WO2004047881A1 - Calcium phosphate carrying protein, process for producing the same and sustained release protein preparation, artificial bone and tissue enginerring scaffold using the same - Google Patents

Calcium phosphate carrying protein, process for producing the same and sustained release protein preparation, artificial bone and tissue enginerring scaffold using the same Download PDF

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Publication number
WO2004047881A1
WO2004047881A1 PCT/JP2003/015040 JP0315040W WO2004047881A1 WO 2004047881 A1 WO2004047881 A1 WO 2004047881A1 JP 0315040 W JP0315040 W JP 0315040W WO 2004047881 A1 WO2004047881 A1 WO 2004047881A1
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Prior art keywords
calcium phosphate
sintered body
protein
calcium
phosphate
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PCT/JP2003/015040
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French (fr)
Japanese (ja)
Inventor
Atsuo Ito
Yu Sogo
Kazuo Onuma
Noboru Ichinose
Koshiro Fukazawa
Atsushi Yamazaki
Nao Kondo
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National Institute Of Advanced Industrial Science And Technology
Waseda University
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Priority to AU2003284691A priority Critical patent/AU2003284691A1/en
Publication of WO2004047881A1 publication Critical patent/WO2004047881A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds

Definitions

  • the present invention relates to a protein-supporting calcium phosphate, a method for producing the same, and a sustained-release protein using the same, an artificial bone and a tissue-engineered scaffold.
  • the present invention relates to calcium phosphate ceramics carrying non-extracellular matrix proteins, such as water-soluble proteins, especially water-soluble growth factors and water-soluble cell adhesion factors.
  • Bioactivators such as growth factors, cell adhesion factors, other proteins, phospholipids, polysaccharides, hormones, etc. for remodeling, tissue induction, and cell differentiation of living tissues
  • biologically active subs tance biologically active subs tance
  • Examples of a method for supporting a protein on a biomaterial include, for example, a method of supporting a trypsin-treated bovine bone matrix (US Pat. No. 4,563,350), and a method of supporting an inorganic component or bone collagen powder if the supporting substrate is an organic substance. Composition consisting of 0 to 98% (EP
  • Bone tissue consists of calcium phosphate and collagen, and calcium phosphate is widely used in human bone materials. Therefore, for bone tissue, it is faster to rebuild the tissue by supporting the biological activator on calcium phosphate than by allowing the cells to produce calcium phosphate from scratch by supporting the biological activator on organic matter. And efficient.
  • a method of immersing and adsorbing (US 6, 113, 993), A method of immersing and adsorbing a calcium phosphate film formed on titanium by spatula coating in a bone morphogenetic solution (Plastic Recon Surgery 108, 434-) 443 (2001)), hydroxyapatite-1 0: Method of immersing calcium phosphate composite sintered body in protein solution and adsorbing it (J. Mater Sci; Mater in Med, 12, 761-766 (2001)), ⁇ A method of adsorbing calcium phosphate by immersing it in a protein solution (J Biomed Mater Res, 59,
  • the method of supporting a biologically active substance on calcium phosphate is carried in pores of a calcium phosphate ceramic, mixed with calcium phosphate cement, cured, combined with an absorbent polymer, or utilizes an adsorption effect.
  • the amount of the biologically activating substance carried was small and it was difficult to perform sustained release over a long period.
  • a sustained-release body having a strength as high as that of calcium phosphate ceramics could not be produced, and when combined with an absorbent polymer, the operation was complicated.
  • the method of co-precipitating calcium phosphate and protein from an aqueous solution is an effective supporting method, and the method of precipitating on a metal surface such as a polymer or titanium (US 6, 136, 369, US 6, 143, 948, US 6, 344, 061, EP96201293).
  • a metal surface such as a polymer or titanium
  • Patent Document 1
  • Patent Document 2
  • Patent Document 3 U.S. Patent No.6344061
  • Patent Document 4
  • the present invention provides a bioactive substance-supported calcium phosphate firing material that has biocompatibility and can be used as a biological tissue replacement material, tissue engineering skid holder, and protein sustained release body that promotes biological tissue reconstruction.
  • the purpose is to provide union.
  • a method of co-precipitating calcium phosphate and protein using an aqueous solution is to use a stable supersaturated solution such as Hanks' solution that does not cause spontaneous nucleation even though calcium phosphate is supersaturated, and utilizes heterogeneous nucleation. Co-precipitate calcium phosphate and protein on the substrate. However, this method results in a small amount of precipitated calcium phosphate and a small amount of protein that can be carried. The use of an unstable phosphoric acid supersaturated solution that causes spontaneous nucleation can increase the amount of precipitated calcium phosphate, which can be expected to increase the amount of protein carried.
  • the time to spontaneous nucleation is artificially controlled, and the co-precipitation on the calcium phosphate substrate is terminated before spontaneous nucleation is formed in the entire solution, or in the vicinity of the substrate. It is necessary only for spontaneous nucleation.
  • a protein-containing unstable phosphocalcium supersaturated solution that causes spontaneous nucleation is used, and the time until spontaneous nucleation is artificially controlled.
  • the present invention provides a calcium phosphate-based sintered body having an increased amount of protein carried by terminating precipitation or forming spontaneous nuclei only in the vicinity of a substrate to increase the amount of precipitated calcium phosphate. Further, the present invention provides calcium phosphate carrying a biologically activating substance, in which the biocompatibility of the drug used for carrying is high and the carrying out operation is simple.
  • the present invention is as follows.
  • the calcium phosphate is a calcium phosphate in which at least one of carbonic acid, silicon, magnesium, zinc, iron, and manganese is dissolved, (1) the calcium phosphate sintered body,
  • calcium phosphate contains at least one oxide or phosphate of a metal selected from calcium, magnesium, zinc, iron, and manganese
  • the sintered body mainly composed of calcium phosphate according to any one of (1) to (11), wherein the sintered body has a Ca / P molar ratio of 0.75 or more and 2.1 or less.
  • the water-soluble protein that is the biological activator to be supported contains at least one of the J3 ⁇ 4 long factor or the cell adhesion factor (1) to (14). Body,
  • Aqueous solutions include one or more solutions selected from medical infusions, dialysisperitoneal perfusion, infusion correction preparations, calcium preparations, dialysisperitoneal perfusion replenishers (17) a method for producing a protein-supported calcium phosphate main component sintered body using an aqueous solution,
  • a solution containing a calcium component and a solution containing a phosphoric acid component are separately prepared in advance, and co-precipitation of the protein is started by mixing the two, and the protein according to any one of (16) to (18) A method for producing a supported calcium phosphate main component sintered body,
  • the solution containing a phosphate component is one or a mixture of two or more selected from phosphate buffered saline or a medical fluid containing no phosphate-containing calcium.
  • an unstable calcium phosphate supersaturated solution that causes spontaneous nucleation is a solution in which calcium phosphate precipitation occurs spontaneously by spontaneous nucleation. Therefore, a stable calcium phosphate supersaturated solution that is supersaturated with calcium phosphate and does not generate calcium phosphate precipitation, that is, a Hanks solution or a 1-fold concentration simulated body fluid, is not included in an unstable calcium phosphate supersaturated solution that causes spontaneous nucleation.
  • spontaneous nucleation means that the components of the solution help the substrate, foreign substances, and the walls of the container. Without borrowing, spontaneous aggregation and visual turbidity of the solution or in this way growing into particles of 1 xm or more in solution.
  • the particle size of the particles in the solution can be measured by a light scattering method.
  • Sedimentation refers to particles generated by spontaneous nucleation.
  • Co-precipitation means that other substances that should not precipitate by themselves precipitate together with the main precipitate.
  • Precipitation of calcium phosphate means not only the generation of calcium phosphate particles due to spontaneous nucleation, but also the heterogeneous nucleation of calcium phosphate on these surfaces with the help of substrates, foreign substances and the walls of containers. Also includes calcium phosphate formation.
  • Controlled delayed co-precipitation with calcium phosphate means that the time to calcium phosphate precipitation is artificially controlled by selecting the solution composition and temperature, and most or all of the calcium phosphate deposition is on the substrate.
  • This is a coprecipitation method in which highly active calcium phosphate nanoparticles immediately after nucleation are co-precipitated by absorbing or chemically bonding surrounding proteins. Specifically, two or more aqueous solutions are mixed in order to produce an unstable supersaturated calcium phosphate solution, and the time until calcium phosphate precipitation by spontaneous nucleation is 10 seconds or more and 7 days or less from the last solution mixing, Preferably, coprecipitation occurs for more than 2 minutes and less than 2 days.
  • the induction time until spontaneous nucleation is controlled by selecting the chemical composition and temperature of the aqueous solution to optimal values.
  • the waiting time for co-precipitation is determined by preparing a supersaturated calcium phosphate solution in the absence of a substrate, and until the solution becomes cloudy visually or precipitated particles with a particle size of 1 m or more are detected by the light scattering method. is there.
  • the solution from which the calcium phosphate supersaturated solution of the present invention can be prepared is a solution containing at least a calcium component, a solution containing at least a phosphoric acid component, or a solution containing both a calcium component and a phosphoric acid component.
  • These solutions themselves have a spontaneous nucleation induction time It may contain a delay component, or may further contain one or more solutions that control and delay the induction time until spontaneous nucleation. It is preferable that the calcium component and the phosphoric acid component are separately dissolved in different containers before mixing.
  • a solution containing at least a calcium component, a solution containing at least a phosphate component, or a solution containing both a calcium component and a phosphate component are not limited.
  • solutions containing a phosphate component include phosphate buffered saline, phosphate solution, dipotassium hydrogen phosphate solution, potassium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium dihydrogen phosphate Solution and the like.
  • examples of the solution containing a calcium component include a calcium chloride solution, a calcium lactate solution, a calcium acetate solution, a calcium dalconate solution, and a calcium citrate solution.
  • examples of the solution containing both the calcium component and the phosphoric acid component include a stable supersaturated solution such as Hanks' solution and a 1-fold simulated body fluid, and a calcium phosphate unsaturated solution.
  • Examples of such a calcium phosphate unsaturated solution include a solution having a calcium chloride concentration of 2.5 mM, a potassium hydrogen phosphate concentration of 1.0 mM, and a pH of less than 5.
  • a suitable alkaline solution such as a potassium hydroxide solution or a sodium hydrogen carbonate solution, or a chloride or sodium chloride solution.
  • the solution containing calcium component, the solution containing phosphoric acid component, the solution containing calcium component and phosphoric acid component, the solution for controlling spontaneous nucleation induction time, each component solution and the control solution may be one type of solution or different in composition. It may consist of a plurality of types of solutions. The order of mixing these solutions is not particularly limited as long as spontaneous nucleation does not occur during or within less than 10 seconds after mixing.
  • a first type of phosphoric acid component solution is mixed with a first type of spontaneous nucleation induction time control solution, then a calcium component solution is mixed, and then a second type of spontaneous nucleation induction
  • the time control solution can be mixed, and finally the second type of phosphoric acid component solution can be mixed.
  • the calcium component solution can be mixed with the phosphoric acid component solution, and then the first type of spontaneous nucleation induction
  • the time control solution can be mixed, and finally the second spontaneous nucleation induction time control solution can be mixed.
  • the protein may be added and dissolved in any of the above solutions, the protein may be added and dissolved in multiple solutions, or all the solutions may be dissolved. After the liquid is mixed, the protein may be added and dissolved. That is, (1) the protein may be added and dissolved in a solution containing both the calcium component and the phosphate component, or (2) the protein may be added to one or both of the solution containing the calcium component and the solution containing the phosphate component. (3) It may be added and dissolved in a solution that controls the induction time of spontaneous nucleation.
  • the protein to be added and dissolved may be a solid protein or a protein that has already been dissolved in a solution.
  • Co-precipitation of protein and calcium phosphate is started by the above operation.
  • the calcium phosphate main component sintered body is brought into contact, and the protein is co-precipitated and precipitated on the calcium phosphate main component sintered body, and the calcium phosphate carrying the protein is precipitated.
  • a main component sintered body is obtained.
  • a soluble calcium-containing phase such as tricalcium phosphate, tetracalcium phosphate, and calcium oxide may be previously formed on the surface of the sintered body of the main component of calcium phosphate, and the protein may be coprecipitated.
  • the pH at which a solution containing both a calcium component and a phosphate component becomes supersaturated with respect to calcium phosphate depends on the calcium ion concentration and the phosphate ion concentration.For example, the calcium chloride concentration is 2.5 ⁇ . However, when the concentration of potassium hydrogen phosphate is 1. O mM, ⁇ 5 or more. Whether a supersaturated solution is stable or unstable is affected by coexisting ions and ionic strength.
  • a solution containing a sodium component or a potassium component as a main component is preferable.
  • a solution is specifically 50 to 200 mM, preferably 100 to 180 mM, more preferably 1 to 200 mM.
  • a 2 OmM potassium chloride solution can be mentioned.
  • a solution having a calcium chloride concentration of 42.9 and a phosphoric acid concentration of 28.6 mM 80 mL of a 12.5 mM KC1 solution and 20 mM hydroxylating solution are used. Addition of 12 mL of the solution results in a final pH of 7.4, a high biocompatibility, and a calcium phosphate supersaturated solution with a delay in precipitation of 10 minutes.
  • Unstable calcium phosphate supersaturated solutions with a delayed waiting time for precipitation are, specifically, Ca—P—Na—K—C 1 system and Ca_P—Na—K—Mg—C 1—CO 3 system Is an aqueous solution having a specific composition range.
  • the Ca component is 0 to 2.5 mM, preferably 0.8 to 2.5 mM
  • the phosphate component is 1.0 to 20 mM, preferably 1.2. ⁇ 10 mM
  • K component 0 ⁇ 4 OmM preferably 0 ⁇ 2 OmM
  • Na component is more preferably 0 ⁇ 20 OmM Is a solution containing 0 to 15 OmM and a C1 component of 0 to 20 OmM, preferably 0 to 15 OmM.
  • the molar ratio of C a ZP of the solution is not particularly specified, but is preferably in the range of 0.1 to 2.5.
  • C a- P- The N a- K- Mg- C 1 _C0 3 based solution of, C a component 1.. 2 to 2. 75 mM preferably 1. from 39 to 2 33 mM, phosphoric acid ingredient 0. 6: L 5 mM preferably 1.1 to 10 mM, K component 0 to 3 OmM, preferably 4 to 2 OmM, Na component 30 to: L 5 OmM, preferably 40 to 145 mM, 1 ⁇ component 0.1 to 3.
  • OmM preferably Contains 0.2 to 2.OmM, C 1 component 30 to 150 mM, preferably 40 to 145 mM, HCO, component 0 to 60 mM, preferably 0 to 45 mM, pH 5.
  • the CaZP molar ratio of the solution is not particularly specified, but is preferably in the range of 2.5 or less.
  • calcium phosphate unstable supersaturated solution of C a- P- N a- K- Mg- C 1- C0 3 system latency is delayed leading to deposition to medical infusion solution, dialysis, peritoneal perfusate, infusion It can also be prepared by mixing one or more selected from supplements for correction, calcium, dialysis and peritoneal perfusate.
  • These soluble liquid or agents, C a come P, Na, K, Mg, in use as a source of C and C_ ⁇ 3, this case has all the solutions or drugs are already approved for medical , And Since it has been sterilized, it is convenient for use in operating rooms.
  • Electrolyte infusion containing Ca component and no P component, electrolyte infusion containing P component and no Ca component, dialysis A sodium hydrogen replenisher is particularly preferably used, but is not limited thereto.
  • electrolyte infusions that contain Ca component and do not contain P component electrolyte infusions that contain P component and do not contain Ca component
  • sodium chloride infusion sodium bicarbonate replenisher for dialysate
  • sodium bicarbonate replenisher for dialysate include, for example, commercially available Ringer's solution (Otsuka Pharmaceutical Co., Ltd.) , Clinizarts B (Kobayashi Yakue), physiological saline (Otsuka Pharmaceutical), and sodium bicarbonate replenisher for Bayfil (Takeda Yakuhin) may be used.
  • the composition is as described in Examples.
  • the solution to which the protein is added is a solution containing at least the calcium component described above, a solution containing at least the phosphate component, a solution containing both the calcium component and the phosphate component, and controls the induction time until spontaneous nucleation. It may be added to any of the solutions. However, from the viewpoint of preventing protein denaturation, it is desirable to add it to a solution with a pH of 5 or more and 8 or less Byone.
  • the amount of protein to be added at this time varies depending on the type of protein and the use of the sintered body supporting the protein, but it is several g / mL to several g / mL, preferably 10 g / mL and 10 g / mL. is there.
  • the amount supported on the sintered body also varies depending on the type of protein and the use of the sintered body supporting the protein, but it is 0.4 g or more, preferably 1 g or more per square cm. No restrictions.
  • the value of the area for calculating the supported amount per unit area is the area of the sintered body before being supported.
  • the lower limit of the supported amount was determined experimentally. That is, the inventors determined the amount of protein that can be supported on calcium phosphate only by adsorption without coprecipitation and found that 0.333 g of protein could be supported per square cm by adsorption alone (Example 5). ). Therefore, the lower limit of the supported amount was set at 0.1 per square cm.
  • each component solution are not particularly limited as long as the temperature and pH can prevent protein denaturation.
  • most proteins are denatured above body temperature, so the temperature of the solution after mixing with each component solution is desirably 37 ° C or less.
  • the denaturation temperature of proteins differs depending on the type of protein, and is not limited to 37 ° C or lower depending on the protein used.
  • the sintered body containing calcium phosphate as the main component is Calcium phosphate, which is a calcium oxide sintered body, and one containing at least one of carbonic acid, silicon, magnesium, zinc, iron, manganese, and calcium oxide.
  • the calcium phosphate in which the element or carbonic acid is dissolved in a solid solution is a calcium phosphate or silicon in which calcium of calcium phosphate is partially substituted as an impurity if the metal element of magnesium, zinc, iron, or manganese is dissolved.
  • it refers to calcium phosphate in which phosphorus of calcium phosphate is partially substituted as an impurity
  • carbonic acid it refers to calcium phosphate in which the phosphate group of calcium phosphate is partially substituted as an impurity.
  • silicon or carbonic acid forms a solid solution
  • a charge inconsistency between the atom or ion group to be replaced so a secondary solid solution of other elements to make up for this and a void in which no atoms exist in the structure Or a hole site is generated.
  • carbonic acid is hydroxyapatite
  • Carbonic acid forms a solid solution by simultaneous substitution of (C0 3 2 ") and (Ca 2 ⁇ , ⁇ ") or by simultaneous substitution of (H ⁇ , C0 3 2 ") and (Ca 2 ⁇ , ⁇ ).
  • Has a solid solution limit and if silicon, magnesium, zinc, iron, and manganese are to be contained in calcium phosphate beyond the solid solution limit, in addition to calcium phosphate in which these elements are dissolved, oxides of these elements phosphate is generated, a composition comprising an oxide or phosphate of these elements.
  • the solid solubility limit, for example calcium phosphate sinter is cold-type Ca 3 (P0 4) if 2, magnesium, Zinc, iron and manganese are all about 12 mol% of the total calcium.
  • the sintered body containing calcium phosphate as a main component contains one or more bio-essential elements selected from zinc, magnesium, iron, manganese, and silicon to enhance bone formation and other biological functions. Can be added.
  • the content of zinc, iron, manganese, and silicon after sintering is at least 1 times the concentration of zinc, iron, manganese, and silicon in bone.10
  • Bone contains zinc, iron, manganese, and silicon in a concentration of zinc: 0.
  • the content of essential biological elements is less than 1 times that of bone, the biological function promoting action unique to these elements Cannot be demonstrated in the bone. If the content is 100 times or more, these elements are excessive in both artificial bone used in bone tissue and tissue engineering skidfold used in cell culture solution, and toxicity is exhibited. When the content is 25 times or more and 100 times or less, toxicity occurs in bone tissue, but no toxicity occurs in a cell culture medium.
  • the content of magnesium after sintering should be between 1 and 50 times the magnesium content of bone.
  • the magnesium content of the bone is between 0.26% by weight and 0.55% by weight. If the magnesium content is less than 1 times the bone content, the biological function promoting effect of magnesium cannot be exerted in bone.
  • the reason why the upper limit of the magnesium content is set to 50 times the bone concentration unlike other essential biological elements is that the bone content of magnesium is 50 times larger than other essential biological elements.
  • the molar number of magnesium becomes larger than the molar number of the calcium hydroxide after sintering, and calcium phosphate is not the main component.
  • Zinc, magnesium, iron, manganese, silicon, and calcium may be dissolved in calcium phosphate, which is the raw material powder before sintering, or mixed as inorganic salts, metals, oxides, hydroxides, and organometallic compounds. You may let it.
  • inorganic salts, metals, oxides, hydroxides, and organometallic compounds are mixed in advance, these elements react with calcium phosphate during sintering to form a solid solution.
  • the amount of the mixed element is equal to or higher than the solid solution limit, the metal oxide or phosphate is generated after sintering in addition to the element-dissolved calcium phosphate.
  • a carbonate or a nitrate in which an anion group is volatilized during sintering, is preferable.
  • Metal chlorides, fluorides and sulphates cannot be used because of the residual chlorine, fluorine and sulphate groups which are poorly biocompatible during sintering.
  • the Ca / P molar ratio of the entire sintered body mainly composed of calcium phosphate is 0.75 or more and 2.1 or less, preferably 1.1 or more and 1.9 or less. Even if the Ca / P molar ratio is in the range of 1.5 or less, a calcium phosphate sintered body containing impurities selected from carbonic acid, silicon, magnesium, zinc, iron, and manganese, for example, a calcium phosphate sintered body containing magnesium In the case of a body, a mixture of magnesium-dissolved tricalcium phosphate and trimagnesium phosphate can be obtained after sintering, and the formation of calcium pyrophosphate having low biocompatibility can be prevented.
  • the Ca / P molar ratio is less than 0.75, impurities selected from carbonic acid, silicon, magnesium, zinc, iron, and manganese are added. In this case, even if the formation of calcium pyrophosphate can be prevented, the number of moles of these impurity components is larger than the number of moles of calcium, so that the sintered body is not a calcium phosphate sintered body. Therefore, the lower limit of the Ca / P molar ratio of the calcium phosphate sintered body was set to 0.75. When the Ca / P molar ratio is 2.1 or more, calcium oxide is generated at a level exceeding the toxic limit, and the biocompatibility of the sintered body is impaired. Therefore, the upper limit of the Ca / P molar ratio of the calcium phosphate sintered body was set to 2.1.
  • the Ca / P molar ratio of the calcium phosphate sintered body not containing carbonic acid, silicon, magnesium, zinc, iron, and manganese is desirably 1.5 or more and 2.0 or less.
  • Specific examples of such a calcium phosphate sintered body include hydroxyapatite having a Ca / P molar ratio of 1.5 or more and 2.0 or less, / 3 tricalcium phosphate, tricalcium phosphate, and the like.
  • a sintered body in which calcium oxide is mixed may be used. These compounds may be of stoichiometric or non-stoichiometric composition.
  • the calcium phosphate sintered body containing carbonic acid examples include a hydroxyapatite sintered body in which carbonic acid is dissolved.
  • the calcium phosphate sintered body containing silicic acid examples include hydroxyapatite in which silicic acid is dissolved, tricalcium phosphate in which silicic acid is dissolved, and a mixed sintered body of these, and calcium silicate or silicic acid.
  • the added sintered body can be mentioned.
  • calcium phosphate sintered body containing magnesium, zinc, iron, and manganese, in addition to hydroxyapatite, tetracalcium phosphate, and tricalcium phosphate in which these metal ions are dissolved, these metal oxides and phosphates are added.
  • Calcium phosphate As a calcium phosphate sintered body containing magnesium, zinc, iron, and manganese, in addition to hydroxyapatite, tetracalcium phosphate, and tricalcium phosphate in which these metal ions are dissolved, these metal oxides and phosphates are added.
  • calcium oxide in the sintered body elutes and raises the pH near the surface of the sintered body, thereby increasing the degree of supersaturation near the surface of the sintered body.
  • This is particularly effective for forming the target in a uniform manner.
  • calcium oxide is present in the sintered body in an amount of 0.1% to 4% by weight, preferably 0.1% to 3.5% by weight, more preferably 0.1% to 3.5% by weight. It can contain 1.8% by weight. If the calcium oxide content is less than 0.1% by weight, the effect of increasing the pH is small, so the lower limit of the calcium oxide content was set to 0.1% by weight. Calcium oxide Reacts with moisture in the air, so that if the content is large, the strength of the sintered body decreases. When the content of calcium oxide is more than 4% by weight, the strength of the sintered body is remarkably reduced, so the upper limit is set to 4% by weight.
  • calcium oxide can be contained only in the surface layer of at least 1 micron from the surface of the sintered body.
  • the reason for setting the lower limit of the depth from the surface to 1 micron is that the particle size of the calcium phosphate sintered body is usually 1 micron to 10 microns, and the outermost surface layer has a thickness of 1 micron or less. This is because they cannot be divided.
  • tricalcium phosphate and tetracalcium phosphate are also effective for increasing the degree of supersaturation near the surface of the sintered body and for forming precipitates intensively on the substrate surface. That is, these calcium phosphates are converted into low-crystalline hydroxyapatite by hydrolysis, and the degree of supersaturation near the surface of the sintered body increases due to release of calcium during the hydrolysis reaction. Highly active, low-crystalline hydroxyapatite produced by hydrolysis also takes up proteins in solution.
  • the content of tricalcium phosphate and tetracalcium phosphate in the sintered body is 90% by weight or more and 20% by weight or less, preferably 95% by weight or more and 15% by weight or less.
  • the inclusion of tricalcium phosphate and tetracalcium phosphate only in the surface layer of at least 1 micron from the surface of the sintered body reduces the strength due to the reaction with moisture in the air. It is preferable from the viewpoint of preventing the occurrence of the above.
  • the reason for setting the lower limit of the depth from the surface to 1 micron is that the particle size of the calcium phosphate sintered body is usually from 1 micron to 10 microns, and the outermost surface layer is divided into a thickness of 1 micron or less. Because it cannot be split.
  • ⁇ -tricalcium phosphate and tetracalcium phosphate may be distributed throughout the sintered body.
  • the extent to which calcium oxide, tricalcium arsenate or tetracalcium phosphate is present from the surface can be determined, for example, by X-ray microscopy. It can be confirmed by observing the cross section with a scanning electron microscope or a transmission electron microscope equipped with a chromaanalysis device.
  • the supported protein is a water-soluble protein, and a biological activator can be used.
  • Water-soluble proteins include water-insoluble proteins such as albumin and other water-soluble carrier proteins or copolymers of polyethylene glycol, ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and poly-1. Water-soluble by binding to water-soluble polymers such as 1,3-dioxolane, poly-1,3,6-trioxane, ethylene Z maleic anhydride copolymer, polyamino acids (homopolymer or random copolymer) Also includes sex proteins.
  • the binding between the water-insoluble protein and the above-mentioned water-soluble carrier protein or water-soluble polymer may be achieved by utilizing the functional groups of both substances, and can be carried out by various known methods.
  • the biologically activating substance refers to a substance having biological activity on an organism, that is, a substance capable of inducing some change in an organism by acting on the organism.
  • Biological activators include bioactive substances such as cytokins and hormones that can regulate the living body and change the function of the living body, such as growth factors and cell adhesion factors.
  • examples of water-soluble and non-bioactive proteins include albumin, cytochrome C, globulin, and the like.
  • water-soluble and biologically active proteins examples include basic fibroblast growth factor, IL-1 (interleukin 1), IL-2, IL_3, IL-4, IL -5, IL-6, IL_7, IL-8, IL-9, IL-10, IL-11, IL-12,
  • IL-13 IL-15
  • IL-17 IL-18
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • erythropoietin erythropoietin
  • CSF-1 colony stimulating factor
  • SCF Stem Cell Factor
  • Thrombopoietin EGF
  • Epidermal Growth Factor Epidermal Growth Factor
  • TGF-H Transforming Growth Factor-H
  • HB-EGF Heparin-binding EGF-like Growth Factor
  • Epiregulin Neuregulin 1, 2, 3, PDGF (platelet-derived growth factor), insulin, HGF
  • Hepatocyte growth factor VEGF (vascular endothelial growth factor), NGF (nerve growth factor), GDNF (glial cell line-derived neurotrophic factor), mitdocaine, TGF- / 3 (transforming growth factor-) 6) , Betadarican, activin, BMP (bone morphogenetic factor), TNF (tumor necrosis factor), IFN-a / jS (interferon- ⁇ /) IFN-r (inyuichi-feron-a), Examples include, but are not limited to, ibronectin, laminin, cadherin, integrins, selectins, and the like. Collagen and gelatin, which are proteins constituting the connective tissue of animal cells, are not included in the biologically activating factor of the present invention.
  • the calcium phosphate sintered body may be a dense sintered body or a porous sintered body.
  • a biologically active protein is carried on a porous sintered body, regenerated tissues such as blood vessels, bones, epithelium, and nerves are allowed to penetrate into the pores of the sintered body after the sintered body is embedded in the body. It can be very effective. If the porosity is lower than 20%, it is practically dense and unsuitable for tissue penetration. If the porosity is higher than 80%, the strength is reduced and the practical value is impaired. Therefore, the porosity of the porous calcium phosphate sintered body was set to 20% or more and 80% or less.
  • the porosity can be measured by the following method. That is, the outer dimensions and weight of the porous sintered body are measured, and the volume V (cm 3 ) and the weight W (g) are determined.
  • P (1-P (W / (VxD)) x 100.
  • the weight is 1.0704 g
  • the pores penetrate in a three-dimensional network. If it is a porous hydroxyapatite sintered body, the theoretical density value of hydroxyapatite 3.16 g
  • the minimum stomatal diameter was set to 70 microns. Since the necessity of penetrating tissue larger than 4 mm in diameter is practically negligible, the upper limit of the pore diameter was set to 4 mm. The pore diameter can be measured by observing the cross section of the porous sintered body using an optical microscope or a scanning electron microscope.
  • the pores penetrate the sintered body, they can penetrate blood vessels and nerves. If the pores penetrate in a three-dimensional network, it is convenient for capillaries and bone tissue to penetrate in a three-dimensional network.
  • the raw material powder of the sintered body containing calcium phosphate as a main component includes calcium phosphate powder, calcium carbonate, carbonic acid, silicon, magnesium, zinc, iron, and manganese. Calcium phosphate powder containing or dissolving at least one of the components can be used.
  • the Ca / P molar ratio of the calcium phosphate raw material powder not containing carbonic acid, silicon, magnesium, zinc, iron and manganese is desirably 1.5 or more and 2.0 or less.
  • Specific examples of such calcium phosphate powder include hydroxyapatite having a Ca / P molar ratio of 1.5 or more and 2.0 or less, tricalcium phosphate, tetracalcium phosphate, amorphous calcium phosphate, and the like.
  • powders having a Ca / P molar ratio of 1.5 or less or 2.0 or more in these single powders or mixtures specifically, calcium hydrogen phosphate, calcium glycerate calcium, and calcium metal Calcium oxide, calcium carbonate, calcium lactate, calcium citrate, calcium nitrate, calcium salt such as calcium alkoxide, or a mixture of ammonium phosphate, phosphoric acid, etc. Good.
  • These compounds may be of stoichiometric or non-stoichiometric composition.
  • Examples of the calcium phosphate raw material powder containing carbonic acid include hydroxyapatite in which carbonic acid is dissolved, amorphous calcium phosphate in which carbonic acid is dissolved, and a mixture thereof, and phosphorus to which sodium carbonate, potassium carbonate, and ammonium carbonate are added. Acid calcium powder can be mentioned.
  • calcium phosphate powder containing silicic acid examples include hydroxyapatite in which silicic acid is dissolved, amorphous calcium phosphate in which silicic acid is dissolved, triphosphate calcium in which silicic acid is dissolved, and mixtures thereof.
  • Calcium silicate powder to which calcium silicate or silicic acid is added can be given.
  • Examples of calcium phosphate powder containing magnesium, zinc, iron, and manganese include hydroxyapatite, amorphous calcium phosphate, tetracalcium phosphate, and tricalcium phosphate in which these metal ions are dissolved, as well as metals and metal oxides. Phosphate powder to which a substance, a hydroxide, a phosphate, a nitrate, or a carbonate is added. Metal chlorides, fluorides, and sulfates cannot be used because sintering results in the retention of poorly biocompatible chlorine, fluorine, and sulfate groups.
  • the raw material powder of the sintered body containing calcium oxide is a phosphorous compound that generates calcium oxide at a high temperature so that the total Ca / P molar ratio exceeds 1.67. It can be manufactured by mixing with calcium acid and sintering. Specific examples of such calcium compounds include calcium nitrate, carbonate, lactate, citrate, hydroxide, and chelates. In order to contain calcium oxide only on the surface, for example, these aqueous solutions may be applied to a molded body before sintering.
  • the particle size of the raw material powder of the sintered body containing calcium phosphate as a main component is not particularly limited, but is preferably from about 0.1 lim to ⁇ .
  • a molded body is prepared by adding a binder such as polyvinyl alcohol to these raw material powders and, in some cases, further including a solvent such as water or alcohol.
  • the sintering step is performed at 500 ° C. or more and 1500 ° C. or less, preferably at 700 ° C. or more and 1400 ° C. or less in an air atmosphere using a normal electric furnace. Below 500 ° C, sintering does not occur, and above 1500 ° C, many calcium phosphates decompose.
  • the optimum sintering temperature depends on the chemical composition of the calcium phosphate powder to be sintered.
  • the optimum sintering temperature of hydroxyapatite containing 3 to 15% by weight of carbonic acid is from 600 ° C to 800 ° C.
  • Hydroxyl Apatai Bok (Ca 1Q (P0 4) 6 (OH) 2: C aZP molar ratio 1.6 7) optimal sintering temperature of 90 0 ° C over 1
  • the optimum sintering temperature of the composite sintered body of hydroxyapatite and tricalcium arsenate is not less than 110 ° C and not more than 1200 ° C.
  • the optimum sintering temperature of the composite sintered body of hydroxyapatite and calcium oxide is not less than 110 ° C and not more than 1200 ° C.
  • the optimum sintering temperature of hydroxyapatite containing silicon is 900 ° C or higher and 1200 ° C or lower.
  • the optimum sintering temperature for tricalcium phosphate is between 1200 ° C and 1500 ° C.
  • the optimum sintering temperature of ⁇ -tricalcium phosphate containing zinc, manganese and magnesium is 900 ° C or more and 1200 ° C or less.
  • the optimum sintering temperature of tricalcium phosphate containing zinc, manganese, and magnesium is between 130 ° C and 1500 ° C.
  • coprecipitation can be confirmed visually or at the nanometer level using dynamic light scattering.
  • the coprecipitate can be evaluated by powder X-ray diffraction and scanning electron microscope observation.
  • the amount of protein can be quantified by colorimetric analysis combining the burette method with Bicinchonic Acid.
  • Sustained release of protein is tan
  • the calcium phosphate sintered body carrying the protein can be immersed in physiological saline, and the amount of protein in the saline can be quantified and evaluated over time.
  • the sustained release of the protein comprising the protein-supported calcium phosphate sintered body of the present invention varies depending on the type of the protein supported and the use of the protein-supported sintered body.
  • the sustained release can be adjusted by changing the amount of calcium phosphate to be coprecipitated, and it can be adapted to various uses.
  • the sintered body mainly composed of calcium phosphate having the water-soluble protein supported on the surface of the present invention can be used as a biomaterial such as an artificial bone.
  • Biological activators which are proteins carried on the surface, are useful for biological tissue reconstruction. Also, since the carried protein is gradually released, it can be used as a sustained-released evening protein.
  • a sintered body supporting the protein of the present invention may be embedded in a living body and used. Further, the sintered body supporting the protein of the present invention can be used as a tissue engineering scanner. That is, cells can be cultured on a sintered body to form human tissues and organs such as skin and bone.
  • FIG. 1 is a graph showing the amount of co-precipitated cytochrome C from a solution of a Ca—P—Na—K—C1 system unstable calcium phosphate supersaturated solution.
  • FIG. 2 is a diagram showing an X-ray diffraction pattern of the surface of a dense hydroxyapatite-calcium oxide composite sintered body supporting cytochrome C.
  • KC1 0 to 0.1192 g was added to ultrapure water, and the mixture was dissolved by stirring with a stirrer for 30 minutes to prepare a 0 to 2 OmM KC1 solution. Then add 50 mM H 3 PO The solution was added. Then l O OmM C aC l 2 solution was added, and slowly added dropwise 20 mM KOH solution was adjusted to pH 7. 4, (3 & ( 31 2 Concentration 5111] ⁇ ,?
  • This solution was called a “CaP solution.”
  • Basic fibroblast growth factor (bFGF) was dissolved in ultrapure water, trishydroxymethylaminomethane was added (5 mM;), and 12.5 ⁇ 15 OmgZL of Tris-buffered protein solution
  • the CaP solution and the protein solution were mixed at a ratio of 1.
  • the growth of the particle size was monitored by dynamic light scattering.
  • KC 1 component 0 ⁇ 5 OmM, C a C 1 2 -component 1. 17 ⁇ 2. 5mM, H 3 P_ ⁇ 4 components
  • KC 1 component 2.
  • KH 2 P_ ⁇ four components 1. 46 mM, N a C 1 component 1 36.
  • the dense sintered body was produced by uniaxially pressing various calcium phosphate powders having a particle size of 7 to which 3% polyvinyl alcohol was added at 10 OMPa and sintering at 1100 ° C to 1200 ° C.
  • the size after sintering was 13 to 14 mm in diameter, 1 to 1.2 mm in thickness, and the relative density was 90 to 95%.
  • the surface area of dense sintered body 3. was 06 ⁇ 3. 60 cm 2.
  • the porous sintered body was manufactured by the following method.
  • the above operation was repeated four times. After the pressure molding, all the long pillar-shaped bosses were extracted to form pores. This was dried at room temperature for 2 days, and sintered at 700 to 1170 for 5 hours to obtain a porous body.
  • the porous body had pores in which straight through pores with a diameter of 400 / m were alternately orthogonalized, and pores with a diameter of 50 to 200 m were formed at the intersection of the pores in the two directions.
  • a porous sintered body having a weight approximately equal to that of the dense sintered body was cut out and used. The porosity is 60-64%. All of these sintered bodies were subjected to dry heat sterilization at 160 ° C for 1 hour.
  • CaP solution used for loading was CaCl 2 component 2.1 mM, H 3 P0 4 component 1.4 mM, KC1 component
  • cytochrome basic fibroblast growth factor bFGF
  • laminin proteins that are calcium-free magnesium-free phosphate buffered saline (PBS (—)), that is, KC 1 component 2 68 mM, KH PC ⁇ component 1.46 mM, NaC 1 component 1 36.89 mM, Na 2 HP ⁇ 4 component
  • the sterilized sintered body was placed in a 24-well plate for cell culture, and 1 mL of CaP solution and 1 mL of protein-containing PBS (-) solution were placed therein. Leave in nitrogen atmosphere for 7 days and release After the incubation, the solution was recovered, the amount of protein was quantified, and the amount of protein carried on the sintered body was determined (Table 2). As a result, it was found that all of the sintered bodies supported the protein. In particular, the amount of the porous sintered body and the sintered body containing calcium oxide were large.
  • Table 1 Table 1 Calcium phosphate main component sintered body used for supporting protein
  • HAP / o TCP dense 20 cytochrome C 2.5 0.7
  • the protein was dissolved in an aqueous solution containing no calcium, and was supported on a sintered body containing calcium oxide that releases calcium.
  • the sintered body has a diameter of 13 mm, a thickness of lmm and a surface area of 3.06 cm 2 . That is, cytochrome C was added to calcium-free magnesium-free phosphate buffered saline (PBS (—)), namely, KC 1 component 2.68 mM, KH1 P4 4 component 1.46 mM, NaC 1 component 1 36. 89mM, ⁇ & 2 ⁇ 4 ingredients
  • Examples of the sintered body that releases calcium ions in water include the dense hydroxyapatite-calcium oxide composite sintered body of Example 3, zinc-containing hydroxyapatite-calcium oxide composite sintered body, and hydroxyapatite- ⁇ -phosphate.
  • the dense hydroxyapatite sintered body of Example 3 and zinc-containing hydroxyapatite The union was used. The sterilized sintered compacts were placed in a 24-well plate for cell culture, and 2 mL of a cytochrome C-containing PBS (-) solution was placed therein.
  • the dense protein-supported calcium phosphate sintered body obtained in Example 3 was washed with calcium-free magnesium-free phosphate buffered saline (PBS (-)) and placed in a 24-well petri dish for cell culture.
  • PBS (-) calcium-free magnesium-free phosphate buffered saline
  • 1.5 mL of physiological saline (0.9 wt% saline) was added dropwise thereto, and the mixture was allowed to stand at 37 ° C under a nitrogen atmosphere.
  • the physiological saline was collected over time, the amount of protein was quantified, and the sustained release rate of protein was determined (Table 4). It was shown that any of the sintered bodies can release protein slowly and can be used as a protein sustained release body. It was also found that the sintered body containing calcium oxide had a slower release rate than other sintered bodies.
  • Table 6 shows the molar ratio of CaZP, the concentration of NaHC ⁇ 3 , and the amount of co-precipitated cytochrome C in the solution in which calcium phosphate spontaneously formed nuclei at room temperature and co-precipitation of cytochrome C occurred.
  • Table 7 shows that calcium phosphate spontaneously nucleated at 37 ° C, Shows the amount of cytochrome C co-precipitated in the solution in which chromium C co-precipitation occurred. That is, it was shown that these solutions were unstable supersaturated solutions of calcium phosphate, and were solutions capable of co-precipitating proteins.
  • Solution P Calcium-free phosphate-containing medical electrolyte infusion
  • C a solution calcium-containing phosphate-free medical electrolyte infusion
  • Carbonate solution diafiltration type artificial kidney dialysate replenisher for sodium bicarbonate
  • Table 6 Table 6 C aP-Na-K- Mg-C ⁇ - C_ ⁇ protein co ⁇ from 3 system unstable calcium phosphate over saturated. Temperature is room temperature.
  • the calcium-free phosphate-containing medical electrolyte infusion and the calcium-containing phosphate-free medical electrolyte infusion whose composition is shown in Table 5 were mixed so that the Ca / P molar ratio was 1.5.
  • This NaHCO 3 concentration were mixed diafiltration artificial kidney dialysis solution dedicated bicarbonate Natoriumu replenisher such that 1 5 mM, was prepared a supersaturated calcium phosphate solution.
  • a 250 g / mL saline solution (154 mM NaCl solution) of cytochrome C was mixed with this solution at a volume ratio of 9: 1, and a calcium phosphate sintered body was added thereto. It was left still.
  • the base material used was a hydroxyapatite-calcium oxide composite sintered body with a diameter of 13 mm and a thickness of 1 mm, with the calcium oxide content varied from 0.00 to 1.65% by weight. Surface area of the sintered body is 3. 0 6 cm 2.
  • Table 8 shows the relationship between the amount of protein co-precipitated in the process of depositing apatite from the unstable supersaturated solution on the surface of the substrate and the calcium oxide content of the substrate.
  • the present invention is a sintered body mainly composed of calcium phosphate carrying a protein which is a biologically activating substance, has biocompatibility, and promotes the reconstruction of a biological tissue by the action of the carried protein. It can be used for biomaterials such as artificial bones, tissue engineering scanners, etc. Furthermore, as shown in the examples, the supported protein exhibits a sustained release property, so that it can be used as a sustained release body.

Abstract

It is intended to provide sintered calcium phosphate carrying a protein which has a high biocompatibility with a drug to be used, by which the protein can be strongly carried in an increased amount owing to enhanced calcium phosphate deposition and which can sustainedly release the protein over a prolonged period of time. Sintered calcium phosphate wherein, using a persaturated solution of unstable calcium phosphate containing a protein capable of undergoing spontaneous nucleation, the period of time until the spontaneous nucleation is artificially controlled so that coprecipitation/deposition on a calcium phosphate substrate is completed before the spontaneous nucleation of the whole solution, or the spontaneous nucleation is effected exclusively around the substrate to thereby increase the amount of the deposited calcium phosphate, thereby increasing the amount of the protein carried thereon.

Description

タンパク質担持リン酸カルシウム、 その製造方法及びそれを用いた ダンパク質徐放体、 人工骨及び組織工学スキヤフォールド 技術分野  TECHNICAL FIELD The present invention relates to a protein-supporting calcium phosphate, a method for producing the same, and a sustained-release protein using the same, an artificial bone and a tissue-engineered scaffold.
本発明は、 水溶性タンパク質特に水溶性成長因子や水溶性細胞接着因子のよう な、 非細胞外基質性タンパク質を担持したリン酸カルシウムセラミックスに関す 明  The present invention relates to calcium phosphate ceramics carrying non-extracellular matrix proteins, such as water-soluble proteins, especially water-soluble growth factors and water-soluble cell adhesion factors.
る。 本発明によって得られるタンパク質担持リン酸カルシウム主成分セラミック 田 You. Protein-supported calcium phosphate-based ceramic obtained by the present invention
スは、 生体適合性を有しており、 生体組織再構築を促進する生体組織代替材料、 書 Is a bio-compatible, bio-tissue replacement material that promotes tissue reconstruction
組織工学スキャホールド、 タンパク質徐放体として利用される。 背景技術 Used as tissue engineering scaffolds and sustained-release proteins. Background art
生体組織の再構築、 組織誘導、 細胞分化を図るために成長因子、 細胞接着因子 その他のタンパク質、 リン脂質、 多糖類、 ホルモン等の生物学的活性化物質 Bioactivators such as growth factors, cell adhesion factors, other proteins, phospholipids, polysaccharides, hormones, etc. for remodeling, tissue induction, and cell differentiation of living tissues
(biological ly act ive subs tance) が使用されていた。 これらの活性化物質はそ のまま生体投与されるほか、 何らかの生体材料等に担持させて、 場合によっては さらにこれら担持材料から徐放させて使用されていた。 (biologically active subs tance) was used. These activators have been used as they are administered to living organisms as they are, supported on some kind of biological material or the like, and in some cases, further released from these supported materials.
タンパク質を生体材料に担持する方法としては、 例えば、 担持基材が有機物で あれば、 トリプシン処理された牛の骨マトリックス （US 4, 563, 350) に担持する 方法、 無機成分あるいは骨コラーゲン粉末 6 0〜 9 8 %からなる組成物 （EP Examples of a method for supporting a protein on a biomaterial include, for example, a method of supporting a trypsin-treated bovine bone matrix (US Pat. No. 4,563,350), and a method of supporting an inorganic component or bone collagen powder if the supporting substrate is an organic substance. Composition consisting of 0 to 98% (EP
309， 241)に担持する方法、再構成されたコラーゲン (US 3, 394, 370、 US 4, 172, 128) に担持する方法、 ポリマーマトリックスに担持する方法 （特開 2001-131086、 特 開平 2001-122800) などがある。 さらに、 タンパク質をゲル形成する高分子中に 保持する方法 （US 6, 410， 645)、 7 0〜8 0 k Da のアロエベラ由来多糖類を主成 分とする組成物と複合化させる方法 （US 6， 395, 311)、 ハイドロゲル又はバッグの 中に保持して徐放させる方法 （特開 2000- 178180)、 乳酸及び/又はグリコール酸 と P-ジォキサンとポリエチレングリコールとを主成分として反応させた共重合 体に含有させる方法 （特開 2000 - 237297)、 乳酸 Zグリコール酸共重合体に含有 させる方法 （J BiomedMater Res, 57, 291-299 (2001) ) などが、 開示されている。 骨組織はリン酸カルシウムとコラーゲンからなり、 リン酸カルシウムは広く人 ェ骨材料に使用されている。 したがって、 骨組織に対しては、 リン酸カルシウム に生物学的活性化物質を担持して組織再構築する方が、 有機物に生物学的活性化 物質を担持して細胞にリン酸カルシウムをゼロからつくらせるより早くて効率的 である。 309, 241), a method of supporting reconstituted collagen (US 3, 394, 370, US 4, 172, 128), a method of supporting a polymer matrix (Japanese Patent Application Laid-Open No. 2001-131086, Tokuhei 2001) -122800). Furthermore, a method of retaining a protein in a polymer that forms a gel (US Pat. No. 6,410,645), and a method of complexing with a composition containing a 70 to 80 kDa polysaccharide derived from aloe vera as a main component (US 6, 395, 311), a method of sustained release by holding in a hydrogel or bag (Japanese Patent Laid-Open No. 2000-178180), in which lactic acid and / or glycolic acid, P-dioxane and polyethylene glycol are reacted as main components Method for including in copolymer (JP-A-2000-237297), including in lactic acid-Z glycolic acid copolymer (J Biomed Mater Res, 57, 291-299 (2001)) and the like. Bone tissue consists of calcium phosphate and collagen, and calcium phosphate is widely used in human bone materials. Therefore, for bone tissue, it is faster to rebuild the tissue by supporting the biological activator on calcium phosphate than by allowing the cells to produce calcium phosphate from scratch by supporting the biological activator on organic matter. And efficient.
そのような観点から、 リン酸カルシウム質生体材料にタンパク質を担持させる 方法としては、 水酸ァパタイトゃ i8リン酸三カルシウム多孔質焼結体の気孔中に 成長因子を物理的に保持する方法 （US 6, 346, 123、 Cl in Orthop, 187, From such a viewpoint, as a method of supporting a protein on a calcium phosphate biomaterial, a method of physically retaining a growth factor in pores of a hydroxyapatite ゃ i8 tricalcium phosphate porous sintered body (US 6, 346, 123, Cl in Orthop, 187,
277-280 (1983) , J Biomed Mater Res 49， 415-421 (2000) ) , リン酸三カルシウム や石膏に接触させて担持する方法 （特開平 2001-131086、 特開平 2001- 122800)、 水酸ァパタイト多孔体や顆粒に吸着させて担持する方法 （Biochem Biophys Res277-280 (1983), J Biomed Mater Res 49, 415-421 (2000)), a method of contacting and supporting tricalcium phosphate or gypsum (JP-A-2001-131086, JP-A-2001-122800), Method for adsorbing and supporting on apatite porous materials and granules (Biochem Biophys Res
Co匪 un, 193, 509-517 (1993) . Biomaterials, 17, 703-709 (1996) , Cl in Orthop 268,Co material un, 193, 509-517 (1993) .Biomaterials, 17, 703-709 (1996), Cl in Orthop 268,
303-312 (1991) , J. Mater Sci iMater in Med, 12, 293-298 (2001) , J Biomed Mater303-312 (1991), J. Mater Sci iMater in Med, 12, 293-298 (2001), J Biomed Mater
Res, 41, 405-411 (1998) )、 水酸アパタイト顆粒にコラーゲンゲルを添加して骨 形成因子を粘着固定する方法 （Biomaterials, 22, 1643-1651 (2001) ) , 水酸ァパ タイトと生体崩壊性ポリマーを含む多孔性複合体に生理活性物質をさらに添加し た組成物 0JS 5, 766, 618) とする方法、 M0CVD 法で金属上に形成したリン酸カル シゥム被膜をタンパク質溶液中に浸漬して吸着させる方法 （US 6, 113, 993)、 スパ ッ夕コ一ティングでチタン上に形成したリン酸カルシウム被膜を骨形成因子溶液 に浸漬して吸着させる方法 (Plast ic Recon Surgery 108, 434-443 (2001) ) , 水酸 ァパタイ卜一 0；リン酸カルシウム複合焼結体をタンパク質溶液に浸潰して吸着さ せる方法 (J. Mater Sci ;Mater in Med, 12, 761-766 (2001) )、 《リン酸カルシゥ ム多孔体をタンパク質溶液に浸漬して吸着させる方法 （J Biomed Mater Res, 59,Res, 41, 405-411 (1998)), a method of adhesively fixing bone morphogenetic factors by adding collagen gel to hydroxyapatite granules (Biomaterials, 22, 1643-1651 (2001)), A method in which a bioactive substance is further added to a porous composite containing a biodegradable polymer 0JS 5, 766, 618), and a calcium phosphate coating formed on metal by the M0CVD method is added to a protein solution. A method of immersing and adsorbing (US 6, 113, 993), A method of immersing and adsorbing a calcium phosphate film formed on titanium by spatula coating in a bone morphogenetic solution (Plastic Recon Surgery 108, 434-) 443 (2001)), hydroxyapatite-1 0: Method of immersing calcium phosphate composite sintered body in protein solution and adsorbing it (J. Mater Sci; Mater in Med, 12, 761-766 (2001)), 《 A method of adsorbing calcium phosphate by immersing it in a protein solution (J Biomed Mater Res, 59,
422-428 (2002) ) , リン酸八カルシウム顆粒を骨形成因子溶液に浸漬後、 凍結乾燥 して担持する方法 (J. Biomed Mater Res, 57, 175-182 (2001) ) , 非晶質リン酸力 ルシゥム、 軟骨形成細胞又はその前駆細胞、 水分又は生理活性物質を含む水分を 複合化し、 硬化させて生理活性物質を保持する方法 （US 6, 277, 151)、 リン酸カル シゥムを含む微粒子表面に薬剤を被覆する方法 （US 5， 958， 458、 M 0695/94)、 生 体吸収性ポリマーや生体吸収性リン酸カルシウムと成長因子を混合して、 金属等 の多孔性インプラントの気孔内に保持する方法 （US 5， 947， 893)、 リン酸カルシゥ ムセメントにタンパク質や生理活性物質や薬剤を混合して硬化させ、 タンパク質 や生理活性物質や薬剤を保持する方法 （US 5， 782， 971、 特開平 9-225020、 特開平 4-248774、特開平 6- 228011、特開平 7-31673, Biomed Mater Eng, 4, 291-307 (1994) , Cl in Ort o Related Res. , 367S, S396-S405 (1999) , J Periodont, 71, 8 - 13 (2000)、 Bioiaterials, 23, 1261-1268 (2002) , J. Biomed Mater Res, 59, 265-271 (2002) ) , 硬化後のリン酸カルシウムセメントに吸収させる方法（J. Biomed Mater Res. , 44, 168-175 (1999) )、 分極処理したリン酸カルシウム上に選択吸着させる方法 （特開 2001 - 187133)、カルシウム成分及び増粘剤が配合されたペーストに混合して骨形 成促進物質を担持させる方法 （特開平 2001-106638) 等が知られている。 422-428 (2002)), immersion of octacalcium phosphate granules in osteogenic factor solution, freeze-drying and supporting (J. Biomed Mater Res, 57, 175-182 (2001)), amorphous phosphorus Acidic calcium, chondrogenic cells or their precursor cells, a method of complexing water or water containing a physiologically active substance and curing it to retain the physiologically active substance (US 6, 277, 151), fine particles containing calcium phosphate Method of coating drugs on surfaces (US 5, 958, 458, M 0695/94), raw A method in which a bioabsorbable polymer or bioabsorbable calcium phosphate is mixed with a growth factor and held in the pores of a porous implant such as a metal (US Pat. No. 5,947, 893). A method of mixing and curing drugs to retain proteins, physiologically active substances and drugs (US Pat. No. 5,782,971, JP-A-9-225020, JP-A-4-248774, JP-A-6-228011, JP-A-7-31673) , Biomed Mater Eng, 4, 291-307 (1994), Cl in Orto Related Res., 367S, S396-S405 (1999), J Periodont, 71, 8-13 (2000), Bioiaterials, 23, 1261-1268 (2002), J. Biomed Mater Res., 59, 265-271 (2002)), a method of absorption into hardened calcium phosphate cement (J. Biomed Mater Res., 44, 168-175 (1999)), and polarization treatment Method of selectively adsorbing on calcium phosphate (Japanese Patent Laid-Open No. 2001-187133), mixed with a paste containing a calcium component and a thickener Method for supporting the osteogenesis promoting substance (JP-A-2001-106638) are known Te.
ところが、 リン酸カルシウムに生物学的活性化物質を担持させる方法は、 上述 のように、 リン酸カルシウムセラミックの気孔内に担持、 リン酸カルシウムセメ ントに混合して硬化、 吸収性ポリマーと複合化、 あるいは吸着作用を利用したも のがほとんどであり、 これらの方法では生物学的活性化物質の担持量が少なかつ たり、 長期間の徐放が困難であった。 さらに、 リン酸カルシウムセメントに混合 する場合には、 リン酸カルシウムセラミックス程の高強度を有する徐放体を作製 できない、 吸収性ポリマーと複合化する場合は操作が複雑になるといった欠点が あった。  However, as described above, the method of supporting a biologically active substance on calcium phosphate is carried in pores of a calcium phosphate ceramic, mixed with calcium phosphate cement, cured, combined with an absorbent polymer, or utilizes an adsorption effect. In most cases, the amount of the biologically activating substance carried was small and it was difficult to perform sustained release over a long period. Furthermore, when mixed with calcium phosphate cement, there was a drawback that a sustained-release body having a strength as high as that of calcium phosphate ceramics could not be produced, and when combined with an absorbent polymer, the operation was complicated.
水溶液からリン酸カルシウムとタンパク質を共沈させる方法は、 有力な担持法 であり、 ポリマー又はチタンなどの金属表面に析出させる方法 （US 6, 136, 369、 US 6， 143， 948、 US 6, 344, 061、 EP96201293) が開示されている。 しかし、 水溶液 からリン酸カルシウムとの共沈でタンパク質をリン酸カルシウムセラミック表面 上に担持する方法は知られていなかった。  The method of co-precipitating calcium phosphate and protein from an aqueous solution is an effective supporting method, and the method of precipitating on a metal surface such as a polymer or titanium (US 6, 136, 369, US 6, 143, 948, US 6, 344, 061, EP96201293). However, no method has been known for supporting proteins on a calcium phosphate ceramic surface by coprecipitation with calcium phosphate from an aqueous solution.
特許文献 1 Patent Document 1
米国特許第 6136369号明細書  U.S. Pat.No. 6,136,369
特許文献 2 Patent Document 2
米国特許第 6143948号明細書  U.S. Pat.No. 6,143,948
特許文献 3 米国特許第 6344061号明細書 Patent Document 3 U.S. Patent No.6344061
特許文献 4 ' Patent Document 4 '
欧州特許第 96201293号明細書 発明の開示  EP 96201293 DISCLOSURE OF THE INVENTION
本発明は、 生体適合性を有しており、 生体組織再構築を促進する生体組織代替 材料、 組織工学スキヤホールド、 タンパク質徐放体として利用し得る、 生物学的 活性化物質を担持したリン酸カルシウム焼結体の提供を目的とする。  The present invention provides a bioactive substance-supported calcium phosphate firing material that has biocompatibility and can be used as a biological tissue replacement material, tissue engineering skid holder, and protein sustained release body that promotes biological tissue reconstruction. The purpose is to provide union.
水溶液を用いてリン酸カルシウムとタンパク質を共沈させる方法は、 リン酸カ ルシゥムに過飽和であるものの、 自発核形成は生じないハンクス液などの安定過 飽和溶液を使用し、 不均一核形成を利用して基板上にリン酸カルシウムとタンパ ク質を共沈させる。 しかしこの方法ではリン酸カルシウムの析出量が少なく、 担 持できるタンパク質の量も少ない結果となる。 自発核形成を生じる不安定リンカ ルシゥム過飽和溶液を使用すれば、 リン酸カルシウムの析出量を増やすことがで き、 その結果タンパク質担持量も増やすことが期待できる。 ただしこの場合は、 自発核形成までの時間を人為的に制御し、 溶液全体で自発核形成する以前に、 リ ン酸カルシウム基板上への共沈析出を終了させるか、 あるいは、 基板のごく近傍 でのみ自発核形成させる必要がある。  A method of co-precipitating calcium phosphate and protein using an aqueous solution is to use a stable supersaturated solution such as Hanks' solution that does not cause spontaneous nucleation even though calcium phosphate is supersaturated, and utilizes heterogeneous nucleation. Co-precipitate calcium phosphate and protein on the substrate. However, this method results in a small amount of precipitated calcium phosphate and a small amount of protein that can be carried. The use of an unstable phosphoric acid supersaturated solution that causes spontaneous nucleation can increase the amount of precipitated calcium phosphate, which can be expected to increase the amount of protein carried. However, in this case, the time to spontaneous nucleation is artificially controlled, and the co-precipitation on the calcium phosphate substrate is terminated before spontaneous nucleation is formed in the entire solution, or in the vicinity of the substrate. It is necessary only for spontaneous nucleation.
本発明では、 自発核形成を生じるタンパク質含有不安定リンカルシウム過飽和 溶液を使用し、 自発核形成までの時間を人為的に制御し、 溶液全体で自発核形成 する以前に、 リン酸カルシウム基板上への共沈析出を終了させるか、 あるいは、 基板のごく近傍でのみ自発核形成させてリン酸カルシウムの析出量を増すことで、 タンパク質担持量を増加させたリン酸カルシウム主成分焼結体を提供する。 さら に、 本発明では担持に使用する薬品の生体適合性が高く、 なおかつ担持操作が単 純な、 生物学的活性化物質担持リン酸カルシウムが提供される。  In the present invention, a protein-containing unstable phosphocalcium supersaturated solution that causes spontaneous nucleation is used, and the time until spontaneous nucleation is artificially controlled. The present invention provides a calcium phosphate-based sintered body having an increased amount of protein carried by terminating precipitation or forming spontaneous nuclei only in the vicinity of a substrate to increase the amount of precipitated calcium phosphate. Further, the present invention provides calcium phosphate carrying a biologically activating substance, in which the biocompatibility of the drug used for carrying is high and the carrying out operation is simple.
すなわち、 本発明は以下の通りである。  That is, the present invention is as follows.
( 1 ) 自発核形成を生じる不安定リン酸カルシウム過飽和溶液中で、 生物学的 活性化物質である水溶性夕ンパク質とリン酸カルシウムとの制御された遅延共沈 を行ない、 生物学的活性化物質である水溶性タンパク質を表面に担持させたリン 酸カルシウムを主成分とする焼結体、 (1) In a supersaturated solution of unstable calcium phosphate that causes spontaneous nucleation, controlled delayed coprecipitation of water-soluble protein, a biologically active substance, and calcium phosphate is carried out. Phosphorus with water-soluble protein supported on the surface Sintered body mainly composed of calcium acid,
(2) リン酸カルシウムが、 炭酸、 珪素、 マグネシウム、 亜鉛、 鉄、 マンガン のうち少なくとも 1種を固溶したリン酸カルシウムである （1) のリン酸カルシ ゥム焼結体、  (2) The calcium phosphate is a calcium phosphate in which at least one of carbonic acid, silicon, magnesium, zinc, iron, and manganese is dissolved, (1) the calcium phosphate sintered body,
(3) リン酸カルシウムの他に、 カルシウム、 マグネシウム、 亜鉛、.鉄、 マン ガンから選ばれた金属の酸化物またはリン酸塩の少なくとも 1種を含んでいる (3) In addition to calcium phosphate, contains at least one oxide or phosphate of a metal selected from calcium, magnesium, zinc, iron, and manganese
(1) のリン酸カルシウム焼結体、 (1) a calcium phosphate sintered body,
(4) 焼結後の亜鉛含有量が 0. 012重量％〜1. 2重量％である （2) ま たは （3) のリン酸カルシウム焼結体、  (4) The calcium phosphate sintered body according to (2) or (3), wherein the zinc content after sintering is 0.012% by weight to 1.2% by weight,
(5) 焼結後の炭酸含有量が 0. 3重量％〜15重量％である （2) のリン酸 カルシウム焼結体、  (5) The calcium phosphate sintered body according to (2), wherein the carbonic acid content after sintering is 0.3 to 15% by weight,
(6) 焼結後のマグネシウム含有量が 0. 26重量％〜 13重量％である （2) または （3) のリン酸カルシウム焼結体、  (6) The calcium phosphate sintered body of (2) or (3), wherein the magnesium content after sintering is 0.26% by weight to 13% by weight,
(7) 焼結後の珪素含有量が 0. 0105重量％〜1. 05重量％である （2) または （3) のリン酸カルシウム焼結体、  (7) The calcium phosphate sintered body according to (2) or (3), wherein the silicon content after sintering is 0.0105% by weight to 1.05% by weight.
(8) 焼結後の鉄含有量が 0. 014重量％〜1. 4重量％である （2) また は （3) のリン酸カルシウム焼結体、  (8) The calcium phosphate sintered body according to (2) or (3), wherein the iron content after sintering is 0.014% by weight to 1.4% by weight.
(9) 焼結後のマンガン含有量が 1重量 P pm〜 100重量 p pmである （2) または （3) のリン酸カルシウム焼結体、  (9) The sintered calcium phosphate according to (2) or (3), wherein the manganese content after sintering is 1 wt Ppm to 100 wt ppm,
(10) 焼結後の酸化カルシウム含有量が 0. 1〜4重量％である （3) のリ ン酸カルシウム焼結体、  (10) The calcium phosphate sintered body according to (3), wherein the calcium oxide content after sintering is 0.1 to 4% by weight.
(11) ひリン酸三カルシウム、 リン酸 4カルシウム、 酸化カルシウム、 または 酸化マグネシウムの少なくとも 1種が、 表面から少なくとも深さ 1ミクロンまで の表面層に含有された （3) のリン酸カルシウム焼結体、  (11) The calcium phosphate sintered body of (3), wherein at least one of tricalcium phosphate, tetracalcium phosphate, calcium oxide, or magnesium oxide is contained in a surface layer at least 1 micron deep from the surface.
(12) 焼結体の Ca/Pモル比が 0. 75以上 2. 1以下である （1) から （1 1) のいずれかのリン酸カルシウムを主成分とする焼結体、  (12) The sintered body mainly composed of calcium phosphate according to any one of (1) to (11), wherein the sintered body has a Ca / P molar ratio of 0.75 or more and 2.1 or less.
(13) リン酸カルシウム焼結体が気孔率 20%〜80%で、 気孔直径 Ί 0 a m〜4mmの多孔質である （1) から （12) のいずれかのリン酸カルシウム焼 結体、 (14) 気孔が人工的に作られた直径 70 m〜 4 mmの三次元網目状で、 焼 結体を貫通している （13) のリン酸カルシウム焼結体、 (13) The calcium phosphate sintered body according to any one of (1) to (12), wherein the calcium phosphate sintered body has a porosity of 20% to 80% and a pore diameter of Ί0 am to 4 mm. (14) The calcium phosphate sintered body of (13), which has a three-dimensional network with a diameter of 70 m to 4 mm and whose pores are artificially created and penetrates the sintered body.
(15) 担持する生物学的活性化物質である水溶性タンパク質が J¾長因子また は細胞接着因子のうちの少なくとも一種を含んでいる （1) から （14) のいず れかのリン酸カルシウム焼結体、  (15) The water-soluble protein that is the biological activator to be supported contains at least one of the J¾ long factor or the cell adhesion factor (1) to (14). Body,
(16) Ca成分 0〜2. 5mM、 リン酸成分 1. 0〜 20 mM、 K成分 0〜 4 0mM、 Na成分 0〜200mM、 C 1成分 0〜 200 mMを含み p Hが 5. 0〜 9. 0の水溶液を用いてリン酸カルシウム主成分焼結体上にタンパク質を共沈析 出させる、 （1)カゝら （15) に記載したタンパク質担持リン酸カルシウム主成分 焼結体の製造方法、  (16) Ca component 0-2.5 mM, phosphate component 1.0-20 mM, K component 0-40 mM, Na component 0-200 mM, C1 component 0-200 mM, pH 5.0- 9. Co-precipitating proteins on the calcium phosphate main component sintered body using the aqueous solution of 9.0, (1) the method for producing a protein-supported calcium phosphate main component sintered body described in (15),
(17) &成分1. 2〜2. 75mM、 リン酸成分 0. 6〜 15 mM、 K成分 (17) & component 1.2-2.75 mM, phosphate component 0.6-15 mM, K component
0〜30mM、 N a成分 30〜： L 50mM、 ¾1 成分0. 1〜3. 0mM、 C 1 成分 30〜150mM、 HC03成分 0〜6 OmMを含み pHが 5. 0〜9. 0の 水溶液を用いてリン酸カルシウム主成分焼結体上にタンパク質を共沈析出させる、 (1) から （15) のいずれかのタンパク質担持リン酸カルシウム主成分焼結体 の製造方法、 0 to 30 mM, Na component 30 to: L 50 mM, ¾1 component 0.1 to 3.0 mM, C 1 component 30 to 150 mM, HC0 3 component 0 to 6 OmM, pH 5.0 to 9.0 aqueous solution (1) a method of producing a protein-supported calcium phosphate-based main component sintered body according to any one of (1) to (15),
(18) 水溶液として、 医療用輸液剤、透析 ·腹膜灌流液、輸液の補正用製剤、 カルシウム製剤、 透析 ·腹膜灌流液の補充液の中から選択される 1種または 2種 以上の溶液を含んだ水溶液を使用する （17) のタンパク質担持リン酸カルシゥ ム主成分焼結体の製造方法、  (18) Aqueous solutions include one or more solutions selected from medical infusions, dialysisperitoneal perfusion, infusion correction preparations, calcium preparations, dialysisperitoneal perfusion replenishers (17) a method for producing a protein-supported calcium phosphate main component sintered body using an aqueous solution,
(19) カルシウム成分を含む溶液とリン酸成分を含む溶液をあらかじめ別々 に作製しておき、 両者を混合することでタンパク質の共沈を開始させる、 （16) から （18) のいずれかのタンパク質担持リン酸カルシウム主成分焼結体の製造 方法、  (19) A solution containing a calcium component and a solution containing a phosphoric acid component are separately prepared in advance, and co-precipitation of the protein is started by mixing the two, and the protein according to any one of (16) to (18) A method for producing a supported calcium phosphate main component sintered body,
(20) リン酸三カルシウム、 リン酸 4カルシウム、 酸化カルシウムから選 ばれる 1種または 2種以上の相をあらかじめリン酸カルシウム主成分焼結体表面 に形成しておき該相からカルシウムを放出させてタンパク質を共沈析出させる、 (20) One or more phases selected from tricalcium phosphate, tetracalcium phosphate, and calcium oxide are previously formed on the surface of the sintered body of calcium phosphate as a main component, and calcium is released from the phase to release proteins. Coprecipitate,
(16) から （18) のいずれかのタンパク質担持リン酸カルシウム主成分焼結 体の製造方法、 (2 1) タンパク質を溶解させたリン酸成分を含む溶液をリン酸カルシウム主 成分焼結体と接触させ、 タンパク質の共沈を開始させる （20) のタンパク質担 持リン酸カルシウム主成分焼結体の製造方法、 Any one of (16) to (18), (21) A method for producing a protein-bearing calcium phosphate-based main component sintered body according to (20), wherein the solution containing the phosphate component in which the protein is dissolved is brought into contact with the calcium phosphate main component sintered body to start coprecipitation of the protein.
(22) リン酸成分を含む溶液がリン酸緩衝生理的食塩水又はリン酸含有カル シゥム不含有医療用輸液剤の中から選択される 1種又は 2種の混合液である （2 0) のタンパク質担持リン酸カルシウム主成分焼結体の製造方法、  (22) The solution containing a phosphate component is one or a mixture of two or more selected from phosphate buffered saline or a medical fluid containing no phosphate-containing calcium. Method for producing protein-supported calcium phosphate main component sintered body,
(23) (1) から （1 5) のいずれかのタンパク質担持担持リン酸カルシゥ ム主成分焼結体を用いた生体材料、  (23) A biomaterial using the protein-supported calcium phosphate main component sintered body according to any one of (1) to (15),
(24) (16) から （22) のいずれかのタンパク質担持リン酸カルシウム 主成分焼結体の製造方法を用いた生体材料の製造方法、  (24) A method for producing a biomaterial using the method for producing a protein-supported calcium phosphate main component sintered body according to any one of (16) to (22),
(25) (1) から （1 5) のいずれかのタンパク質担持リン酸カルシウム主 成分焼結体を用いたタンパク質徐放体、  (25) A protein sustained-release body using the protein-supported calcium phosphate main component sintered body according to any one of (1) to (15),
(26) (16) カゝら （22) のいずれかのタンパク質担持リン酸カルシウム 主成分焼結体の製造方法を用いたタンパク質徐放体の製造方法、  (26) (16) A method for producing a sustained-release protein body using the method for producing a protein-supported calcium phosphate-based sintered body according to any of (22),
(27) (1) から （1 5) のいずれかのタンパク質担持リン酸カルシウム主 成分焼結体を用いた人工骨、  (27) An artificial bone using the protein-supported calcium phosphate main component sintered body of any one of (1) to (15),
(28) (1 6) から （22) のいずれかのタンパク質担持リン酸カルシウム 主成分焼結体の製造方法を用いた人工骨の製造方法、  (28) A method for producing an artificial bone using the method for producing a protein-supported calcium phosphate main component sintered body according to any one of (16) to (22),
(29) (1) から （1 5) のいずれかのタンパク質担持リン酸カルシウム主 成分焼結体を用いた組織工学スキヤフォ一ルド、 および  (29) A tissue-engineered skifold using the protein-supported calcium phosphate main component sintered body according to any one of (1) to (15), and
(30) (1 6) から （22) のいずれかのタンパク質担持リン酸カルシウム 主成分焼結体の製造方法を用いた組織工学スキヤフォールドの製造方法。  (30) A method for producing a tissue-engineered scaffold using the method for producing a protein-supported calcium phosphate main component sintered body according to any one of (16) to (22).
本発明において、自発核形成を生じる不安定リン酸カルシウム過飽和溶液とは、 リン酸カルシウム沈殿が自発核形成で自然に生じてしまう溶液のことである。 したがって、 リン酸カルシウムに過飽和でありながら、 リン酸カルシウム沈殿を 生成しない安定リン酸カルシウム過飽和溶液、 すなわち、 ハンクス溶液や 1倍濃 度擬似体液などは自発核形成を生じる不安定リン酸カルシウム過飽和溶液に含ま れない。  In the present invention, an unstable calcium phosphate supersaturated solution that causes spontaneous nucleation is a solution in which calcium phosphate precipitation occurs spontaneously by spontaneous nucleation. Therefore, a stable calcium phosphate supersaturated solution that is supersaturated with calcium phosphate and does not generate calcium phosphate precipitation, that is, a Hanks solution or a 1-fold concentration simulated body fluid, is not included in an unstable calcium phosphate supersaturated solution that causes spontaneous nucleation.
本発明では、 自発核形成とは、 溶液の成分が基板や異物質や容器の壁の助けを 借りずに、 自然発生的に集合して溶液が目視で白濁すること又はこのようにして 溶液中で粒径 1 x m以上の粒子に成長することである。 溶液中の粒子の粒径は光 散乱法で測定することができる。沈殿とは自発核形成で生じた粒子のことである。 共沈とは、 単独では沈殿しないはずのほかの物質が同時に主沈殿とともに沈殿す ることである。 リン酸カルシウムの析出とは、 自発核形成によるリン酸カルシゥ ム粒子の発生だけでなく、 基板や異物質や容器の壁 p助けを借りて、 これらの表 面上にリン酸カルシウムが集合する不均一核形成によるリン酸カルシウム形成を も含む。 In the present invention, spontaneous nucleation means that the components of the solution help the substrate, foreign substances, and the walls of the container. Without borrowing, spontaneous aggregation and visual turbidity of the solution or in this way growing into particles of 1 xm or more in solution. The particle size of the particles in the solution can be measured by a light scattering method. Sedimentation refers to particles generated by spontaneous nucleation. Co-precipitation means that other substances that should not precipitate by themselves precipitate together with the main precipitate. Precipitation of calcium phosphate means not only the generation of calcium phosphate particles due to spontaneous nucleation, but also the heterogeneous nucleation of calcium phosphate on these surfaces with the help of substrates, foreign substances and the walls of containers. Also includes calcium phosphate formation.
リン酸カルシウムとの制御された遅延共沈とは、 溶液組成と温度の選択によつ て、 リン酸カルシウムの析出に至るまでの時間を人工的に制御遅延させ、 リン酸 カルシウム析出のほとんど又は全てが基板上だけに生ずるようにし、 核発生直後 の高活性リン酸カルシウムナノ粒子が周囲のタンパク質を吸着又は化学結合でと り込んで共沈するようにした共沈である。 具体的には、 2種類以上の水溶液を順 番に混合して不安定な過飽和リン酸カルシウム溶液を作製し、 自発核形成による リン酸カルシウム析出までの時間が最後の溶液混合から 1 0秒以上 7日以下、 好 ましくは 2分以上 2日以下となる共沈を生じせしめる。 自発核形成までの誘導時 間は水溶液の化学組成と温度を、 至適値に選択することで制御する。 共沈に至る 待ち時間は、 基板がない状態で過飽和リン酸カルシウム溶液を作製し、 目視で溶 液が白濁するか又は粒径 1 m以上の沈殿粒子が光散乱法で検出されるまでの時 間である。  Controlled delayed co-precipitation with calcium phosphate means that the time to calcium phosphate precipitation is artificially controlled by selecting the solution composition and temperature, and most or all of the calcium phosphate deposition is on the substrate. This is a coprecipitation method in which highly active calcium phosphate nanoparticles immediately after nucleation are co-precipitated by absorbing or chemically bonding surrounding proteins. Specifically, two or more aqueous solutions are mixed in order to produce an unstable supersaturated calcium phosphate solution, and the time until calcium phosphate precipitation by spontaneous nucleation is 10 seconds or more and 7 days or less from the last solution mixing, Preferably, coprecipitation occurs for more than 2 minutes and less than 2 days. The induction time until spontaneous nucleation is controlled by selecting the chemical composition and temperature of the aqueous solution to optimal values. The waiting time for co-precipitation is determined by preparing a supersaturated calcium phosphate solution in the absence of a substrate, and until the solution becomes cloudy visually or precipitated particles with a particle size of 1 m or more are detected by the light scattering method. is there.
析出に至る待ち時間が短い通常の沈殿形成の場合は、 最後に添加した溶液液滴 と周囲の溶液の界面での沈殿形成が卓越し、 基板上へ沈殿を析出することは出来 ない。 しかし、 自発核形成までの誘導時間を遅延させ、 さらに界面自由エネルギ —の低いリン酸カルシウムを主成分とする基板を共存させる場合は、 リン酸カル シゥムの生成は界面自由エネルギーの低い基板上での析出が卓越し、 リン酸カル シゥムと共存タンパク質を基板上に固定することが可能となる。  In the case of ordinary precipitate formation with a short waiting time until precipitation, the precipitate formed at the interface between the last added solution droplet and the surrounding solution is predominant, and the precipitate cannot be deposited on the substrate. However, if the induction time to spontaneous nucleation is delayed and a substrate containing calcium phosphate having a low interfacial free energy as a main component is coexisted, calcium phosphate is generated on a substrate having a low interfacial free energy. It is possible to immobilize calcium phosphate and coexisting proteins on a substrate.
本発明のリン酸カルシウム過飽和溶液を作製し得る溶液は少なくともカルシゥ ム成分を含む溶液、 少なくともリン酸成分を含む溶液、 またはカルシウム成分と リン酸成分の両者を含む溶液である。 これらの溶液自身が自発核形成誘導時間の 遅延成分を含んでいても良いし、 自発核形成までの誘導時間を制御遅延する 1種 または 2種以上の溶液をさらに混合してもよい。カルシウム成分とリン酸成分は、 混合前は異なる容器に別々に溶解させておくことが好ましい。 少なくともカルシ ゥム成分を含む溶液、 少なくともリン酸成分を含む溶液、 またはカルシウム成分 とリン酸成分の両者を含む溶液は限定されない。 リン酸成分を含む溶液の例とし ては、 リン酸緩衝生理的食塩水、 リン酸溶液、 リン酸水素二カリウム溶液、 リン 酸二水素カリウム溶液、 リン酸水素ニナトリウム溶液、 リン酸二水素ナトリウム 溶液などが挙げられる。 カルシウム成分を含む溶液の例としては、 塩化カルシゥ ム溶液、乳酸カルシウム溶液、酢酸カルシウム溶液、ダルコン酸カルシウム溶液、 クェン酸カルシウム溶液などが挙げられる。 カルシウム成分とリン酸成分の両者 を含む溶液としては、 ハンクス液や 1倍擬似体液のような安定過飽和溶液や、 リ ン酸カルシゥム不飽和溶液を挙げることができる。 このようなリン酸カルシウム 不飽和溶液の例としては、 例えば、 塩化カルシウム濃度 2 . 5 mM、 リン酸水素力 リウム濃度 1 . 0 mM、 pH 5未満の溶液を挙げることができる。 自発核形成の誘導 時間を制御する溶液としては、 水酸化カリウム溶液、 炭酸水素ナトリウム溶液な どの適当なアル力リ性溶液、 または塩化力リゥム溶液または塩化ナトリゥム溶液 を使用する。 The solution from which the calcium phosphate supersaturated solution of the present invention can be prepared is a solution containing at least a calcium component, a solution containing at least a phosphoric acid component, or a solution containing both a calcium component and a phosphoric acid component. These solutions themselves have a spontaneous nucleation induction time It may contain a delay component, or may further contain one or more solutions that control and delay the induction time until spontaneous nucleation. It is preferable that the calcium component and the phosphoric acid component are separately dissolved in different containers before mixing. A solution containing at least a calcium component, a solution containing at least a phosphate component, or a solution containing both a calcium component and a phosphate component are not limited. Examples of solutions containing a phosphate component include phosphate buffered saline, phosphate solution, dipotassium hydrogen phosphate solution, potassium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium dihydrogen phosphate Solution and the like. Examples of the solution containing a calcium component include a calcium chloride solution, a calcium lactate solution, a calcium acetate solution, a calcium dalconate solution, and a calcium citrate solution. Examples of the solution containing both the calcium component and the phosphoric acid component include a stable supersaturated solution such as Hanks' solution and a 1-fold simulated body fluid, and a calcium phosphate unsaturated solution. Examples of such a calcium phosphate unsaturated solution include a solution having a calcium chloride concentration of 2.5 mM, a potassium hydrogen phosphate concentration of 1.0 mM, and a pH of less than 5. As a solution for controlling the induction time of spontaneous nucleation, use a suitable alkaline solution such as a potassium hydroxide solution or a sodium hydrogen carbonate solution, or a chloride or sodium chloride solution.
カルシウム成分を含む溶液、 リン酸成分を含む溶液、 カルシウム成分とリン酸 成分を含む溶液、 自発核形成誘導時間制御溶液の各成分溶液と制御溶液は各々 1 種類の溶液でも良いし、 組成の異なる複数種の溶液から成っていても良い。 これ らの溶液の混合順序は、 混合中又は混合後 1 0秒未満で自発核形成しない限り、 特に制限はない。具体的には例えば、 1種類目の自発核形成誘導時間制御溶液に 1 種類目のリン酸成分溶液を混合し、 次にカルシウム成分溶液を混合し、 次に 2種 類目の自発核形成誘導時間制御溶液を混合し、 最後に 2種類目のリン酸成分溶液 を混合することもできるし、 例えば、 リン酸成分溶液にカルシウム成分溶液を混 合し、 次に 1種類目の自発核形成誘導時間制御溶液を混合し、 最後に 2種類目の 自発核形成誘導時間制御溶液を混合することもできる。  The solution containing calcium component, the solution containing phosphoric acid component, the solution containing calcium component and phosphoric acid component, the solution for controlling spontaneous nucleation induction time, each component solution and the control solution may be one type of solution or different in composition. It may consist of a plurality of types of solutions. The order of mixing these solutions is not particularly limited as long as spontaneous nucleation does not occur during or within less than 10 seconds after mixing. Specifically, for example, a first type of phosphoric acid component solution is mixed with a first type of spontaneous nucleation induction time control solution, then a calcium component solution is mixed, and then a second type of spontaneous nucleation induction The time control solution can be mixed, and finally the second type of phosphoric acid component solution can be mixed.For example, the calcium component solution can be mixed with the phosphoric acid component solution, and then the first type of spontaneous nucleation induction The time control solution can be mixed, and finally the second spontaneous nucleation induction time control solution can be mixed.
タンパク質の変成や失活が生じなければ、 上述のどの溶液にタンパク質を添加 溶解してもよいし、 複数の溶液にタンパク質を添加溶解しても良いし、 全ての溶 液が混合された後にタンパク質を添加溶解しても良い。すなわち、 （1 ) カルシゥ ム成分とリン酸成分の両方を含む溶液にタンパク質を添加溶解しても良いし、 ( 2 ) カルシウム成分を含む溶液、 リン酸成分を含む溶液の一方または両方に夕 ンパク質を添加溶解しても良いし、 （3 ) 自発核形成の誘導時間を制御する溶液に 添加溶解してもよい。 添加溶解するタンパク質は、 固体状のタンパク質でも良い し、 すでに溶液に溶解しているものでも構わない。 If protein denaturation or inactivation does not occur, the protein may be added and dissolved in any of the above solutions, the protein may be added and dissolved in multiple solutions, or all the solutions may be dissolved. After the liquid is mixed, the protein may be added and dissolved. That is, (1) the protein may be added and dissolved in a solution containing both the calcium component and the phosphate component, or (2) the protein may be added to one or both of the solution containing the calcium component and the solution containing the phosphate component. (3) It may be added and dissolved in a solution that controls the induction time of spontaneous nucleation. The protein to be added and dissolved may be a solid protein or a protein that has already been dissolved in a solution.
以上の操作によりタンパク質とリン酸カルシウムの共沈が開始され、 その際に リン酸カルシウム主成分焼結体を接触させることにより、 リン酸カルシウム主成 分焼結体上にタンパク質が共沈析出し、 タンパク質を担持したリン酸カルシウム 主成分焼結体が得られる。  Co-precipitation of protein and calcium phosphate is started by the above operation. At this time, the calcium phosphate main component sintered body is brought into contact, and the protein is co-precipitated and precipitated on the calcium phosphate main component sintered body, and the calcium phosphate carrying the protein is precipitated. A main component sintered body is obtained.
この際、 リン酸成分を含む溶液のみ、 またはリン酸成分を含む溶液と自発核形 成誘導時間制御溶液を用い、 該溶液にタンパク質を添加溶解させておき、 これと リン酸カルシウム主成分焼結体を接触させた場合でも、 焼結体に含有されている ひリン酸三カルシウム、 リン酸 4カルシウム、 酸化カルシウムの溶解によりカル シゥムが溶液中に放出され、 タンパク質の共沈析出が開始され得る。  At this time, using only a solution containing a phosphoric acid component or a solution containing a phosphoric acid component and a spontaneous nucleation induction time control solution, a protein is added and dissolved in the solution. Even in the case of contact, calcium is released into the solution due to dissolution of tricalcium phosphate, tetracalcium phosphate, and calcium oxide contained in the sintered body, and coprecipitation of proteins can be started.
さらに、 リン酸三カルシウム、 リン酸 4カルシウム、 酸化カルシウム等の溶 解性カルシウム含有相をあらかじめリン酸カルシウム主成分焼結体表面に形成し ておき、 タンパク質を共沈析出させてもよい。  Furthermore, a soluble calcium-containing phase such as tricalcium phosphate, tetracalcium phosphate, and calcium oxide may be previously formed on the surface of the sintered body of the main component of calcium phosphate, and the protein may be coprecipitated.
カルシウム成分とリン酸成分が同時に含まれている溶液が、 リン酸カルシウム に対して過飽和になる pHは、カルシウムイオン濃度とリン酸イオン濃度に依存す るが、 例を挙げれば塩化カルシウム濃度 2 . 5 πιΜ、 リン酸水素カリウム濃度 1 . O mMの場合は ρΗ 5以上である。 過飽和溶液が、 安定な過飽和状態か、 不安定な 過飽和状態かは、 共存イオンとイオン強度によって影響される。  The pH at which a solution containing both a calcium component and a phosphate component becomes supersaturated with respect to calcium phosphate depends on the calcium ion concentration and the phosphate ion concentration.For example, the calcium chloride concentration is 2.5 πιΜ. However, when the concentration of potassium hydrogen phosphate is 1. O mM, ρΗ5 or more. Whether a supersaturated solution is stable or unstable is affected by coexisting ions and ionic strength.
自発核形成の誘導時間を遅延させる成分は、 自発核形成時間までの誘導時間を The component that delays the induction time of spontaneous nucleation
1 0秒以上遅延させるものであれば特に制限はないが、 担持溶液の生体適合性の 上から、 ナトリウム成分かカリウム成分を主成分とする溶液が好ましい。 このよ うな溶液は具体的には 50〜200mM、好ましくは 100〜180謹、 さらに好ましくは 1There is no particular limitation as long as the delay is 10 seconds or more, but from the viewpoint of the biocompatibility of the carrier solution, a solution containing a sodium component or a potassium component as a main component is preferable. Such a solution is specifically 50 to 200 mM, preferably 100 to 180 mM, more preferably 1 to 200 mM.
6 6 mM炭酸水素ナトリウム溶液、 5〜50mM、 好ましくは 10〜30mM、 さらに好ま しくは 2 O mM水酸化カリウム溶液、 O〜40mM、 好ましくは O〜20mM、 さらに 好ましくは 2 OmM塩化カリウム溶液を挙げることができ、 例えば、 塩化カルシ ゥム濃度 42.9 、 リン酸濃度 28.6mMの溶液 4.9mLに対しては、 12.5mMの KC1溶 液 80mLと 20 mM水酸化力リゥム溶液 12 mLを添加すれば、 最終 pHが 7. 4 になり生体適合性が高く、 析出に至る待ち時間が 1 0分に遅延されたリン酸カル シゥム過飽和溶液となる。 66 mM sodium bicarbonate solution, 5-50 mM, preferably 10-30 mM, more preferably 2 OmM potassium hydroxide solution, O-40 mM, preferably O-20 mM, more Preferably, a 2 OmM potassium chloride solution can be mentioned.For example, for 4.9 mL of a solution having a calcium chloride concentration of 42.9 and a phosphoric acid concentration of 28.6 mM, 80 mL of a 12.5 mM KC1 solution and 20 mM hydroxylating solution are used. Addition of 12 mL of the solution results in a final pH of 7.4, a high biocompatibility, and a calcium phosphate supersaturated solution with a delay in precipitation of 10 minutes.
析出に至る待ち時間が遅延された不安定リン酸カルシウム過飽和溶液とは具体 的には、 C a— P— Na— K— C 1系及び、 C a_P— Na— K— Mg— C 1— C O 3系のある特定組成範囲の水溶液である。 Unstable calcium phosphate supersaturated solutions with a delayed waiting time for precipitation are, specifically, Ca—P—Na—K—C 1 system and Ca_P—Na—K—Mg—C 1—CO 3 system Is an aqueous solution having a specific composition range.
C a— P— Na— K— C 1系の水溶液としては、 C a成分 0〜2. 5mM好ま しくは 0. 8〜2. 5mM、 リン酸成分 1. 0〜 20 mM好ましくは 1. 2〜1 0 mM、 K成分 0〜4 OmM好ましくは 0〜2 OmMを含み、 11カ 5. 0〜9. 0 好ましくは 5. 8〜8. 5であり、 Na 成分がさらに 0〜 20 OmM好ましくは 0〜1 5 OmM、 C 1成分が 0〜 20 OmM好ましくは 0〜1 5 OmM含まれた 溶液である。溶液の C a ZPモル比は特に規定しないが、好ましくは 0 · 1〜 2. 5の範囲である。  As an aqueous solution of a Ca—P—Na—K—C1 system, the Ca component is 0 to 2.5 mM, preferably 0.8 to 2.5 mM, and the phosphate component is 1.0 to 20 mM, preferably 1.2. ~ 10 mM, K component 0 ~ 4 OmM, preferably 0 ~ 2 OmM, 11 5.0 ~ 9.0, preferably 5.8 ~ 8.5, Na component is more preferably 0 ~ 20 OmM Is a solution containing 0 to 15 OmM and a C1 component of 0 to 20 OmM, preferably 0 to 15 OmM. The molar ratio of C a ZP of the solution is not particularly specified, but is preferably in the range of 0.1 to 2.5.
C a— P— N a— K— Mg— C 1 _C03系の溶液としては、 C a成分 1. 2〜 2. 75mM好ましくは 1. 39〜2. 33mM、 リン酸成分 0. 6〜： L 5 mM 好ましくは 1. 1 7〜10mM、 K成分 0〜3 OmM好ましくは 4〜2 OmM、 Na成分 30〜： L 5 OmM好ましくは 40〜145mM、 1^ 成分0. 1〜3. OmM好ましくは 0. 2〜2. OmM, C 1成分 30〜 1 50 mM好ましくは 4 0-145 mM、 HCO,成分 0〜 60 mM好ましくは 0〜 45 mM含み、 pH 5.. C a- P- The N a- K- Mg- C 1 _C0 3 based solution of, C a component 1.. 2 to 2. 75 mM preferably 1. from 39 to 2 33 mM, phosphoric acid ingredient 0. 6: L 5 mM preferably 1.1 to 10 mM, K component 0 to 3 OmM, preferably 4 to 2 OmM, Na component 30 to: L 5 OmM, preferably 40 to 145 mM, 1 ^ component 0.1 to 3. OmM, preferably Contains 0.2 to 2.OmM, C 1 component 30 to 150 mM, preferably 40 to 145 mM, HCO, component 0 to 60 mM, preferably 0 to 45 mM, pH 5.
0〜9. 0好ましくは 5. 8〜8. 5の溶液である。 溶液の C aZPモル比は特 に規定しないが、 好ましくは 2. 5以下の範囲である。 0 to 9.0, preferably 5.8 to 8.5. The CaZP molar ratio of the solution is not particularly specified, but is preferably in the range of 2.5 or less.
さらに、 析出に至る待ち時間が遅延された C a— P— N a— K— Mg— C 1― C03系のリン酸カルシウム不安定過飽和溶液は、 医療用輸液剤、 透析 ·腹膜灌流 液、 輸液の補正用製剤、 カルシウム製剤、 透析 ·腹膜灌流液の補充液の中から選 ばれた 1種または 2種以上を混合することで作製することもできる。 これらの溶 液または薬剤は、 C a、 P、 Na、 K、 Mg、 Cし C〇3の供給源として使用で き、 この場合は、 全ての溶液または薬剤が既に医療用に認可されており、 しかも 滅菌済みであるため、 手術室等で使用するのに好都合である。 医療用輸液の、 C a成分を含み P成分を含まない電解質輸液、 P成分を含み C a成分を含まない電 解質輸液、 塩化ナトリゥム輸液、 透析 ·腹膜灌流液の補充液の透析液専用炭酸水 素ナトリウム補充液は特に好適に使用されるが、これらに限定するものではない。 C a成分を含み P成分を含まない電解質輸液、 P成分を含み C a成分を含まない 電解質輸液、 塩化ナトリウム輸液、 透析液専用炭酸水素ナトリウム補充液として は、例えばそれぞれ市販のリンゲル液（大塚製薬）、 クリニザルツ B (小林薬ェ）、 生理食塩液 （大塚製薬）、 バイフィル専用炭酸水素ナトリウム補充液 （武田薬品） を用いれば良い。 また、 その組成は実施例に記載の通りである。 Furthermore, calcium phosphate unstable supersaturated solution of C a- P- N a- K- Mg- C 1- C0 3 system latency is delayed leading to deposition to medical infusion solution, dialysis, peritoneal perfusate, infusion It can also be prepared by mixing one or more selected from supplements for correction, calcium, dialysis and peritoneal perfusate. These soluble liquid or agents, C a, come P, Na, K, Mg, in use as a source of C and C_〇 3, this case has all the solutions or drugs are already approved for medical , And Since it has been sterilized, it is convenient for use in operating rooms. Electrolyte infusion containing Ca component and no P component, electrolyte infusion containing P component and no Ca component, dialysis A sodium hydrogen replenisher is particularly preferably used, but is not limited thereto. Examples of electrolyte infusions that contain Ca component and do not contain P component, electrolyte infusions that contain P component and do not contain Ca component, sodium chloride infusion, and sodium bicarbonate replenisher for dialysate include, for example, commercially available Ringer's solution (Otsuka Pharmaceutical Co., Ltd.) , Clinizarts B (Kobayashi Yakue), physiological saline (Otsuka Pharmaceutical), and sodium bicarbonate replenisher for Bayfil (Takeda Yakuhin) may be used. The composition is as described in Examples.
タンパク質を添加しておく溶液は、 上述の少なくともカルシウム成分を含む溶 液、 少なくともリン酸成分を含む溶液、 カルシウム成分とリン酸成分の両方を含 む溶液、 自発核形成までの誘導時間を制御する溶液のいずれかに添加しておいて も良い。ただし、タンパク質の変性を防止する観点から p H 5以上 8以卞の溶液に 添加しておくことが望ましい。 この際のタンパク質の添加量は、 タンパク質の種 類やタンパク質を担持する焼結体の用途により異なるが、 数 g/mL〜数 g/mL、 好 ましくは lO g/mL lOO g/mLである。 焼結体への担持量もタンパク質の種類や タンパク質を担持する焼結体の用途により異なるが、 1平方 c m当り 0 . 4 g 以上、 好ましくは 1 g以上であり、 担持量の上限には特に制限はない。 単位面 積当りの担持量を計算するための面積の値は担持前の焼結体面積のことである。 担持量の下限は実験的に求めた。 すなわち、 発明者らは共沈を伴わず吸着のみで リン酸カルシウムに担持できるタンパク質の量を求めた結果、 吸着だけで 1平方 c m当り 0 . 3 3 gのタンパク質が担持できることがわかった （実施例 5 )。そ こで、 担持量の下限は 1平方 c m当り 0 . とした。  The solution to which the protein is added is a solution containing at least the calcium component described above, a solution containing at least the phosphate component, a solution containing both the calcium component and the phosphate component, and controls the induction time until spontaneous nucleation. It may be added to any of the solutions. However, from the viewpoint of preventing protein denaturation, it is desirable to add it to a solution with a pH of 5 or more and 8 or less Byone. The amount of protein to be added at this time varies depending on the type of protein and the use of the sintered body supporting the protein, but it is several g / mL to several g / mL, preferably 10 g / mL and 10 g / mL. is there. The amount supported on the sintered body also varies depending on the type of protein and the use of the sintered body supporting the protein, but it is 0.4 g or more, preferably 1 g or more per square cm. No restrictions. The value of the area for calculating the supported amount per unit area is the area of the sintered body before being supported. The lower limit of the supported amount was determined experimentally. That is, the inventors determined the amount of protein that can be supported on calcium phosphate only by adsorption without coprecipitation and found that 0.333 g of protein could be supported per square cm by adsorption alone (Example 5). ). Therefore, the lower limit of the supported amount was set at 0.1 per square cm.
各成分溶液の温度と p Hは、タンパク質の変性を防止できる温度と p Hであれば 特に制限はない。 一般的には、 多くのタンパク質は体温以上では変性することが 多いので、各成分溶液と混合後の溶液の温度は 3 7 °C以下であることが望ましい。 しかし、 タンパク質の変性温度は、 タンパク質の種類によって異なるので、 使用 するタンパク質によっては 3 7 °C以下に限るものではない。  The temperature and pH of each component solution are not particularly limited as long as the temperature and pH can prevent protein denaturation. In general, most proteins are denatured above body temperature, so the temperature of the solution after mixing with each component solution is desirably 37 ° C or less. However, the denaturation temperature of proteins differs depending on the type of protein, and is not limited to 37 ° C or lower depending on the protein used.
本発明において、 リン酸カルシウムを主成分とする焼結体とは、 焼結後にリン 酸カルシウム焼結体となるリン酸カルシウム及び、 これに炭酸、 珪素、 マグネシ ゥム、 亜鉛、 鉄、 マンガン、 酸化カルシウムの少なくとも 1種類を含んだものを レ う。 In the present invention, the sintered body containing calcium phosphate as the main component is Calcium phosphate, which is a calcium oxide sintered body, and one containing at least one of carbonic acid, silicon, magnesium, zinc, iron, manganese, and calcium oxide.
本発明において、元素や炭酸が固溶したリン酸カルシウムとは、マグネシウム、 亜鉛、 鉄、 マンガンの金属元素の固溶であればリン酸カルシウムのカルシウムを 当該金属元素が不純物として一部置換したリン酸カルシウム、 珪素であればリン 酸カルシウムのリンを不純物として一部置換したリン酸カルシウム、 炭酸の場合 はリン酸カルシウムのリン酸基を不純物として一部置換したリン酸カルシウムの ことである。 珪素や炭酸が固溶する場合は置換される原子またはイオン団との間 で電荷の不一致があるため、 これを補うための他元素の 2次的固溶や、 構造中に 原子が存在しない空孔サイトが生成したりする。 例えば、 炭酸が水酸アパタイト  In the present invention, the calcium phosphate in which the element or carbonic acid is dissolved in a solid solution is a calcium phosphate or silicon in which calcium of calcium phosphate is partially substituted as an impurity if the metal element of magnesium, zinc, iron, or manganese is dissolved. For example, it refers to calcium phosphate in which phosphorus of calcium phosphate is partially substituted as an impurity, and in the case of carbonic acid, it refers to calcium phosphate in which the phosphate group of calcium phosphate is partially substituted as an impurity. When silicon or carbonic acid forms a solid solution, there is a charge inconsistency between the atom or ion group to be replaced, so a secondary solid solution of other elements to make up for this and a void in which no atoms exist in the structure Or a hole site is generated. For example, carbonic acid is hydroxyapatite
Ca1()(P04)fi(OH)2に固溶する場合、 （Na†， Co}') と （Ca2 、 PO ) の同時置換、 （K+, Ca 1 () (P0 4) When the solid solution fi (OH) 2, (Na †, Co} ') simultaneous substitution with (Ca 2, PO), ( K +,
C03 2") と （Ca2} 、 Ρθ ") の同時置換、 や （H†， C03 2") と （Ca2† 、 Ρθ の同時置換 で炭酸が固溶する。各元素やイオン団には固溶限界があり、固溶限界以上に珪素、 マグネシウム、亜鉛、鉄、マンガンをリン酸カルシウムに含有させようとすると、 これらの元素を固溶したリン酸カルシウムの他に、 これらの元素の酸化物ゃリン 酸塩が生成して、 これら元素の酸化物やリン酸塩を含む組成物となる。 固溶限界 は、 例えばリン酸カルシウム焼結体が低温型 Ca3(P04)2であれば、 マグネシウム、 亜鉛、 鉄、 マンガンはいずれも全カルシウムの約 12mol%である。 Carbonic acid forms a solid solution by simultaneous substitution of (C0 3 2 ") and (Ca 2} , Ρθ") or by simultaneous substitution of (H † , C0 3 2 ") and (Ca 2 † , Ρθ). Has a solid solution limit, and if silicon, magnesium, zinc, iron, and manganese are to be contained in calcium phosphate beyond the solid solution limit, in addition to calcium phosphate in which these elements are dissolved, oxides of these elements phosphate is generated, a composition comprising an oxide or phosphate of these elements. the solid solubility limit, for example calcium phosphate sinter is cold-type Ca 3 (P0 4) if 2, magnesium, Zinc, iron and manganese are all about 12 mol% of the total calcium.
リン酸カルシウムを主成分とする焼結体には、 骨形成やその他の生体機能を強 化するために、 亜鉛， マグネシウム、 鉄、 マンガン、 珪素のなかから選ばれた 1 種または複数の生体必須元素を添加することができる。 亜鉛、 鉄、 マンガン、 珪 素の焼結後の含有量は、 骨の亜鉛、 鉄、 マンガン、 珪素含有濃度の 1倍以上 1 0 The sintered body containing calcium phosphate as a main component contains one or more bio-essential elements selected from zinc, magnesium, iron, manganese, and silicon to enhance bone formation and other biological functions. Can be added. The content of zinc, iron, manganese, and silicon after sintering is at least 1 times the concentration of zinc, iron, manganese, and silicon in bone.10
0倍までの範囲とする。 骨の亜鉛， 鉄、 マンガン、 ケィ素含有濃度は亜鉛： 0.The range is up to 0 times. Bone contains zinc, iron, manganese, and silicon in a concentration of zinc: 0.
0 12重量％〜0. 0217重量％、 鉄： 0. 0 14重量％〜0. 02重量％、 マンガン： 1 p pm〜4 p pm、 珪素： 0. 01 05重量％である。 生体必須元 素含有量が骨の含有量の 1倍以下では、 これら元素特有の持つ生体機能促進作用 を骨中で発揮することは出来ない。 また、 1 0 0倍以上の含有量では、 骨組織中 で使用する人工骨においても、 細胞培養液中で使用する組織工学スキヤフォール ドにおいても、 これらの元素が過剰となり毒性を発現する。 2 5倍以上 1 0 0倍 以下の含有量の場合は、 骨組織中では毒性を発現するものの、 細胞培養液中では 毒性を発現しない。 マグネシウムの焼結後の含有量は、 骨のマグネシウム含有濃 度の 1倍以上 5 0倍までの範囲とする。 骨のマグネシウム含有濃度は 0 . 2 6重 量％〜0 . 5 5重量％である。マグネシウム含有量が骨の含有量の 1倍以下では、 マグネシウムの生体機能促進作用を骨中で発揮することは出来ない。 マグネシゥ ム含有量の上限だけ他の生体必須元素と違って骨の含有濃度の 5 0倍までとした 理由は、 マグネシウムの骨含有量が他の生体必須元素よりけた違いに多いため、 5 0倍以上の含有量では焼結後の力ルシゥムモル数よりマグネシウムモル数のほ うが多くなり、 リン酸カルシウムが主成分とならないからである。 0.12% to 0.0217% by weight, iron: 0.014% to 0.02% by weight, manganese: 1 ppm to 4 ppm, silicon: 0.0105% by weight. When the content of essential biological elements is less than 1 times that of bone, the biological function promoting action unique to these elements Cannot be demonstrated in the bone. If the content is 100 times or more, these elements are excessive in both artificial bone used in bone tissue and tissue engineering skidfold used in cell culture solution, and toxicity is exhibited. When the content is 25 times or more and 100 times or less, toxicity occurs in bone tissue, but no toxicity occurs in a cell culture medium. The content of magnesium after sintering should be between 1 and 50 times the magnesium content of bone. The magnesium content of the bone is between 0.26% by weight and 0.55% by weight. If the magnesium content is less than 1 times the bone content, the biological function promoting effect of magnesium cannot be exerted in bone. The reason why the upper limit of the magnesium content is set to 50 times the bone concentration unlike other essential biological elements is that the bone content of magnesium is 50 times larger than other essential biological elements. At the above content, the molar number of magnesium becomes larger than the molar number of the calcium hydroxide after sintering, and calcium phosphate is not the main component.
亜鉛、 マグネシウム、 鉄、 マンガン、 珪素、 カルシウムは、 焼結前の原料粉末 であるリン酸カルシウムに固溶させておいてもよいし、 無機塩、 金属、 酸化物、 水酸化物、 有機金属化合物として混合させておいてもよい。 無機塩、 金属、 酸化 物、 水酸化物、 有機金属化合物をあらかじめ混合させておく場合は、 焼結時にこ れら元素はリン酸カルシウムと反応して固溶する。 混合元素量が固溶限界以上の 場合は、 焼結後当該元素固溶リン酸カルシウムの他に当該金属酸化物またはリン 酸塩が生成する。 無機塩として混合させる場合は、 焼結時に陰イオン団が揮発す る、 炭酸塩や硝酸塩が好ましい。 金属の塩化物、 弗化物、 硫酸塩は焼結時に生体 適合性の低い塩素、 フッ素、 硫酸基が残存する結果となるため、 使用できない。  Zinc, magnesium, iron, manganese, silicon, and calcium may be dissolved in calcium phosphate, which is the raw material powder before sintering, or mixed as inorganic salts, metals, oxides, hydroxides, and organometallic compounds. You may let it. When inorganic salts, metals, oxides, hydroxides, and organometallic compounds are mixed in advance, these elements react with calcium phosphate during sintering to form a solid solution. When the amount of the mixed element is equal to or higher than the solid solution limit, the metal oxide or phosphate is generated after sintering in addition to the element-dissolved calcium phosphate. When mixed as an inorganic salt, a carbonate or a nitrate, in which an anion group is volatilized during sintering, is preferable. Metal chlorides, fluorides and sulphates cannot be used because of the residual chlorine, fluorine and sulphate groups which are poorly biocompatible during sintering.
リン酸カルシウムを主成分とする焼結体の全体としての Ca/Pモル比は 0 . 7 5 以上 2 . 1以下、 好ましくは 1 . 1以上 1 . 9以下である。 Ca/Pモル比 1 . 5以 下の範囲にあっても、 炭酸、 珪素、 マグネシウム、 亜鉛、 鉄、 マンガンから選ば れた不純物を含むリン酸カルシウム焼結体、 例えばマグネシウムを含有するリン 酸カルシウム焼結体であれば、 焼結後にマグネシウム固溶リン酸三カルシウムと リン酸三マグネシウムの混合物とすることができ、 生体適合性の低いピロリン酸 カルシウムの生成を防ぐことができる。 しかし、 Ca/P モル比 0 . 7 5未満では、 炭酸、 珪素、 マグネシウム、 亜鉛、 鉄、 マンガンから選ばれた不純物を添加した 場合は、 ピロリン酸カルシウムの生成を防ぐことができても、 これら不純物成分 の含有モル数のほうがカルシウムの含有モル数より多い結果となり、 リン酸カル シゥム質焼結体でなくなる。従って、 リン酸カルシウム焼結体の Ca/Pモル比の下 限は 0 . 7 5とした。 Ca/Pモル比が 2 . 1以上では毒性限界以上の酸化カルシゥ ム生成を伴い、 焼結体の生体適合性が損なわれる。 そこでリン酸カルシウム焼結 体の Ca/Pモル比の上限は 2 . 1とした。 The Ca / P molar ratio of the entire sintered body mainly composed of calcium phosphate is 0.75 or more and 2.1 or less, preferably 1.1 or more and 1.9 or less. Even if the Ca / P molar ratio is in the range of 1.5 or less, a calcium phosphate sintered body containing impurities selected from carbonic acid, silicon, magnesium, zinc, iron, and manganese, for example, a calcium phosphate sintered body containing magnesium In the case of a body, a mixture of magnesium-dissolved tricalcium phosphate and trimagnesium phosphate can be obtained after sintering, and the formation of calcium pyrophosphate having low biocompatibility can be prevented. However, if the Ca / P molar ratio is less than 0.75, impurities selected from carbonic acid, silicon, magnesium, zinc, iron, and manganese are added. In this case, even if the formation of calcium pyrophosphate can be prevented, the number of moles of these impurity components is larger than the number of moles of calcium, so that the sintered body is not a calcium phosphate sintered body. Therefore, the lower limit of the Ca / P molar ratio of the calcium phosphate sintered body was set to 0.75. When the Ca / P molar ratio is 2.1 or more, calcium oxide is generated at a level exceeding the toxic limit, and the biocompatibility of the sintered body is impaired. Therefore, the upper limit of the Ca / P molar ratio of the calcium phosphate sintered body was set to 2.1.
炭酸、 珪素、 マグネシウム、 亜鉛、 鉄、 マンガンを含まないリン酸カルシウム 焼結体の Ca/Pモル比は 1 . 5以上 2 . 0以下であることが望ましい。 このような リン酸カルシウム焼結体としては、具体的には単独でも Ca/Pモル比が 1 . 5以上 2 . 0以下である水酸アパタイト、 /3リン酸三カルシウム、 ひリン酸三カルシゥ ム、 リン酸 4カルシウム、 及びこれらの混合焼結体のほか、 酸化カルシウムが混 合された焼結体でもよい。 これらの化合物は、 化学量論組成のものであっても良 いし、 非化学量論組成のものでもよい。  The Ca / P molar ratio of the calcium phosphate sintered body not containing carbonic acid, silicon, magnesium, zinc, iron, and manganese is desirably 1.5 or more and 2.0 or less. Specific examples of such a calcium phosphate sintered body include hydroxyapatite having a Ca / P molar ratio of 1.5 or more and 2.0 or less, / 3 tricalcium phosphate, tricalcium phosphate, and the like. In addition to tetracalcium phosphate and a mixed sintered body thereof, a sintered body in which calcium oxide is mixed may be used. These compounds may be of stoichiometric or non-stoichiometric composition.
炭酸を含むリン酸カルシウム焼結体としては、 具体的には炭酸を固溶した水酸 ァパタイ卜焼結体を挙げることができる。  Specific examples of the calcium phosphate sintered body containing carbonic acid include a hydroxyapatite sintered body in which carbonic acid is dissolved.
珪酸を含むリン酸カルシウム焼結体としては、 具体的には珪酸を固溶した水酸 アパタイト、 珪酸を固溶したリン酸三カルシウム、 及びこれらの混合焼結体のほ か、 ケィ酸カルシウム又は珪酸を添加した焼結体を挙げることができる。  Specific examples of the calcium phosphate sintered body containing silicic acid include hydroxyapatite in which silicic acid is dissolved, tricalcium phosphate in which silicic acid is dissolved, and a mixed sintered body of these, and calcium silicate or silicic acid. The added sintered body can be mentioned.
マグネシウム、 亜鉛、 鉄、 マンガンを含むリン酸カルシウム焼結体としては、 これらの金属イオンを固溶した水酸アパタイト、 リン酸 4カルシウム、 リン酸三 カルシウムのほか、 これら金属酸化物及びリン酸塩を添加したリン酸カルシウム を挙げることができる。  As a calcium phosphate sintered body containing magnesium, zinc, iron, and manganese, in addition to hydroxyapatite, tetracalcium phosphate, and tricalcium phosphate in which these metal ions are dissolved, these metal oxides and phosphates are added. Calcium phosphate.
また、 本発明において焼結体中の酸化カルシウムは、 溶出して焼結体表面近傍 の p H を上昇させ、 その結果焼結体表面近傍の過飽和度を上昇させるため、 沈殿 を基板表面で集中的に形成させるために特に効果的である。 この目的のため、 酸 化カルシウムを焼結体中に 0 . 1重量％から 4重量％まで、 好ましくは 0 . 1重 量％から 3 . 5重量％まで、 さらに好ましくは 0 . 1重量％から 1 . 8重量％含 ませることができる。 酸化カルシウム 0 . 1重量％未満では p H上昇の効果が小 さいため、 酸化カルシウム含有量の下限を 0 . 1重量％とした。 酸化カルシウム は空気中の水分と反応するため、 含有量が多いと焼結体の強度が低下する。 4重 量％より酸化カルシウム含有量が多いと焼結体の強度が著しく低下するため、 上 限を 4重量％とした。 Further, in the present invention, calcium oxide in the sintered body elutes and raises the pH near the surface of the sintered body, thereby increasing the degree of supersaturation near the surface of the sintered body. This is particularly effective for forming the target in a uniform manner. For this purpose, calcium oxide is present in the sintered body in an amount of 0.1% to 4% by weight, preferably 0.1% to 3.5% by weight, more preferably 0.1% to 3.5% by weight. It can contain 1.8% by weight. If the calcium oxide content is less than 0.1% by weight, the effect of increasing the pH is small, so the lower limit of the calcium oxide content was set to 0.1% by weight. Calcium oxide Reacts with moisture in the air, so that if the content is large, the strength of the sintered body decreases. When the content of calcium oxide is more than 4% by weight, the strength of the sintered body is remarkably reduced, so the upper limit is set to 4% by weight.
酸化カルシウムと空気中の水分との反応による強度劣化を防止する目的で、 酸 化カルシウムを焼結体の表面から少なくとも 1ミクロンの表面層内だけに含有さ せておくこともできる。 表面からの深さの下限を 1ミクロンとした理由は、 リン 酸カルシウム焼結体の粒径が通常は 1ミクロンから 1 0ミクロンの大きさであり、 最表面層を 1ミクロン以下の厚さに分割できないからである。 表面からの深さの 上限は特に無く、 焼結体の強度に影響がない含有量範囲であれば、 全体に酸化力 ルシゥムが分布していても差し支えない。  In order to prevent strength deterioration due to the reaction between calcium oxide and moisture in the air, calcium oxide can be contained only in the surface layer of at least 1 micron from the surface of the sintered body. The reason for setting the lower limit of the depth from the surface to 1 micron is that the particle size of the calcium phosphate sintered body is usually 1 micron to 10 microns, and the outermost surface layer has a thickness of 1 micron or less. This is because they cannot be divided. There is no particular upper limit for the depth from the surface, and oxidizing calcium may be distributed throughout as long as the content does not affect the strength of the sintered body.
酸化カルシウムと同様に、 リン酸三カルシウムとリン酸 4カルシウムも、 焼 結体表面近傍の過飽和度を上昇させるため、 沈殿を基板表面で集中的に形成させ るために効果的である。 すなわち、 これらのリン酸カルシウムは加水分解によつ て低結晶性水酸ァパタイトに変化し、 加水分解反応時のカルシウム放出により、 焼結体表面近傍の過飽和度が上昇する。 また、 加水分解で生じる高活性な低結晶 性水酸ァパタイ卜も、 溶液中のタンパク質を取り込む。  Like calcium oxide, tricalcium phosphate and tetracalcium phosphate are also effective for increasing the degree of supersaturation near the surface of the sintered body and for forming precipitates intensively on the substrate surface. That is, these calcium phosphates are converted into low-crystalline hydroxyapatite by hydrolysis, and the degree of supersaturation near the surface of the sintered body increases due to release of calcium during the hydrolysis reaction. Highly active, low-crystalline hydroxyapatite produced by hydrolysis also takes up proteins in solution.
リン酸三カルシウムとリン酸 4カルシウムの焼結体中での含有量は 9 0重 量％以上か 2 0重量％以下、好ましくは 9 5重量％以上か 1 5重量％以下である。  The content of tricalcium phosphate and tetracalcium phosphate in the sintered body is 90% by weight or more and 20% by weight or less, preferably 95% by weight or more and 15% by weight or less.
リン酸三カルシウムゃリン酸 4カルシウムの含有量が 2 0重量％より高く 9 0 重量％未満の範囲では、 焼結体の強度は著しく小さい。  When the content of tricalcium phosphate / tetracalcium phosphate is more than 20% by weight and less than 90% by weight, the strength of the sintered body is extremely low.
酸化カルシウムと同様に、 ひリン酸三カルシウムとリン酸 4カルシウムを焼結 体の表面から少なくとも 1ミクロンの表面層内のみに含有させておくことは、 空 気中の水分と反応して強度劣化するのを防ぐ観点から好ましい。 表面からの深さ の下限を 1ミクロンとした理由は、 リン酸カルシウム焼結体の粒径が通常は 1ミ クロンから 1 0ミクロンの大きさであり、 最表面層を 1ミクロン以下の厚さに分 割できないからである。 表面からの深さの上限は特に無く、 強度に影響のない含 有量範囲であれば、 焼結体全体に αリン酸三カルシウムとリン酸 4カルシウムが 分布していても差し支えない。 酸化カルシウム、 ひリン酸三カルシウムまたはリ ン酸 4カルシウムが表面からどの程度内部に存在しているかは、 例えば X線マイ クロアナリシス装置を備えた走査型電子顕微鏡や透過型電子顕微鏡で断面観察す ることにより確認することができる。 As with calcium oxide, the inclusion of tricalcium phosphate and tetracalcium phosphate only in the surface layer of at least 1 micron from the surface of the sintered body reduces the strength due to the reaction with moisture in the air. It is preferable from the viewpoint of preventing the occurrence of the above. The reason for setting the lower limit of the depth from the surface to 1 micron is that the particle size of the calcium phosphate sintered body is usually from 1 micron to 10 microns, and the outermost surface layer is divided into a thickness of 1 micron or less. Because it cannot be split. There is no particular upper limit on the depth from the surface, and as long as the content does not affect the strength, α-tricalcium phosphate and tetracalcium phosphate may be distributed throughout the sintered body. The extent to which calcium oxide, tricalcium arsenate or tetracalcium phosphate is present from the surface can be determined, for example, by X-ray microscopy. It can be confirmed by observing the cross section with a scanning electron microscope or a transmission electron microscope equipped with a chromaanalysis device.
担持する夕ンパク質は水溶性の夕ンパク質であり、 生物学的活性化物質を使用 することができる。 水溶性タンパク質には、 非水溶性のタンパク質をアルブミン などの水溶性担体タンパク質またはポリェチレングリコール、 エチレングリコー ル Zプロピレングリコールのコポリマー、 カルポキメチルセルロース、 デキスト ラン、 ポリビニルアルコール、 ポリビニルピロリドン、 ポリ— 1， 3—ジォキソ ラン、ポリ一 1， 3 , 6—トリオキサン、エチレン Z無水マレイン酸コポリマー、 ポリアミノ酸類 （ホモポリマ一またはランダムコポリマー） などの水溶性ポリマ 一に結合させることで水可溶性とした非水溶性タンパク質も含む。 非水溶性の夕 ンパク質と上記水溶性担体タンパク質または水溶性ポリマーとの結合は両方の物 質の官能基を利用すればよく、 種々の公知の方法で結合させることができる。 ここで生物学的活性化物質は生物に対して生物活性を有する、 すなわち生物に作 用することで生物体に何らかの変化を誘起し得る物質をいう。 生物学的活性化物 質として、 生体の調節や生体の機能を変化させ得るサイト力イン、 ホルモン等の 生理活性物質が含まれ、 例えば成長因子や細胞接着因子がある。 水溶性で生物活 性を持たないタンパク質の例としては、アルブミン、チトクロム C、グロブリン、 などを挙げることができる。 水溶性で生物活性を持つタンパク質、 すなわち生物 学的活性化因子の例としては、 塩基性繊維芽細胞成長因子、 IL-1 (インターロイ キン 1 )、 IL- 2、 IL_3、 IL- 4、 IL - 5、 IL- 6、 IL_7、 IL- 8、 IL-9、 IL- 10、 IL- 11、 IL- 12、 The supported protein is a water-soluble protein, and a biological activator can be used. Water-soluble proteins include water-insoluble proteins such as albumin and other water-soluble carrier proteins or copolymers of polyethylene glycol, ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and poly-1. Water-soluble by binding to water-soluble polymers such as 1,3-dioxolane, poly-1,3,6-trioxane, ethylene Z maleic anhydride copolymer, polyamino acids (homopolymer or random copolymer) Also includes sex proteins. The binding between the water-insoluble protein and the above-mentioned water-soluble carrier protein or water-soluble polymer may be achieved by utilizing the functional groups of both substances, and can be carried out by various known methods. Here, the biologically activating substance refers to a substance having biological activity on an organism, that is, a substance capable of inducing some change in an organism by acting on the organism. Biological activators include bioactive substances such as cytokins and hormones that can regulate the living body and change the function of the living body, such as growth factors and cell adhesion factors. Examples of water-soluble and non-bioactive proteins include albumin, cytochrome C, globulin, and the like. Examples of water-soluble and biologically active proteins, i.e., biological activators, include basic fibroblast growth factor, IL-1 (interleukin 1), IL-2, IL_3, IL-4, IL -5, IL-6, IL_7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13、 IL- 15、 IL- 17、 IL-18, GM-CSF (顆粒球マクロファージコロニー刺激因子）、IL-13, IL-15, IL-17, IL-18, GM-CSF (granulocyte macrophage colony stimulating factor),
G- CSF (顆粒球コロニー刺激因子）、エリスロポエチン、 CSF- 1 (コロニー刺激因子）、G-CSF (granulocyte colony stimulating factor), erythropoietin, CSF-1 (colony stimulating factor),
SCF (幹細胞因子）、 トロンポポェチン、 EGF (上皮増殖因子）、 TGF-ひ （トランス フォ一ミング増殖因子 -ひ）、 HB-EGF (へパリン結合性 EGF様増殖因子）、 ェピレグ リン、 ニューレグリン 1、 2、 3、 PDGF (血小板由来増殖因子）、 インスリン、 HGFSCF (Stem Cell Factor), Thrombopoietin, EGF (Epidermal Growth Factor), TGF-H (Transforming Growth Factor-H), HB-EGF (Heparin-binding EGF-like Growth Factor), Epiregulin, Neuregulin 1, 2, 3, PDGF (platelet-derived growth factor), insulin, HGF
(肝細胞増殖因子）、 VEGF (血管内皮増殖因子）、 NGF (神経成長因子）、 GDNF (グ リア細胞株由来神経栄養因子）、 ミツドカイン、 TGF- /3 (トランスフォーミング増 殖因子- ) 6 )、 ベータダリカン、 ァクチビン、 BMP (骨形成因子）、 TNF (腫瘍壊死因 子）、 IFN- a/ jS (インターフェロン - α/ ) IFN- r (イン夕一フエロン-ァ）、 フ イブロネクチン、 ラミニン、 カドヘリン、 インテグリン、 セレクチンなどを挙げ ることができるが、 これらに限定はされない。 動物細胞の結合組織を構成する夕 ンパク質であるコラーゲン、 ゼラチンは本発明の生物学的活性化因子には含まれ ない。 (Hepatocyte growth factor), VEGF (vascular endothelial growth factor), NGF (nerve growth factor), GDNF (glial cell line-derived neurotrophic factor), mitdocaine, TGF- / 3 (transforming growth factor-) 6) , Betadarican, activin, BMP (bone morphogenetic factor), TNF (tumor necrosis factor), IFN-a / jS (interferon-α /) IFN-r (inyuichi-feron-a), Examples include, but are not limited to, ibronectin, laminin, cadherin, integrins, selectins, and the like. Collagen and gelatin, which are proteins constituting the connective tissue of animal cells, are not included in the biologically activating factor of the present invention.
リン酸カルシウム焼結体は、 緻密質焼結体であっても良いし、 多孔質焼結体で あってもよい。 多孔質焼結体に生物活性を有するタンパク質を担持する場合は、 焼結体を体内に埋入した後に、 血管、 骨、 上皮、 神経等の再生組織を焼結体の気 孔内に侵入させることができるので効果が大きい。 気孔率が 20 %より低いと実 質的に緻密質であり組織侵入に適さず、 気孔率が 80%より高いと強度が低下し 実用上の価値が損なわれる。 そのため、 多孔質リン酸カルシウム焼結体の気孔率 は 20 %以上 80 %以下とした。 気孔率は、 以下の方法により測定することが可 能である。 すなわち、 多孔質焼結体の外寸法と重量を測定し、 体積 V (cm3) と 重量 W (g) を求める。 多孔質焼結体を構成するリン酸カルシウムの理論密度をThe calcium phosphate sintered body may be a dense sintered body or a porous sintered body. When a biologically active protein is carried on a porous sintered body, regenerated tissues such as blood vessels, bones, epithelium, and nerves are allowed to penetrate into the pores of the sintered body after the sintered body is embedded in the body. It can be very effective. If the porosity is lower than 20%, it is practically dense and unsuitable for tissue penetration. If the porosity is higher than 80%, the strength is reduced and the practical value is impaired. Therefore, the porosity of the porous calcium phosphate sintered body was set to 20% or more and 80% or less. The porosity can be measured by the following method. That is, the outer dimensions and weight of the porous sintered body are measured, and the volume V (cm 3 ) and the weight W (g) are determined. The theoretical density of calcium phosphate constituting the porous sintered body
D (gZcm3) とすれば、 気孔率 （％) は P= (1— P (W/ (VxD)) x 1 00で求めることができる。 例えば、 縦 1. 560 cm、 横 1. 550 c m、 高 さ 0. 340 cm、 重量 1. 0704 gで、 気孔が 3次元網目状に貫通している 水酸ァパタイト多孔質焼結体であれば、 水酸ァパタイ卜の理論密度値 3. 1 6 gIf D (gZcm 3 ), the porosity (%) can be obtained by P = (1-P (W / (VxD)) x 100. For example, 1.560 cm long and 1.550 cm wide The height is 0.340 cm, the weight is 1.0704 g, and the pores penetrate in a three-dimensional network. If it is a porous hydroxyapatite sintered body, the theoretical density value of hydroxyapatite 3.16 g
Zcm3を用いて、 P=58. 8%となる。 Using Zcm 3 , P = 58.8%.
組織が侵入するためには、 少なくとも 2個以上細胞が気孔内に侵入する必要が ある。 細胞 1個の大きさは 30ミクロンであるため、 気孔直径の最小値は 70ミ クロンとした。 また、 直径 4 mmより大きい組織を侵入させる必要性は実用上皆 無に等しいため、 気孔直径の上限は 4mmとした。 気孔直径は光学顕微鏡や走査 型電子顕微鏡を用いて多孔質焼結体の断面を観察することで測定することができ る。  In order for a tissue to invade, at least two or more cells must enter the stomata. Since the size of a single cell is 30 microns, the minimum stomatal diameter was set to 70 microns. Since the necessity of penetrating tissue larger than 4 mm in diameter is practically negligible, the upper limit of the pore diameter was set to 4 mm. The pore diameter can be measured by observing the cross section of the porous sintered body using an optical microscope or a scanning electron microscope.
気孔が焼結体を貫通していれば、 血管や神経を貫通ささせることもできる。 気 孔が 3次元網目状に貫通していれば、 毛細血管や骨組織が三次元網目状に貫通す るのに都合が良い。  If the pores penetrate the sintered body, they can penetrate blood vessels and nerves. If the pores penetrate in a three-dimensional network, it is convenient for capillaries and bone tissue to penetrate in a three-dimensional network.
リン酸カルシウムを主成分とする焼結体の原料粉末としては、 リン酸カルシゥ ム粉末、 及びリン酸カルシウムに炭酸、 珪素、 マグネシウム、 亜鉛、 鉄、 マンガ ンのうち少なくとも 1種が含有されるか固溶したリン酸カルシウム粉末を使用す ることができる。 The raw material powder of the sintered body containing calcium phosphate as a main component includes calcium phosphate powder, calcium carbonate, carbonic acid, silicon, magnesium, zinc, iron, and manganese. Calcium phosphate powder containing or dissolving at least one of the components can be used.
炭酸、 珪素、 マグネシウム、 亜鉛、 鉄、 マンガンを含まないリン酸カルシウム 原料粉末の Ca/Pモル比は 1 . 5以上 2 . 0以下であることが望ましい。 このよう なリン酸カルシウム粉末としては、具体的には単独でも Ca/Pモル比が 1 . 5以上 2 . 0以下である水酸アパタイト、 リン酸三カルシウム、 リン酸 4カルシウム、 非晶質リン酸カルシウム、 及びこれらの混合物のほか、 これらの単独粉末又は混 合物に Ca/Pモル比が 1 . 5以下または 2 . 0以上の粉末、 具体的には、 リン酸水 素カルシウム、 グリセ口リン酸カルシウム、 金属カルシウム、 酸化カルシウム、 炭酸カルシウム、 乳酸カルシウム、 クェン酸カルシウム、 硝酸カルシウム、 カル シゥムアルコキサイド等のカルシウム塩が混合されたものでもよいし、 リン酸ァ ンモニゥム、 リン酸等が混合されたものでもよい。 これらの化合物は、 化学量論 組成のものであってもいし、 非化学量論組成のものでもよい。  The Ca / P molar ratio of the calcium phosphate raw material powder not containing carbonic acid, silicon, magnesium, zinc, iron and manganese is desirably 1.5 or more and 2.0 or less. Specific examples of such calcium phosphate powder include hydroxyapatite having a Ca / P molar ratio of 1.5 or more and 2.0 or less, tricalcium phosphate, tetracalcium phosphate, amorphous calcium phosphate, and the like. In addition to these mixtures, powders having a Ca / P molar ratio of 1.5 or less or 2.0 or more in these single powders or mixtures, specifically, calcium hydrogen phosphate, calcium glycerate calcium, and calcium metal Calcium oxide, calcium carbonate, calcium lactate, calcium citrate, calcium nitrate, calcium salt such as calcium alkoxide, or a mixture of ammonium phosphate, phosphoric acid, etc. Good. These compounds may be of stoichiometric or non-stoichiometric composition.
炭酸を含むリン酸カルシウム原料粉末としては、 具体的には炭酸を固溶した水 酸アパタイト、 炭酸を固溶した非晶質リン酸カルシウム、 及びこれらの混合物、 さらに炭酸ナトリウム、 炭酸カリウム、 炭酸アンモニゥムを添加したリン酸カル シゥム粉末を挙げることができる。  Examples of the calcium phosphate raw material powder containing carbonic acid include hydroxyapatite in which carbonic acid is dissolved, amorphous calcium phosphate in which carbonic acid is dissolved, and a mixture thereof, and phosphorus to which sodium carbonate, potassium carbonate, and ammonium carbonate are added. Acid calcium powder can be mentioned.
珪酸を含むリン酸カルシウム粉末としては、 具体的には珪酸を固溶した水酸ァ パタイト、 珪酸を固溶した非晶質リン酸カルシウム、 珪酸を固溶したリン酸三力 ルシゥム、 及びこれらの混合物のほか、 ケィ酸カルシウム又は珪酸を添加したリ ン酸カルシウム粉末を挙げることができる。  Specific examples of the calcium phosphate powder containing silicic acid include hydroxyapatite in which silicic acid is dissolved, amorphous calcium phosphate in which silicic acid is dissolved, triphosphate calcium in which silicic acid is dissolved, and mixtures thereof. Calcium silicate powder to which calcium silicate or silicic acid is added can be given.
マグネシウム、 亜鉛、 鉄、 マンガンを含むリン酸カルシウム粉末としては、 こ れらの金属イオンを固溶した水酸アパタイト、 非晶質リン酸カルシウム、 リン酸 4カルシウム、 リン酸三カルシウムのほか、 これら金属及び金属酸化物、 水酸化 物、 リン酸塩、 硝酸塩、 炭酸塩を添加したリン酸カルシウム粉末を挙げることが できる。 金属の塩化物、 弗化物、 硫酸塩は焼結時に生体適合性の低い塩素、 フッ 素、 硫酸基が残存する結果となるため、 使用できない。  Examples of calcium phosphate powder containing magnesium, zinc, iron, and manganese include hydroxyapatite, amorphous calcium phosphate, tetracalcium phosphate, and tricalcium phosphate in which these metal ions are dissolved, as well as metals and metal oxides. Phosphate powder to which a substance, a hydroxide, a phosphate, a nitrate, or a carbonate is added. Metal chlorides, fluorides, and sulfates cannot be used because sintering results in the retention of poorly biocompatible chlorine, fluorine, and sulfate groups.
酸化カルシウムを含有する焼結体の原料粉末は、 高温で酸化カルシウムを生成 するカルシウム化合物を、 全体の C a / Pモル比が 1 . 6 7を超えるようにリン 酸カルシウムと混合して燒結することで作製することができる。 このようなカル シゥム化合物としては具体的には、 カルシウムの硝酸塩、 炭酸塩、 乳酸塩、 クェ ン酸塩、 水酸化物、 キレ一卜等を挙げることができる。 表面だけ酸化カルシウム を含有するようにするには、 例えば、 これらの水溶液を焼結前の成型体に塗布し ておけば良い。 The raw material powder of the sintered body containing calcium oxide is a phosphorous compound that generates calcium oxide at a high temperature so that the total Ca / P molar ratio exceeds 1.67. It can be manufactured by mixing with calcium acid and sintering. Specific examples of such calcium compounds include calcium nitrate, carbonate, lactate, citrate, hydroxide, and chelates. In order to contain calcium oxide only on the surface, for example, these aqueous solutions may be applied to a molded body before sintering.
リン酸カルシウムを主成分とする焼結体の原料粉末の粒子径には特に制限はな いが、 約 0. l imから ΙΟΟ ΠΙであることが望ましい。 これらの原料粉末にポリビ ニルアルコールなどのバインダーを含有させ、 場合によってはさらに水やアルコ ールなどの溶剤を含有させて成型体を作製する。 焼結工程は、 通常の電気炉を用 い大気雰囲気下で、 500°C以上 1 50 0°C以下、 好ましくは 700°C以上 140 0で 以下で行う。 50 0°C以下では焼結は起こらず、 1 50 0°C以上では多くのリン 酸カルシウムが分解する。 最適焼結温度は、 焼結させるリン酸カルシウム粉末の 化学組成によって異なる。 例をあげれば、 炭酸を 3— 1 5重量％含有する水酸ァ パタイトの最適焼結温度は 6 0 0°C以上 8 0 0°C以下である。 水酸ァパタイ卜 (Ca1Q(P04) 6 (OH) 2： C aZPモル比 = 1. 6 7) の最適焼結温度は 90 0 °C以上 1The particle size of the raw material powder of the sintered body containing calcium phosphate as a main component is not particularly limited, but is preferably from about 0.1 lim to ΙΟΟ. A molded body is prepared by adding a binder such as polyvinyl alcohol to these raw material powders and, in some cases, further including a solvent such as water or alcohol. The sintering step is performed at 500 ° C. or more and 1500 ° C. or less, preferably at 700 ° C. or more and 1400 ° C. or less in an air atmosphere using a normal electric furnace. Below 500 ° C, sintering does not occur, and above 1500 ° C, many calcium phosphates decompose. The optimum sintering temperature depends on the chemical composition of the calcium phosphate powder to be sintered. For example, the optimum sintering temperature of hydroxyapatite containing 3 to 15% by weight of carbonic acid is from 600 ° C to 800 ° C. Hydroxyl Apatai Bok (Ca 1Q (P0 4) 6 (OH) 2: C aZP molar ratio = 1.6 7) optimal sintering temperature of 90 0 ° C over 1
2 0 0°C以下である。 水酸ァパタイトとひリン酸三カルシウムの複合焼結体の最 適焼結温度は 1 1 3 0°C以上 1 2 0 0°C以下である。 水酸アパタイトと酸化カル シゥムの複合焼結体の最適焼結温度は 1 1 3 0°C以上 1 2 00°C以下である。 ケ ィ素を含む水酸ァパタイトの最適焼結温度は 9 0 0°C以上 1 2 0 0°C以下である。 <8リン酸三カルシウム （Ca3(P04),： C aZPモル比 = 1. 50) の最適焼結温度 は、 9 00°C以上 1 1 0 0°C以下である。 リン酸三カルシウムの最適焼結温度 は 1 2 00°C以上 1 500°C以下である。 亜鉛、 マンガン、 マグネシウムを含有 する βリン酸三カルシウムの最適焼結温度は 9 00°C以上 1 2 0 0°C以下である。 亜鉛、 マンガン、 マグネシウムを含有する リン酸三カルシウムの最適焼結温度 は 1 3 0 0°C以上 1 50 0°C以下である。 It is below 200 ° C. The optimum sintering temperature of the composite sintered body of hydroxyapatite and tricalcium arsenate is not less than 110 ° C and not more than 1200 ° C. The optimum sintering temperature of the composite sintered body of hydroxyapatite and calcium oxide is not less than 110 ° C and not more than 1200 ° C. The optimum sintering temperature of hydroxyapatite containing silicon is 900 ° C or higher and 1200 ° C or lower. Optimal sintering temperature of <8 tricalcium phosphate (Ca 3 (P0 4) ,: C aZP molar ratio = 1.50) is 9 00 ° C or more 1 1 0 0 ° C or less. The optimum sintering temperature for tricalcium phosphate is between 1200 ° C and 1500 ° C. The optimum sintering temperature of β-tricalcium phosphate containing zinc, manganese and magnesium is 900 ° C or more and 1200 ° C or less. The optimum sintering temperature of tricalcium phosphate containing zinc, manganese, and magnesium is between 130 ° C and 1500 ° C.
共沈するかどうかは目視で確認することもできるし、 動的光散乱法を用いてナ ノメーターレベルで確認することもできる。共沈物は粉末 X線回折法 、走查型電 子顕微鏡観察で評価できる。 タンパク質量はビュレット法に Bicinchonic Acid を組み合わせた、 比色分析で定量することができる。 タンパク質の徐放性はタン パク質担持リン酸カルシウム焼結体を生理的食塩水に浸し、 経時的に食塩水中の タンパク質量を定量して評価することができる。 本発明のタンパク質担持リン酸 カルシウム焼結体からなるタンパク質の徐放性は、 担持するタンパク質の種類、 タンパク質を担持する焼結体の用途により異なる。 共沈させるリン酸カルシウム の量を変えることにより徐放性を調節することができ、 様々な用途に対応させる ことが可能である。 Whether or not coprecipitation can be confirmed visually or at the nanometer level using dynamic light scattering. The coprecipitate can be evaluated by powder X-ray diffraction and scanning electron microscope observation. The amount of protein can be quantified by colorimetric analysis combining the burette method with Bicinchonic Acid. Sustained release of protein is tan The calcium phosphate sintered body carrying the protein can be immersed in physiological saline, and the amount of protein in the saline can be quantified and evaluated over time. The sustained release of the protein comprising the protein-supported calcium phosphate sintered body of the present invention varies depending on the type of the protein supported and the use of the protein-supported sintered body. The sustained release can be adjusted by changing the amount of calcium phosphate to be coprecipitated, and it can be adapted to various uses.
本発明の水溶性タンパク質を表面に担持させたリン酸カルシウムを主成分とす る焼結体は、 人工骨等の生体材料として用いることができる。 表面に担持してい るタンパク質である生物学的活性化因子が生体組織再構築に有用である。 また、 担持している夕ンパク質が徐々に放出されるので、 夕ンパク質徐放体として用い ることができる。 例えば、 本発明のタンパク質を担持した焼結体を生体内に埋め 込んで用いればよい。 さらに、 本発明のタンパク質を担持した焼結体を組織工学 スキヤホールドとして用いることができる。 すなわち、 焼結体上で細胞を培養し て皮膚、 骨等のヒトの組織や臓器を形成させることが可能である。  The sintered body mainly composed of calcium phosphate having the water-soluble protein supported on the surface of the present invention can be used as a biomaterial such as an artificial bone. Biological activators, which are proteins carried on the surface, are useful for biological tissue reconstruction. Also, since the carried protein is gradually released, it can be used as a sustained-released evening protein. For example, a sintered body supporting the protein of the present invention may be embedded in a living body and used. Further, the sintered body supporting the protein of the present invention can be used as a tissue engineering scanner. That is, cells can be cultured on a sintered body to form human tissues and organs such as skin and bone.
本明細書は本願の優先権の基礎である日本国特許出願 2002- 341464号の明細書 および/または図面に記載される内容を包含する。 図面の簡単な説明  This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2002-341464, which is a priority document of the present application. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 C a— P— N a—K— C 1系不安定リン酸カルシウム過飽和溶液溶液 からのチトクロム C共沈量を示す図である。  FIG. 1 is a graph showing the amount of co-precipitated cytochrome C from a solution of a Ca—P—Na—K—C1 system unstable calcium phosphate supersaturated solution.
図 2は、 チトクロム Cを担持した緻密質水酸ァパタイトー酸化カルシウム複合 焼結体表面の X線回折パターンを示す図である。 発明を実施するための最良の形態  FIG. 2 is a diagram showing an X-ray diffraction pattern of the surface of a dense hydroxyapatite-calcium oxide composite sintered body supporting cytochrome C. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を実施例に基づいて説明する。 本発明はこの実施例に限定される ものではない。  Hereinafter, the present invention will be described based on examples. The present invention is not limited to this embodiment.
実施例 1 Example 1
超純水に K C 1を 0〜0 . 1 1 9 2 gを加えてスターラ一で 3 0分間攪拌して 溶解し、 0〜2 O mMK C 1溶液を作製した。 次にこの溶液に 5 0 mM H3 P O 溶液を加えた。 次に l O OmM C aC l 2溶液を加え、 20 mM KOH溶液を 徐々に滴下して pH 7. 4に調整し、 （3 &(312濃度5111]^、 ？〇4濃度1]11 の 溶液を得た。 これを C a P溶液と呼ぶ。 塩基性繊維芽細胞増殖因子 （bFGF) を超 純水に溶解しトリスヒドロキシメチルァミノメタンを加えて （5 mM；)、 1 2. 5 〜 1 5 OmgZLのトリス緩衝タンパク質溶液とした。 C a P溶液とタンパク質 溶液を 1 ： 1で混合した。 動的光散乱法で粒子サイズが成長するのを追跡したと ころ、 KC120mM以下で析出が確認された。 析出に至る待ち時間は KC1濃度に強く 依存し、 KC1 OmMで約 1時間、 KC1 lOmMでは 10時間程度であった。 析出物は粉末 X線回折法で同定を行い、 走查型電子顕微鏡で形態観察を行なった。 更に、 析出 物を濾過後の溶液についてタンパク質濃度を定量した。 その結果、 析出物は bFGF とァパタイトの共沈物であることが確認された。析出物のサイズは数 100〜1ミク ロン程度であった。 すなわち、 KC1 の濃度を適当に選択することで、 リン酸カル シゥムの析出までの時間を制御でき、 しかもリン酸カルシウムにタンパク質 bFGF を結合させて共沈させることが示された。 0 to 0.1192 g of KC1 was added to ultrapure water, and the mixture was dissolved by stirring with a stirrer for 30 minutes to prepare a 0 to 2 OmM KC1 solution. Then add 50 mM H 3 PO The solution was added. Then l O OmM C aC l 2 solution was added, and slowly added dropwise 20 mM KOH solution was adjusted to pH 7. 4, (3 & ( 31 2 Concentration 5111] ^,? 〇 4 concentration 1] 11 This solution was called a “CaP solution.” Basic fibroblast growth factor (bFGF) was dissolved in ultrapure water, trishydroxymethylaminomethane was added (5 mM;), and 12.5 ~ 15 OmgZL of Tris-buffered protein solution The CaP solution and the protein solution were mixed at a ratio of 1. The growth of the particle size was monitored by dynamic light scattering. The waiting time until precipitation strongly depended on the KC1 concentration, which was about 1 hour for KC1 OmM and about 10 hours for KC1 lOmM. Morphological observation was performed with an electron microscope, and the protein concentration of the solution after filtration of the precipitate was quantified. It was confirmed that it was a co-precipitate of patite, and the size of the precipitate was about several hundred to one micron, that is, until the precipitation of calcium phosphate by appropriately selecting the KC1 concentration. It was shown that the time can be controlled and that protein bFGF is bound to calcium phosphate for co-precipitation.
実施例 2 Example 2
KC 1成分 0〜 5 OmM、 C a C 12成分 1. 17〜2. 5mM、 H3P〇4成分KC 1 component 0~ 5 OmM, C a C 1 2 -component 1. 17~2. 5mM, H 3 P_〇 4 components
1. 0mM〜2. 33mMの種々の組成の溶液を作製した。 すなわち、 クリーン ベンチ内で 80 m 1の超純水に KC 1を 0〜0. 2982 gを加えてスターラー で 30分間攪拌して溶解し、 0〜5 OmMKC 1溶液を作製した。 次にこの溶液 に 50mMH3PO4溶液を 1. 6〜3. 72 mLを加えた。 次に l O OmM C a C l9溶液を 0. 936〜2. OmLを加え、 20 mM K OH溶液を徐々に滴下 して pH7. 4に調整した。 これを C a P溶液と呼ぶ。 チトクロム Cをカルシゥ ム不含マグネシウム不含リン酸緩衝生理的食塩水 （PBS (—））、 すなわちSolutions of various compositions from 1.0 mM to 2.33 mM were made. That is, in a clean bench, 0 to 0.2982 g of KC1 was added to 80 ml of ultrapure water and dissolved by stirring with a stirrer for 30 minutes to prepare a 0 to 5 OmM KC1 solution. Next, 1.6 to 3.72 mL of a 50 mM 3 PO 4 solution was added to this solution. Then l O OmM C a C l 9 solution 0. 936-2. OML was added and adjusted by slowly added dropwise 20 mM K OH solution pH 7. 4. This is called a CaP solution. Cytochrome C with calcium-free magnesium-free phosphate buffered saline (PBS (—)),
、 KC 1成分 2. 68mM、 KH2P〇4成分 1. 46 mM、 N a C 1成分 1 36., KC 1 component 2. 68 mM, KH 2 P_〇 four components 1. 46 mM, N a C 1 component 1 36.
89mM、 Na2HP04成分 8. 10 mMの水溶液に 50 g /mLの濃度で溶 解させた。 さきの CaP溶液 lmLとタンパク質含有 PBS (―) lmLを混合して遅 延共沈水溶液とした。 窒素雰囲気下にこの水溶液を 7日間放置し、 孔径 0. 22 mのフィルターでろ過し、 ろ液のチトクロム C含有量を定量した。 その結果、 溶液の C aZPモル比 1. 0〜2. 5の範囲、 KC 1濃度 0〜2 OmMでチトク ロム Cとリン酸カルシウムの共沈が生じた （図 1)。 89 mM, was dissolve at a concentration of 50 g / mL in an aqueous solution of Na 2 HP0 4 component 8. 10 mM. The CaP solution (1 mL) and the protein-containing PBS (-) (1 mL) were mixed to obtain a delayed coprecipitation aqueous solution. This aqueous solution was allowed to stand for 7 days in a nitrogen atmosphere, and filtered through a filter having a pore size of 0.22 m, to determine the content of cytochrome C in the filtrate. As a result, it was found that the molar ratio of CaZP in the solution ranged from 1.0 to 2.5, and the KC1 concentration was 0 to 2 OmM. Co-precipitation of rom C and calcium phosphate occurred (Figure 1).
実施例 3 Example 3
リン酸カルシウムを主成分とする種々の緻密質及び多孔質焼結体を作製した (表 1)。 緻密質焼結体は、 3 %ポリビニルアルコールを添加した粒径 7 ァ ンダ一の各種リン酸カルシウム粉末を 10 OMPa で一軸加圧成形し、 1 100°C 〜 1200°Cで焼結して作製した。 焼結後の大きさは直径 1 3〜14mm、 厚さ 1〜1. 2 mmであり、 相対密度 90〜 95 %であった。 緻密質焼結体の表面積 は 3. 06〜3. 60 cm2であった。 多孔質焼結体は以下のような方法で作製し た。 すなわち、 3 %ポリビニルアルコールを添加した粒径 75 m アンダーの各 種リン酸カルシウム粉末を 0. 175 gを秤量し、 超純水 40〜65 L 添加し て鍊和し、直径 0. 5 mm長さ 28 mmのステンレス製長柱体状ォス型 1 3本を 0. 3 mm間隔に平行に配列し、 この上に、 これと直交する方向で同一寸法のステン レス製長柱体状ォス型 14本を配列した。 この長柱体状ォス型配列物に上記粉末 鍊和物を詰め込み、 36MPa で加圧した。 加圧後、 長柱体状ォス型を被覆してい る粉末をブラチック製スクレーパーで取り除いた。 上記操作を 4回繰り返した。 加圧成型後、 長柱体状ォス型を全部抜き取って、 気孔を形成させた。 これを室温 で、 2日間乾燥させ、 700〜1 170でで 5時間燒結して多孔体とした。 多孔 体は直径 400 /mの直線状貫通気孔を交互に直交させた気孔を有し、 2方向の 気孔の交点は直径 50〜200 m の気孔が形成されていた。 多孔質焼結体は、 緻密質焼結体とほぼ同重量のものを切り出して使用した。 気孔率は 60〜64% である。 これらの焼結体は全て 160°C、 1時間乾熱滅菌した。 Various dense and porous sintered bodies mainly composed of calcium phosphate were produced (Table 1). The dense sintered body was produced by uniaxially pressing various calcium phosphate powders having a particle size of 7 to which 3% polyvinyl alcohol was added at 10 OMPa and sintering at 1100 ° C to 1200 ° C. The size after sintering was 13 to 14 mm in diameter, 1 to 1.2 mm in thickness, and the relative density was 90 to 95%. The surface area of dense sintered body 3. was 06~3. 60 cm 2. The porous sintered body was manufactured by the following method. That is, 0.175 g of each type of calcium phosphate powder having a particle size of 75 m under which 3% polyvinyl alcohol was added was weighed, added with 40 to 65 L of ultrapure water, and adjusted to a diameter of 0.5 mm and a length of 28 mm. 13 mm long stainless steel rod-shaped dies are arranged in parallel at 0.3 mm intervals, and a stainless steel long rod-shaped dies with the same dimensions in the direction perpendicular to this are placed on top of this. Books were arranged. The powdered hydrate was packed in the columnar os-type array, and pressurized at 36 MPa. After pressurization, the powder covering the long columnar os-type was removed with a scraper made of bratic. The above operation was repeated four times. After the pressure molding, all the long pillar-shaped bosses were extracted to form pores. This was dried at room temperature for 2 days, and sintered at 700 to 1170 for 5 hours to obtain a porous body. The porous body had pores in which straight through pores with a diameter of 400 / m were alternately orthogonalized, and pores with a diameter of 50 to 200 m were formed at the intersection of the pores in the two directions. A porous sintered body having a weight approximately equal to that of the dense sintered body was cut out and used. The porosity is 60-64%. All of these sintered bodies were subjected to dry heat sterilization at 160 ° C for 1 hour.
担持に用いた CaP溶液は CaCl2成分 2. 1 mM、 H3P04成分 1. 4mM、 KC1成分CaP solution used for loading was CaCl 2 component 2.1 mM, H 3 P0 4 component 1.4 mM, KC1 component
1 0または 2 OmMである。タンパク質はチトクロム 塩基性繊維芽細胞増殖因 子 （bFGF)、 ラミニンの 3種類で、 これらのタンパク質はカルシウム不含マグネ シゥム不含リン酸緩衝食塩水 （PBS (—））、 すなわち、 KC 1成分 2. 68mM、 KH PC^成分1. 46mM、 N a C 1成分 1 36. 89 mM、 N a 2H P〇4成分10 or 2 OmM. There are three types of proteins: cytochrome basic fibroblast growth factor (bFGF) and laminin. These proteins are calcium-free magnesium-free phosphate buffered saline (PBS (—)), that is, KC 1 component 2 68 mM, KH PC ^ component 1.46 mM, NaC 1 component 1 36.89 mM, Na 2 HP〇 4 component
8. 1 OmMの水溶液に 25又は 50 g ZmLの濃度で溶解させた。 8. Dissolved in an aqueous solution of 1 OmM at a concentration of 25 or 50 g ZmL.
滅菌した焼結体を細胞培養用 24穴プレートに入れ、 これに CaP溶液 lmLと タンパク質含有 PBS (—） 溶液 lmLを入れた。 窒素雰囲気中に 7日間放置し、 放 置後溶液を回収してタンパク質量を定量し、 焼結体へのタンパク質担持量を求め た（表 2 )。その結果、 いずれの焼結体にもタンパク質が担持されていることがわ かった。 特に、 多孔質焼結体や酸化カルシウムを含有する焼結体で、 担持量が多 かった。 The sterilized sintered body was placed in a 24-well plate for cell culture, and 1 mL of CaP solution and 1 mL of protein-containing PBS (-) solution were placed therein. Leave in nitrogen atmosphere for 7 days and release After the incubation, the solution was recovered, the amount of protein was quantified, and the amount of protein carried on the sintered body was determined (Table 2). As a result, it was found that all of the sintered bodies supported the protein. In particular, the amount of the porous sintered body and the sintered body containing calcium oxide were large.
表 1 表 1 夕ンパク質担持に用いたリン酸カルシウム主成分焼結体 Table 1 Table 1 Calcium phosphate main component sintered body used for supporting protein
HAP 水酸アパタイ ト Ca10 (P04) 6 (OH) 2 HAP water acid apatite Ca 10 (P0 4) 6 ( OH) 2
β TCP j8 リン酸三カルシウム /3 33 (1>04) 2 β TCP j8 Tricalcium phosphate / 3 3 3 (1> 0 4 ) 2
CHAP C037. 1重量％固溶炭酸水酸アパタイ ト CHAP C0 3 7.1 1% by weight solid solution carbonated hydroxyapatite
HAP/CaO 水酸アパタイ ト一酸化カルシウム複合焼結体、 C a O O . 5 6 — 3. 2 5 w t %■。  HAP / CaO Hydroxyapatite calcium monoxide composite sintered compact, CaO O .56 — 3.25 wt% ■.
HAP/ a TCP 水酸ァパタイ ト— α リ ン酸三カルシウム複合焼結体、 CaZPモル比 = 1. 6 4。  HAP / a TCP hydroxyapatite-α-tricalcium phosphate composite sintered body, CaZP molar ratio = 1.64.
ZnTCP/HAP 亜鉛含有リ ン酸 3カルシウム一水酸アパタイ ト複合焼結体、  ZnTCP / HAP Zinc-containing tricalcium phosphate monocalcium apatite composite sintered body,
(Ca+Zn) /P= 1. 6 4、 亜鉛 0. 8 4 w t %。  (Ca + Zn) /P=1.64, zinc 0.84 wt%.
ZnHAP 亜鉛含有水酸アパタイ ト、 亜鉛 0. 3 4 w t  ZnHAP Zinc-containing hydroxyapatite, zinc 0.3 4 wt
ZnHAP/CaO 亜鉛含有水酸ァパタイ トー酸化カルシウム複合焼結体、  ZnHAP / CaO zinc-containing hydroxyapatite-to-calcium oxide composite sintered body,
亜鉛 0. 3 2 w t %、 Ca03. 2 5 w t %。  Zinc 0.32 wt%, Ca03.25 wt%.
ZnllAPX aTCP 亜鉛含有水酸ァパタイ トー α リ ン酸三カルシウム複合焼結体、  ZnllAPX aTCP Zinc-containing hydroxyapatite α-tricalcium phosphate composite sintered body,
亜鉛 0. 3 4 w t %、 （Ca+Zn) /"Pモル比 = 1. 6 7、  Zinc 0.34 wt%, (Ca + Zn) / "P molar ratio = 1.67,
a T C Ρ含有量 2. 7 8重量％。 a T C Ρ content 2.78% by weight.
gTCP マグネシウム含有リ ン酸三カルシウム、  gTCP Magnesium-containing tricalcium phosphate,
マグネシウム 2. 5 w t %。  Magnesium 2.5 wt%.
MgTCP/HAP マグネシウム含有リ ン酸三カルシウム一水酸ァパタイ ト複合焼結体, マグネシウム 2. 2 w t %  MgTCP / HAP Magnesium-containing tricalcium phosphate monohydrate apatite composite, magnesium 2.2 wt%
(Ca+Mg ) ZPモル比 = 1. 5 5  (Ca + Mg) ZP molar ratio = 1.55
FeTCPXHAP 鉄含有リン酸酸カルシウム—水酸アパタイ ト焼結体、  FeTCPXHAP Iron-containing calcium phosphate-hydroxyapatite sintered body,
鉄 0. 5 w t %、 （Ca+Fe) /Pモル比 = 1. 6 0 表 2 0.5 wt% iron, (Ca + Fe) / P molar ratio = 1.60 Table 2
表 2 C a-P-Na-K-C 1系不安定リン酸カルシウム過 和溶液によるリ ン酸カルシウム主成分焼結体上への夕ンパク担持結果 焼結体 κα濃度 タンパク 担持量 ^g) 担持量 (μ§ /cm2) jS CP 多孔質 10 チ卜クロム C 6. 8 Table 2 CaP-Na-KC 1-based unstable calcium phosphate solution containing calcium phosphate on sintered body containing calcium phosphate Sintered body κα concentration Protein supported amount ^ g) Supported amount (μ§ / cm 2 ) jS CP porous 10 cytochrome C 6.8
HAP 緻密質 10 チトクロム C 3. 8 1. 3  HAP Dense 10 Cytochrome C 3.8.1.3
HAP 緻密質 10 bFGF 9. 4 3. 1  HAP dense 10 bFGF 9.4 3.1
HAP 緻密質 10 ラミニン 7. 3 2. 4  HAP Dense 10 Laminin 7. 3 2. 4
HAP 緻密質 20 チトクロム C 2. 5 0. 8  HAP Dense 20 Cytochrome C 2.5 0.5
HAP 多孔質 10 チトクロム C 6. 8  HAP porous 10 cytochrome C 6.8
CHAP 多孔質 10 チ卜クロム C 7. 8  CHAP porous 10 cytochrome C 7.8
HAP/CaO 緻密質 10 チ卜クロム C 12. 9 4. 3  HAP / CaO dense 10 cytochrome C 12.9 4.3
HAP/CaO 緻密質 20 チトクロム C 13. 0 4. 3  HAP / CaO Dense 20 Cytochrome C 13.0.4.3
HAP/ a; TCP 緻密質 10 チトクロム C 5. 4 1. 7  HAP / a; TCP dense 10 cytochrome C 5.4 1.7
HAP/ o: TCP 緻密質 20 チ卜クロム C 2. 5 0. 7  HAP / o: TCP dense 20 cytochrome C 2.5 0.7
ZnTCP/ΉΑΡ 多孔質 10 チトクロム c 15. 0 ―  ZnTCP / ΉΑΡ porous 10 cytochrome c15.0-
ZnHAP 緻密質 10 チ卜グロム C 6. 1 2. 0  ZnHAP Dense 10 C troglom C 6.1.1.20
ZnHAP 緻密質 20 チトクロム C 3. 0 1. 0  ZnHAP dense 20 cytochrome C 3.0 1.0
ZnHAP/CaO 緻密質 10 チトクロム C 11. 8 3. 9  ZnHAP / CaO dense 10 cytochrome C 11.8 3.9
ZnHAP/CaO 緻密質 10 bFGF 12. 3 4. 1  ZnHAP / CaO dense 10 bFGF 12.34.1
ZnHAP/CaO 緻密質 20 チ卜クロム C 10. 1 3. 4  ZnHAP / CaO Dense 20 cytochrome C 10.1 3.4
ZnHAP/ a TCP 緻密質 10 チトクロム C 5. 0 1. 3  ZnHAP / a TCP dense 10 cytochrome C 5.0 1.3
ZnHAP/ TCP 緻密質 20 チ卜クロム C 3. 6 1. 0  ZnHAP / TCP dense 20 cytochrome C 3.6 1.10
MgTCP 多孔質 10 チトクロム C 1 1. 8 ―  MgTCP porous 10 cytochrome C 1 1.8-
M TCP/HAP 多孔質 10 チ卜クロム C 20. 3 ―  M TCP / HAP porous 10 cytochrome C 20.3-
FeTCP/HAP 多孔質 10 チトクロム C 15. 3 一  FeTCP / HAP porous 10 cytochrome C 15.3
実施例 4 Example 4
実施例 3で得られた、 チトクロム Cを担持した緻密質水酸ァパタイトー酸化力 ルシゥム複合焼結体表面の X線回折パターンを測定した （図 2)。その結果、 焼結 体表面に酸化カルシウムは検出されず、 担持溶液から析出生成した低結晶性ァパ タイトに覆われていることがわかった。 すなわち、 水酸アパタイト一酸化カルシ ゥム複合焼結体は水溶液中でカルシウムを放出し、 放出されたカルシウムが焼結 体表面近傍の過飽和度を更に上昇させ、 焼結体表面で低結晶性ァパタイトが大量 に析出し、 この時、 大量のタンパク質を共沈させたものと推察された。 An X-ray diffraction pattern of the surface of the dense hydroxyapatite-oxidizing power of the composite calcium oxide obtained carrying cytochrome C obtained in Example 3 was measured (FIG. 2). As a result, no calcium oxide was detected on the surface of the sintered body, and it was found that the surface was covered with low-crystalline apatite precipitated and generated from the supporting solution. That is, hydroxyapatite calcium oxide The calcium-based sintered body releases calcium in the aqueous solution, and the released calcium further increases the degree of supersaturation near the surface of the sintered body, and a large amount of low-crystalline apatite precipitates on the surface of the sintered body. It was presumed that a large amount of protein was coprecipitated.
実施例 5 Example 5
カルシウムを含有しない水溶液にタンパク質を溶解し、 カルシウムを放出する 酸化カルシウム含有焼結体上に担持させた。 焼結体は直径 13mm、 厚さ lmm で表面積は 3. 06 cm2である。 すなわち、 チトクロム Cをカルシウム不含マグ ネシゥム不含リン酸緩衝食塩水（PBS (—））、すなわち、 KC 1成分 2. 68mM、 KHり P04成分 1. 46mM、 N a C 1成分 1 36. 89mM、 Ν&2ΗΡ〇4成分The protein was dissolved in an aqueous solution containing no calcium, and was supported on a sintered body containing calcium oxide that releases calcium. The sintered body has a diameter of 13 mm, a thickness of lmm and a surface area of 3.06 cm 2 . That is, cytochrome C was added to calcium-free magnesium-free phosphate buffered saline (PBS (—)), namely, KC 1 component 2.68 mM, KH1 P4 4 component 1.46 mM, NaC 1 component 1 36. 89mM, Ν & 2 ΗΡ〇 4 ingredients
8. 1 OmMの水溶液に 25 gZmLの濃度で溶解させた。 8. Dissolved in an aqueous solution of 1 OmM at a concentration of 25 gZmL.
水中でカルシウムイオンを放出する焼結体として、 実施例 3の緻密質水酸ァパ タイトー酸化カルシウム複合焼結体、 亜鉛含有水酸ァパタイトー酸化カルシウム 複合焼結体、 水酸アパタイト一 αリン酸三カルシウム複合焼結体、 亜鉛含有水酸 ァパタイト _ひリン酸三カルシウム複合焼結体、 カルシウムイオンを放出しない 焼結体として実施例 3の緻密質水酸ァパタイト焼結体と亜鉛含有水酸ァパタイト 焼結体を用いた。 滅菌したこれら緻密質焼結体を細胞培養用 24穴プレートに入 れ、 これにチトクロム C含有 PBS (—） 溶液 2mLを入れた。 窒素雰囲気中に 7日 間放置し、 放置後溶液を回収してタンパク質量を定量し、 焼結体へのタンパク質 担持量を求めた（表 3)。カルシウムを放出しない水酸ァパタイト焼結体と亜鉛含 有水酸ァパタイト焼結体では、 タンパク質の担持は受動的吸着効果のみによるも ので、 タンパク質担持量は僅かであった。 一方、 カルシウムを放出する焼結体上 では、 放出カルシウムと PBS (—） 中のリン酸との反応で、 リン酸カルシウム が表面に生成し、 この時 PBS (—） 中のタンパク質を能動的に共沈で取込むた め、 カルシウムを放出しない焼結体の 2〜 7倍量のタンパク質を担持することが できた。 タンパク含有カルシウム不含水溶液によるカルシウム放出性リン酸カル シゥム主成分焼結体上へのタンパク担持結果 焼結体 担持液 タンパク 担持量 （μ8) 担持量 （pgZcm2)Examples of the sintered body that releases calcium ions in water include the dense hydroxyapatite-calcium oxide composite sintered body of Example 3, zinc-containing hydroxyapatite-calcium oxide composite sintered body, and hydroxyapatite-α-phosphate. Calcium composite sintered body, zinc-containing hydroxyapatite _ Tricalcium arsenate composite sintered body, which does not release calcium ions As a sintered body, the dense hydroxyapatite sintered body of Example 3 and zinc-containing hydroxyapatite The union was used. The sterilized sintered compacts were placed in a 24-well plate for cell culture, and 2 mL of a cytochrome C-containing PBS (-) solution was placed therein. After standing in a nitrogen atmosphere for 7 days, the solution was recovered and the amount of protein was quantified to determine the amount of protein carried on the sintered body (Table 3). In the hydroxyapatite sintered body that does not release calcium and the zinc-containing hydroxyapatite sintered body, the amount of protein carried was small because the protein was supported only by the passive adsorption effect. On the other hand, on the sintered body that releases calcium, calcium phosphate is generated on the surface by the reaction between the released calcium and phosphoric acid in PBS (—), and at this time, the protein in PBS (—) is actively co-precipitated. As a result, it was possible to carry 2 to 7 times as much protein as a sintered body that does not release calcium. Results of protein loading on calcium-releasing calcium phosphate main component sintered body using protein-containing calcium-free aqueous solution Sintered body Supported liquid Protein supported amount (μ8) Supported amount (pgZcm 2 )
HAP 緻密質 PBS (-) チトクロム C 1. 0 0. 3HAP dense PBS (-) cytochrome C 1.0 0.3
ZnHAP 緻密質 PBS (-) チトクロム C 1. 1 0. 3ZnHAP dense PBS (-) cytochrome C 1.10.3
HAP/CaO 緻密質 PBS (-) チトクロム C 7. 8 2. 6HAP / CaO dense PBS (-) cytochrome C 7.8.2.6
ZnHAP/CaO 緻密質 PBS (-) チトクロム C 7. 2 2. 4ZnHAP / CaO dense PBS (-) cytochrome C 7.2.2.4
HAP/ TCP 緻密質 PBS (-) チトクロム C 3. 3 1. 1HAP / TCP dense PBS (-) cytochrome C 3.3.1.1
ZnHAP/ a TCP 緻密質 PBS (-) チトクロム C 2. 0. 8 ZnHAP / a TCP Compact PBS (-) cytochrome C 2.0.8
実施例 6 Example 6
実施例 3で得られた緻密質タンパク質担持リン酸カルシウム焼結体をカルシゥ ム不含マグネシウム不含リン酸緩衝生理的食塩水 （PBS (—）） で洗い、 細胞培養 用 24穴シャーレに入れた。 ここに生理食塩水 （0. 9wt %食塩水） を 1. 5 mL滴下し、 37°C、 窒素雰囲気下に放置した。 経時的に生理食塩水を回収して タンパク質量を定量し、 タンパク質の徐放率を求めた（表 4)。 いずれの焼結体も タンパク質を徐放することができ、 タンパク質徐放体として使用できることが示 された。 また酸化カルシウムを含有する焼結体は他の焼結体に比較して徐放速度 が小さいことがわかった。 The dense protein-supported calcium phosphate sintered body obtained in Example 3 was washed with calcium-free magnesium-free phosphate buffered saline (PBS (-)) and placed in a 24-well petri dish for cell culture. 1.5 mL of physiological saline (0.9 wt% saline) was added dropwise thereto, and the mixture was allowed to stand at 37 ° C under a nitrogen atmosphere. The physiological saline was collected over time, the amount of protein was quantified, and the sustained release rate of protein was determined (Table 4). It was shown that any of the sintered bodies can release protein slowly and can be used as a protein sustained release body. It was also found that the sintered body containing calcium oxide had a slower release rate than other sintered bodies.
表 4 表 4 タンパクの徐放性 κα濃度 タンパク 放出率 (%) Table 4 Table 4 Sustained release of protein κα concentration Protein release rate (%)
(共沈時） 1曰後 3曰後 10曰後 (During co-precipitation) 1 after 3 after 10 after
ΗΑΡ 緻密質 1 0 チトクロム C 1 7. 0 25. 2 5 6. 2ΗΑΡ Dense 10 Cytochrome C 17.0 0 25.2 56.2
ΗΑΡ 緻密質 1 0 bFGF 1 3. 7 20. 0 ΗΑΡ Dense 1 0 bFGF 1 3.7 20.0
ΗΑΡ 緻密質 2 0 チ卜クロム C 3 0. 4 ― 5 2. 9 ΗΑΡ Dense 20 C-chromium C 3 0.4-5 2.9
HAP/CaO 緻密質 1 0 チ卜クロム C 1 0. 5 ― 1 2. 2HAP / CaO Dense 10 Cytochrome C 10.5-12.2
HAP/CaO 緻密質 2 0 チ卜クロム C 1 1. 9 ― 1 4. 9HAP / CaO dense 20 cytochrome C 1 1.9 ― 1 4.9
HAP/" a TCP 緻密質 1 0 チ卜クロム C 2 7. 0 — 4 0. 0HAP / "a TCP Dense 1 0 Cytochrome C 2 7.0 — 4 0.0
HAP/ α TCP 緻密質 2 0 チ卜クロム C 5 4. 8 一 5 9. 8HAP / α TCP dense 20 cytochrome C 5 4.8 1 5 9.8
ZnHAP 緻密質 1 0 チ卜クロム C 2 7. 7 3 4. 1ZnHAP Dense 10 Cytochrome C 2 7. 7 3 4. 1
ZnHAP 緻密質 2 0 チ卜クロム C 4 5. 7 6 0. 4ZnHAP Dense 20 Cytochrome C 4 5.7 6 0.4
ZnHAP/CaO 緻密質 1 0 チ卜クロム C 1 3. 8 1 7. 1 4 1. 4ZnHAP / CaO Dense 10 Cytochrome C 1 3.8 1 7. 1 4 1. 4
ZnHAP/CaO 緻密質 1 0 bFGF 1 2. 0 16. 0 ZnHAP / CaO Dense 1 0 bFGF 1 2.0 16.0
ZnHAP/CaO 緻密質 2 0 チトクロム C 1 6. 7 ― 2 5. 5 ZnHAP / CaO Dense 20 Cytochrome C 16.7-25.5
ZnHAP/ a TCP 緻密質 1 0 チ卜クロム C 2 5. 8 ― 4 6. 7ZnHAP / a TCP Dense 10 Cytochrome C 2 5.8 ― 4 6.7
ZnHAP/ a TCP 緻密質 2 0 チ卜クロム C 5 0. 0 ― 6 6. 2 ZnHAP / a TCP Dense 20 C cytochrome C 50.0-66.2
実施例 7 Example 7
表 5に組成を示したカルシウム不含有リン酸塩含有医療用電解質輸液とカルシ ゥム含有リン酸塩不含有医療用電解質輸液を Ca/Pモル比 =0.5〜2.5となるように 混合した。 これに NaHC0¾濃度 =0〜47.43 mMとなるように透析ろ過型人工腎臓用透 析液専用炭酸水素ナトリゥム補充液を混合し、 過飽和リン酸カルシウム溶液を調 製した。 この溶液 1. 8mLに、 チトクローム C濃度 250 zg/mLの生理食塩水溶 液（154 mM NaCl溶液）を体積比で 9:1 となるように混合して全体を 2 mLとし、 室温又は 37°Cで 2日間、 静置した。 できあがった溶液の組成は CaCl2成分 1. 2 0〜2. 3 9mM、 H3P(¾成分 0. 6 9〜3. 37 mM、 KC 1成分 0〜 40 mMを含 み pH が 5. 8〜9. 0であった。 表 6に室温でリン酸カルシウムが自発核形成 し、 チトクロム Cの共沈が生じた溶液の C a ZPモル比、 NaHC〇3濃度、 チト クロム C共沈量を示す。 表 7に 37°Cでリン酸カルシウムが自発核形成し、 チト クロム Cの共沈が生じた溶液における、 チトクロム C共沈量を示す。 すなわち、 これらの溶液はリン酸カルシウムの不安定過飽和溶液であり、 タンパク質を共沈 析出できる溶液であることが示された。 The calcium-free phosphate-containing medical electrolyte infusion and the calcium-containing phosphate-free medical electrolyte infusion whose compositions are shown in Table 5 were mixed so that the Ca / P molar ratio was 0.5 to 2.5. This was mixed with NaHCO ¾ concentration = from 0 to 47.43 mM become as diafiltration artificial kidney for magnetic析液dedicated bicarbonate Natoriumu replenisher was a supersaturated calcium phosphate solution was made tone. To 1.8 mL of this solution, mix a physiological saline solution (154 mM NaCl solution) with a cytochrome C concentration of 250 zg / mL (volume ratio of 9: 1) to a total volume of 2 mL, and mix at room temperature or at 37 ° C. For 2 days. The composition of the resulting solution CaCl 2 component 1. 2 0~2. 3 9mM, H 3 P (¾ component 0. 6 9~3. 37 mM, KC 1 components 0 to 40 mM unrealized pH is 5.8 Table 6 shows the molar ratio of CaZP, the concentration of NaHC 量3 , and the amount of co-precipitated cytochrome C in the solution in which calcium phosphate spontaneously formed nuclei at room temperature and co-precipitation of cytochrome C occurred. Table 7 shows that calcium phosphate spontaneously nucleated at 37 ° C, Shows the amount of cytochrome C co-precipitated in the solution in which chromium C co-precipitation occurred. That is, it was shown that these solutions were unstable supersaturated solutions of calcium phosphate, and were solutions capable of co-precipitating proteins.
表 5 表 5 医療用輸液の化学組成 （mEqZL) Table 5 Table 5 Chemical composition of medical infusion (mEqZL)
Na+ K+ Mg2+ cr Ca2+ H2P04" HCV GH3COO_ Na + K + Mg 2+ cr Ca 2+ H 2 P0 4 "HCV GH 3 COO _
P液 45 25 5 45 10 20 P liquid 45 25 5 45 10 20
Ca液 147 4 155.7 4.5  Ca solution 147 4 155.7 4.5
炭酸液 166 166  Carbonated liquid 166 166
N a C 1液 154 154  NaC 1 solution 154 154
P液：カルシウム不含有リン酸塩含有医療用電解質輸液Solution P: Calcium-free phosphate-containing medical electrolyte infusion
C a液：カルシウム含有リン酸塩不含有医療用電解質輸液 炭酸液：透析ろ過型人工腎臓用透析液専用炭酸水素ナトリゥム補充液C a solution: calcium-containing phosphate-free medical electrolyte infusion Carbonate solution: diafiltration type artificial kidney dialysate replenisher for sodium bicarbonate
N a C 1液：生理的食塩水 N a C 1 solution: physiological saline
表 6 表 6 C a-P-Na-K-Mg-C ί— C〇 3系不安定リン酸カルシウム過飽 からのタンパク共沈量。 温度は室温。 Table 6 Table 6 C aP-Na-K- Mg-C ί- C_〇 protein co沈量from 3 system unstable calcium phosphate over saturated. Temperature is room temperature.
Ca/Pモル比 NaHC03濃度 I mM Ca / P molar ratio of NaHCO 3 concentration I mM
0.00 7.90 15.09 27.67 47.43 +  0.00 7.90 15.09 27.67 47.43 +
0.5 7.27±3.81 8.85 ±4.28 8.30 ±3.45 6.85 ±3.46 5.49 ±3.74  0.5 7.27 ± 3.81 8.85 ± 4.28 8.30 ± 3.45 6.85 ± 3.46 5.49 ± 3.74
1.0 9.39±3.68 8.51 ±3.95 6.43 ±3.79 4.39+4.27 1·18±3.93  1.0 9.39 ± 3.68 8.51 ± 3.95 6.43 ± 3.79 4.39 + 4.27 1 ・ 18 ± 3.93
1.5 6.91 ±3.55 7.94 ±3.69 7.96 ±3.49 6.41 ±3.87 4.78 ±4.41  1.5 6.91 ± 3.55 7.94 ± 3.69 7.96 ± 3.49 6.41 ± 3.87 4.78 ± 4.41
2.0 9·10±4·05 7.98 ±3.63 8.87±3.61 5.87 ±3.63 2.78士 3.58  2.0 9 ± 10 ± 4.5 7.98 ± 3.63 8.87 ± 3.61 5.87 ± 3.63 2.78 士 3.58
2.5 8.15±3.82 8.58 ±3.61 8.31 ±4.59 6.22+3.80 2.70 ±4.13 表 7 表 7 C a - P -N a -K-M g - C 1—C 03系不安定リン酸カルシウム過飽 和溶液からのタンパク共沈量。 温度は 3 7 °C 2.5 8.15 ± 3.82 8.58 ± 3.61 8.31 ± 4.59 6.22 + 3.80 2.70 ± 4.13 Table 7 Table 7 C a - P -N a -KM g - C 1-C 0 3 based protein co沈量from unstable phosphate over saturated solution. Temperature is 37 ° C
Ca/Pモル比 NaHCO3濃度 I mM Ca / P molar ratio NaHCO 3 concentration I mM
0.00 7.90 15.09 27.67 47.43  0.00 7.90 15.09 27.67 47.43
0.5 17·14±3·47 18.25 ±1.60 17.16± 1.60 15·06±2.18 12.12 ±2.40  0.5 1714 ± 3 47 18.25 ± 1.60 17.16 ± 1.60 1506 ± 2.18 12.12 ± 2.40
1.0 16.41 + 3.23 13.88±2·04 10.51 ±2.28 14.46 ±2.93 13.74±6.24  1.0 16.41 + 3.23 13.88 ± 204 10.51 ± 2.28 14.46 ± 2.93 13.74 ± 6.24
1.5 17.54±2.81 15.48±4.27 12.95 ±3.91 9.67 ±2.53 8.72±2.04  1.5 17.54 ± 2.81 15.48 ± 4.27 12.95 ± 3.91 9.67 ± 2.53 8.72 ± 2.04
2.0 12·82±4.92 7.87 ±4.27 5.23±2.82 7.09 ±1.96 7.65 ±2.34  2.0 1282 ± 4.92 7.87 ± 4.27 5.23 ± 2.82 7.09 ± 1.96 7.65 ± 2.34
2.5 8.22+2.05 10.69±2.05 10.27 ±3.19 7.34 ±1.92 6.90 ±2.53  2.5 8.22 + 2.05 10.69 ± 2.05 10.27 ± 3.19 7.34 ± 1.92 6.90 ± 2.53
実施例 8 Example 8
表 5に組成を示したカルシウム不含有リン酸塩含有医療用電解質輸液とカルシ ゥム含有リン酸塩不含有医療用電解質輸液を Ca/Pモル比 = 1 . 5となるように混 合した。これに NaHC03濃度が 1 5 mMとなるように透析ろ過型人工腎臓用透析液専 用炭酸水素ナトリゥム補充液を混合し、過飽和リン酸カルシウム溶液を調製した。 この溶液に、 チトクローム Cの 250 g/mLの生理食塩水溶液（154 mM NaCl溶液） を体積比で 9 : 1となるように混合し、 リン酸カルシウム焼結体を投入し、 37°Cで 2日間、 静置した。 基材には、 酸化カルシウム含有量を 0. 00〜1. 65 重量％で変化 させた直径 13 mm、 厚さ 1 匪の円盤上の水酸ァパタイトー酸化カルシウム複合焼 結体を使用した。 焼結体の表面積は 3 . 0 6 c m2である。 不安定過飽和溶液から 基材表面へのァパタイトの析出過程で共沈したタンパク質量と基材の酸化カルシ ゥム含有量との関係は表 8の通りである。 表 8 The calcium-free phosphate-containing medical electrolyte infusion and the calcium-containing phosphate-free medical electrolyte infusion whose composition is shown in Table 5 were mixed so that the Ca / P molar ratio was 1.5. This NaHCO 3 concentration were mixed diafiltration artificial kidney dialysis solution dedicated bicarbonate Natoriumu replenisher such that 1 5 mM, was prepared a supersaturated calcium phosphate solution. A 250 g / mL saline solution (154 mM NaCl solution) of cytochrome C was mixed with this solution at a volume ratio of 9: 1, and a calcium phosphate sintered body was added thereto. It was left still. The base material used was a hydroxyapatite-calcium oxide composite sintered body with a diameter of 13 mm and a thickness of 1 mm, with the calcium oxide content varied from 0.00 to 1.65% by weight. Surface area of the sintered body is 3. 0 6 cm 2. Table 8 shows the relationship between the amount of protein co-precipitated in the process of depositing apatite from the unstable supersaturated solution on the surface of the substrate and the calcium oxide content of the substrate. Table 8
表 8 C a - P -N a -K -M g - C 1— C〇3系不安定リン酸カルシウム過飽 和溶液によるリン酸カルシウム主成分焼結体上へのタンパク担持結果。 温度は 3 Table 8 C a - P -N a -K -M g - C 1- C_〇 3 system unstable calcium peroxide saturated solution with a protein bearing a result of the calcium phosphate main component sintered body on. Temperature is 3
CaO含有 タンパク担持量 CaO-containing protein loading
(重量％)  (% By weight)
0.00 6.66 ±0.06  0.00 6.66 ± 0.06
0.56 7.70 ±0.05  0.56 7.70 ± 0.05
1.11 8.40+0.06  1.11 8.40 + 0.06
1.65 9.69 ±0.06  1.65 9.69 ± 0.06
本明細書で引用した全ての刊行物、 特許および特許出願をそのまま参考として 本明細書にとり入れるものとする。 産業上の利用の可能性 All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial potential
本発明は、 生物学的活性化物質であるタンパク質を担持したリン酸カルシウム を主成分とする焼結体であり、 生体適合性を有しており、 担持したタンパク質の 作用により、 生体組織再構築が促進され、 人工骨等の生体材料、 組織工学スキヤ ホールド等に用いることができる。 さらに、 実施例に示すように、 担持したタン パク質は徐放性を示すので、 徐放体として利用することもできる。  The present invention is a sintered body mainly composed of calcium phosphate carrying a protein which is a biologically activating substance, has biocompatibility, and promotes the reconstruction of a biological tissue by the action of the carried protein. It can be used for biomaterials such as artificial bones, tissue engineering scanners, etc. Furthermore, as shown in the examples, the supported protein exhibits a sustained release property, so that it can be used as a sustained release body.

Claims

請求の範囲 The scope of the claims
1. 自発核形成を生じる不安定リン酸カルシウム過飽和溶液中で、 生物学的 活性化物質である水溶性夕ンパク質とリン酸カルシウムとの制御された遅延共沈 を行ない、 生物学的活性化物質である水溶性タンパク質を表面に担持させたリン 酸カルシウムを主成分とする焼結体。 1. In a supersaturated solution of unstable calcium phosphate that causes spontaneous nucleation, controlled delayed co-precipitation of water-soluble protein, a biological activator, and calcium phosphate is performed, and aqueous solution, a biological activator, is dissolved. A sintered body mainly composed of calcium phosphate with a surface carrying a hydrophilic protein.
2. リン酸カルシウムが、 炭酸、 珪素、 マグネシウム、 亜鉛、 鉄、 マンガン のうち少なぐとも 1種を固溶したリン酸カルシウムである請求項 1に記載したリ ン酸カルシウム焼結体。  2. The calcium phosphate sintered body according to claim 1, wherein the calcium phosphate is calcium phosphate in which at least one of carbonate, silicon, magnesium, zinc, iron and manganese is dissolved.
3. リン酸カルシウムの他に、 カルシウム、 マグネシウム、 亜鉛、 鉄、 マン ガンから選ばれた金属の酸化物またはリン酸塩の少なくとも 1種を含んでいる請 求項 1に記載したリン酸カルシウム焼結体。  3. The calcium phosphate sintered body according to claim 1, which further comprises at least one oxide or phosphate of a metal selected from calcium, magnesium, zinc, iron, and manganese, in addition to calcium phosphate.
4. 焼結後の亜鉛含有量が 0. 0 1 2重量％〜1. 2重量％である請求項 2 または請求項 3に記載したリン酸カルシウム焼結体。  4. The calcium phosphate sintered body according to claim 2 or 3, wherein the zinc content after sintering is from 0.01 to 1.2% by weight.
5. 焼結後の炭酸含有量が 0. 3重量％〜1 5重量％である請求項 2に記載 したリン酸カルシウム焼結体。  5. The calcium phosphate sintered body according to claim 2, wherein the carbonic acid content after sintering is from 0.3% by weight to 15% by weight.
6. 焼結後のマグネシウム含有量が 0. 26重量％〜 13重量％である請求 項 2または請求項 3に記載したリン酸カルシウム焼結体。  6. The calcium phosphate sintered body according to claim 2 or 3, wherein the magnesium content after sintering is 0.26% by weight to 13% by weight.
7. 焼結後の珪素含有量が 0. 0105重量％〜1. 05重量％である請求 項 2または請求項 3に記載したリン酸カルシウム焼結体。  7. The calcium phosphate sintered body according to claim 2 or 3, wherein the silicon content after sintering is 0.0105% by weight to 1.05% by weight.
8. 焼結後の鉄含有量が 0. 0 14重量％〜1. 4重量％である請求項 2ま たは請求項 3に記載したリン酸カルシウム焼結体。  8. The calcium phosphate sintered body according to claim 2 or 3, wherein the iron content after sintering is 0.014% by weight to 1.4% by weight.
9. 焼結後のマンガン含有量が 1重量 p pm〜l 00重量 p pmである請求 項 2または請求項 3に記載したリン酸カルシウム焼結体。  9. The calcium phosphate sintered body according to claim 2 or 3, wherein the manganese content after sintering is 1 weight ppm to 100 weight ppm.
1 0. 焼結後の酸化カルシウム含有量が 0. 1〜4重量％である請求項 3に 記載したリン酸カルシウム焼結体。  10. The calcium phosphate sintered body according to claim 3, wherein the calcium oxide content after sintering is 0.1 to 4% by weight.
1 1. リン酸三カルシウム、 リン酸 4カルシウム、 酸化カルシウム、 また は酸化マグネシウムの少なくとも 1種が、 表面から少なくとも深さ 1ミクロンま での表面層に含有された請求項 3に記載のリン酸カルシウム焼結体。 1 1. The calcium phosphate calcination according to claim 3, wherein at least one of tricalcium phosphate, tetracalcium phosphate, calcium oxide, or magnesium oxide is contained in a surface layer from the surface to at least 1 micron in depth. Union.
12. 焼結体の Ca/Pモル比が 0. 75以上 2. 1以下である請求項 1から 1 1に記載したリン酸カルシウムを主成分とする焼結体。 12. The sintered body containing calcium phosphate as a main component according to claim 1, wherein the Ca / P molar ratio of the sintered body is 0.75 or more and 2.1 or less.
13. リン酸カルシウム焼結体が気孔率 20 %〜 80 %で、 気孔直径 70 m〜4mmの多孔質である請求項 1から 12に記載したリン酸カルシウム焼結体。  13. The calcium phosphate sintered body according to any one of claims 1 to 12, wherein the calcium phosphate sintered body is porous with a porosity of 20% to 80% and a pore diameter of 70m to 4mm.
14. 気孔が人工的に作られた直径 70 /m〜 4 mmの三次元網目状で、 焼 結体を貫通している請求項 13のリン酸カルシウム焼結体。  14. The calcium phosphate sintered body according to claim 13, wherein the pores are formed in a three-dimensional network having a diameter of 70 / m to 4 mm and artificially created and penetrate the sintered body.
15. 担持する生物学的活性化物質である水溶性タンパク質が成長因子また は細胞接着因子のうちの少なくとも一種を含んでいる請求項 1から 14に記載し たリン酸カルシウム焼結体。  15. The calcium phosphate sintered body according to claim 1, wherein the water-soluble protein which is a biologically activating substance to be carried contains at least one of a growth factor or a cell adhesion factor.
16. C a成分 0〜 2. 5 mM、 リン酸成分 1. 0〜 20 mM、 K成分 0〜 4 0mM、 Na成分 0〜200mM、 C 1成分 0〜 200 mMを含み pHが 5. 0〜 9. 0の水溶液を用いてリン酸カルシウム主成分焼結体上にタンパク質を共沈析 出させる、 請求項 1から 15に記載したタンパク質担持リン酸カルシウム主成分 焼結体の製造方法。  16.Ca component 0-2.5 mM, phosphate component 1.0-20 mM, K component 0-40 mM, Na component 0-200 mM, C1 component 0-200 mM, pH 5.0- 16. The method for producing a protein-supported calcium phosphate-based sintered body according to claim 1, wherein the protein is co-precipitated on the calcium phosphate-based sintered body using the aqueous solution of 9.0.
17. 〇 &成分1. 2〜2. 75mM、 リン酸成分 0. 6〜； L 5 mM、 K成分 17.〇 & Component 1.2 ~ 2.75mM, Phosphate component 0.6 ~; L 5mM, K component
0〜30mM、 N a成分 30〜 150mM、 Mg成分0. 1〜3. 0mM、 C 1 成分 30〜150mM、 HC〇3成分 0〜6 OmMを含み pHが 5. 0〜9. 0の 水溶液を用いてリン酸カルシウム主成分焼結体上にタンパク質を共沈析出させる、 請求項 1から 15に記載したタンパク質担持リン酸カルシウム主成分焼結体の製 造方法。 0~30mM, N a component. 30 to 150 mM, Mg component 0. 1~3. 0mM, C 1 component 30 to 150 mm, pH include HC_〇 3 ingredients Less than six Omm is 5. 0 to 9.0 of the aqueous solution 16. The method for producing a protein-supported calcium phosphate-based main component sintered body according to claim 1, wherein the protein is co-precipitated and precipitated on the calcium phosphate-based main component sintered body.
18. 水溶液として、 医療用輸液剤、透析 ·腹膜灌流液、 輸液の補正用製剤、 カルシウム製剤、 透析 ·腹膜灌流液の補充液の中から選択される 1種または 2種 以上の溶液を含んだ水溶液を使用する請求項 17に記載したタンパク質担持リン 酸カルシウム主成分焼結体の製造方法。  18. The aqueous solution contains one or more solutions selected from medical infusions, dialysis · peritoneal perfusion, infusion correction preparations, calcium preparations, dialysis · peritoneal perfusion replenishers 18. The method for producing a protein-supported calcium phosphate main component sintered body according to claim 17, wherein an aqueous solution is used.
19. カルシウム成分を含む溶液とリン酸成分を含む溶液をあらかじめ別々 に作製しておき、 両者を混合することでタンパク質の共沈を開始させる、 請求項 16から請求項 18に記載したタンパク質担持リン酸カルシウム主成分焼結体の 製造方法。  19. The protein-supported calcium phosphate according to claim 16, wherein a solution containing a calcium component and a solution containing a phosphate component are separately prepared in advance, and the two are mixed to start coprecipitation of the protein. Manufacturing method of main component sintered body.
20. ひリン酸三カルシウム、 リン酸 4カルシウム、 酸化カルシウムから選 ばれる 1種または 2種以上の相をあらかじめリン酸カルシウム主成分焼結体表面 に形成しておき該相からカルシウムを放出させてタンパク質を共沈析出させる、 請求項 1 6から請求項 1 8に記載したタンパク質担持リン酸カルシウム主成分焼 結体の製造方法。 20. Select from tricalcium arsenate, tetracalcium phosphate and calcium oxide The method according to any one of claims 16 to 18, wherein one or two or more phases are formed in advance on the surface of the calcium phosphate main component sintered body, and calcium is released from the phase to co-precipitate the protein. A method for producing a protein-supported calcium phosphate-based sinter.
2 1 . タンパク質を溶解させたリン酸成分を含む溶液をリン酸カルシウム主 成分焼結体と接触させ、 タンパク質の共沈を開始させる請求項 2 0に記載した夕 ンパク質担持リン酸カルシウム主成分焼結体の製造方法。  21. The protein-supported calcium phosphate main component sintered body according to claim 20, wherein a solution containing a phosphate component in which the protein is dissolved is brought into contact with the calcium phosphate main component sintered body to start coprecipitation of the protein. Production method.
2 2 . リン酸成分を含む溶液がリン酸緩衝生理的食塩水又はリン酸含有カル シゥム不含有医療用輸液剤の中から選択される 1種又は 2種の混合液である請求 項 2 0のタンパク質担持リン酸カルシウム主成分焼結体の製造方法。  22. The solution according to claim 20, wherein the solution containing a phosphate component is one or a mixture of two or more selected from phosphate-buffered physiological saline and a phosphate-containing calcium-free medical infusion. A method for producing a protein-supported calcium phosphate main component sintered body.
2 3 . 請求項 1から請求項 1 5のいずれかひとつに記載されたタンパク質担 持リン酸カルシウム主成分焼結体を用いた生体材料。  23. A biomaterial using the protein-supported calcium phosphate main component sintered body according to any one of claims 1 to 15.
2 4 . 請求項 1 6から請求項 2 2のいずれかひとつに記載されたタンパク質 担持リン酸カルシウム主成分焼結体の製造方法を用いた生体材料の製造方法。  24. A method for producing a biomaterial using the method for producing a protein-supported calcium phosphate main component sintered body according to any one of claims 16 to 22.
2 5 . 請求項 1から請求項 1 5のいずれかひとつに記載されたタンパク質担 持リン酸カルシウム主成分焼結体を用いたタンパク質徐放体。  25. A protein sustained-release body using the protein-bearing calcium phosphate main component sintered body according to any one of claims 1 to 15.
2 6 . 請求項 1 6から請求項 2 2のいずれかひとつに記載されたタンパク質 担持リン酸カルシウム主成分焼結体の製造方法を用いたタンパク質徐放体の製造 方法。  26. A method for producing a sustained-release protein body using the method for producing a protein-supported calcium phosphate main component sintered body according to any one of claims 16 to 22.
2 7 . 請求項 1から請求項 1 5のいずれかひとつに記載されたタンパク質担 持リン酸カルシウム主成分焼結体を用いた人工骨。  27. An artificial bone using the protein-bearing calcium phosphate main component sintered body according to any one of claims 1 to 15.
2 8 . 請求項 1 6から請求項 2 2のいずれかひとつに記載されたタンパク質 担持リン酸カルシウム主成分焼結体の製造方法を用いた人工骨の製造方法。  28. A method for producing an artificial bone using the method for producing a protein-supported calcium phosphate main component sintered body according to any one of claims 16 to 22.
2 9 . 請求項 1から請求項 1 5のいずれかひとつに記載されたタンパク質担 持リン酸カルシウム主成分焼結体を用いた組織工学スキヤフォールド。  29. A tissue engineering scaffold using the protein-bearing calcium phosphate main component sintered body according to any one of claims 1 to 15.
3 0 . 請求項 1 6から請求項 2 2のいずれかひとつに記載されたタンパク質 担持リン酸カルシウム主成分焼結体の製造方法を用いた組織工学スキヤフォール ドの製造方法。  30. A method for producing a tissue-engineered skifold using the method for producing a protein-supported calcium phosphate main component sintered body according to any one of claims 16 to 22.
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