阅读说明：本技术 无机盐类蛋白复合医疗器械 (Inorganic salt protein composite medical appliance ) 是由 大野忠夫 安永茉由 伊藤敦夫 十河友 小林文子 于 2020-01-30 设计创作，主要内容包括：本发明提供了一种医疗器械,其配置成将包埋了肽类激素等的磷灰石等无机盐类固体涂敷于金属等,上述无机盐类固体通过在不稳定磷酸钙过饱和溶液中的受控的延迟共沉淀等来提供,上述医疗器械暴露于足以进行灭菌的剂量的电离辐射。(The present invention provides a medical device configured to apply an inorganic salt solid such as apatite having a peptide hormone or the like embedded therein, provided by controlled delayed co-precipitation or the like in a supersaturated solution of unstable calcium phosphate, to a metal or the like, the medical device being exposed to an ionizing radiation in a dose sufficient for sterilization.)

1. A medical device, which is a medical device for mammals including humans,

the medical device is configured to coat a part or all of metal, ceramic or both with an inorganic salt solid in which a biologically active protein is embedded,

(a) the inorganic salt-based solid embedded with a biologically active protein is provided by a process of controlled delayed co-precipitation or sandwich coating or drying in a supersaturated solution of neutral or weakly basic labile calcium phosphate which produces spontaneous nucleation,

(b) the medical device is manufactured by a process of exposing the medical device to ionizing radiation in a dose sufficient for sterilization to thereby prepare a terminally sterilized medical device having one or more biological activities selected from the group consisting of cell proliferation activity, blood vessel proliferation activity, soft tissue formation activity, bone differentiation promoting activity, reactivity to an antibody, agonistic activity, and antagonistic activity,

(c) the inorganic salt is one or more inorganic salts selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate and calcium carbonate

(d) The protein with biological activity is one or more than two proteins selected from peptide hormone, growth factor and osteogenic protein.

2. The medical device of claim 1, wherein the apatite is a low crystalline apatite.

3. The medical device according to claim 1 or 2, wherein the inorganic salt-based solid in which the biologically active protein is embedded is further embedded with a polysaccharide derived from an extracellular matrix, which itself does not have a direct cell proliferation and differentiation activity.

4. The medical device of claim 3, wherein the polysaccharide is heparin.

5. The medical device according to any one of claims 1 to 4, wherein the metal is one or two or more metals selected from titanium, a titanium alloy, stainless steel, and a cobalt-chromium alloy.

6. The medical device according to any one of claims 1 to 5, wherein the ceramic is one or more ceramics selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, alumina, zirconia, and composites thereof.

7. The medical device of any one of claims 1-6, wherein the ionizing radiation is gamma rays and/or electron beams.

8. The medical instrument of claim 7, wherein the gamma and/or electron beam based sterilization is performed under conditions that inhibit free radical generation,

the conditions are one or more conditions selected from the group consisting of (a) sterilization in a deaerated state at an atmospheric pressure of 50kPa or less, (b) sterilization in a state in which air is exchanged with nitrogen or an inert gas, (c) sterilization at a low temperature in the range of 0 ℃ to-196 ℃, and (d) sterilization in a state in which ascorbic acid or an ascorbate is further added to an inorganic salt-based solid in which a biologically active protein is embedded.

9. The medical device according to claim 8, wherein the ascorbic acid or ascorbate is selected from ascorbic acid, sodium ascorbate, calcium ascorbate dihydrate, magnesium ascorbyl phosphate n-hydrate.

10. The medical device of any one of claims 7-9, wherein the dose of gamma rays is 3-40 kGy.

11. The medical device according to any one of claims 1 to 10, wherein the peptide hormone is one or more peptide hormones selected from the group consisting of a peptide hormone derived from hypothalamus, vasopressin, oxytocin, pituitary hormone, gonadotropin, growth hormone, parathyroid hormone, inhibin, activin, relaxin, insulin, glucagon, somatostatin, cholecystokinin, secretin, motilin, atrial natriuretic peptide, erythropoietin, leptin, endothelin, gastrohunin, adiponectin, insulin-like growth factor, and calcitonin gene-related peptide.

12. The medical device according to any one of claims 1 to 10, wherein the growth factor is one or more growth factors selected from FGF-2 and functional equivalents thereof.

13. The medical device according to any one of claims 1 to 10, wherein the osteogenic protein is one or more than two osteogenic proteins selected from the group consisting of OP-1, OP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, DPP, Vg1, Vgr-1, and functional equivalents thereof.

14. The medical device according to any one of claims 1 to 13, wherein the biological activity is one or more biological activities selected from the group consisting of cell proliferation activity, vascular proliferation activity, soft tissue formation activity, bone differentiation promoting activity, reactivity to an antibody, agonistic activity, and antagonistic activity.

15. The medical device of any one of claims 1 to 14, wherein the biological activity after terminal sterilization by ionizing radiation at a dose sufficient for sterilization is 13% or more relative to the biological activity before sterilization.

16. The medical device according to any one of claims 1 to 15, wherein the biologically active protein is embedded in the inorganic salt solid by a process comprising,

the step is selected from one or more of (a) a coprecipitation precipitation method using a sodium chloride solution, a sodium phosphate solution, a calcium chloride solution, a sodium carbonate solution, and a sodium hydrogen carbonate solution, (b) a sandwich coating method, and (c) a drying method.

17. The medical device according to any one of claims 1 to 15, wherein the biologically active protein is embedded in the inorganic salt-based solid by one or more steps selected from (a) a coprecipitation method based on controlled delayed coprecipitation and (b) a cover-sandwich method,

the controlled delayed co-precipitation is a controlled delayed co-precipitation of a biologically active protein with calcium phosphate in a supersaturated solution of unstable calcium phosphate that produces spontaneous nucleation using a neutral or weakly basic solution.

18. The medical device of claim 17, wherein delaying co-precipitation comprises:

controlling the KCl concentration in an aqueous solution containing 0.5 to 2.5mM of Ca ions, 1.0 to 20mM of phosphate ions, 0 to 40mM of K ions, 0 to 200mM of Na ions, and 0 to 200mM of Cl ions and having a pH of 7.0 to 9.0 as a supersaturated solution of unstable calcium phosphate, and

the calcium phosphate precipitation time is artificially controlled and delayed.

19. The medical device of claim 17, wherein delaying co-precipitation comprises:

the supersaturated solution of unstable calcium phosphate contains 1.2-2.75 mM of Ca ions and 0.6-15 mM of Ca ionsPhosphate radical ion, 0-30mM K ion, 30-150 mM Na ion, 0.1-3.0 mM Mg ion, 30-150 mM Cl ion, 0-60 mM HCO₃Control of KCl concentration in an ionic aqueous solution having a pH of 7.0 to 9.0, and

the calcium phosphate precipitation time is artificially controlled and delayed.

20. The medical device of any one of claims 1-19, for use in tissue repair.

21. The medical device according to claim 20, which is one or more selected from the group consisting of an internal fixation pin, an internal fixation screw, an artificial bone, a bone filler, a dental intraosseous implant, an internal spinal fixation device, an internal marrow nail, and a spinal cage.

22. The medical device of any one of claims 1-19 for use as an artificial joint.

23. A method for producing the medical device according to any one of claims 1 to 22, comprising the step of embedding the biologically active protein in an inorganic salt-based solid,

the step is selected from one or more of (a) a coprecipitation precipitation method using a sodium chloride solution, a sodium phosphate solution, a calcium chloride solution, a sodium carbonate solution, and a sodium hydrogen carbonate solution, (b) a sandwich coating method, and (c) a drying method.

Technical Field

The present invention relates to a medical device having resistance to sterilization by ionizing radiation, which comprises a crystal of an inorganic salt or an amorphous solid of an inorganic salt (hereinafter, may be referred to as "inorganic salt solid" in the present specification) and a biologically active protein.

More specifically, the present invention relates to a medical device for mammals including humans, which comprises an inorganic salt solid in which a biologically active protein that is not recognized as a foreign substance by the animal is embedded, and which is sterilized by ionizing radiation while maintaining the biological activity of the protein, using the resistance to sterilization by ionizing radiation obtained by the embedding.

Background

Embedding (embedding) is a term used in pathology. Embedding refers to embedding and solidifying a living tissue to be examined in a curable liquid paraffin or resin, and fixing the living tissue. In the pathological examination, the embedded tissue block is sliced with a microtome or the like to prepare a section (specimen) for tissue staining, and the section is examined with a microscope or the like. In the present specification, the concept is expanded, and embedding all or a part of a protein molecule in an inorganic salt-based solid or wrapping all or a part of a protein molecule with an inorganic salt-based solid to immobilize the protein molecule in the inorganic salt-based solid is referred to as "embedding". "entrapping" is different from the state where the protein molecules are merely "adsorbed" or "contacted" or "mixed" in the inorganic salt-based solid.

An example of "embedding" of protein molecules in an inorganic salt solid is a composition in which the protein molecules and calcium phosphate are coprecipitated from a supersaturated solution of calcium phosphate in which the protein molecules coexist, and the protein molecules are dispersed and arranged in a calcium phosphate matrix at intervals of a nanometer order (non-patent document 1).

On the other hand, products and raw materials such as medicines and medical instruments used in medical fields requiring sterility are sterilized in various ways in the production process. Among the sterilization methods, a sterilization method utilizing the characteristic of radiation that easily permeates substances is an ionizing radiation sterilization method. In particular, the ionizing radiation sterilization method is easily applied to the final step of production, and is widely used as a terminal sterilization method in the case where there is no problem of denaturation or inactivation of an object by irradiation with ionizing radiation.

The type and dose of ionizing radiation used vary depending on the species, number, and form of bacteria expected to be present in the object to be sterilized, and gamma rays having high substance permeability are often used for medical instruments, and the doses around and above 25kGy are widely used as an appropriate sterilization dose. In addition, when the surface of an object is to be sterilized, an electron beam which has a high dose rate and can be irradiated for a short time is widely used.

However, all sterilization methods are liable to cause inactivation of the biologically active substance. In particular, nucleic acids and proteins are easily inactivated by ionizing radiation sterilization because of their large molecular size, and for example, a radiation inactivation method has been established as a method for measuring the molecular weight of an active domain of a protein in a living body (non-patent document 2). In addition, in general, "a biological agent is a product whose sterility control is very difficult when it is produced by aseptic production process because it cannot be subjected to terminal sterilization" (non-patent document 3), and a concept of "For high sensitive products with biological products and biological products technical basis of the drug product manufacture based on aseptic process under controlled conditions" is common knowledge, and a published draft of guidelines For sterilization of a drug by European Medicines Agency activities (non-patent document 4) "describes" For high sensitive products with non-biological products having the drug product manufacture of the drug product manufacture, and processing based on aseptic process under controlled conditions can make the drug have satisfactory quality ". "guidance on the production of a sterile drug by a terminal sterilization method" is disclosed (non-patent document 5). However, there is no mention at all of a method for protecting the activity of a drug requiring sterilization.

Regardless of the type of radiation, ionizing radiation non-specifically hits various compounds to generate free radicals. The radical generation site may transfer a radical electron, and a radical reaction may occur in a compound completely different from the original compound. As a result of the radical reaction, when unnatural changes occur in DNA, membrane lipids, and compounds important to cells, the changes are harmful to the cells (non-patent document 6). If such harmful effects act on bacteria and viruses, sterilization is performed. Likewise, if a biologically active protein reacts with a free radical, the protein is inactivated.

As radioprotectors that inactivate harmful free radicals, thiols such as cysteine and glutathione are known, and typical examples thereof include aminothiol derivatives. Conventionally, cysteamine (mercaptoethylamine), WR-2721(S-2- (3-aminopropylamino) ethyl thiophosphoric acid), and the like have been known (non-patent document 7). In addition, 2-mercaptoethylamine and alcohol (ethanol) are also described in the literature (non-patent document 8). In addition, in screening using cytotoxicity as an index, (+) catechin, curcumin, vitamin C, resveratrol, caffeic acid, and quercetin are described as substances exhibiting radioprotective effects (non-patent document 9). Further, as a substance exhibiting a radioprotective effect, a nitrogen-containing compound is described (non-patent document 10). In addition, it is known that amino acid mixtures also have radioprotective effects on biologically active proteins (patent documents 1 and 2). Further, as a method for suppressing inactivation of a protein during sterilization using ionizing radiation, there are disclosed a method for suppressing the inactivation of a protein in the presence of a cellulose ether derivative and a specific amino acid group (patent documents 3 and 4), a method for allowing an aliphatic polyester to coexist (patent document 5), and a method for allowing a foreign protein such as gelatin to coexist (patent document 6). However, these are organic radioprotectors. Further, although a method of allowing a bioactive protein to coexist with a collagen sponge and an absorbent polymer is disclosed (patent documents 7 and 8), a method of suppressing inactivation when sterilization is performed using ionizing radiation is not disclosed.

On the other hand, as radioprotectors of inorganic substances, selenium (non-patent document 11), sodium vanadate (non-patent document 12), zinc sulfate (non-patent document 13), and manganese compounds (patent documents 9 and 10) are known. However, many inorganic salts have a problem in toxicity as medical instruments for mammals, and therefore, inorganic salts have not been developed as radioprotectors for medical use.

Among inorganic substances, calcium phosphate has high safety and biocompatibility, and Ca ions and PO ions₄Apatite and amorphous calcium phosphate (Ca/P molar ratio 1 to 1.8) having different ion molar ratios such as tricalcium phosphate (Ca/P molar ratio 1.67) and tricalcium phosphate are used for medical instruments.

A dosimetry method using sintered apatite has been developed, which analyzes radicals generated when calcium phosphate is irradiated with ionizing radiation by electron spin resonance (patent document 11). However, the dose range described is about 0 to 60Gy, and there is no mention of the radioprotective effect of other coexisting bioactive molecules when irradiated in a dose range suitable for ionizing radiation sterilization up to 1000 times or more (several kGy or more, usually 10 to 30kGy in most cases).

Further, as a document which mentions a radiation dose suitable for sterilization when apatite or hydroxyapatite which is an inorganic salt is irradiated with radiation and a protective effect against a bioactive protein, japanese patent application laid-open No. h 11-506360 (patent document 12) is known. In this document, "any one or combination of materials that can be advantageously used (without limitation) including ceramics (e.g., hydroxyapatite, tricalcium phosphate and other calcium phosphates and combinations thereof") are described as one of the insoluble synthetic polymeric carrier materials containing biologically active osteogenic proteins, "a terminally sterilized osteogenic device for implantation in mammals is described.

However, there is no mention whatsoever in this document of how "any one or combination" of biologically active osteogenic proteins with ceramic containing materials should be. There are various combinations of bioactive osteogenic proteins and materials, such as "mixing", "adsorption", "contact" and "embedding" (hereinafter collectively referred to as "combination type"), and there is no disclosure of how the bioactivity of the final sterilized proteins is greatly different depending on the combination type, and there is no disclosure or teaching of what combination is an optimal combination that can be used to perform final sterilization while maintaining the bioactivity of the osteogenic proteins.

Japanese Kokai publication No. 2007-513083 (patent document 13) discloses a sterilizing composition containing an inorganic salt and a bioactive protein as a medical graft. However, there is no disclosure or teaching as to which combination of the inorganic salt and the bioactive protein is different from each other in terms of the biological activity of the bioactive protein after the terminal sterilization and which combination is the most suitable combination for the terminal sterilization while maintaining the activity of the bioactive protein.

Jp 2007-51515196 a (patent document 14) discloses a composition containing hydroxyapatite as an inorganic salt and a bone growth-inducing substance as a filler for controlling bone bleeding, but it is described that terminal sterilization cannot be performed when the bone growth-inducing substance is a radiation-sensitive bioactive protein such as demineralized bone matrix or bone morphogenetic protein.

Japanese patent application laid-open No. 2002-501786 (patent document 15) discloses a bone paste composition comprising heat-or radiation-sterilized gelatin, a bone-forming component such as a regenerative growth factor, and an inorganic salt such as a calcium phosphate ceramic, but does not describe or teach sterilization by radiation irradiation and heat treatment finally after forming the composition.

Jp 2002-529201 a (patent document 16) discloses a graft containing a bioactive substance such as a ceramic or a growth factor as an inorganic salt and an allograft/autograft/xenograft tissue containing the same, which is implanted into a human body, but final sterilization is performed by "irradiation with gamma rays or other types at a known dose which does not adversely affect tissue characteristics", or "electron beam sterilization or ethylene oxide sterilization which does not produce toxicity or reduce desired bioactivity". That is, it is considered that the sterilization effect of radiation is allowed to be sacrificed for maintaining the biological activity, and the harmful effect and the reduction of the biological activity are prevented by limiting the irradiation dose. As a solution, only "injecting a desired bioactive substance into a graft as a further enhancement before terminal sterilization" is described as necessary.

Jp-a-10-511957 (patent document 17) discloses radiation-sterilized nanoparticles comprising a bioactive agent and apatite and bone ceramics, wherein the bioactive agent is formed of a biocompatible and biodegradable polymer core having an average particle diameter of less than about 300nm, but does not relate to a method for preventing inactivation of the bioactive agent by radiation sterilization, and does not relate to any combination method which is the most suitable combination method for final sterilization while maintaining the activity.

Although japanese patent application laid-open No. 2003-503423 (patent document 18) describes a carrier in which biological activity is incorporated into a matrix of an inorganic, organic or organic and inorganic substance-containing carrier and sterilization is performed after synthesis, it neither describes nor suggests whether biological activity is maintained after sterilization in the case of any combination of types of the matrix of the inorganic, organic or organic and inorganic substance-containing carrier or whether biological activity is maintained after sterilization in any sterilization method.

Japanese patent No. 5221132 (patent document 19) describes a method of obtaining a coated substrate by bringing an acidified composition comprising a brine mixture containing calcium, magnesium, phosphoric acid, bicarbonate ions and a bioactive substance into contact with the substrate, increasing the pH to produce co-precipitation of the salts and bioactive substance. Although not mentioned in the invention described in the claims, it is described in the column of the detailed description of the invention that the gamma irradiation can be performed after the final step (in paragraph [0020], it is described that "in the final stage after step c)," the method can be performed using the gamma irradiation under the conditions of non-aseptic (aseptic) and non-sterile (sterize) conditions "). However, the gamma irradiation is mentioned only as a possibility as an example of a general sterilization method, and a specific experimental example of sterilization by irradiation with gamma rays is not disclosed. In addition, although there are substrates of different materials such as metal, ceramic, and polymer in the substrate, there is no description nor suggestion as to whether the biological activity is maintained after irradiation of all the substrates, or the biological activity is maintained after irradiation only when a substrate of a specific material is irradiated with gamma rays, or the biological activity is maintained after irradiation in which manner the gamma rays are irradiated.

International publication WO2006/004778 (patent document 33) describes an implant having a coating layer, in which a peptide having a cell adhesion promoting effect is incorporated into a nanocrystalline apatite layer, and examples describe that hydroxyapatite is electrochemically deposited on a titanium disk in the presence of the peptide, and the implant incorporating the peptide in the apatite layer does not have a decrease in the cell adhesion promoting effect after gamma-ray irradiation of 25kgrey, while the implant adsorbing the peptide only on the surface of the apatite layer loses the cell adhesion promoting effect after gamma-ray irradiation (examples 1 and 2).

However, the implants disclosed in the above patent documents are formed by electrochemical deposition of hydroxyapatite and are not composites produced by coprecipitation with peptides in a supersaturated solution of calcium phosphate. The incorporation of peptides into apatite layers by electrochemical deposition is completely different from the microstructure and crystallinity of a composite obtained by coprecipitating peptides together with apatite.

That is, the apatite formed by the electrochemical deposition has high crystallinity, and the diffraction lines of (002) (211) (112) (300) are separated by the powder X-ray diffraction method (for example, non-patent document 18), and even in a crystal form, a hexagonal needle-like or hexagonal plate-like form characteristic to apatite crystals is easily formed. The apatite has high crystallinity and thus has low solubility (patent document 33). Apatite having low solubility is suitable for the composition of peptides and proteins that do not require slow release and are fixed only on the surface. An example of peptides and proteins that do not require sustained release and are only emphasized to be immobilized on a surface is peptides and proteins having a cell adhesion promoting effect. In fact, this patent document also describes that the formed acicular apatite, and a peptide having low solubility, stability and a cell adhesion promoting effect of the apatite are stably and strongly bound to the surface. On the other hand, peptides and proteins having cell proliferation activity, tissue formation activity, cell differentiation-promoting activity, reactivity to antibodies, agonistic activity and antagonistic activity exert effects on surrounding tissues by sustained release, and therefore, are not suitable for incorporation of apatite having high crystallinity and low solubility. Instead, the solubility of apatite and calcium phosphate needs to be properly reduced to allow the slow release of incorporated peptides and proteins.

Fundamentally, coatings based on electrochemical deposition can be applied to conductive metals and not to non-conductive ceramics. Further, this patent document does not disclose an example in which a polysaccharide (e.g., heparin) derived from an extracellular matrix and having no direct cell growth and differentiation activity by itself coexists with a peptide and the peptide is incorporated into a layer of hydroxyapatite.

On the other hand, in a vaccine for the purpose of inducing an immune response in an organism, an immunological adjuvant of an inorganic salt and an antigen are sometimes used together. As the immunological adjuvant of inorganic salts, aluminum salts such as aluminum chloride, aluminum phosphate and aluminum sulfate have been used in general for a long time in addition to aluminum hydroxide, and calcium phosphate has also been used. As calcium phosphate immunoadjuvants, vaccines and adjuvants in which cancer-specific antigens derived from living organisms are combined with β -tricalcium phosphate as an inorganic salt to form a cancer-specific antigen vaccine (patent documents 20 and 21) and calcium phosphate immunoadjuvants used together with antigens (patent documents 22 and 23) are described, but there is no mention of radiation sterilization after combination with antigens as proteins. There is a long history of use of aluminum salt immunoadjuvants in vaccines for preventing infectious diseases in combination with antigens such as bacteria and viruses, and among them, there is a vaccine produced by terminal sterilization using radiation (non-patent document 14).

However, antigens such as bacteria and viruses are xenogeneic organisms or biomolecules of xenogeneic organisms to animals to which vaccines are administered, and are proteins recognized as foreign substances by animals to which the vaccines are administered or transplanted. Therefore, these documents do not disclose radiation sterilization of proteins that are not recognized as foreign matter by administration or transplantation of the subject animal. Fundamentally, antigens such as bacteria and viruses are foreign to the animal to which they are administered and are eliminated by the immune system. Therefore, even unnatural molecular changes due to radiation sterilization remain foreign substances, and thus the adverse effect on the intended effect (clearance by the immune system) is considered to be small. On the other hand, when an effect such as tissue regeneration is to be obtained using a protein that is not recognized as a foreign substance by administration or a transplant target animal, it is considered that the protein is recognized as a foreign substance due to unnatural molecular changes caused by radiation sterilization, binding to a receptor does not occur, and the function of the protein is adversely affected, and it is difficult to obtain a desired effect.

Under such circumstances, only chymotrypsin and papain are known as examples of biological agents to which a radiation-based terminal sterilization method is applied, particularly a long-chain protein having an amino acid residue number of 50 or more, rather than a short-chain peptide having an amino acid residue number of less than about 50 (non-patent documents 15 and 16).

Documents of the prior art

Patent document

Patent document 1: international publications WO 01/043143;

patent document 2: japanese patent laid-open publication No. 2016-53063;

patent document 3: japanese patent No. 5872032;

patent document 4: japanese patent No. 6317307;

patent document 5: japanese patent No. 5746430;

patent document 6: U.S. patent No. 5730933;

patent document 7: european patent publication 0562864a 1;

patent document 8: european patent 0562864B 1;

patent document 9: japanese patent laid-open publication No. 2016-;

patent document 10: japanese patent laid-open publication No. 2017-222687;

patent document 11: japanese patent laid-open publication No. H09-133770;

patent document 12: japanese patent laid-open publication No. 11-506360;

patent document 13: japanese Kokai publication Nos. 2007 and 513083;

patent document 14: japanese Kokai publication No. 2007-515196;

patent document 15: japanese Kohyo publication No. 2002-501786;

patent document 16: japanese Kohyo publication No. 2002-529201;

patent document 17: japanese Kohyo publication Hei 10-511957;

patent document 18: japanese Kokai publication Nos. 2003-503423;

patent document 19: japanese patent No. 5221132;

patent document 20: japanese patent No. 6082901;

patent document 21: international publication WO 2012/105224;

patent document 22: international publications WO 2017/047095;

patent document 23: japanese patent No. 4569946;

patent document 24: japanese laid-open patent application No. 2016-173357;

patent document 25: european patent publication 806212;

patent document 26: japanese patent laid-open publication No. 2000-93503;

patent document 27: european patent 1786483B1 (corresponding european patent of patent document 17);

patent document 28: international publications WO 2006/016807;

patent document 29: japanese patent No. 4478754;

patent document 30: U.S. patent No. 6136369;

patent document 31: U.S. patent No. 6143948;

patent document 32: U.S. patent No. 6344061;

patent document 33: international publication WO 2006/004778.

Non-patent document

Non-patent document 1: biomaterials, 27, pp.167-175, 2006;

non-patent document 2: radiochemistry, 57, 3, 1994;

non-patent document 3: the GMP Committee for PDA aseptic products, "manufacturing and quality management of sterile drugs that have contributed to society," PDA Journal of GMP and Validation in Japan, 16, pp.9-14, 2014;

non-patent document 4: european Medicines Agency, Guideline on the simulation of the medial product, active substance, exposure and primary container _ Draft, April 11, 2016;

non-patent document 5: "guideline for manufacturing aseptic drug by terminal sterilization method", which is in average 23 years of science research on Caucasian (supervision science comprehensive research projects such as drug, medical instrument, etc.), Proc. department of medicine and food safety administration supervision and guidance of Caucasian province, department of drug countermeasures, act, liaison 2011;

non-patent document 6: RADIIOISOTOPES, 24, pp.894-901, 1975;

non-patent document 7: japan society of atomic mechanics, 35, pp.688-693, 1993;

non-patent document 8: journal of the university of Suzu medical science, 9, pp.87-96, 2002;

non-patent document 9: isotoppe News, 710, pp.2-6, 2013;

non-patent document 10: RADIIOISOTOPES, 30, pp.258-262, 1981;

non-patent document 11: space res, 12, pp.223-231, 1992;

non-patent document 12: cancer res, 70, pp.257-265, 2010;

non-patent document 13: ertekin MV, et al, j.radial.res., 45, pp.543-548, 2004;

non-patent document 14: non-compliance Report of EMA-DICM/INSP/AMG/MBP/ACS, GMP information abstract, virtual pharmaceutical factory 2017.1.8, http:// ncogmp. com/blog/emannomultiplicacross Report-vaccine-dichinpalmbpacs/;

non-patent document 15: "new trend of sterilization of drugs and medical instruments", "radiation sterilization" [3 rd round ], 2015.09.17 http: com/topics _ detail1/id 1010;

non-patent document 16: "effect of gamma radiation on drug substances" -radiation utilization technology database (RADA), 1996;

non-patent document 17: "revision of the guideline for manufacturing sterile drugs by the terminal sterilization method", the labor-saving and trouble-saving liaison of the university of heaven, which is as long as 24 years, 11 months and 9 days;

non-patent document 18: materials, 11, 1897, 2018.

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a medical device for animals, which contains a biologically active protein that retains biological activity even when radiation sterilization is performed.

Another object of the present invention is to provide a method for producing a medical device for mammals, which contains a bioactive protein that retains a biological activity even when radiation sterilization is performed.

It is still another object of the present invention to provide a medical device for mammalian use, for example, a medical device for bone tissue repair, an artificial joint, or the like, which contains a bioactive protein that retains a biological activity even when radiation sterilization is performed.

It is still another object of the present invention to provide a method for manufacturing a medical device, such as a medical device for repairing bone tissue or an artificial joint, as a medical device for mammals, which contains a bioactive protein that retains a biological activity even when radiation sterilization is performed.

Means for solving the problems

As a result of intensive studies to solve the above problems, the present inventors have found that when a bioactive protein is embedded in an inorganic salt-based solid, inactivation of the protein can be significantly suppressed even by irradiation with radiation at a dose suitable for sterilization by coprecipitating an inorganic salt and the bioactive protein to form a complex. That is, it was found that the inorganic salt solid itself has a useful radiation protective effect.

For example, calcium phosphate can be easily prepared as a supersaturated solution depending on conditions. When some (physical or chemical) stimulus is applied to the supersaturated solution, the supersaturated state is rapidly eliminated and calcium phosphate precipitates to produce a precipitate. In this case, by adding a bioactive protein as a solute to the supersaturated solution, precipitated calcium phosphate is involved in the protein, and a calcium phosphate coprecipitated composition such as apatite is produced in which the protein is embedded. The composition obtained by freeze-drying the deposit can be greatly inhibited from inactivation even when irradiated with gamma rays at a dose suitable for sterilization. Similarly, in a composition obtained by embedding a bioactive protein in sodium chloride and vacuum-drying the same, inactivation of the bioactive protein by irradiation with gamma rays at a dose suitable for sterilization is also greatly suppressed. These findings indicate that inactivation of a biologically active protein can be prevented if the generation of free radicals upon irradiation is suppressed, and the present invention has been completed based on these findings.

That is, the present invention includes the following inventions.

[1] A medical device, which is a medical device for mammals including humans,

the medical device is configured to apply an inorganic salt-based solid in which a biologically active protein is embedded to a portion or all of a metal, a ceramic, or both,

(a) the inorganic salt solid embedded with the protein with biological activity is provided by the procedures of Controlled Delayed Coprecipitation (Controlled Delayed Coprecipitation) or coating sandwich method or drying method in neutral or weakly alkaline supersaturated solution of unstable calcium phosphate which generates spontaneous nucleation,

(b) the medical device is produced by a process of exposing the medical device to ionizing radiation in a dose sufficient for sterilization to thereby produce a terminally sterilized medical device having 1 or 2 or more biological activities selected from the group consisting of cell proliferation activity, blood vessel proliferation activity, soft tissue formation activity, bone differentiation promoting activity, antibody-reactive activity, agonistic activity and antagonistic activity,

(c) the inorganic salt is 1 or more than 2 inorganic salts selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate and calcium carbonate

(d) The above-mentioned protein with biological activity is 1 or more than 2 kinds of protein selected from peptide hormone, growth factor and osteogenic protein.

[2] The medical device according to the above [1], wherein the apatite is a low-crystalline apatite.

[3] The medical device according to the above [1] or [2], wherein the delayed co-precipitation comprises artificially controlling a delayed calcium phosphate precipitation time by controlling a KCl concentration in an aqueous solution having a pH of 7.0 to 9.0 as an unstable calcium phosphate supersaturated solution, the aqueous solution containing 0.5 to 2.5mM of Ca ions, 1.0 to 20mM of phosphate ions, 0 to 40mM of K ions, 0 to 200mM of Na ions, and 0 to 200mM of Cl ions.

[4]According to the above [1]Or [2]]The medical device, wherein the delayed co-precipitation comprises artificially controlling the delayed calcium phosphate precipitation time by controlling the KCl concentration in an aqueous solution having a pH of 7.0 to 9.0 as an unstable calcium phosphate supersaturated solution, the aqueous solution containing 1.2 to 2.75mM of Ca ions, 0.6 to 15mM of phosphate ions, 0 to 30mM of K ions, 30 to 150mM of Na ions, 0.1 to 3.0mM of Mg ions, 30 to 150mM of Cl ions, 0 to 60mM of HCO₃Ions.

[5] The medical device according to any one of the above [1] to [4], wherein the inorganic salt-based solid in which the biologically active protein is embedded further embeds a polysaccharide, preferably heparin, derived from an extracellular matrix and having no direct cell growth and differentiation activity by itself.

[6] The medical device according to any one of the above [1] to [5], wherein the metal is 1 or 2 or more metals selected from titanium, titanium alloy, stainless steel and cobalt-chromium alloy.

[7] The medical device according to any one of the above [1] to [5], wherein the ceramic is 1 or 2 or more kinds of ceramics selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, alumina, zirconia, and a composite thereof.

[8] The medical device according to any one of the above [1] to [7], wherein the ionizing radiation is gamma rays and/or electron beams.

[9] The medical device according to the above [8], wherein the sterilization based on gamma rays and/or electron beams is performed under conditions of suppressing generation of radicals, the conditions being selected from: (a) sterilizing in a degassed state at an atmospheric pressure of 50kPa or less; (b) sterilization in a state where air is exchanged for nitrogen or an inert gas; (c) sterilization at a low temperature in the range of 0 ℃ to-196 ℃; (d) sterilizing the inorganic salt solid embedded with the protein with biological activity by adding ascorbic acid or ascorbate; 1 or 2 or more conditions.

[10] The medical device according to the above [9], wherein the ascorbic acid or ascorbate is selected from ascorbic acid, sodium ascorbate, calcium ascorbate dihydrate and magnesium ascorbyl phosphate n-hydrate.

[11] The medical device according to any one of the above [8] to [10], wherein the dose of the gamma ray is 3 to 40 kGy.

[12] The medical device according to any one of the above [1] to [11], wherein the peptide hormone is 1 or 2 or more selected from the group consisting of hypothalamic peptide hormones, vasopressin, oxytocin, pituitary mesenchymal hormone, gonadotropin, growth hormone, parathyroid hormone, inhibin, activin, relaxin, insulin, glucagon, somatostatin, cholecystokinin, secretin, motilin, atrial natriuretic peptide, erythropoietin, leptin, endothelin, ghrelin, adiponectin, insulin-like growth factor, and calcitonin gene-related peptide.

[13] The medical device according to any one of the above [1] to [11], wherein the growth factor is 1 or 2 or more growth factors selected from FGF-2 and functional equivalents thereof.

[14] The medical device according to any one of the above [1] to [11], wherein the osteogenic protein is 1 or 2 or more selected from the group consisting of OP-1, OP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, DPP, Vg1, Vgr-1, and functional equivalents thereof.

[15] The medical device according to any one of the above [1] to [14], wherein a biological activity after terminal sterilization by ionizing radiation at a dose sufficient for sterilization is 13% or more relative to a biological activity before sterilization.

[16] The medical device according to any one of the above [1] to [15], which is used for tissue repair.

[17] The medical device according to the item [16], which is 1 or 2 or more selected from the group consisting of an internal fixation pin, an internal fixation screw, an artificial bone, a bone filler, a dental intraosseous implant, an internal spinal fixation device, an internal marrow nail, and a spinal cage.

[18] The medical device according to any one of the above [1] to [15], which is used as an artificial joint.

[19] A method of manufacturing a medical device for a mammal, including a human,

the medical device is configured to coat a part or all of metal, ceramic or both of them with an inorganic salt solid in which a bioactive protein is embedded,

the method comprises the following steps:

(a) a step of producing the above inorganic salt-type solid in which a protein having biological activity is embedded by controlled delayed coprecipitation or a coating sandwich method or a drying method in a supersaturated solution of neutral or weakly basic unstable calcium phosphate which causes spontaneous nucleation; and

(b) exposing the medical device to ionizing radiation in a dose sufficient for sterilization, thereby producing a terminally sterilized medical device having 1 or 2 or more biological activities selected from the group consisting of cell proliferation activity, vascular proliferation activity, soft tissue formation activity, bone differentiation promoting activity, reactivity to an antibody, agonistic activity, and antagonistic activity.

[20] A method of manufacturing a medical device for a mammal, including a human,

the medical device is configured to coat a part or all of metal, ceramic or both of them with an inorganic salt solid in which a bioactive protein is embedded,

the method comprises the following steps:

(a) a step of producing the above inorganic salt-type solid in which a protein having biological activity is embedded by controlled delayed coprecipitation or a coating sandwich method or a drying method in a supersaturated solution of neutral or weakly basic unstable calcium phosphate which causes spontaneous nucleation; and

(b) exposing the medical device to ionizing radiation in a dose sufficient for sterilization, thereby producing a terminally sterilized medical device having 1 or 2 or more biological activities selected from the group consisting of cell proliferation activity, vascular proliferation activity, soft tissue formation activity, bone differentiation-promoting activity, reactivity to an antibody, agonistic activity, and antagonistic activity, and having a biological activity of at least about 13% or more of that before sterilization.

[21] The method according to the above [19] or [20], wherein the inorganic salt is 1 or 2 or more inorganic salts selected from the group consisting of low crystalline apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate and calcium carbonate.

[22] The method according to any one of the above [19] to [21], wherein the delayed coprecipitation comprises artificially controlling a delayed calcium phosphate precipitation time by controlling a KCl concentration in an aqueous solution having a pH of 7.0 to 9.0 as an unstable supersaturated solution of calcium phosphate, the aqueous solution containing 0.5 to 2.5mM of Ca ions, 1.0 to 20mM of phosphate ions, 0 to 40mM of K ions, 0 to 200mM of Na ions, and 0 to 200mM of Cl ions.

[23]According to [19] above]～[21]The method according to any one of the above methods, wherein the delayed co-precipitation comprises artificially controlling the delayed calcium phosphate precipitation time by controlling the KCl concentration in an aqueous solution having a pH of 7.0 to 9.0, which is an unstable supersaturated solution of calcium phosphate, the aqueous solution containing 1.2 to 2.75mM of Ca ions and 0.6 to 15mM of Ca ionsPhosphate radical ion, 0-30mM K ion, 30-150 mM Na ion, 0.1-3.0 mM Mg ion, 30-150 mM Cl ion, 0-60 mM HCO₃Ions.

[24] The method according to any one of the above [19] to [23], wherein the step of producing the inorganic salt-based solid in which the biologically active protein is embedded comprises a step of producing an inorganic salt-based solid,

the inorganic salt solid is embedded with a polysaccharide, preferably heparin, derived from an extracellular matrix and having no direct cell growth and differentiation activity by itself, in addition to a biologically active protein.

[25] The method according to any one of the above [19] to [24], wherein the metal is 1 or 2 or more metals selected from titanium, titanium alloy, stainless steel and cobalt-chromium alloy.

[26] The method according to any one of the above [19] to [24], wherein the ceramic is 1 or 2 or more kinds selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, alumina, zirconia, and a composite thereof.

[27] The method according to any one of the above [19] to [26], wherein the ionizing radiation is gamma rays and/or electron beams.

[28] The method according to [27], wherein the sterilization by the gamma ray and/or the electron beam is performed under a condition of suppressing generation of radicals, the condition being 1 or 2 or more selected from (a) sterilization in a deaerated state at an atmospheric pressure of 50kPa or less, (b) sterilization in a state in which air is exchanged for nitrogen or an inert gas, (c) sterilization at a low temperature in a range of 0 ℃ to-196 ℃, and (d) sterilization in a state in which an ascorbic acid or an ascorbate is further added to an inorganic salt-based solid in which a protein having a biological activity is embedded.

[29] The process according to [28], wherein the ascorbic acid or ascorbate is selected from ascorbic acid, sodium ascorbate, calcium ascorbate dihydrate and magnesium ascorbyl phosphate n-hydrate.

[30] The method according to any one of the above [27] to [29], wherein the dose of the gamma ray is 3 to 40 kGy.

[31] The method according to any one of [19] to [30], wherein the peptide hormone is 1 or 2 or more selected from the group consisting of hypothalamic peptide hormones, vasopressin, oxytocin, pituitary mesenchymal hormone, gonadotropin, growth hormone, parathyroid hormone, inhibin, activin, relaxin, insulin, glucagon, somatostatin, cholecystokinin, secretin, motilin, atrial natriuretic peptide, erythropoietin, leptin, endothelin, ghrelin, adiponectin, insulin-like growth factor, and calcitonin gene-related peptide.

[32] The method according to any one of the above [19] to [30], wherein the growth factor is 1 or 2 or more growth factors selected from FGF-2 and functional equivalents thereof.

[33] The method according to any one of [19] to [30], wherein the osteogenic protein is 1 or 2 or more selected from the group consisting of OP-1, OP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, DPP, Vg1, Vgr-1 and functional equivalents thereof.

[34] A method for manufacturing a medical device for tissue repair, the method comprising any one of the methods of [19] to [33 ].

[35] The method according to the above [34], wherein the medical device for tissue repair is 1 or 2 or more selected from an intracorporeal fixation pin, an intracorporeal fixation screw, an artificial bone, a bone filler, a dental intraosseous implant, an intraspinal fixation device, an intramedullary nail, and a spinal cage.

[36] A method for producing a medical device used as an artificial joint, the method comprising the method according to any one of [19] to [33 ].

Effects of the invention

The medical device of the present invention can avoid an aseptic manufacturing process in which process management is complicated, and one embodiment of the medical device of the present invention is a medical device sterilized by a radiation-based terminal sterilization method (a sterilization method in which irradiation with radiation is performed in a state in which a material to be sterilized is contained in a terminal package, and death of microorganisms after the sterilization can be quantitatively measured or estimated).

In the medical device of the present invention in which the inorganic salt-based solid in which the biologically active protein is embedded is coated on a part or all of the metal, the ceramic, or both, inactivation of the biological activity of the protein due to radiation sterilization can be suppressed, and therefore, a simple terminal sterilization method by radiation can be applied to the manufacturing process of various medical devices utilizing the biological activity of the protein, and an aseptic manufacturing method can be avoided, so that the cost can be greatly reduced.

Drawings

FIG. 1 is a view showing that when FGF-2 embedded with apatite is applied to a titanium External fixation pin (External fixation pin) for bone repair, the cell growth activity of FGF-2 is maintained and protected from radiation inactivation even by irradiation with gamma rays at a dose suitable for sterilization. The cell proliferation rate of FGF-2 after gamma ray irradiation has no significant difference from that of FGF-2 without gamma ray irradiation.

FIG. 2 is a view showing that FGF-2 loses cell growth activity and is inactivated by radiation without being protected when gamma rays are irradiated at a dose suitable for sterilization in the case where FGF-2 is adsorbed to a titanium outer fixation pin for bone repair coated with apatite. The cell proliferation rate of FGF-2 after gamma irradiation is statistically significantly lower than that of FGF-2 without gamma irradiation.

FIG. 3 is a view showing that FGF-2 loses cell growth activity and is inactivated by radiation without being protected when gamma rays are irradiated at a dose suitable for sterilization in the case where FGF-2 embedded with gelatin is applied to a titanium external fixation pin for bone repair. The cell proliferation rate of FGF-2 after gamma irradiation is statistically significantly lower than that of FGF-2 without gamma irradiation.

FIG. 4 is a view showing a member of a medical device capable of maintaining cell proliferation activity of FGF-2 using a ceramic for transplantation when FGF-2 embedded with apatite is coated and irradiated with gamma rays at a dose suitable for sterilization. The cell proliferation rate of FGF-2 after gamma ray irradiation has no significant difference from that of FGF-2 without gamma ray irradiation.

FIG. 5 is a view showing that when FGF-2 embedded with apatite is applied to a titanium external fixation pin for bone repair and gamma-ray irradiation is performed at a dose suitable for sterilization, the disappearance of the reactivity of FGF-2 against FGF-2 antibodies can be suppressed when gamma-ray irradiation is performed after degassing.

FIG. 6 is a view showing that when FGF-2 embedded with apatite is applied to a titanium external fixation pin for bone repair and gamma-ray irradiation is performed at a dose suitable for sterilization, the disappearance of the reactivity of FGF-2 against FGF-2 antibodies can be suppressed when the gamma-ray irradiation is performed at a low temperature in the presence of dry ice after degassing.

FIG. 7 is a graph showing the differentiation-inducing ability of BMP-2 remaining in the mesenchymal stem cells in the presence of Dex when BMP-2 embedded with apatite is applied to an apatite disk and irradiated with gamma rays at a dose suitable for sterilization.

FIG. 8 is a view showing that when FGF-2 embedded with apatite is applied to a titanium external fixation pin for bone repair and gamma-ray irradiation is performed at a dose suitable for sterilization, the disappearance of the reactivity of FGF-2 against FGF-2 antibodies can be suppressed when ascorbate is added to the application and the gamma-ray irradiation is performed.

FIG. 9 is a graph showing that when the cell growth activity of FGF-2 is compared with that of a coating layer of apatite having both FGF-2 and heparin adsorbed thereon, which is a coating layer having both FGF-2 and heparin embedded thereon and a coating layer of apatite having both FGF-2 and heparin adsorbed thereon are produced on a titanium outer fixing pin, and gamma rays of a dose suitable for sterilization are irradiated thereon, the disappearance of the cell growth activity of FGF-2 can be suppressed by the coating layer of apatite having both FGF-2 and heparin embedded thereon.

FIG. 10 is a graph showing that when a coating layer in which both FGF-2 and heparin are embedded with apatite and a coating layer in which both FGF-2 and heparin are adsorbed are prepared on zirconia for artificial joints/bones and the cell growth activity of FGF-2 is compared after irradiation with gamma rays at a dose suitable for sterilization, the coating layer in which both FGF-2 and heparin are embedded with apatite can suppress the disappearance of the cell growth activity of FGF-2 compared to the coating layer in which both FGF-2 and heparin are adsorbed.

FIG. 11 is a powder X-ray diffraction chart showing that a coprecipitate obtained when a coating layer in which FGF-2 and heparin are both encapsulated with apatite is formed on zirconia is an inorganic salt solid containing low-crystalline apatite, amorphous calcium phosphate, and calcium carbonate as main components.

Detailed Description

The medical device and the manufacturing method thereof provided by the invention are characterized in that the medical device is used for mammals including human and the manufacturing method thereof, the medical device is configured that inorganic salt solid embedded with protein with bioactivity is coated on a part or all of metal, ceramic or both of the metal and the ceramic,

(a) the above inorganic salt-based solid embedded with a biologically active protein is provided by a process of controlled delayed co-precipitation or sandwich coating or drying in a supersaturated solution of neutral or weakly basic labile calcium phosphate which produces spontaneous nucleation,

(b) the medical device is produced by a process of exposing the medical device to ionizing radiation in a dose sufficient for sterilization to produce a terminally sterilized medical device having 1 or 2 or more biological activities selected from the group consisting of cell proliferation activity, blood vessel proliferation activity, soft tissue formation activity, bone differentiation promoting activity, antibody-reactive activity, agonistic activity and antagonistic activity,

(c) the inorganic salt is 1 or more than 2 inorganic salts selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate and calcium carbonate

(d) The above-mentioned protein with biological activity is 1 or more than 2 kinds of protein selected from peptide hormone, growth factor and osteogenic protein.

As described above, the term "embedding" generally means that a biological tissue to be a pathological examination object is embedded in curable liquid paraffin or resin and cured to fix the biological tissue, but in the present specification, "embedding" means that all or a part of a protein molecule is embedded in an inorganic salt-based solid, or that all or a part of the protein molecule is densely covered with an inorganic salt-based solid to fix the protein molecule in the inorganic salt-based solid. Thus, "entrapping" is different from a state in which the protein molecule is merely "adsorbed" or "contacted" in the inorganic salt-based solid, or a state in which the protein molecule is "mixed". An example of "embedding" of protein molecules in an inorganic salt solid is a composition in which the protein molecules and calcium phosphate are coprecipitated from a supersaturated solution of calcium phosphate in which the protein molecules coexist, and the protein molecules are dispersed and arranged in a matrix of calcium phosphate at intervals of a nanometer order (non-patent document 1).

In more detail, in the present specification, the term "embedded" includes: the inorganic salt and the protein molecule are both crystallized or precipitated from the liquid phase for solid phase solidification, so that the whole or a part of the protein molecule is embedded into the inorganic salt solid, or the whole or a part of the protein molecule is densely covered by the inorganic salt solid, and the protein molecule is fixed in the inorganic salt solid.

Likewise, the term "embedded" also includes: both an organic substance such as gelatin and a protein molecule are crystallized or precipitated from a liquid phase at the same time to form a solid phase, whereby the whole or a part of the protein molecule is embedded in the solid organic substance, or the whole or a part of the protein molecule is densely covered with the solid organic substance to fix the protein molecule in the solid organic substance.

However, the term "embedded" should be interpreted in its broadest sense to include the above definitions, and the above definitions should not be interpreted as limiting in any sense.

On the other hand, "adsorption" or "contact" means a state obtained as follows: an organic substance such as an inorganic salt or gelatin is previously crystallized or precipitated to be solid-phased, and then protein molecules present in the liquid phase are immobilized on the solid inorganic salt or organic substance. Therefore, usually, when protein molecules are merely in the "adsorbed" or "contact" state, the immobilized protein molecules are specifically present on the surface of an inorganic salt-like solid or solid organic substance.

Further, "mixed" is any of the following states: a state in which both the inorganic salt and the protein molecule are previously crystallized or precipitated to form a solid phase and then brought close to each other, or a state in which both an organic substance such as gelatin and the protein molecule are previously crystallized or precipitated to form a solid phase and then brought close to each other. Examples of the "mixing" include a state in which powder particles of an inorganic salt and powder particles of a protein are macroscopically homogeneous and a state in which particles of an organic solid powder and powder particles of a protein are macroscopically homogeneous.

Whether or not a protein is embedded in an inorganic salt-based solid can be easily confirmed by, for example, an immunoelectron microscope. That is, if the protein in the inorganic salt-based solid is stained/visualized for observation by an electron microscope using an antibody labeled with a substance having a high electron density such as gold colloid or ferritin or a precursor thereof, whether or not the protein is embedded in the inorganic salt-based solid can be confirmed by an electron microscope. If the protein is embedded in the inorganic salt-based solid, it can be confirmed that the separated protein is dispersed in the inorganic salt-based solid matrix (non-patent document 1).

In the present specification, the inorganic salt-based solid is a biocompatible inorganic salt-based solid suitable for use as a medical device, and specifically, 1 or 2 or more inorganic salts selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, and calcium carbonate.

The inorganic salt solid may be either a crystalline inorganic salt solid or an amorphous inorganic salt solid. Further, the inorganic salt-containing solid may be a mixture of an amorphous inorganic salt-containing solid and a crystalline inorganic salt-containing solid, or a mixture of a plurality of inorganic salt-containing solids having different compositions may be present. Whether crystalline or amorphous is generally easily distinguished by powder X-ray diffraction, which shows a broad diffraction halo in the powder X-ray diffraction pattern if the solid inorganic salt is completely amorphous, and a plurality of diffraction peaks if crystalline.

In the present specification, the term "low-crystalline apatite" means an apatite having low crystallinity, and is characterized in that 3 diffraction lines (211), (112), and (300) appear as one peak or diffraction halo without being separated in a powder X-ray diffraction pattern. These 3 diffraction lines appear as 3 lines separated at the positions of diffraction angles of 31.8 °, 32.2 °, and 32.9 ° when measured by CuK α rays in apatite having high crystallinity (for example, pure crystalline hydroxyapatite).

Calcium phosphate, calcium carbonate, calcium hydrogen carbonate, sodium phosphate, sodium carbonate, sodium hydrogen carbonate, sodium chloride, apatite, tricalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate may be solid-dissolved substances in which other inorganic elements and ion groups are dissolved as impurities. Examples of such a solid solution include, but are not limited to, calcium carbonate containing magnesium dissolved therein, calcium phosphate containing carbonic acid dissolved therein, sodium phosphate, calcium phosphate containing zinc dissolved therein, and sodium chloride containing potassium dissolved therein. Examples of the solid-dissolved elements and ion groups include magnesium, iron, zinc, potassium, hydrogen ions, hydroxide ions, carbonate ions, sulfate ions, nitrate ions, and the like, and these elements and ion groups can be solid-dissolved in inorganic salts by allowing them to coexist with the raw materials used in the embedding.

In the present specification, 1 or 2 or more proteins selected from peptide hormones, growth factors, and osteogenic proteins can be used as the biologically active protein. The protein having biological activity may be any protein that is not recognized as a foreign substance by a mammal using a medical device and is not biologically rejected by the mammal. Including for example: a genetically modified protein having the same physiological function, which is artificially prepared based on these proteins originally present in mammals using medical instruments, a protein which is modified by physical or chemical treatment without losing the original biological activity, and the like. The term biologically active protein as used in this specification is not to be construed in any sense as limiting, but rather in the broadest sense. Examples of the biological activity include, but are not limited to, 1 or 2 or more biological activities selected from the group consisting of cell proliferation activity, vascular proliferation activity, soft tissue formation activity, bone differentiation promoting activity, antibody reactivity, agonistic activity, and antagonistic activity.

In the present specification, a medical device means a device used for diagnosis, treatment, and prevention of diseases in mammals including humans, and means a device that affects, for example, the structure, function, and the like of the body of a mammal including humans. In the present specification, mammals include humans and non-human mammals, and as the non-human mammals, include, for example: and rodents such as monkeys, felines, canines, equines, lagomorphs, and guinea pigs, but the present invention is not limited to these specific animals. Some of the medical instruments are designated by the government code, but the medical instrument of the present invention includes medical instruments other than the government code, for example, a mask. Including for example: pacemaker, coronary stent, artificial blood vessel, PTCA catheter, central venous catheter, bolt for intra-absorbent body fixation, nonwoven fabric for operation, and the like, but the present invention is not limited to these specific embodiments. The medical device for tissue repair and the medical device for joint function restoration are preferable, and examples thereof include, but are not limited to, an artificial joint. Medical devices that are delivered and indwelling in the body, for example, for use in surgeries other than injection, including puncture, are preferred. The term in vivo also includes, for example, teeth. The period of retention is not particularly limited, and may be, for example, temporary retention within 24 hours, short/medium retention of about 1 to 30 days, or long-term retention of 30 days or more.

According to a preferred embodiment of the present invention, there can be mentioned, for example: an instrument in which a metal rod portion in direct contact with a bone is coated with an inorganic salt solid in which a bioactive protein is embedded, the metal rod portion being an artificial hip joint using a metal rod, a ceramic bone, and an ultra-high molecular weight polyethylene lining. In addition, as other examples, there can be mentioned: a device in which an inorganic salt solid in which a bioactive protein is embedded is coated only on the ridge portion of a screw, which is a metal screw for bone fixation; a device in which a solid inorganic salt containing a bioactive protein is coated only on a portion in contact with a bone and a periodontal tissue as an intraosseous dental implant; an apparatus for coating inorganic salt solid embedded with protein with bioactivity only on the part contacting with bones as an internal spinal fixation device and a spinal cage; or an apparatus in which an inorganic salt solid in which a bioactive protein is embedded is coated on the whole of an artificial bone as an artificial bone made of ceramic for bone filling; and devices in which an inorganic salt solid in which a bioactive protein is embedded is coated on the entire artificial bone, which is a composite material of metal and ceramic. However, the scope of the present invention is not limited to these specific embodiments.

A method of sterilizing a physiologically active substance by coprecipitation with an inorganic salt, applying it to a substrate and irradiating it with gamma rays at the final stage is disclosed (patent document 19), but in this document, there is no description or teaching of retaining a specific biological activity of the physiologically active substance after sterilization, and further, there is no description or teaching of an optimal substrate for retaining the specific biological activity of the physiologically active substance after sterilization. The present inventors have found that, in the case of coating on a substrate of a polymer material, it is difficult to retain the biological activity of a physiologically active substance after sterilization, but by coating on a substrate of metal or ceramic an inorganic salt-based solid in which a protein having biological activity is embedded, it is possible to highly retain the specific biological activity of the protein even after sterilization by ionizing radiation.

The ceramic means a non-metallic inorganic solid material produced by artificial heat treatment in a narrow sense, but in the present specification, it is also referred to as a ceramic including a non-heat-treated non-metallic inorganic solid material which can be used in the field of medical and medical instruments. In the present specification, the ceramics may be any non-metallic inorganic solid material, and include materials obtained by any preparation method, such as materials prepared by artificial heat treatment or materials prepared without heat treatment.

In a preferred embodiment of the present invention, there is provided a medical device comprising a structure in which a solid of inorganic salts, in which not only a bioactive protein but also a polysaccharide, preferably heparin, is embedded, is coated on a part or all of a metal for transplantation, a ceramic for transplantation, or both of them, and a method for manufacturing the same. Polysaccharides derived from extracellular matrix such as heparin are biopolymers which do not have direct cell proliferation and differentiation activity, but are valuable because they contribute to maintaining the biological activity of biologically active proteins.

In a preferred embodiment of the present invention, there is provided a medical device comprising a structure in which a solid inorganic salt in which a bioactive protein is embedded is coated on a part or the whole of a metal for implantation selected from titanium, a titanium alloy, stainless steel, and a cobalt-chromium alloy, and a method for manufacturing the same. Titanium, titanium alloys, stainless steel, and cobalt-chromium alloys are metals having high biocompatibility and are widely used in orthopedics and dentistry, and therefore have high value as medical instruments in the fields of orthopedics and dentistry.

In a further preferred embodiment of the present invention, there is provided a medical device and a method for manufacturing the same, wherein the medical device is configured such that a part or the whole of a ceramic for transplantation selected from apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, and a composite thereof is coated with an inorganic salt solid in which a bioactive protein is embedded. The ceramics for transplantation may contain alumina and/or zirconia. These ceramics are also highly biocompatible materials and are widely used in orthopedics and dentistry, and therefore have high value as medical instruments in the fields of orthopedics and dentistry. These ceramics also include ceramics in which other inorganic elements and ion groups are solid-dissolved as impurities. Examples of such a material include, but are not limited to, apatite having carbon dioxide and silicon dissolved therein, tricalcium phosphate having silicon dissolved therein, amorphous calcium phosphate having magnesium dissolved therein, and zirconia having yttrium dissolved therein. Examples of the composite include a biphasic ceramic made of apatite and tricalcium phosphate, and a composite ceramic made of alumina and zirconia, but the composite is not limited to these specific embodiments.

The medical device comprising the inorganic salt solid in which the biologically active protein is embedded can be sterilized by irradiating the medical device with a sufficient dose of ionizing radiation suitable for sterilization while substantially maintaining the activity of the biologically active protein, and can be finally sterilized.

As ionizing radiation for sterilization, gamma rays and/or electron beams are preferred. Even inorganic salt-based solids in which biologically active proteins are embedded can be easily sterilized by gamma rays which are easily transmitted through the material. In the protein embedded in the inorganic salt-based solid, the exposed portion of the solid surface and the like can be sterilized by electron beam irradiation according to a method known to those skilled in the art. If the entire medical device is appropriately packaged in a sealed state in advance, and then gamma rays or electron rays are irradiated by a method known to those skilled in the art, a product in which the sterilized medical device is hermetically encapsulated can be obtained, and the product can be supplied to a medical field as a sterile hermetically packaged medical device (non-patent document 17). However, the embodiments of the present invention are not limited to these specific embodiments.

The dose required for sterilization is generally a SAL (Sterility assessment Level)10^-6The lowest dosage required is enough. The sterility assurance level is determined by standards such as ISO (ISO 11137-1, 1137-2) and JIS (JIS T0806-1, 0806-2), and is adopted by regulatory agencies in various countries such as the United states FDA and the Japanese PMDA (pharmaceutical and medical device integration organization).

In the case of sterilization using gamma rays, a level 10 is guaranteed as assurance of sterility^-6The dose for sterilizing the medical device of the present invention can be selected, for example, from about 10 to 40kGy, preferably from 15 to 30 KGy. As a radiation dose to achieve a sterility assurance level, about 25kGy is sometimes preferred. However, the radiation dose is not limited to these specific radiation doses.

In a further preferred embodiment of the present invention, in the sterilization step using gamma rays and/or electron beams, sterilization can be performed in a degassed state at an atmospheric pressure of 50kPa or less, preferably less than 50Pa, and more preferably 20Pa or less, in order to suppress generation of radicals. Alternatively, it is also preferable to sterilize the mixture in a state where the air is replaced with nitrogen or an inert gas. Further, sterilization may be performed at a low temperature of preferably from 0 ℃ to-196 ℃, preferably from-20 ℃ to-80 ℃, and more preferably from-20 ℃ to-80 ℃ in the presence of dry ice. Alternatively, it may be preferable to add ascorbic acid or an ascorbate to the protein-embedded inorganic salt solid and sterilize the mixture. The ascorbic acid and ascorbate are added to uniformly disperse the ascorbic acid and ascorbate by immersing the mixture in a solution of 5 to 50mM, preferably 10 to 30mM, ascorbic acid and ascorbate, and drying the solution.

From another viewpoint, a preferred embodiment of the present invention provides a medical device for mammals, which comprises an inorganic salt solid having embedded therein a peptide hormone selected from the group consisting of a peptide hormone derived from hypothalamus, vasopressin, oxytocin, pituitary hormone, gonadotropin, growth hormone, parathyroid hormone, inhibin, activin, relaxin, insulin, glucagon, somatostatin, cholecystokinin, secretin, motilin, atrial natriuretic peptide, erythropoietin, leptin, endothelin, gastric ghrelin, adiponectin, insulin-like growth factor, and calcitonin gene-related peptide.

In another preferred embodiment, there is provided a medical device for mammals, which comprises a solid inorganic salt in which FGF-2 (fibroblast growth factor-2) as a growth factor is embedded, and a method for manufacturing the same. FGF-2 is a growth factor useful for soft tissue regeneration, angiogenesis, and bone formation, and thus such a medical device is useful for promoting tissue regeneration.

In still another preferred embodiment, there is provided a medical device for mammals and a method for manufacturing the same, comprising an inorganic salt solid in which 1 or 2 or more kinds of osteogenic proteins selected from the group consisting of OP-1, OP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, DPP, Vg1, Vgr-1 and functional equivalents thereof are embedded as osteogenic proteins. Such medical devices are useful in applications that promote bone regeneration.

From another viewpoint, a preferred embodiment of the present invention provides a medical device and a method for producing the same, wherein the biological activity of the biologically active protein is a biological activity selected from the group consisting of a cell proliferation activity, a blood vessel proliferation activity, a soft tissue formation activity, a bone differentiation promoting activity, an antibody response activity and an agonistic activity. The biological activities of proteins and medical devices can be evaluated by the cell aggregation state such as the number of cells in cell culture, differentiation markers, production substances, gene expression, cell morphology, and formation of vascular-like structures. In animal experiments, evaluation can be performed by tissue morphology observation using tissue specimens, tissue images such as X-rays and MRI, gene expression, and the like. When a biologically active protein functions as an antigen, agonist or antagonist, it can be evaluated by various methods such as western blotting using a monoclonal antibody related to the biologically active site of the protein or a labeled monoclonal antibody. When the protein having biological activity is an antibody, the antibody is labeled in advance and then bound to an antigen, and the amount and binding activity of the antibody bound to the antigen are evaluated using the activity and amount of the labeling substance, whereby the biological activity can be evaluated. When the protein having biological activity is an enzyme, the biological activity can also be evaluated by a method of evaluating a reaction product of the enzyme and an enzyme substrate using the enzyme substrate. However, the method is not limited to this, as long as it is an evaluation method corresponding to a specific biological activity to be evaluated. In either evaluation method, for example, by comparing a specific biological activity between a group of inorganic salt-based solids containing a protein and a group of inorganic salt-based solids not containing a protein, it can be verified whether or not the protein or the medical device has a biological activity. The samples for comparing the biological activities of the two groups may be obtained by directly using inorganic salt solids containing or not containing proteins, medical instruments, or implants, or by using an appropriate extraction solution to dissolve inorganic salt solids to extract proteins.

The method of quantitatively evaluating the biological activity and the evaluation method thereof can be used for evaluating the biological activity of the medical instrument sterilized by ionizing radiation at a dose sufficient for sterilization. The medical device of the present invention ensures sterility at a level of 10^-6After sterilization, the composition has a biological activity of at least about 13% or more, preferably 65% or more, and more preferably 70% or more of the biological activity before sterilization.

As a preferable method for embedding the biologically active protein in the inorganic salt solid, for example, a coprecipitation method using a sodium chloride solution, a sodium phosphate solution, a calcium solution containing carbonate ions, a sodium carbonate solution, or a sodium bicarbonate solution, a sandwich coating method, or a drying method can be used.

The coprecipitation precipitation method is as follows: the protein is entrapped by allowing a desired bioactive protein to coexist in a supersaturated solution of a desired inorganic salt, and simultaneously capturing the protein when a crystalline or amorphous solid of the inorganic salt is precipitated from the supersaturated solution, or by allowing the protein molecule to be densely encapsulated by a solid of the inorganic salt while precipitating the protein. The coprecipitation deposition also includes a method of depositing a crystalline or amorphous solid of an inorganic salt in which the protein is embedded so as to coat the surface of another solid.

The cover sandwich method is a method in which a desired bioactive protein is adsorbed to or brought into contact with the surface of a desired inorganic salt-based solid, and the surface is coated with the same or different kinds of inorganic salt-based solid in which the bioactive protein is embedded. As another method, a drying method can be mentioned, and for example, a method can be mentioned in which a desired bioactive protein is dissolved in a solution of a desired inorganic salt, and the solution is freeze-dried or concentrated and dried to thereby densely entrap the protein in an inorganic salt solid. These methods may be used alone, or 2 or more kinds may be appropriately combined, and repeated as many times as necessary. By using these methods, it is possible to form a plurality of layers of the inorganic salt-based solid protein-encapsulating composition, preferably in a state in which a desired bioactive protein can be protected from radiation sterilization. The inorganic salt-based solid entrapping bioactive protein thus produced can be obtained as a state precipitated from a solution or suspended in a solution, can also be obtained as a form coated with a layer of a metal for transplantation or a ceramic for transplantation as described above, and can be appropriately separated from a solution or dried.

As a preferred method for embedding the biologically active protein in the inorganic salt-based solid, a coprecipitation method or a coating sandwich method can be employed in which controlled delayed coprecipitation of the biologically active protein and calcium phosphate is carried out in an unstable supersaturated solution of calcium phosphate which generates spontaneous nucleation in a neutral or weakly alkaline solution. As a high concentration of phosphoric acid in a protein-containing mediumIn a calcium solution, a method of suppressing spontaneous nucleation, crystallization, or precipitation of calcium phosphate, stabilizing the calcium phosphate in the form of a solution in a high concentration state, and coprecipitating protein and calcium phosphate at a desired timing is known as follows: the supersaturation degree is lowered by lowering the pH by bubbling of carbon dioxide or addition of an acid, and OH is generated by degassing of carbon dioxide, addition of an alkali, and electrochemical reduction of water molecules after complete dissolution^－The pH is gradually increased by the ions to increase the supersaturation degree, and calcium phosphate is gradually crystallized to cause coprecipitation with the protein (patent document 19, patent document 25, patent document 26, patent document 27, and patent document 33). In this method, it is considered that the supersaturation degree is increased by raising the pH of the stable supersaturated solution of calcium phosphate which is completely dissolved in acidic conditions and does not cause spontaneous nucleation, thereby causing coprecipitation.

However, most proteins have a problem of being denatured at acidic pH, high alkaline pH and thus losing activity. In order to avoid the above problems, as an unstable supersaturated solution of calcium phosphate which causes spontaneous nucleation, a solution in which a large amount of K ions and Na ions are added to a neutral or weakly alkaline high-concentration calcium phosphate solution which can form a large amount of precipitates at once is prepared, and the activation energy for crystallization is increased, whereby the nucleation frequency can be reduced, the crystallization time can be delayed, calcium phosphate is slowly crystallized, and coprecipitation with protein can be caused. The method is not a method of adjusting the supersaturation degree of a solution, but a method of performing controlled delayed coprecipitation of a biologically active protein and calcium phosphate by changing the activation energy of crystallization, and more specifically, a method of controlling and extending the time of spontaneous nucleation by adjusting the concentration of calcium phosphate, pH, and KCl concentration regardless of the supersaturation degree, thereby enabling delayed coprecipitation.

For example, the delayed coprecipitation is an aqueous solution of pH 7.0 to 9.0 containing 0.5 to 2.5mM of Ca ions, 1.0 to 20mM of phosphate ions, 0 to 40mM of K ions, 0 to 200mM of Na ions, and 0 to 200mM of Cl ions, preferably 1.2 to 2.75mM of Ca ions, 0.6 to 15mM of phosphate ions, 0 to 30mM of K ions, 30 to 150mM of Na ions, 0.1 to 3.0mM of Mg ions, 30 to 150mM of Cl ions, and 0 to 60mHCO of M₃Preferably, the time for delaying the precipitation of calcium phosphate is artificially controlled by controlling the KCl concentration in an aqueous solution of ions having a pH of 7.0 to 9.0. Due to Mg ions, HCO₃Since the ions are inhibitors of calcium phosphate crystallization, Mg ions and HCO ions are added in addition to K ions and Na ions₃In the ionic unstable supersaturated solution of calcium phosphate, the delayed calcium phosphate precipitation time can be further controlled manually (patent document 29).

Examples

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. The terms and concepts in the present embodiment are based on the meanings of terms commonly used in the art, and the techniques used to implement the present invention are easily and reliably implemented by those skilled in the art based on publicly known documents, except for the techniques specifically indicated. Various analyses and the like are performed by using methods described in the instructions, catalogs and the like of the analytical instruments, reagents and kits used.

Example 1: cell proliferation Activity after radiation Sterilization with FGF-2 Encapsulated Apatite coated external fixation pins

The titanium pins for internal fixation used for fracture fixation were coated with an inorganic salt solid in which FGF-2 having cell-proliferating activity was embedded, and after the whole was sterilized by ionizing radiation, whether FGF-2 has cell-proliferating activity was examined.

A mixture containing 4.89mM of Ca ions, 1.28mM of phosphate ions, 6.13mM of K ions, 138.8mM of Na ions, 0.23mM of Mg ions, 136.6mM of Cl ions, and 15.09mM of HCO was used₃An unstable supersaturated calcium phosphate solution which is ionic and has a pH of 7.8 and which can crystallize calcium phosphate by spontaneous nucleation even when left at 37 ℃ for about 4 to 5 hours (this unstable supersaturated calcium phosphate solution is different from the solution of patent document 19). Fibroblast growth factor-2 (FGF-2) was added to the unstabilized supersaturated solution of calcium phosphate at concentrations of 4. mu.g/ml and 0. mu.g/ml. Immersing a titanium inner fixing pin (Synthesis, Inc. cell Drall 4.0/3.0mmTi, 20mm-80mm) in the solution at 37 deg.C for 48 hours to allow FGF-2 and apatite to be mixed6 or 8 coprecipitated apatite FGF-2 (coprecipitated ApFGF) pins were produced by coprecipitating and coating the same. Likewise, 6 or 8 Ap pins were prepared using unstable supersaturated solutions of calcium phosphate without FGF-2. The prepared coprecipitated ApFGF pin was put into a tube and vacuum-dried at 12.4Pa for 2 hours at room temperature. Immediately after drying, the tube with the pin was capped and packaged using an oxygen-free/dry storage system (ISO corporation) consisting of a gas barrier storage bag, an oxygen absorbent, and a synthetic zeolite desiccant.

To be provided with⁶⁰Half of these ApFGF pins were gamma irradiated with Co as radiation source at a dose of 25 + -0.5 kGy. Irradiation was carried out at ambient temperature, including storage at 4 ℃ during transport. The coprecipitated ApFGF pin which has not been irradiated with gamma rays is stored at 4 ℃. To evaluate the cell growth activity of FGF-2 carried on the coprecipitated ApFGF pins irradiated and not irradiated with gamma rays, the pins were immersed in 10mM sodium citrate for 30 minutes for dissolution. As a control, Ap pins without FGF-2 were also immersed in a 10mM sodium citrate solution for 30 minutes to dissolve the coating layer. Since calcium in the dissolution solution promoted Cell proliferation, ICP emission spectroscopy was performed, and then the calcium concentration between samples was adjusted to be equal to each other, and the proliferation rate was measured using Cell Counting Kit-8 in a mouse fibroblast Cell line NIH3T 3. The Ap pin containing no FGF-2 was used as a control, and the growth rate was set to 1, and the coprecipitated Ap FGF pin having a statistically significant growth rate was judged to be "active". The operations of coating by coprecipitation, vacuum drying, irradiation or non-irradiation with gamma rays, and measurement of the proliferation rate were repeated 4 times.

Table 1 shows the number of co-precipitated ApFGF pins judged to be "active" in 4 replicates. Fig. 1 shows the values of the measured proliferation rates.

[ Table 1]

As shown in table 1, in 4 replicates, 10/13 of the γ -ray-irradiated groups and 10/13 of the non-irradiated groups of the coprecipitated ApFGF pins were judged to have cell growth activity, and the number of the pins having cell growth activity was the same in the γ -ray-irradiated groups and the non-irradiated groups. As shown in fig. 1, in the coprecipitated ApFGF pins, the proliferation rate of the Ap pin was about 1.5 times that of both the gamma-ray irradiated group and the non-irradiated group, the proliferation rate of the non-irradiated gamma-ray group was statistically significantly high (p ═ 0.021) relative to the proliferation rate of the Ap pin (proliferation rate ═ 1), and the difference between the proliferation rate of the gamma-ray irradiated group and the proliferation rate of the Ap pin (proliferation rate 1) was a level extremely close to a significant level (p ═ 0.058). No significant difference was observed between the proliferation rates of the irradiated and non-irradiated groups (p ═ 0.436). That is, it was found that when a composition in which FGF-2 was embedded with apatite was applied to a titanium intrabody fixation pin as a metal for transplantation, the protein acquired resistance to ionizing radiation sterilization by the embedding, in other words, the protein exhibited a radioprotective effect against bioactive proteins by the embedding with apatite. In addition, under the condition of gamma ray irradiation of 25 + -0.5 kGy, the encapsulation with apatite exhibits a radioprotective effect on the bioactive protein, and therefore, of course, the radioprotective effect is also exhibited on gamma ray irradiation of less than 25 + -0.5 kGy.

Example 2: cell proliferation Activity after radiation Sterilization with FGF-2 adsorbed Apatite coated external fixation pins

A titanium pin for internal fixation used for fracture fixation was coated with an inorganic salt solid having FGF-2 having cell proliferating activity adsorbed thereon, and the whole was sterilized by ionizing radiation to investigate whether FGF-2 has cell proliferating activity.

Using the same unstable supersaturated solution of calcium phosphate as in example 1, a supersaturated solution of calcium phosphate containing no FGF-2 was prepared, and 6 or 8 titanium pins for intracorporeal fixation (Synthesis, Inc. cell Drill4.0/3.0mmTi, 20mm to 80mm) were immersed therein at 37 ℃ for 48 hours to prepare Ap pins with apatite coated surfaces. The Ap pin was immersed in supersaturated calcium phosphate containing 12. mu.g/ml of FGF-2 for several seconds, and frozen at-18 ℃ to prepare an apatite-adsorbed FGF-2 (ApFGF-adsorbed) pin coated with FGF-2-adsorbed apatite. Vacuum drying, irradiation or non-irradiation with γ -ray, storage, and evaluation of cell growth activity were carried out at room temperature under exactly the same conditions as in example 1. The procedure of coating the FGF-2-adsorbed apatite, irradiation with or without gamma ray, and measurement of the proliferation rate was repeated 5 times.

Table 2 shows the number of "active" adsorbing ApFGF pins in 5 replicates. Fig. 2 shows the measured proliferation rate values.

[ Table 2]

As shown in table 2, in 5 experiments, 2/16 of the γ -ray irradiated groups and 11/16 of the non-irradiated groups, to which ApFGF pins were adsorbed, were judged to have cell growth activity, and the number of pins having cell growth activity in the γ -ray irradiated groups was about 1/5 of the non-irradiated groups. The chi-square test was performed on both groups, and it was confirmed that since a significant difference (p ═ 0.001) was present between the gamma-irradiated group and the non-irradiated group, it was found that the biological activity of FGF-2 was lost by gamma-irradiation sterilization of the ApFGF-adsorbed gene. As shown in fig. 2, the non-irradiated group showed a growth rate of about 1.7 times (p 0.003) that of the Ap pin in the case of adsorbing the Ap FGF pins, but the γ -irradiated group reduced the growth rate of 1.1 times that of the Ap pin, and it was found that the cell growth activity of FGF-2 was remarkably lost (p 0.0004) by γ -irradiation, which was about 35% of that of the Ap pin (p 0.087). On the other hand, as shown in fig. 1, in the coprecipitated ApFGF pins, both the gamma-irradiated group and the non-irradiated group showed a growth rate of about 1.5 times that of the Ap pins, and no significant difference was observed between the irradiated group and the non-irradiated group (p ═ 0.436). That is, although patent documents 12, 13, and 17 describe that compositions in which a biologically active protein and an inorganic salt are combined are subjected to radiation sterilization, as a combination method, there are methods such as "mixing", "adsorption", "contact", and "entrapment", and it is known that a large difference occurs in the biological activity of the protein after final sterilization depending on the combination method, and that the biological activity of the protein can be maintained with high efficiency in the method such as "entrapment".

Example 3: cell proliferation Activity after radiation Sterilization with FGF-2 Encapsulated gelatin coated external fixation pins

The same titanium pin for in vivo fixation as in example 1 was immersed in a 1% gelatin solution containing 4. mu.g/ml of FGF-2 for several seconds, and frozen at-18 ℃ to prepare a pin (gelatin FGF) coated with FGF-2-embedded gelatin. Vacuum drying, irradiation or non-irradiation with γ -ray, storage, and evaluation of cell growth activity were carried out at room temperature under exactly the same conditions as in example 1. The procedure of coating FGF-2-embedded gelatin, irradiation with or without gamma irradiation, and proliferation rate measurement was repeated 4 times.

Table 3 shows the number of pins of gelatin FGF judged to be "active" in 4 replicates. Fig. 3 shows the measured proliferation rate values.

[ Table 3]

As shown in table 3, in 4 experiments, 13/13 of the gamma-ray irradiated groups and 13/13 of the non-irradiated groups, which were each coated with FGF-2 gelatin-coated gelatin FGF pins, were judged to have cell growth activity, and the number of pins having cell growth activity was the same between the gamma-ray irradiated groups and the non-irradiated groups. However, as shown in fig. 3, in the gelatin FGF-based pellet, the proliferation rate of the non-irradiated group was about 12 times that of the Ap-based pellet, and the proliferation rate of the γ -irradiated group was reduced to about 8 times that of the Ap-based pellet, and it was determined that the cell proliferation activity of FGF-2 was statistically significant (p ═ 0.011) and disappeared by about 30%. That is, it is found that when the substrate for embedding the bioactive protein is an organic substance such as gelatin, the radioprotective effect is small, unlike the case of fig. 1 in example 1 in which the substrate is embedded with an inorganic salt solid. Patent document 18 describes a carrier in which a biological activity is incorporated into a matrix of an inorganic, organic or carrier containing an organic and inorganic substance and which is sterilized after synthesis, but does not describe what combination and material the matrix of the inorganic, organic or carrier containing an organic and inorganic substance is made of, and in what sterilization method, the biological activity can be maintained after sterilization, or in what sterilization method, the biological activity can be maintained after sterilization. The examples of the present invention show that the case of embedding a biologically active protein in an inorganic salt solid matrix shows an extremely excellent radioprotective effect than the case of incorporating it in an organic matrix. It is presumed that the organic substance may generate a large amount of radicals upon irradiation with radiation unlike the inorganic salt-based solid.

Example 4: cell proliferation Activity of apatite ceramics for artificial bone coated with FGF-2 Encapsulated apatite after radiation Sterilization

A compact apatite disk (oval diameter 5mm, width 3mm, thickness 1mm) is produced by press molding a hydroxyapatite powder having a particle size of 70 μm or less containing 3% of polyvinyl alcohol, and sintering the molded product at 1150 ℃ for 1 hour. This manufacturing method is essentially the same as the method for manufacturing an apatite ceramic for artificial bone. In the same manner as in example 1, the coprecipitated ApFGF was applied to a plate made of this apatite, vacuum-dried, gamma-irradiated, and then NIH3T3 cells were cultured on the plate to measure the cell growth rate. When NIH3T3 cells were cultured directly on the plate, the cell adhesion was changed by the influence of the protein, and therefore, the apatite plates of the control non-irradiated group were immersed in an unstable calcium phosphate supersaturated solution containing Bovine Serum Albumin (BSA) in place of FGF-2, and coated with ApBSA. The procedure of coating, irradiation or non-irradiation with gamma rays, and measurement of growth rate was repeated three times.

Table 4 shows the number of disks of coprecipitated ApFGF apatite which were judged to be "active" in 3 replicates. Fig. 4 shows the measured proliferation rate values.

[ Table 4]

As shown in Table 4, in 3 replicates, 18/18 cells in the gamma-irradiated group and 18/18 cells in the non-irradiated group of the co-precipitated ApFGF apatite trays were judged to have cell growth activity, and the apatite trays having cell growth activity were counted in the same number in the gamma-irradiated group and the non-irradiated group. As shown in fig. 4, in the coprecipitated ApFGF apatite disks, the growth rate was about 1.4 times that of the Ap pin in both the gamma-ray irradiated group and the non-irradiated group, and there was no significant difference between the irradiated group and the non-irradiated group (p is 0.415). That is, even when a composition in which FGF-2 is embedded with apatite is applied to a ceramic for transplantation, it is known that the protein can be protected from ionizing radiation sterilization by embedding, in other words, bioactive protein by embedding with apatite, similarly to the case of applying the composition to a metal for transplantation.

Example 5: cell proliferation Activity after radiation Sterilization of polymers coated with FGF-2 Encapsulated Apatite

Using a round bar (diameter: 6 mm. times.8 cm long) made of polyether ether ketone (PEEK) as a polymer, the coprecipitated ApFGF was applied to the surface under the same conditions as in example 1, and vacuum-dried, stored, and measured for cell growth rate with or without irradiation of γ rays. Polyetheretherketone is a polymer used for grafting.

Table 5 shows the number of round rods made of coprecipitated ApFGF-PEEK which were judged to be "active" in 3 replicates.

[ Table 5]

As shown in table 5, in 3 experiments, 6/9 of the gamma-irradiated groups and 9/9 of the unirradiated groups of the coprecipitated ApFGF-PEEK round rods were judged to have cell growth activity, and the number of pins having cell growth activity in the gamma-irradiated groups was 2/3 in the unirradiated groups. It is known that the coprecipitated ApFGF-PEEK round bar loses the bioactivity of FGF-2 due to gamma-ray irradiation sterilization. Patent document 19 describes a method in which an acidified composition comprising a brine mixture containing calcium, magnesium, phosphoric acid, bicarbonate ions, and a bioactive substance is brought into contact with a substrate, and the pH is increased to cause coprecipitation of the salts and the bioactive substance, thereby obtaining a coated substrate. Although not mentioned in the invention described in the claims, it is described in the text that the gamma ray irradiation may be performed after the final step. However, the examples of the present invention show that sufficient radioprotective effect cannot be achieved in the case of coprecipitating bioactive proteins onto a polymer for transplantation, and that extremely excellent radioprotective effect is exhibited in the case of coprecipitating onto metals and ceramics for transplantation. Presumably, the polymer generates a large amount of radicals upon irradiation with radiation, unlike metals and ceramics.

Example 6: effect of the Environment upon radiation Sterilization on the biological Activity of proteins Encapsulated in inorganic salt solids

Under the same conditions as in example 1, the apatite having FGF-2 embedded therein was coated on a titanium pin for external fixation as a metal for transplantation, and gamma-rays were irradiated at a dose of 25. + -. 0.5kGy, to examine how the environment at the time of gamma-ray irradiation exerts an influence on FGF-2 having reactivity against an FGF-2 antibody.

A coprecipitated ApFGF pin was produced using a titanium pin for internal fixation in the same manner as in example 1, and hermetically packaged. At this time, 3 environmental conditions of (i) the same oxygen-free/dry packaging as in example 1, (ii) the deaeration packaging, and (iii) the nitrogen-filled packaging were applied. Degassing the vacuum was degassed at 29.2kPa for 5 seconds. The nitrogen substitution is performed by flowing high-purity nitrogen gas. Thereafter, gamma irradiation was performed at a dose of 25. + -. 0.5 kGy. The same oxygen-free/dry packed gamma-ray unirradiated ApFGF pins (unirradiated group) as in example 1 were used as a control. The gamma-irradiated/unirradiated coprecipitated ApFGF pins were immersed in 10mM sodium citrate for 30 minutes to dissolve the coating layer, and FGF-2 carried on the pins was detected by Western blotting using an anti-FGF-2 antibody. The lysate was concentrated 20-fold by freeze-drying and then subjected to western blotting. The antibody used was a human FGF-2 mouse monoclonal antibody (Thermo Fisher Scientific Inc.) that is related to the biological activity of FGF-2. The obtained image data was subjected to quantification of signal intensity detected at the position of 17kDa (FGF-2 molecular weight: 17000) using an Imge Lab (Bio-Rad Co.) and compared.

In the non-irradiated group, the signal of the band was clearly detected at the 17kDa position. When the signal intensity of the non-irradiated group as a control was set to 100%, the signal intensity of the irradiated group was: (i) the same oxygen-free/dry package irradiation as in example 1 was 13%, (ii) degassed package irradiation was 19%, (iii) nitrogen-filled package irradiation was 14% (fig. 5). Thus, it was found that the decrease of FGF-2 reactive with an anti-FGF-2 antibody can be suppressed by gamma-ray irradiation in a degassed state or a nitrogen atmosphere, and particularly that the protective effect of FGF-2 in gamma-ray irradiation is higher in a degassed state than in a nitrogen atmosphere. In addition, in the case of irradiation with an anaerobic/dry package, even if FGF-2, which is reactive to an anti-FGF-2 antibody, is reduced to 13%, the cell proliferation activity of FGF-2 is equivalent to that of the non-irradiated group as described in example 1.

Example 7: effect of temperature during radiation Sterilization on the biological Activity of proteins Encapsulated in inorganic salt solids

Under the same conditions as in example 1, the apatite having FGF-2 embedded therein was coated on a titanium pin for external fixation as a metal for transplantation, and gamma-ray irradiation was performed at a dose of 25. + -. 0.5kGy at room temperature and low temperature, and it was examined what effect the temperature at the time of gamma-ray irradiation had on the reactivity of FGF-2 against FGF-2 antibody.

A coprecipitated ApFGF pin was produced using a titanium inner fixing pin in the same manner as in example 1, and hermetically packaged in the same degassed package as in example 6. The pin subjected to gamma-ray irradiation at a low temperature coexisted with about 3.8kg of dry ice, and the pin subjected to gamma-ray irradiation at room temperature was subjected to gamma-ray irradiation without dry ice. The sublimation temperature of the dry ice was-78.5 ℃ at atmospheric pressure. Therefore, the temperature of the dry ice itself is minus 78.5 ℃. The irradiation dose was the same as in example 6. Thereafter, FGF-2 carried on the pins after gamma irradiation was detected by Western blotting in the same manner as in example 6.

FIG. 6 shows the effect of temperature during radiation sterilization on 17kDaFGF-2 embedded in apatite, which is reactive with anti-FGF-2 antibodies. A band at 17kDa was detected in both groups. Further, it was found that the signal intensity was increased by 2 times in the low temperature irradiation with dry ice at about-80 ℃ as compared with the room temperature irradiation (FIG. 6). It was found that the decrease of FGF-2, which is reactive to an anti-FGF-2 antibody, can be suppressed by gamma-ray irradiation at a low temperature in which dry ice coexists in addition to the degassed package, and that FGF-2 protective effect is higher in gamma-ray irradiation than in irradiation at room temperature.

Example 8: bone differentiation promoting activity of apatite ceramic for artificial bone coated with apatite embedded with human recombinant BMP-2(rhBMP-2) after radiation sterilization

FGF-2 of example 1 was replaced with rhBMP-2, and the compact apatite disks were coated with ApBMP coprecipitated, and gamma-rays were irradiated at a dose of 25. + -. 0.5kGy to examine the presence or absence of the protective effect of BMP-2 against gamma-ray irradiation.

Using a compact apatite disk prepared in the same manner as in example 4, FGF-2 of example 4 was changed to human recombinant BMP-2(rhBMP-2) which is an osteogenic protein, and then ApBMP coating was performed by coprecipitation. As a control, co-precipitated ApBSA coating was performed using Bovine Serum Albumin (BSA) instead of rhBMP-2. After the compact apatite disk coated with the coprecipitated ApBMP and coprecipitated ApBSA was vacuum-dried and irradiated with gamma rays, the mesenchymal stem cells from the rat bone marrow were seeded on the disk, and the bone differentiation marker was measured 12 days after the induction of bone differentiation. Primary mesenchymal stem cells isolated from bone marrow of 7-week-old F344/nscl rats were used as mesenchymal stem cells, and the mesenchymal stem cells seeded on the disc were cultured in an osteoblast differentiation-inducing matrix supplemented with 10mM β glycerophosphate and 0.28mM ascorbic acid for 12 days immediately after seeding without or with 10nM dexamethasone (Dex). The medium was changed in half amount every 2 days for 1 time. After 12 days of culture, the cells were freeze-thawed in PBS containing 0.1% Triron-X, and alkaline phosphatase (ALP) activity as a marker of bone differentiation was quantified using laboratory analysis ALP (Wako Co.). To evaluate the activity per cell number, the amount of DNA in the cell lysate was quantified using Quant-iT (registered trademark) PicoGreen (registered trademark) dsDNA Reagent and Kits (Thermo Fisher Scientific Inc.).

FIG. 7 shows ALP activity per DNA amount after differentiation induction in the absence or presence of Dex in a case of an apatite disk coated with BMP-2-embedded apatite by gamma-ray irradiation. As shown in fig. 7, in the case of the coprecipitated ApBMP apatite disks, the ALP activity per DNA amount after differentiation induction in the absence of Dex was significantly higher than that of ApBSA in the non-irradiated γ -ray group (p 0.02), and the activity equivalent to that of ApBSA was exhibited in the irradiated group (p 0.46). On the other hand, the ALP activity per unit DNA amount after the induction of differentiation in the presence of Dex was significantly higher than ApBSA (p ═ 0.02, 0.0001) in both the γ -ray irradiated group and the non-irradiated group. That is, when BMP-2, which is an osteogenic protein, is embedded with apatite, the effect of promoting bone differentiation in the presence of Dex, which is one of the biological activities of BMP-2, can be maintained even when gamma-ray sterilization is performed. That is, even when a composition in which BMP-2 is embedded with apatite is applied to a ceramic for transplantation, it has been found that the composition maintains a specific biological activity of a protein by obtaining resistance to sterilization by ionizing radiation by embedding, as in the case of embedding FGF-2 with apatite, in other words, it shows radioprotective effect against the specific biological activity of a protein by embedding with apatite.

Example 9: effect of coexistence of L-ascorbic acid phosphate magnesium salt n-hydrate on bioactivity of protein embedded in inorganic salt solid during radiation sterilization

An apatite having FGF-2 embedded was coated on a titanium pin for external fixation as a metal for transplantation under the same conditions as in example 1, and immersed in a solution of magnesium phosphate-L-ascorbate n hydrate (AsMg solution) for several seconds, followed by vacuum drying. The prepared coprecipitated ApFGF pins were subjected to gamma-ray irradiation at a dose of 25. + -. 0.5kGy, and the effect of coexistence of AsMg on FGF-2 reactive with an anti-FGF-2 antibody upon gamma-ray irradiation was examined.

After apatite having FGF-2 embedded was coated with titanium intracorporeal immobilization pins in the same manner as in example 1, the coated product was immersed twice in 25mM AsMg solution for several seconds and vacuum-dried, and AsMg was added to the apatite having FGF-2 embedded. The prepared coprecipitated ApFGF pin was hermetically packaged in the same degassed package as in example 6, and irradiated with gamma rays at a dose of 25. + -. 0.5 kGy. Thereafter, FGF-2 carried on the pins after gamma irradiation was detected by Western blotting in the same manner as in example 6.

FIG. 8 shows the effect of the addition of AsMg to FGF-2 embedded apatite on the reactivity of embedded FGF-2 against FGF-2 antibodies upon radiation sterilization. In any of the groups of no irradiation with γ rays, γ -ray irradiation in the presence of AsMg, and γ -ray irradiation in the absence of AsMg, a band was detected at the position of 17 kDa. When the signal intensity of the gamma ray non-irradiated group was set to 100%, the signal intensity of the gamma ray irradiated group in the presence of AsMg was 55%. However, the signal intensity of the gamma-ray irradiation group in the absence of AsMg was 13%, which is about 1/4 of the gamma-ray irradiation group in the presence of AsMg. Thus, it was found that gamma-irradiation with AsMg added to FGF-2-embedded apatite suppressed the decrease in FGF-2 reactive with an anti-FGF-2 antibody, and that FGF-2 protective effect is higher in gamma-irradiation than in the absence of AsMg. Since AsMg is one of the compounds of ascorbic acid having an antioxidant effect, it is considered that the excellent protective effect is due to the inhibition of the generation of radicals by irradiation with radiation.

Example 10: cell proliferation Activity after radiation Sterilization of external fixation titanium pins coated with Apatite in which heparin is embedded or adsorbed in addition to FGF-2

Apatite-coated external-fixed titanium pins in which both FGF-2 and heparin were embedded and apatite-coated external-fixed titanium pins in which both FGF-and heparin were adsorbed were prepared, and gamma irradiation was performed at a dose of 25 ± 0.5kGy to investigate whether FGF-2 had cell growth activity after gamma irradiation.

Heparin sodium was added at a concentration of 0.5 units/ml to the unstable supersaturated solution of calcium phosphate of example 1 to which FGF-2 was added at concentrations of 4. mu.g/ml and 0. mu.g/ml. Then, an external fixed titanium pin (synthesis, inc. cell drill4.0/3.0mmTi, 20mm to 80mm) was immersed in the supersaturated unstable calcium phosphate solution under the same conditions as in example 1, and FGF-2 and heparin were co-precipitated together with apatite to coat the pin (co-precipitated ApFGF heparin pin). On the other hand, heparin sodium was added to the supersaturated calcium phosphate solution containing 12. mu.g/ml of FGF-2 of example 2 at a concentration of 0.5 units/ml. Thereafter, the pin of the external fixed titanium was immersed in the supersaturated unstable calcium phosphate solution for several seconds under the same conditions as in example 2, and coated with apatite having FGF-2 and heparin adsorbed thereon (ApFGF heparin adsorbed). Vacuum drying, irradiation with or without γ -ray, storage, and evaluation of cell growth activity were carried out under the same conditions as in example 1.

Table 6 shows the number of co-precipitated or ApFGF heparin-adsorbed pins judged to be "active" in 3 replicates.

[ Table 6]

As shown in table 6, in 3 experiments, 9/9 of the γ -ray-irradiated groups and 9/9 of the non-irradiated groups of the coprecipitated ApFGF heparin pins were judged to have cell growth activity, and the number of the pins having cell growth activity was the same in the γ -ray-irradiated group and the non-irradiated group. On the other hand, 3/9 of the gamma-ray irradiated groups having ApFGF heparin pins adsorbed thereon and 9/9 of the non-irradiated groups were judged to have cell growth activity, and the number of pins having cell growth activity in the gamma-ray irradiated groups was about 1/3 in the non-irradiated groups. Chi-square test was performed on both groups, and then it was confirmed that there was a significant difference between the gamma-ray irradiated group and the non-irradiated group (p ═ 0.003). That is, it was found that the ApFGF heparin-adsorbing pin had a strong tendency to lose the biological activity of FGF-2 by gamma irradiation sterilization.

As shown in fig. 9, the cell growth rate of the gamma-ray irradiated group normalized with the cell growth rate of the non-irradiated gamma-ray group being 100% was about 23% in the coprecipitated ApFGF heparin pin and only 4% in the ApFGF heparin-adsorbed pin, and the values of both were statistically significantly different (p is 0.0002). That is, when a composition in which both FGF-2 and heparin are embedded with apatite is coated on a titanium in vivo fixation pin as a metal for transplantation, the composition has higher resistance to ionizing radiation sterilization than when apatite having both FGF-2 and heparin adsorbed thereto is coated, in other words, apatite exhibits a radiation protective effect not only when a biologically active protein is embedded but also when biologically inactive heparin is embedded.

Example 11: comparison of cell proliferation Activity after radiation Sterilization of external fixed titanium Pin coated with Apatite Embedded FGF-2 and external fixed titanium Pin coated with Apatite Embedded both FGF-2 and heparin

The cell growth rate of the co-precipitated ApFGF pins of example 1 and the cell growth rate of the co-precipitated ApFGF heparin pins of example 10 after radiation sterilization were compared, and the effect of embedding heparin in addition to FGF-2 was examined.

In example 1, a solution prepared by dissolving the coating layer of the coprecipitated ApFGF pin after radiation sterilization in a 10mM sodium citrate solution was added to a mouse fibroblast cell line NIH3T3, and the cell growth rate was quantitatively evaluated. As a result, the cell growth rate was 1.53. + -. 0.27 (FIG. 1). On the other hand, when a solution prepared by dissolving a coating layer of the radiation-sterilized coprecipitated ApFGF heparin in a 10mM sodium citrate solution was added to the mouse fibroblast cell line NIH3T3, the cell growth rate was as high as the limit of quantitation or more. Therefore, the lysate was diluted 30-fold and added to the mouse fibroblast cell line NIH3T3 to quantitatively evaluate the cell growth rate, and as a result, the value of the cell growth rate was 1.67 ± 0.07. That is, it is shown that when a polysaccharide such as heparin, which is derived from an extracellular matrix and does not have a direct cell growth and differentiation activity by itself, is embedded together with a bioactive protein, the bioactivity after radiation sterilization can be maintained higher.

Example 12: cell proliferation Activity of zirconium oxide for Artificial Joint/Artificial bone after radiation sterilization coated with apatite having heparin-Encapsulated or adsorbed in addition to FGF-2

Zirconium oxide for artificial joints/artificial bones coated with apatite having both FGF-2 and heparin embedded therein and zirconium oxide for artificial joints/artificial bones coated with apatite having both FGF-and heparin adsorbed thereon were prepared, and they were subjected to gamma-ray irradiation at a dose of 25 ± 0.5kGy, and it was investigated whether FGF-2 had cell proliferation activity after gamma-ray irradiation.

Using a zirconia square bar (2.4mm square bar activity, extracellular phase adsorbed), coating, vacuum drying, irradiation or non-irradiation with gamma rays, storage, and measurement of cell growth rate were carried out under the same conditions as in example 10.

Table 7 shows the number of co-precipitated or ApFGF heparin-adsorbed zirconia that was judged to be "active" in 2 replicates.

[ Table 7]

As shown in table 7, in 2 experiments, 6/6 of the gamma-irradiated groups and 6/6 of the non-irradiated groups of the co-precipitated ApFGF heparin zirconia were judged to have cell growth activity, and the number of the zirconia having cell growth activity was the same in the gamma-irradiated group and the non-irradiated group. On the other hand, 2/6 of the gamma-ray irradiated groups and 6/6 of the non-irradiated groups, which adsorbed ApFGF heparin zirconia, were judged to have cell growth activity, and the number of pins having cell growth activity in the gamma-ray irradiated groups was about 1/3 of the non-irradiated groups. Chi-square test was performed on both groups, and then significant difference was confirmed between the gamma-irradiated group and the non-irradiated group (p ═ 0.014). That is, it is found that zirconium oxide adsorbing ApFGF heparin has a strong tendency to lose the biological activity of FGF-2 by gamma irradiation sterilization.

As shown in fig. 10, the cell growth rate of the gamma-ray irradiated group normalized with the cell growth rate of the non-irradiated gamma-ray group as 100% was about 76% in the coprecipitated ApFGF heparin zirconia, and was only 29% in the ApFGF heparin zirconia adsorbed, and the values of both were statistically significantly different (p ═ 0.008). That is, when a composition in which both FGF-2 and heparin are embedded with apatite is applied to zirconia which is a ceramic for transplantation, the composition has higher resistance to ionizing radiation sterilization than when apatite in which both FGF-2 and heparin are adsorbed is applied, in other words, apatite exhibits a radioprotective effect not only when a bioactive protein is embedded but also when biologically inactive heparin is embedded.

Example 13: compositional analysis of coating layer after radiation sterilization of outer fixing pin coated with FGF-2-Encapsulated Apatite

The co-precipitated ApFGF pins prepared in example 1 and irradiated with γ rays and the adsorbed ApFGF pins prepared in example 2 were immersed in a 10mM sodium citrate solution for 30 minutes to dissolve the co-precipitated ApFGF and adsorbed ApFGF as a coating layer. The dissolved solution was chemically analyzed by ICP emission spectrometry, and calcium and phosphorus coprecipitated and adsorbed with ApFGF were quantified. The results are shown in Table 8.

[ Table 8]

As can be seen from Table 8, the coprecipitated ApFGF as a coating layer is mainly calcium phosphateAnd (3) components. The Ca/P molar ratio (1.70-1.71) of the coprecipitated ApFGF is close to that of apatite (Ca) containing no impurity element₁₀(PO₄)₆(OH)₂) The theoretical Ca/P molar ratio (1.67). It is known that when the phosphate of apatite is replaced with an impurity, the Ca/P molar ratio becomes higher than 1.67, and a carbonate is a representative impurity for replacing phosphate. Since the co-precipitated ApFGF of examples 1 and 2 was prepared in a solution containing carbonate ions, it is considered that apatite containing carbonate is co-precipitated with FGF-2 to entrap FGF-2, or apatite and calcium carbonate are co-precipitated with FGF-2 to entrap FGF-2.

Example 14: in Ca-PO₄Production of supersaturated solution of unstable calcium phosphate of-K-Na-Cl system

A solution containing 1.00mM of Ca ions, 1.00mM of phosphate ions, 2.00mM of K ions, 16.7mM of Na ions, 2.00mM of Cl ions, and 16.7mM of HCO was used₃An unstable supersaturated solution of calcium phosphate which is ionic and has a pH of 8.3 and which can crystallize calcium phosphate by spontaneous nucleation within about 4 to 5 hours even when left at 37 ℃ as it is. Except that the dipping time was 24 hours, the coprecipitated ApFGF heparin zirconia was produced under the same conditions as in example 12, and vacuum drying, γ -ray irradiation, storage and cell growth rate measurement were performed.

Table 9 shows the number of co-precipitated ApFGF heparin zirconias judged to be "active".

[ Table 9]

As shown in table 9, 3/3 of the gamma-ray-irradiated groups and 3/3 of the non-irradiated groups of the co-precipitated ApFGF heparin zirconia were judged to have cell growth activity, and the amounts of the zirconia having cell growth activity were the same in the gamma-ray-irradiated groups and the non-irradiated groups. The Ca-PO was used for identification₄When a supersaturated solution of unstable calcium phosphate of the-K-Na-Cl system, in which apatite is embedded in both FGF-2 and heparin, is applied to zirconia as a ceramic for transplantation, the radiation shielding effect is also exhibited by the embedding of apatite.

Example 15: analysis of zirconia coating layer coated with apatite embedding both FGF-2 and heparin

The composition of the coprecipitated ApFGF heparin-coated layer prepared in example 14 was analyzed in the same manner as in example 13. In addition, the coprecipitate obtained in the production of coprecipitated ApFGF heparin zirconia in example 14 was placed on a silicon non-reflecting plate and analyzed by a powder X-ray diffraction method. Powder X-ray diffraction was carried out using CuK α rays under conditions of 40kV and 100 mA. Table 10 shows the results of the composition analysis.

[ Table 10]

As can be seen from table 10, the coprecipitated ApFGF as a coating layer contains calcium phosphate. The Ca/P molar ratio of the coprecipitated ApFGF is 1.70 to 1.82 in the gamma ray non-irradiated group and 1.51 to 1.79 in the irradiated group.

From the Ca/P ratio, it is considered that, in the same manner as in example 13, FGF-2 and heparin are embedded in carbonate-containing apatite, or FGF-2 and heparin are embedded in apatite and calcium carbonate. Further, the Ca/P molar ratio of the amorphous calcium phosphate was usually 1.5, and this also indicates that the amorphous calcium phosphate was precipitated.

Fig. 11 shows the results of analysis by powder X-ray diffraction method. Amorphous calcium phosphate is known to have a broad peak with a large half-value width near 30 °. As shown in fig. 11, in the powder X-ray diffraction pattern of the coprecipitate, a broad peak having a large half-value width corresponding to amorphous calcium phosphate was observed in the vicinity of 30 °. In addition, 3 strong diffraction lines ((211), (112), and (300)) unique to crystalline apatite appearing at 31.8 °, 32.2 °, and 32.9 ° are not separated and form one broad peak, and appear in the vicinity of 32 °. Thus, the coprecipitate apatite is a low-crystalline apatite. Also, a calcium carbonate peak was observed at 29.4 °. That is, the coprecipitate contains amorphous calcium phosphate, low-crystalline apatite, and calcium carbonate as main components. The coprecipitate does not contain crystalline apatite having low solubility, and is suitable for sustained-release entrapping of proteins because it contains amorphous calcium phosphate, low-crystalline apatite, and calcium carbonate having high solubility as main components.

Industrial applicability

Since the medical device of the present invention, in which the metal or ceramic is coated with the inorganic salt solid such as apatite disposed so that the protein such as a growth factor having biological activity is embedded therein, can suppress inactivation of the biological activity of the protein by radiation sterilization, a simple terminal sterilization method using radiation can be applied to the manufacturing process of various medical devices utilizing the biological activity of the protein, and a sterile manufacturing method can be avoided, so that the cost can be greatly reduced.