 was dissolved in 10 mmol / L phosphate buffered saline to prepare a phosphate buffered saline solution, the content of SDF-1 in the phosphate buffered saline solution was adjusted to 0.5 μg per 5 mg of OCP granules (OCP 0.5 μg SDF-1), 1.0 μg per 5 mg of OCP granules (OCP 1.0 μg SDF-1), and 5.0 μg per 5 mg of OCP granules (OCP 5.0 μg SDF-1). The above phosphate buffered saline solution was added dropwise to 5 mg of OCP granules (particle size 300 μm to 500 μm) to allow SDF-1 to be adsorbed onto the OCP granules. The SDF-1-adsorbed OCP granules were pre-frozen at -20°C. The pre-frozen OCP granules were freeze-dried to obtain bone regenerative materials with SDF-1 adsorbed onto the OCP granules. Three types of bone regenerative materials with different SDF-1 contents were prepared.

[0069] A standardized defect measuring 3 mm in diameter and 3 mm in depth was created in the femur of Sprague-Dawley rats (12 weeks old, male). 5 mg of OCP with 0.5 μg SDF-1 (n=4), OCP with 1.0 μg SDF-1 (n=4), or OCP with 5.0 μg SDF-1 (n=4) were implanted into the standardized defects. In addition, 5 mg of OCP without SDF-1 (n=2) was implanted into the standardized defects as a control. Two weeks after implantation, the femurs were excised and decalcified tissue sections were prepared. Decalcified tissue sections were stained with hematoxylin-eosin (HE) and the obtained tissue specimens were observed under an optical microscope. The areas of new bone and remaining OCP granules were measured from the HE tissue images, and the ratios of the new bone area and remaining OCP granule area to the defect area before implantation were calculated. The results are shown in Figures 4 and 5.

[0070] Optical microscopy of tissue specimens revealed that new bone tissue and residual OCP granules were observed in the defect area, regardless of whether SDF-1 was adsorbed. Figure 4 shows the ratio of new bone area to defect area. Figure 5 shows the ratio of residual OCP granule area to defect area. The results shown in Figure 4 indicate that the ratio of new bone area to defect area was highest with SDF-1 at 1.0 μg OCP. Furthermore, the results shown in Figure 5 indicate that the ratio of remaining OCP granule area to defect area was lowest with SDF-1 at 1.0 μg OCP and highest with SDF-1 at 5.0 μg OCP. These results indicate that there is an optimal amount (content) of SDF-1 adsorbed into OCP granules for absorption and replacement with new bone.

[0071] [Example 2] SDF-1 was dissolved at 200 ng / mL in a buffer saturated with respect to OCP (Ca concentration 0.5 mmol / L, inorganic phosphate concentration 0.5 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4, hereafter referred to as "0.5Ca0.5Pi buffer") to prepare SDF-1-containing buffer A. SDF-1 was dissolved at 200 ng / mL in a buffer supersaturated with respect to OCP (Ca concentration 3.0 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4, hereafter referred to as "3.0Ca1.0Pi buffer") to prepare SDF-1-containing buffer B. Furthermore, bovine serum albumin was dissolved as a carrier protein in the SDF-1-containing buffer A and the SDF-1-containing buffer B at a concentration of 1.0 mg / mL. 5 mg of OCP granules (particle size less than 53 μm) were immersed in each of SDF-1-containing buffer solution A and SDF-1-containing buffer solution B in which bovine serum albumin had been dissolved, to allow SDF-1 to be adsorbed onto the OCP granules. The OCP granules to which SDF-1 was adsorbed were washed with pure water. The OCP granules with SDF-1 adsorbed were then freeze-dried for 24 hours to obtain bone regeneration materials with SDF-1 adsorbed onto the OCP granules. Two types of bone regeneration materials were prepared: one using 0.5Ca0.5Pi buffer (0.5Ca0.5Pi group) and one using 3.0Ca1.0Pi buffer (3.0Ca1.0Pi group).

[0072] D1 cell line, which is a mouse bone marrow-derived undifferentiated mesenchymal stem cell (MSC), was placed in a 48-well plate at 4 × 10 4 The mice were seeded at 4 × 10 cells / well and cultured in osteogenic differentiation medium. In addition, mouse bone marrow-derived undifferentiated mesenchymal stem cells (MSCs) were seeded at 4 × 10 cells / well in a 48-well plate together with 5 mg of OCP granules not adsorbed with SDF-1. 4 The control group consisted of cells seeded at 100 cells / well and cultured in osteogenic differentiation medium. The DNA concentration and alkaline phosphatase (ALP) activity of the cells were measured on the 14th or 21st day after the start of culture. ALP is one of the early differentiation markers of osteoblasts. The measured ALP activity was normalized by the DNA concentration. Data are shown as the mean ± standard deviation, and statistically significant differences were analyzed by the Turkey-Kramer test. A significant difference was considered to exist when p was less than 0.05. The results are shown in Figures 6 and 7.

[0073] The results shown in Figure 6 indicate that the DNA concentration on day 14 from the start of culture was significantly higher in the 0.5Ca0.5Pi and 3.0Ca1.0Pi groups than in the control group. The DNA concentration increased from day 14 to day 21 from the start of culture in all groups, but no significant differences were observed among the groups. From the results shown in Figure 7, ALP activity on the 14th day after the start of culture was significantly higher in the 3.0Ca1.0Pi group than in the 0.5Ca0.5Pi group. The 0.5Ca0.5Pi group also tended to have higher ALP activity compared to the control group, but no significant difference was observed. ALP activity increased in all groups from the 14th to 21st days after the start of culture, but no significant difference was observed between the groups. These results indicate that the adsorption of SDF-1 to OCP granules promotes the early proliferation of MSCs on OCP. Furthermore, SDF-1 adsorbed to OCP granules in Tris-HCl buffer that is supersaturated with respect to OCP promotes the early osteoblast differentiation of MSCs compared with SDF-1 adsorbed to OCP granules in Tris-HCl buffer that is saturated with respect to OCP.

[0074] [Example 3] The bone regenerative material was produced using the bone regenerative material production device shown in FIG. The reaction liquid was removed from the OCP precipitate synthesized by the wet method, and the precipitate was washed. Pure water was added to the OCP precipitate to prepare a slurry. An aqueous lysozyme solution was previously supplied to the slurry so that the concentration of lysozyme in the slurry was 0.1 mg / mL, and the total amount of the slurry in the reaction tank 2 was 1000 mL. Pump 12 was operated to supply 40 mmol / L of calcium ions from calcium ion supply unit 4 to the slurry containing lysozyme, and pump 13 was operated to supply 80 mmol / L of inorganic phosphate ions from inorganic phosphate ion supply unit 5. The slurry was stirred at 25° C. to allow lysozyme to be adsorbed onto the OCP. During this, the pH of the slurry in reaction tank 2 was continuously measured by pH electrode 8, and the calcium ion concentration of the slurry in reaction tank 2 was continuously measured by calcium ion electrode 9. The total volume of the slurry after the supply of calcium ions and inorganic phosphate ions was 2000 mL. After lysozyme was adsorbed onto the OCP, the OCP and the supernatant were recovered from the slurry. The amount of lysozyme adsorbed on the OCP was estimated by measuring the concentration of lysozyme in the supernatant, and the crystal structure of the OCP with lysozyme adsorbed was analyzed by X-ray diffraction.

[0075] In the production of bone regeneration material, the calcium ion concentration in the slurry increased for about 500 seconds after the supply of calcium ions to the slurry started, and then decreased. The calcium ion concentration when the supply of calcium ions to the slurry was stopped was 4.84 mmol / L. In addition, the calcium ion concentration 1 hour after the supply of calcium ions to the slurry was stopped was 2.16 mmol / L. When the supply of calcium ions to the slurry was started, the pH of the slurry was about 7. From the start of the supply of calcium ions to the slurry until about 700 seconds later, the pH of the slurry decreased to 5.5 and maintained that value. Of the amount of lysozyme added, 24% was adsorbed onto the surface of the OCP, and 76% remained in the supernatant. It was also confirmed that the OCP maintained its crystalline structure after lysozyme was adsorbed. From the above results, it was confirmed that lysozyme can be adsorbed onto OCP while controlling the degree of supersaturation of the slurry by measuring the pH and calcium ion concentration of the slurry using the bone regeneration material manufacturing device shown in Figure 1.

[0076] [Example 4] Cytochrome c was dissolved at 0-10 mg / mL in a buffer saturated with OCP (Ca concentration 0.5 mmol / L, inorganic phosphate concentration 0.5 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4) to prepare cytochrome c-containing buffer C. Cytochrome c was also dissolved at 0-10 mg / mL in a buffer supersaturated with OCP (Ca concentration 3.0 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4) to prepare cytochrome c-containing buffer D. 5.0 mg of OCP granules (particle size less than 53 μm) were immersed in each of cytochrome c-containing buffer solution C and cytochrome c-containing buffer solution D, to allow cytochrome c to be adsorbed onto the OCP granules. After cytochrome c was adsorbed onto the OCP, the OCP and the supernatant were collected from cytochrome c-containing buffer C and cytochrome c-containing buffer D. The amount of cytochrome c adsorbed to OCP was estimated by measuring the concentration of cytochrome c in the supernatant. The results are shown in Figure 8. In addition, the crystal structure of OCP with adsorbed cytochrome c was analyzed by X-ray diffraction. The results are shown in Figure 9.

[0077] The results shown in FIG. 8 indicate that when the initial concentration of cytochrome c was in the range of 0 to 3.0 mg / mL, the amount of cytochrome c adsorbed was greater in a buffer solution that was supersaturated with respect to OCP than in a buffer solution that was saturated with respect to OCP. From the results shown in FIG. 9, it was confirmed that the crystal structure as OCP was maintained even after adsorption of cytochrome c, regardless of the initial concentration of cytochrome c.

[0078] [Example 5] Cytochrome c was dissolved at 0.1 mg / mL in a buffer solution supersaturated with respect to OCP (Ca concentration 2.5 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4) to prepare cytochrome c-containing buffer solution E. In addition, bovine serum albumin was added at 1.0 mg / mL to cytochrome c-containing buffer solution E to prepare cytochrome c-containing buffer solution F. 5.0 mg of OCP granules (particle size less than 53 μm) were immersed in each of cytochrome c-containing buffer E and cytochrome c-containing buffer F to adsorb cytochrome c to the OCP granules. The OCP granules were immersed in each buffer for 3 days. The buffer was replaced with a new buffer every day to adsorb cytochrome c to the OCP granules. Using a transmission electron microscope (JEOL JEM-2100F, manufactured by JEOL Ltd.), OCP granules before adsorption of cytochrome c, OCP granules to which cytochrome c was adsorbed using cytochrome c-containing buffer solution E, and OCP granules to which cytochrome c was adsorbed using cytochrome c-containing buffer solution F were observed. The results are shown in Figs. 10 to 12. Fig. 10 is a transmission electron microscope image of OCP granules before adsorption of cytochrome c. Fig. 11 is a transmission electron microscope image of OCP granules to which cytochrome c was adsorbed using cytochrome c-containing buffer solution E. Fig. 12 is a transmission electron microscope image of OCP granules to which cytochrome c was adsorbed using cytochrome c-containing buffer solution F. In Figures 11 and 12, it was found that new OCP crystals were formed at the ends of the OCP granules, as indicated by the arrows.

[0079] [Example 6] Fetuin was dissolved in a buffer saturated with OCP (Ca concentration 0.5 mmol / L, inorganic phosphate concentration 0.5 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4, hereafter referred to as "0.5Ca0.5Pi buffer") to prepare fetuin-containing 0.5Ca0.5Pi buffer solutions with fetuin concentrations of 0 mg / mL, 0.10 mg / mL, 0.25 mg / mL, 0.50 mg / mL, 0.75 mg / mL, 1.0 mg / mL, or 1.5 mg / mL. In addition, fetuin was dissolved in a buffer solution supersaturated with OCP (Ca concentration 3.0 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4, hereafter referred to as "3.0Ca1.0Pi buffer") to prepare fetuin-containing 3.0Ca1.0Pi buffer solutions with fetuin concentrations of 0 mg / mL, 0.10 mg / mL, 0.25 mg / mL, 0.50 mg / mL, 0.75 mg / mL, 1.0 mg / mL, or 1.5 mg / mL. 5 mg of OCP granules (particle size less than 53 μm) were immersed in each of fetuin-containing 0.5Ca0.5Pi buffer and fetuin-containing 3.0Ca1.0Pi buffer in which fetuin was dissolved at various concentrations, and fetuin was adsorbed onto the OCP granules. The results are shown in Figure 13. Figure 13 shows the adsorption isotherms of fetuin to OCP in fetuin-containing 0.5Ca0.5Pi buffer and fetuin-containing 3.0Ca1.0Pi buffer. As shown in FIG. 13, the amount of fetuin adsorption increased in the equilibrium concentration range from 0 to about 3.0 mg / mL in the fetuin-containing 0.5Ca0.5Pi buffer solution and in the fetuin-containing 3.0Ca1.0Pi buffer solution. In both adsorption isotherms, the adsorption of fetuin tended to saturate at equilibrium concentrations exceeding 3 mg / mL. The amount of fetuin adsorbed onto OCPs immersed in 3.0Ca1.0Pi buffer was greater than that immersed in 0.5Ca0.5Pi buffer, regardless of the equilibrium concentration of fetuin. The adsorption parameters of fetuin on OCP were calculated by fitting to the Langmuir model. The correlation coefficients were greater than 0.99 for both adsorption isotherms. The adsorption equilibrium constant of the 0.5Ca0.5Pi buffer was 33.5 mL / mg, and that of the 3.0Ca1.0Pi buffer was 69.4 mL / mg. The saturated adsorption amount of 0.5Ca0.5Pi buffer solution was 2.9 mg / m 2 The saturated adsorption amount of 3.0Ca1.0Pi buffer was 4.0mg / m 2 It was. Thus, the adsorption equilibrium constant and the saturated adsorption amount were higher in the 3.0Ca1.0Pi buffer solution than in the 0.5Ca0.5Pi buffer solution.

[0080] Circular dichroism (CD) spectra of fetuin in 0.5Ca0.5Pi buffer and 3.0Ca1.0Pi buffer containing 0.2 mg / mL fetuin were measured before and after immersion in OCP. The CD spectrum was measured using a circular dichroism spectrometer (J-805, manufactured by JASCO Corporation). The results of the CD spectrum measurement are shown in FIG. As shown in Figure 14, the mean residual ellipticity decreased with decreasing concentration of fetuin due to adsorption to OCP.

[0081] Furthermore, as shown in FIG. 15, the secondary structural elements of fetuin in the 0.5Ca0.5Pi buffer and the 3.0Ca1.0Pi buffer were similar before immersion in OCP. As shown in Figures 14 and 15, the secondary structure elements of fetuin were retained after immersion in OCP in 0.5Ca0.5Pi and 3.0Ca1.0Pi buffers. [Explanation of symbols]

[0082] 1 Bone regeneration material manufacturing equipment 2. Reactor 3. Acid / Base Supply Unit 4. Calcium ion supply unit 5. Inorganic phosphate ion supply unit 6. Biofunctional Molecular Supply Division 7. Biofunctional Molecular Concentration Section 8 pH electrode 9. Calcium ion electrode 10 Control section 11~16 Pump 21~25 Piping 31~36 Wiring

Claims

1. A bone regeneration material comprising a composite material comprising a substrate containing octacalcium phosphate and a biofunctional molecule adsorbed to the octacalcium phosphate.

2. The bone regeneration material according to claim 1 , wherein the biofunctional molecule is at least one selected from the group consisting of a growth factor, a serum-derived protein, an antibacterial molecule and an antibacterial compound.

3. The bone regeneration material according to claim 1 , wherein the base material comprises a lactic acid-glycolic acid copolymer.

4. A step of dissolving a biofunctional molecule in phosphate buffered saline to prepare a phosphate buffered saline solution; contacting the phosphate buffered saline solution with a substrate containing octacalcium phosphate to adsorb the biofunctional molecule onto the octacalcium phosphate; and freeze-drying the substrate containing the octacalcium phosphate having the biofunctional molecule adsorbed thereon.

5. A step of dissolving a biofunctional molecule in a buffer solution saturated or supersaturated with respect to octacalcium phosphate to prepare a buffer solution containing the biofunctional molecule; a step of immersing a substrate containing octacalcium phosphate in the buffer solution containing the biofunctional molecule, thereby allowing the biofunctional molecule to be adsorbed onto the octacalcium phosphate; and freeze-drying the substrate containing the octacalcium phosphate having the biofunctional molecule adsorbed thereon.

6. a reaction vessel containing a slurry containing a substrate comprising octacalcium phosphate; an acid / base supply unit for supplying an acid or a base to the slurry in the reaction vessel; a calcium ion supply unit that supplies calcium ions to the slurry in the reaction tank; an inorganic phosphate ion supply unit that supplies inorganic phosphate ions to the slurry in the reaction tank; a biofunctional molecule supplying unit for supplying a biofunctional molecule to the slurry in the reaction tank; a biofunctional molecule concentration unit that recovers and concentrates the biofunctional molecules remaining in the supernatant of the slurry after the biofunctional molecules are adsorbed onto the substrate in the reaction tank, and supplies the biofunctional molecules again to the reaction tank; a pH electrode for measuring the pH of the slurry in the reaction vessel; a calcium ion electrode for measuring a calcium ion concentration of the slurry in the reaction tank; a control unit that calculates a degree of supersaturation of the slurry with respect to octacalcium phosphate based on the pH of the slurry in the reaction tank measured by the pH electrode and the calcium ion concentration of the slurry in the reaction tank measured by the calcium ion electrode, and issues instructions to the acid / base supply unit, the calcium ion supply unit, and the inorganic phosphate ion supply unit to supply an acid or base, calcium ions, and inorganic phosphate ions to the slurry in the reaction tank based on the degree of supersaturation so that the degree of supersaturation of the slurry in the reaction tank becomes a set value.

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