Patent Description:
More particularly, the present invention relates to a method for producing a medical instrument for use in mammals including human beings, wherein the medical instrument includes a coating comprising an inorganic salt solid into which a protein having bioactivity, not to be recognized as any foreign substance by the mammals, and heparin are embedded, and the medical instrument is sterilized by ionizing radiation with bioactivity of the protein being kept, by means of ionizing radiation sterilization resistance attained by embedding.

Embedding is a term for use in pathology. The term refers to fixation of living tissues serving as subjects for pathological examinations, by implanting and solidifying of the living tissues in liquid paraffin or resin which can be solidified. In pathological examinations, tissue-embedded blocks are sliced by microtome or the like to thereby provide sections (prepared slides) for histological stain and such sections are subjected to microscopic examinations or the like. In the present description, such a concept is expanded, and implanting the entire protein molecule or a part thereof into an inorganic salt solid, or densely surrounding the entire protein molecule or a part thereof and fixing such a protein molecule into an inorganic salt solid is called "embedding". The state of "embedding" is different from the state of simple "adsorption" or "contact" of the protein molecule to or with the inorganic salt solid, or the state of "mixing" of the protein molecule and the inorganic salt solid.

Actual examples of "embedding" of a protein molecule in an inorganic salt solid may include a composition obtained by co-precipitating a protein molecule and calcium phosphate from a supersaturated calcium phosphate solution with which a protein coexist, and dispersing and disposing the protein molecule in a calcium phosphate matrix at an interval in the order of nanometers (Non-Patent Document <NUM>).

Products and raw materials, such as drug products and medical instruments, for use in the medical field where sterility is required, are sterilized by various methods in production processes. Among sterilization methods, a sterilization method utilizing characteristics of radiation which easily passes through substances corresponds to ionizing radiation sterilization. Such ionizing radiation sterilization is easily applied particularly in a terminal step of production and thus is widely used as a terminal sterilization method in a case where modification and deactivation of objects by ionizing radiation irradiation do not matter.

While the type and the dose of ionizing radiation used are varied depending on the type, the number, and the presence form of bacteria which are expected to be present in objects to be sterilized, gamma rays, which have high permeability, are often used for medical tools, and an appropriate dose thereof for sterilization is representatively <NUM> kGy and a dose around this dose is widely used. When surfaces of objects are to be sterilized, electron beams are also widely used because of their high dose rate and short irradiation time.

However, any sterilization method easily causes deactivation of substances having bioactivity. In particular, nucleic acids and proteins are large in molecular sizes and thus are easily deactivated in ionizing radiation sterilization, and, that is shown by the fact that a radiation deactivation method is established as a method of measuring the molecular weight of an active domain of protein in vivo (Non-Patent Document <NUM>). The concept "biological formulations are products which are very difficult to aseptically control in production by application of an aseptic production process because no terminal sterilization method can be applied" (Non-Patent Document <NUM>) is commonly taken for granted, and the published Draft of Guideline on the sterilization of the medicinal product in European Medicines Agency (Non-Patent Document <NUM>) also describes "For highly sensitive products such as biological products where terminal sterilization of the drug product is not possible, aseptic processing under controlled conditions provides a satisfactory quality of the drug product". "Guidance for Manufacturing of Aseptic Drug Product by Terminal Sterilization Method" (Non-Patent Document <NUM>) is disclosed. However, there is no mention about any method of protecting the activity of a drug product to be sterilized.

Ionizing radiation non-specifically hits various compounds to make radicals, regardless of the type thereof. Transition of radical electrons may occur on radical generation sites to lead to the occurrence of a radical reaction of any compound completely different from the original compound. If such a radical reaction causes unnatural changes in compounds important for DNA, membrane lipids and cells, cells will be harmed (Non-Patent Document <NUM>). Such harmful action, if exerted on bacteria and viruses, would be sterilization. Similarly, a protein having bioactivity, if reacted with any radical, would be deactivated.

Thiols like cysteine and glutathione are known as radioprotective agents for deactivating harmful radicals, and representative examples thereof include aminothiol derivatives. Cysteamine (mercaptoethylamine), WR-<NUM> (S-<NUM>-(<NUM>-Aminopropylamino) ethylphosphorothioic acid), and the like have been known for a long time (Non-Patent Document <NUM>). Additionally, <NUM>-mercaptoethylamine and alcohol (ethanol) are also described in Document (Non-Patent Document <NUM>). Moreover, (+)catechin, curcumin, vitamin C, resveratrol, caffeine acid, and quercetin are described as substances exerting radioprotective effects in screening where cytotoxicity is evalutated as an index (Non-Patent Document <NUM>). Furthermore, nitrogen-containing compounds are described as substances exerting radioprotective effects (Non-Patent Document <NUM>). Additionally, amino acid mixtures are also known to have radioprotective effect on bioactive proteins (Patent Documents <NUM> and <NUM>). Furthermore, as methods of suppressing deactivation of proteins in sterilization by ionizing radiation, a method of suppression by coexistence with a cellulose ether derivative and/or a specific group of amino acids (Patent Documents <NUM> and <NUM>), a method of suppression by coexistence with an aliphatic polyester (Patent Document <NUM>), and a method of suppression by coexistence with an exogenous protein such as gelatin (Patent Document <NUM>) are disclosed. However, all the foregoing are radioprotective agents of organic substances. While a method involving coexistence of a bioactive protein with collagen sponge and/or an absorbable polymer is disclosed (Patent Documents <NUM> and <NUM>), a method for suppressing deactivation in sterilization by ionizing radiation is not disclosed.

On the other hand, there are known radioprotective agents of inorganic substances, for example, selenium (Non-Patent Document <NUM>), vanadate (Non-Patent Document <NUM>), zinc sulfate (Non-Patent Document <NUM>), and manganese compounds (Patent Documents <NUM> and <NUM>). However, many inorganic salts, when adopted in medical instruments for mammals, are feared to have toxicity, and therefore are not subjects to be developed as radioprotective agents for medical use.

Among inorganic substances, calcium phosphate has high safety and biocompatibility, and, for example, respective crystals of apatite (Ca/P molar ratio <NUM>) and tricalcium phosphate (Ca/P molar ratio <NUM>) which are different in molar ratio of Ca ion to PO<NUM> ion, and amorphous calcium phosphate (Ca/P molar ratio <NUM> to <NUM>) are used for medical instruments and the like.

A method of measuring a dose by using fired apatite, according to electron spin resonance analysis of radical generated in irradiation of calcium phosphate with ionizing radiation (Patent Document <NUM>). However, the dose range described is about <NUM> to <NUM> Gy, and there is no mention of irradiation in a dose range suited for ionizing radiation sterilization (several kGy or more, usually often <NUM> to <NUM> kGy) which is more than <NUM> times higher than that, or radioprotective effect on other bioactive molecules coexisting.

<CIT> (Patent Document <NUM>) is known as a document which mentions the protective effect on a bioactive protein in the case of irradiation of apatite or hydroxyapatite as an inorganic salt at any irradiance suitable for sterilization. This document mentions that "any one or a combination of materials including ceramics (for example, hydroxyapatite, tricalcium phosphate, or a combination thereof with any other calcium phosphate (without limitation), can be advantageously used" as one insoluble synthetic polymer carrier material containing a bioactive osteogenic protein, and describes a terminally sterilized osteogenic device for transplantation into mammals.

However, this document does not refer which modes of "any one or a combination" of a bioactive osteogenic protein and materials including ceramics are to be adopted, at all. Examples of the type of the combination mode of the bioactive osteogenic protein and the material include "mixing", "adsorption", "contact", and "embedding" (hereinafter, collectively referred to as "type of combination mode"), and not only the document does not mention a large difference in bioactivity of the protein after terminal sterilization due to the difference in type of combination mode, but also it neither describes nor indicates which combination mode is an optimal combination mode in which terminal sterilization can be performed with bioactivity of the osteogenic protein being maintained.

<CIT> (Patent Document <NUM>) discloses a sterilizable composition which is a medical graft and which includes an inorganic salt and a bioactive protein. However, this document also neither describes nor indicates that a large difference in bioactivity of a protein after terminal sterilization is caused due to the difference in type of combination mode of an inorganic salt and a bioactive protein, and which combination mode can be adopted as an optimal combination mode in which terminal sterilization can be performed with bioactivity of the bioactive protein being maintained.

<CIT> (Patent Document <NUM>) discloses a composition as a putty for control of bone bleeding, the composition including hydroxyapatite as an inorganic salt, and a bone growth inducing substance, but mentions that terminal sterilization cannot be performed in a case where the bone growth inducing substance is a radiosensitive bioactive protein such as a demineralized bone matrix or a bone morphogenetic protein.

<CIT> (Patent Document <NUM>) discloses a bone paste composition including gelatin sterilized by heating or radiation, an osteogenic component such as a regenerative/proliferative factor, and an inorganic salt such as calcium phosphate ceramic, but neither describes nor indicates that the composition is formed and then finally sterilized by radiation irradiation and heat treatment.

<CIT> (Patent Document <NUM>) discloses a graft to be transplanted into human, the graft including ceramics as an inorganic salt, a bioactive substance as a proliferative factor, and furthermore a allogeneic, autologous or xenogeneic graft tissue, but terminal sterilization is performed by "irradiation with γ-ray or another type of ray at a dose which is well-known not to have any harmful influence on tissue characteristics", or by electron beam sterilization or ethylene oxide sterilization "as long as it causes neither any toxicity nor deterioration in desired bioactivity". In other words, it is considered that sacrifice of the sterilization effect of radiation is accepted in order to allow bioactivity to be maintained, and a harmful influence and deterioration in bioactivity are prevented by limiting the dose of irradiation. A solution here only described is "to inject a desirable bioactive substance to the graft, as a further enhancement", if necessary, before terminal sterilization.

<CIT> (Patent Document <NUM>) discloses a radiation-sterilized nanoparticle having an average size of less than <NUM> and including a core made of a biocompatible-biodegradable polymer, in which the nanoparticle contains a bioactive agent and is combined with apatite and/or bone ceramics, but gives no mention about any method of preventing deactivation of the bioactive agent by radiation sterilization and gives no mention which combination mode can be adopted as an optimal combination mode in which terminal sterilization can be performed with activity being maintained.

<CIT> (Patent Document <NUM>) describes a carrier to be sterilized after synthesis, in which bioactivity is incorporated into a carrier matrix including an inorganic, organic, or organic and inorganic substance, but neither describes nor indicates which type of combination mode of the carrier matrix including an inorganic, organic, or organic and inorganic substance allows bioactivity to be maintained also after sterilization, or which sterilization method allows bioactivity to be maintained also after sterilization.

<CIT> (Patent Document <NUM>) describes a method of obtaining a substrate coated, by contacting a substrate with an acidified composition including a brine mixture including calcium, magnesium, phosphoric acid, hydrogen carbonate ion and a bioactive substance, to result in an increase in pH to thereby allow for co-precipitation of a salt and the bioactive substance. Although there is no mention in the invention recited in claims, the detailed description describes gamma-ray irradiation which can also be performed after the last step (paragraph [<NUM>] describes "the method can also be performed with gamma-ray irradiation at the last stage following step c), in a condition of no aseptic and sterile state"). However, such gamma-ray irradiation is here merely mentioned as one example of a common sterilization procedure which can be taken, and there is not disclosed any specific experimental example for performing sterilization by such gamma-ray irradiation. Moreover, the substrate is here any of substrates made of different materials such as a metal, ceramic, and a polymer, and there is neither described nor indicated whether bioactivity is maintained after gamma-ray irradiation in respective cases of all such substrates made of these materials or bioactivity is maintained after gamma-ray irradiation in only a case of such a substance made of a specific material, or which mode is adopted to allow bioactivity to be maintained also after gamma-ray irradiation.

International Publication No. <CIT> (Patent Document <NUM>) describes an implant having a coating layer in which a peptide having a cell adhesion promotion effect is incorporated in a nanocrystalline apatite layer, and Examples therein describe no deterioration in cell adhesion promotion effect after irradiation with γ-rays at <NUM> kgrey in an implant where hydroxyapatite is electrochemically sedimented in a disc made of titanium in the presence of peptide to incorporate an apatite layer into the peptide, and, on the other hand, loss in cell adhesion promotion effect after irradiation with γ-rays in an implant where peptide merely adsorbs onto the surface of an apatite layer (Examples <NUM> and <NUM>).

However, the implant disclosed in the above Patent Document is obtained by electrochemical sedimentation of hydroxyapatite, and is not a composite produced by co-precipitation with peptide in a supersaturated calcium phosphate solution. One obtained by incorporating peptide into an apatite layer by electrochemical sedimentation and a composite obtained by co-precipitation of peptide with apatite are fully different in microscopic structure and crystallinity.

In other words, apatite formed by electrochemical sedimentation has high crystallinity to such an extent that (<NUM>) (<NUM>) (<NUM>) (<NUM>) diffraction lines are separated in a powder X-ray diffraction method (for example, Non-Patent Document <NUM>), and easily takes, as a crystal form, a hexagonal needle-like or hexagonal platelike form characteristic of an apatite crystal. Apatite has high crystallinity and thus has low solubility (Patent Document <NUM>). Apatite with low solubility is suitable for providing a composite of a peptide or protein which is not required to be gradually released and for which it is only important to be fixed on a surface. One example of such a peptide or protein which is not required to be gradually released and for which it is only important to be fixed on a surface is a peptide or protein having a cell adhesion promotion effect. In fact, this Patent Document also mentions that needle-like apatite is formed, the apatite is low in solubility and stable, and a peptide having a cell adhesion promotion effect is stably and strongly bound to a surface. In this regard, a peptide or protein having cell proliferation activity, tissue formation activity, cell differentiation promotion activity, reaction activity with an antibody, agonistic action activity, and antagonistic action activity is gradually released and thus exerts the effect on surrounding tissues, and thus incorporation thereof into apatite with high crystallinity and low solubility is not suitable. It is rather necessary to appropriately reduce the solubility of apatite or calcium phosphate to thereby allow a peptide or protein incorporated to be gradually released.

Coatings by electrochemical sedimentation originally can be applied to conductive metals, but cannot be applied to non-conductive ceramics. This Patent Document does not disclose any example where polysaccharide (for example heparin) which is derived from an extracellular matrix and which, by itself, does not have direct cell proliferation/differentiation activity is allowed to coexist with peptide and the peptide is thus incorporated into a layer of hydroxyapatite.

Immunoadjuvants of inorganic salts may be sometimes used together with antigens in vaccination aiming induction of the body's immune reaction. Immunoadjuvants of inorganic salts, widely used for a long period, are aluminum salts such as aluminum chloride, aluminum phosphate, and aluminum sulfate, in addition to aluminum hydroxide, and calcium phosphate is also used. Calcium phosphate immunoadjuvants include a vaccine adjuvant designated as a cancer-specific antigen vaccine, which is obtained by combining a cancer-specific antigen derived from an organism with β-tricalcium phosphate as an inorganic salt (Patent Documents <NUM> and <NUM>), and a calcium phosphate immunoadjuvant to be used with an antigen (Patent Documents <NUM> and <NUM>), but all the Patent Documents do not mention any radiation sterilization after combining with the antigen which is a protein. Aluminum salt immunoadjuvants have a long history of being combined with antigens such as bacteria or viruses and used as vaccines for preventing infection, and among them, and some of such vaccines are produced through terminal sterilization by radiation (Non-Patent Document <NUM>).

However, such antigens such as bacteria or viruses are biomolecules of organisms different from animals as subjects of vaccine administration, or xenogeneic organisms, and are proteins recognized as exogenous by such animals to be administrated or transplanted. Therefore, these Documents do not disclose radiation sterilization of a protein not recognized as exogenous by such animals to be administrated or transplanted. Antigens such as bacteria or viruses are originally exogenous for animals to be administrated and thus eliminated by the immune system. Thus, such antigens, even if unnaturally changed in their molecules by radiation sterilization, are still exogenous, and are accordingly considered to have a relatively small adverse influence on the intended effect (elimination by the immune system). In this regard, when an effect such as tissue regeneration is obtained by using a protein not recognized to be exogenous by animals to be administrated or transplanted, an unnatural change in molecule due to radiation sterilization has an adverse influence on functions of the protein, for example, recognized to be exogenous and no occurrence of binding with any receptor, and the intended effect is considered to be hardly obtained.

In view of such circumstances, known examples of a bioactive protein which is, in particular, not a short chain peptide having up to about <NUM> amino acid residues, but a long chain protein having even <NUM> or more amino acid residues, and corresponds to a biological formulation to which a terminal sterilization method with radiation is applied, include only chymotrypsin and papain (Non-Patent Documents <NUM> and <NUM>).

International Publication No. <CIT> (Patent Document <NUM>) discloses an implant having sustained therapeutic agent delivery and which includes a base and an hydroxyapatite seed layer disposed on a surface of the base. A co-precipitated hydroxyapatite coating is disposed on the seed layer. The coating includes a therapeutic agent, wherein the therapeutic agent is provided in a solution of therapeutic agent.

Patent Application Publication No. <CIT> (Patent Document <NUM>) discloses orthopedic bone screws/spinal pedicle screws and implants that include coatings to help promote a structurally stable interface between the implant and the patient's bone/tissue, and methods of coating such screws and implants. The implants and methodologies described involve at least a coating that facilitates osseous integration, and additionally at least one coating that either reduces the risk of infection in immunologically suppressed patients and/or which is for utilization in patients who have an infection, but who require stabilization, or coatings that permit the use of dissimilar metals while preventing galvanic corrosive reactions.

An object of the present invention is to provide a method of producing a medical instrument for use in mammals, in which the medical instrument includes a bioactive protein which retains bioactivity even after radiation-sterilized.

The present inventors have made intensive studies in order to solve the above problem, and as a result, have found that a composite where an inorganic salt and a bioactive protein are co-precipitated is formed in embedding of the bioactive protein into an inorganic salt solid, to thereby allow deactivation of the protein to be considerably suppressed even in irradiation at a dose suitable for sterilization. In other words, it has been found that the inorganic salt solid by itself has useful radioprotective effect.

For example, calcium phosphate can easily form a supersaturated solution depending on conditions. Any (physical or chemical) stimulation immediately disrupt the supersaturated state, and calcium phosphate is deposited to generate a precipitate. If a bioactive protein is here added as a solute into the supersaturated solution, calcium phosphate will be precipitated with entangling the protein, and will produce a composition where calcium phosphate such as apatite is co-precipitated and deposited with the protein being embedded. In a composition obtained by freeze-drying the precipitate, deactivation of the bioactive protein was considerably suppressed even after gamma-ray irradiation at a dose suitable for sterilization. In a composition obtained by embedding the bioactive protein into sodium chloride and vacuum-drying the resultant, deactivation of the bioactive protein due to gamma-ray irradiation at a dose suitable for sterilization was considerably suppressed similarly. These findings indicate that deactivation of the bioactive protein can be prevented by suppressing generation of radical in irradiation, and the present invention has been completed based on the findings.

The invention relates to a method for producing a medical instrument for use in mammals, including human beings, comprising a step of coating a part or the entirety of a substrate with a crystalline or an amorphous form of an inorganic salt into which a protein having bioactivity and heparin are embedded and a step of exposing the coated substrate to ionizing radiation at a dose sufficient for sterilization, wherein.

In an embodiment of the invention, the apatite is low crystalline apatite characterized by appearance of three diffraction lines (<NUM>), (<NUM>), and (<NUM>) not separated, in a powder X-ray diffraction pattern, as one peak or diffraction halo.

In an embodiment of the invention, the substrate includes a metal, and the metal is one or more metals selected from the group consisting of titanium, a titanium alloy, stainless steel, and a cobalt/chromium alloy.

In an embodiment of the invention, the substrate includes a ceramic, and the ceramic is one or more ceramics selected from the group consisting of apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, alumina, zirconia, and a composite thereof.

In an embodiment of the invention, the ionizing radiation is a gamma ray and/or an electron beam.

In an embodiment of the invention, the method at least comprises a step of exposing the coated substrate to a gamma ray, wherein the dose of the gamma ray is <NUM> to <NUM> kGy.

In an embodiment of the invention, the protein is a growth factor.

In an embodiment of the invention, the growth factor is FGF-<NUM>.

The medical instrument prepared according to the method of the present invention can avoid an aseptic production process of which process management is complicated, and one aspect thereof relates to a method for producing a medical instrument sterilized by a terminal sterilization method with radiation (a sterilization method where radiation irradiation is performed in a state where an article to be sterilized is packed in a final package, and where microbial death after such sterilization can be quantitatively measured or estimated).

The medical instrument produced by the method of the present invention, in which an inorganic salt solid into which a protein having bioactivity is embedded is placed so that a metal, a ceramic or both thereof is partially or entirely coated with the inorganic salt solid, can suppress deactivation of bioactivity of the protein due to radiation sterilization, and thus allows a simple terminal sterilization method with radiation to be applied for various production processes of medical instruments utilizing bioactivity of the protein, and thereby an aseptic production method can be avoided, resulting in a significant decrease in cost.

A method of producing a medical instrument, provided by the present invention, provides a medical instrument for use in mammals including human beings, in which an inorganic salt solid into which a protein having bioactivity is embedded is placed, so that a metal, a ceramic or both thereof is partially or entirely coated with the inorganic salt solid, in which.

The term "embedding" commonly means, as described above, to implant and solidify a living tissue serving as a subject for pathological examination in liquid paraffin or resin which can be solidified, and to thereby fix the living tissue, and the "embedding" herein means to implant the entire protein molecule or a part thereof in an inorganic salt solid, or to cover the entire protein molecule or a part thereof densely with an inorganic salt solid and to fix the protein molecule in the inorganic salt solid. Accordingly, "embedding" is different from a state of simple "adsorption" or "contact" of the protein molecule with the inorganic salt solid, or a state of "mixing" of the protein molecule therewith. An example where the protein molecule is "embedded" into the inorganic salt solid can be a composition where a protein molecule and calcium phosphate are co-precipitated from a supersaturated calcium phosphate solution in which a protein coexists and the protein molecule is arranged in a calcium phosphate matrix with being dispersed at an interval in the order of nanometers (Non-Patent Document <NUM>).

More specifically, the term "embedding" herein encompasses formation of a solid phase by simultaneous crystallization or deposition of both an inorganic salt and a protein molecule from a liquid phase and thus implantation of the entire protein molecule or a part thereof in the inorganic salt solid or covering the entire protein molecule or a part thereof with the inorganic salt solid and fixation of the protein molecule in the inorganic salt solid.

The term "embedding" also similarly encompasses formation of a solid phase by simultaneous crystallization or deposition of both an organic substance such as gelatin and a protein molecule from a liquid phase and thus implantation of the entire protein molecule or a part thereof in the solid organic substance or covering the entire protein molecule or a part thereof by the solid organic substance and fixation of the protein molecule in the solid organic substance.

The term "embedding" should be construed in its broadest sense so as to encompass the above definitions, and should not be construed in a limited way in any sense.

In contrast, the "adsorption" or "contact" means a state obtained by formation of a solid phase of an inorganic salt or an organic substance such as gelatin by crystallization or deposition in advance and thereafter fixation of a protein molecule in a liquid phase to a solid inorganic salt or organic substance. Thus, in a state of simple "adsorption" or "contact" of a protein molecule, a protein molecule fixed is usually specifically present on the surface of the inorganic salt solid or the solid organic substance.

The "mixing" means a state obtained by formation of a solid phase of both an inorganic salt and a protein molecule by crystallization or deposition in advance and thereafter approaching of both the salt and the molecule, or a state obtained by formation of a solid phase of both an organic substance such as gelatin and a protein molecule by crystallization or deposition in advance and thereafter approaching of both the substance and the molecule. Examples of the "mixing" include a state where an inorganic salt powder particle and a protein powder particle coexist in a macroscopically uniform manner, and a state where an organic substance solid powder particle and a protein powder particle coexist in a macroscopically uniform manner.

Whether or not a protein is embedded into an inorganic salt solid can be easily confirmed by, for example, immunoelectron microscopy. In other words, whether or not a protein is embedded into an inorganic salt solid can be confirmed with an electron microscope, by utilizing an antibody labelled with a substance having high electron density, such as gold colloid or ferritin, or a precursor substance thereof and staining and visualizing the protein in the inorganic salt solid for observation under electron microscope. In a case where a protein is embedded into an inorganic salt solid, it can be confirmed that a protein isolated is dispersed and present in an inorganic salt solid matrix (Non-Patent Document <NUM>).

The inorganic salt solid herein means an inorganic salt solid which has biocompatibility so as to be suitable for the medical instrument, specifically, one or more inorganic salts selected from the group consisting of apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, and calcium carbonate.

The inorganic salt is in a crystalline or an amorphous form. Whether the inorganic salt solid is crystalline or amorphous can be easily distinguished generally by a powder X-ray diffraction method, and one broad diffraction halo appears in a powder X-ray diffraction pattern when the inorganic salt solid is completely amorphous, and a plurality of diffraction peaks appear when the inorganic salt solid is crystalline.

The "low crystalline apatite" herein means apatite having low crystallinity, characterized by appearance of three diffraction lines (<NUM>), (<NUM>), and (<NUM>) not separated, in a powder X-ray diffraction pattern, as one peak or diffraction halo. Such three diffraction lines appear with being separated into three lines at positions of diffraction angles of <NUM>°, <NUM>°, and <NUM>°, in measurement with CuKα ray, in the case of apatite with high crystallinity (for example, pure crystalline hydroxyapatite).

The inorganic salt is one or more selected from the group consisting of calcium carbonate, apatite, tricalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate which may be each a solid solution with any other inorganic element and/or ion group, as impurities. Examples thereof include a solid solution of magnesium in calcium carbonate, a solid solution of carbonic acid in calcium phosphate, and a solid solution of zinc in calcium phosphate, but are not limited thereto. Examples of such any element and/or ion group in the solid solution can include magnesium, iron, zinc, potassium, hydrogen ion, hydroxide ion, carbonate ion, sulfate ion, and nitrate ion, and the element and/or ion group can be incorporated into the solid solution with the inorganic salt by added to a raw material in embedding.

The protein having bioactivity used herein may be one or more proteins selected from the group consisting of a peptide hormone, a growth factor, and an osteogenic protein. The protein having bioactivity may be a protein which is not recognized as an exogenous substance by any mammal for which the medical instrument is used and which is not biologically rejected by the mammal. For example, the protein encompasses a gene recombinant protein which is artificially prepared based on such a protein intrinsically present in mammals for which the medical instrument is used, and which has similar physiological functions, and a protein which, while modified by a physical or chemical treatment, does not lack essential bioactivity. The term "protein having bioactivity" used herein should not be construed to be in a limited way in any sense, and should be construed in its broadest sense. Examples of the bioactivity may include one or more bioactivities selected from the group consisting of cell proliferation activity, vascular proliferation activity, soft tissue formation activity, bone tissue formation activity, bone differentiation promotion activity, reaction activity with an antibody, agonistic action activity, and antagonistic action activity, but are not limited thereto.

The medical instrument herein means an instrument for use in diagnosis, therapy, and/or prophylaxis of diseases of mammals including human beings, and means, for example, an instrument which has an influence on the structure and function of the body of mammals including human beings. Mammals herein includes human and non-human mammals, and examples of non-human mammals include monkey, felid animals, canine animals, equine animals, leporid animals, and murine animals such as guinea pig, but are not limited to these particular animals. Some medical instruments are specified by government ordinances, and the medical instrument of the present invention further encompasses those other than the medical instruments specified by government ordinance, in either instance to the extent that the medical instrument is as defined in the appended claims. Examples include a bolt for absorbable intracorporeal fixation, a medical instrument for use in tissue repair, and a medical instrument for joint function repair, such as an artificial joint, but are not limited thereto. For example, a medical instrument is preferably delivered by surgical stress other than an injection, encompassing needling, and then indwelled. The term "intracorporeal" also encompasses, for example, teeth. The period of indwelling is not particularly limited, and may be, for example, not only temporary indwelling within <NUM> hours, but also short to medium-term indwelling for about <NUM> to <NUM> days or long indwelling for <NUM> days or more.

Examples of a medical instrument produced by the method of the present invention include an artificial hip joint using a metallic stem, a ceramic bone head, and a liner made of ultrahigh molecular weight polyethylene, in which a metallic stem portion which is to be contacted with the bone is coated with the inorganic salt solid into which the protein having bioactivity is embedded. Other examples thereof may include a metallic screw for bone fixation, in which only a screw head portion is coated with the inorganic salt solid into which the protein having bioactivity is embedded, a dental endosseous implant, in which only a portion of the implant, which is to be contacted with the bone and periodontal tissues, is coated with the inorganic salt solid into which the protein having bioactivity is embedded, a spinal fixation device or a spinal cage, in which only a portion of the device or cage is coated with the inorganic salt solid into which the protein having bioactivity is embedded, a ceramic artificial bone for bone prosthesis, in which the entire artificial bone is coated with the inorganic salt solid into which the protein having bioactivity is embedded, and a composite product of metal and ceramic, in which the entire artificial bone is coated with the inorganic salt solid into which the protein having bioactivity is embedded.

A method is disclosed which involves co-precipitating a physiologically active substance with an inorganic salt to thereby coat a substrate with the coprecipitate and sterilizing the resultant with gamma-ray irradiation at the final stage (Patent Document <NUM>), but this Document neither describes nor indicates the remaining of particular bioactivity of the physiologically active substance after sterilization and furthermore an optimal substrate allowing particular bioactivity of the physiologically active substance to remain after sterilization. The present inventors have found that, although bioactivity of a physiologically active substance hardly remains after sterilization in the case of coating of a substrate with a polymer material, and that particular bioactivity of protein highly remains after ionizing radiation sterilization by coating a metal or ceramic substrate with an inorganic salt solid into which the protein having bioactivity is embedded.

While the ceramic means a non-metal inorganic solid material made by an artificial heat treatment in a narrow sense, a non-metal inorganic solid material not subjected to any heat treatment available in the medical and medical instrument fields is also herein called "ceramic". The ceramic may be herein a non-metal inorganic solid material, and encompasses one obtained by any preparation method, for example, one prepared by an artificial heat treatment and one prepared without any heat treatment.

The present invention provides a method of producing a medical instrument including a structure placed so that a metal for transplantation, a ceramic for transplantation, or both thereof is partially or entirely coated with an inorganic salt solid into which not only a protein having bioactivity, but also polysaccharide, which is heparin, is embedded. Heparin is valuable because it contributes to maintaining bioactivity of a protein having bioactivity, while the polysaccharide itself is a biological polymer having no direct cell proliferation/differentiation activity.

A preferable embodiment of the present invention provides a method of producing a medical instrument including a structure placed so that a metal for transplantation, selected from the group consisting of titanium, a titanium alloy, stainless steel, and a cobalt/chromium alloy, is partially or entirely coated with an inorganic salt solid into which a protein having bioactivity is embedded. Titanium, a titanium alloy, stainless steel, and a cobalt/chromium alloy are each a metal with high biocompatibility and widely used in orthopedics and/or dentistry, and thus are each valuable as a medical instrument in the orthopedic and dentistry fields.

Another further preferable embodiment of the present invention provides a method of producing a medical instrument placed so that a ceramic for transplantation, selected from the group consisting of apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, and a composite thereof, is partially or entirely coated with an inorganic salt solid into which a protein having bioactivity is embedded. The ceramic for transplantation may include, for example, alumina and/or zirconia. These ceramics are also materials with high biocompatibility and widely used in orthopedics and/or dentistry, and thus are valuable as a medical instrument in the orthopedic and dentistry fields. These ceramics also encompass a solid solution of other inorganic element and/or ion group as impurities. Examples thereof include a solid solution of carbonic acid or silicon in apatite, a solid solution of silicon in tricalcium phosphate, a solid solution of magnesium in amorphous calcium phosphate, and a solid solution of yttrium in zirconia, but are not limited thereto. Examples of the composite may include a biphasic ceramic made of apatite and tricalcium phosphate, and a composite ceramic of alumina and zirconia, but are not limited to these particular modes.

The medical instrument comprising an inorganic salt solid into which a protein having bioactivity is embedded can be sterilized at a sufficient dose of ionizing radiation for sterilization with maintaining the activity of the bioactive protein substantially, and thus can be a medical instrument terminally sterilized.

The ionizing radiation for use in sterilization is preferably a gamma ray and/or an electron beam. By utilizing the gamma ray which easily penetrates through a substance, the inorganic salt solid into which the protein having bioactivity is embedded can be easily sterilized. For example, a portion of the protein embedded into the inorganic salt solid, the portion being exposed on the surface of the solid, can also be sterilized by electron beam irradiation according to a method well-known by those skilled in the art. By packaging the entire medical instrument appropriately and thus sealed at first, and thereafter irradiating gamma ray or electron beam by a method well-known by those skilled in the art, a product in which the medical instrument sterilized is sealed and included can be obtained, and the product can be provided as a medical instrument aseptically sealed and packaged, to the medical workplace (Non-Patent Document <NUM>). Aspects of the present invention are not limited to these particular aspects.

The dose necessary for sterilization may be typically minimum dose necessary for ensuring a sterility assurance level (SAL) of <NUM>-<NUM>. The sterility assurance level is prescribed by the standard such as ISO (ISO <NUM>-<NUM>, <NUM>-<NUM>) and JIS (JIS T <NUM>-<NUM>, <NUM>-<NUM>), and is adopted by the regulatory agency of each country, for example, FDA in U. A and PMDA (Pharmaceuticals and Medical Devices Agency) in Japan.

In a case where a gamma ray is used for sterilization, for example, a dose of about <NUM> to <NUM> kGy, preferably <NUM> to <NUM> kGy may be selected as the dose for ensuring a sterility assurance level of <NUM>-<NUM> and sterilizing the medical instrument in the method of the present invention. The dose of radiation for achieving the sterility assurance level may be preferably about <NUM> kGy. It is noted that the dose is not limited to such a particular dose of radiation.

In another further preferable embodiment of the present invention, sterilization may be performed in a degassing state at an atmospheric pressure of <NUM> kPa or less, preferably less than <NUM> Pa, further preferably <NUM> Pa or less in order to suppress the generation of radical in the sterilization step with a gamma ray and/or an electron beam. Alternatively, sterilization is also preferably performed in a state where air is replaced with a nitrogen or inert gas. Furthermore, sterilization may also be preferably performed at a low temperature of <NUM> to -<NUM>, preferably - <NUM> to -<NUM>, further preferably -<NUM> to -<NUM> under the coexistence of dry ice. Alternatively, sterilization may also be preferably performed after addition of ascorbic acid or ascorbate to the inorganic salt solid into which the protein is embedded. Uniform dispersion and addition of ascorbic acid or ascorbate can be achieved by immersion in a solution of <NUM> to <NUM>, preferably <NUM> to <NUM> of ascorbic acid or ascorbate and then drying, but is not limited thereto.

One preferable embodiment of the present invention, from another viewpoint, provides a method of producing a medical instrument for use in mammals, comprising an inorganic salt solid into which a peptide hormone selected from the group consisting of a hypothalamus-derived peptide hormone, vasopressin, oxytocin, intermedin, a gonadotrophic hormone, a growth hormone, a parathyroid hormone, inhibin, activin, relaxin, insulin, glucagon, somatostatin, cholecystokinin, secretin, motilin, atrial natriuretic peptide, erythropoietin, leptin, endothelin, ghrelin, adiponectin, an insulin-like growth factor, and calcitonin gene-related peptide is embedded.

Another preferable embodiment provides a method of producing a medical instrument for use in mammals, comprising an inorganic salt solid into which an FGF-<NUM> (fibroblast growth factor-<NUM>) is embedded as a growth factor. FGF-<NUM> is a growth factor useful for soft tissue regeneration, blood vessel formation, and bone formation, and thus such a medical instrument is useful for an application where tissue regeneration is promoted.

Still another preferable embodiment provides a method of producing a medical instrument for use in mammals, comprising an inorganic salt solid into which one or more osteogenic proteins selected from the group consisting of OP-<NUM>, OP-<NUM>, BMP-<NUM>, BMP-<NUM>, BMP-<NUM>, BMP-<NUM>, BMP-<NUM>, BMP-<NUM>, BMP-<NUM>, DPP, Vg1, Vgr-<NUM>, and functional equivalents thereof is/are embedded as osteogenic protein(s). Such a medical instrument is useful for an application where tissue regeneration is promoted.

One preferable embodiment of the present invention, from still another viewpoint, provides a method of producing a medical instrument in which bioactivity of a protein having bioactivity is bioactivity selected from the group consisting of cell proliferation activity, vascular proliferation activity, soft tissue formation activity, bone tissue formation activity, bone differentiation promotion activity, reaction activity with an antibody, and agonistic action activity. The bioactivity in the protein or the medical instrument can be evaluated by the number of cells in cell culturing, a differentiation marker, a substance produced, gene expression, a cell form, and/or the collective form of cells, for example, formation of a vascular-like structure. In animal testing, the bioactivity can be evaluated by, for example, observation of a tissue form with a tissue specimen, a tissue image by X-ray, MRI, or the like, and/or gene expression. In a case where the protein having bioactivity behaves as an antigen, an agonist, or an antagonist, the bioactivity can be evaluated by any of various methods such as western blotting with a monoclonal antibody involving a bioactive moiety of the protein or a monoclonal antibody labelled. In a case where the protein having bioactivity is an antibody, the bioactivity can be evaluated by labelling the antibody in advance and then binding it to an antigen, and evaluating the amount and binding activity of the antibody bound to the antigen, by utilizing the activity and amount of such a substance labelled. In a case where the protein having bioactivity is an enzyme, the bioactivity can also be evaluated by using an enzymatic substrate and evaluating a reaction product derived from the enzymatic substrate by an enzyme. It is noted that the evaluation method is not limited thereto as long as it can evaluate the particular bioactivity to be evaluated. Any evaluation method can verify whether or not the protein and the medical instrument have bioactivity, by, for example, comparison of the particular bioactivity between a group of the inorganic salt solid including the protein and a group of the inorganic salt solid including no protein. As samples for bioactivity comparison between both the groups, the inorganic salt solid, the medical instrument or the graft containing or not containing the protein can be used as it is or as in the form of an extraction liquid obtained by dissolving the inorganic salt solid with an appropriate extraction liquid and thus extracting the protein.

A procedure for quantitative evaluation, utilizing the above bioactivity and the evaluation method thereof, can be used in order to evaluate the bioactivity of the medical instrument sterilized by ionizing radiation at a dose sufficient for sterilization. In some embodiments, the medical instrument prepared according to the method of the present invention has at least about <NUM>% or more, preferably <NUM>% or more, further preferably <NUM>% or more of bioactivity relative to that before sterilization, after sterilization for ensuring a sterility assurance level of <NUM>-<NUM>.

Embedding the protein having bioactivity into the inorganic salt solid includes a controlled delay co-precipitation deposition method in a neutral or weak alkaline unstable supersaturated calcium phosphate solution which generates spontaneous nucleation , a coverage sandwich method, or a drying method.

The co-precipitation deposition method is a method wherein a desired bioactive protein is allowed to coexist in a desired supersaturated inorganic salt solution and a crystal or amorphous form of the inorganic salt is deposited from the supersaturated solution with capturing the protein or densely surrounding the protein molecule by the inorganic salt solid. The co-precipitation deposition is also a method where a crystalline or amorphous form of the inorganic salt into which the protein is embedded is deposited so that another solid surface is coated therewith.

The coverage sandwich method is a method involving allowing a desired bioactive protein to adsorb to or contact with a surface of a desired inorganic salt solid in advance, and covering the surface with the same one as or different one from the inorganic salt solid into which the bioactive protein is embedded. Other methods may include a drying method in which, for example, the protein can be densely embedded into the inorganic salt solid by dissolving a desired bioactive protein in a desired inorganic salt solution and freeze-drying or concentrating and drying the solution. Such a method can be adopted singly or in appropriate combination of two or more kinds thereof, or may be, if necessary, repeated for an appropriate number of times. By performing these methods, a plurality of layers each including a composition of the protein embedded into the inorganic salt solid can be placed, preferably in a state where a desired bioactive protein can be protected from radiation sterilization. The inorganic salt solid thus produced, into which the bioactive protein is embedded, is obtained as a precipitate from the solution or in the state of being suspended in the solution, and can also be obtained as a layer coating a metal for transplantation or a ceramic for transplantation, as described above, and can be appropriately separated from the solution and then dried.

As a preferable method of embedding the protein having bioactivity into the inorganic salt solid a co-precipitation deposition method involving controlled delay co-precipitation of the protein having bioactivity and calcium phosphate in an unstable supersaturated calcium phosphate solution which uses a neutral or weak alkaline solution and which generates spontaneous nucleation, or a coverage sandwich method. As a method of co-precipitating a protein and calcium phosphate at a desired time point by suppressing spontaneous nucleation, crystallization, or precipitation of calcium phosphate in a protein-containing high-concentration calcium phosphate solution and thus stabilizing the solution at a high concentration as it is, a method involving decreasing the pH by bubbling of carbon dioxide and/or addition of acid to thereby decrease the degree of supersaturation to result in complete dissolution and then gradually increasing the pH by degassing of carbon dioxide, addition of alkali, or OH- ion generation by electrochemical reduction of a water molecule to thereby increase the degree of supersaturation, and gradually crystallizing calcium phosphate to thereby allow co-precipitation with the protein (Patent Document <NUM>, Patent Document <NUM>, Patent Document <NUM>, Patent Document <NUM>, and Patent Document <NUM>). The method is considered to allow co-precipitation to occur, by increasing the pH of a stable supersaturated calcium phosphate solution which is acidic and completely dissolved and which generates no spontaneous nucleation, to thereby increase the degree of supersaturation.

However, many proteins have the problem of being denatured and thus lose their activity at an acidic pH or a high-alkaline pH. In order to avoid this problem, a solution where a large amount of K ion or Na ion is added to a neutral or weak alkaline high-concentration calcium phosphate solution which may rapidly form a large amount of a precipitate is prepared as an unstable supersaturated calcium phosphate solution which generates spontaneous nucleation, the time until crystallization is delayed by an increase in activation energy of crystallization and thus a decrease in frequency of nucleation, and calcium phosphate is gradually crystallized to enable co-precipitation with a protein to occur. This method is a method of performing controlled delay co-precipitation of a protein having bioactivity and calcium phosphate by not adjusting the degree of supersaturation of the solution, but by changing the activation energy of crystallization, and, more specifically, delay co-precipitation can be performed by adjusting the KCl concentration, which does not relate to the concentration of calcium phosphate, pH, and the degree of supersaturation, and controlling and extending the time until spontaneous nucleation.

For example, the above delay co-precipitation is preferably performed by using an aqueous solution including <NUM> to <NUM> of Ca ion, <NUM> to <NUM> of phosphate ion, <NUM> to <NUM> of K ion, <NUM> to <NUM> of Na ion, and <NUM> to <NUM> of Cl ion, and having a pH of <NUM> to <NUM>, preferably an aqueous solution including <NUM> to <NUM> of Ca ion, <NUM> to <NUM> of phosphate ion, <NUM> to <NUM> of K ion, <NUM> to <NUM> of Na ion, <NUM> to <NUM> of Mg ion, <NUM> to <NUM> of Cl ion, and <NUM> to <NUM> of HCO<NUM> ion, and having a pH of <NUM> to <NUM>, as the unstable supersaturated calcium phosphate solution, and controlling the KCl concentration in the aqueous solution to thereby artificially control and delay the time until calcium phosphate deposition. Since Mg ion and HCO<NUM> ion are inhibitors of calcium phosphate crystallization, an unstable supersaturated calcium phosphate solution to which not only K ion and Na ion, but also Mg ion and HCO<NUM> ion are added can allow the time until calcium phosphate deposition to be further controlled and delayed artificially (Patent Document <NUM>).

Hereinafter, the present invention will be more specifically described with reference to Examples, but the scope of the present invention is not limited to the following Examples. The terms and concepts in the Examples are based on the terms conventionally used in the art, and any technique for carrying out the present invention, except for any technique whose reference is specifically and clearly shown, can be easily and certainly carried out by those skilled in the art based on a known document or the like. Various analyses and the like are performed using methods described in instructions, catalogs, and the like of analysis instruments, reagents, or kits used.

A titanium pin for intracorporeal fixation, which may be used for fracture fixation, was coated with an inorganic salt solid into which FGF-<NUM> having cell proliferation activity was embedded, the resultant was entirely subjected to ionizing radiation sterilization, and thereafter whether or not FGF-<NUM> had cell proliferation activity was examined.

An unstable supersaturated calcium phosphate solution including <NUM> of Ca ion, <NUM> of phosphate ion, <NUM> of K ion, <NUM> of Na ion, <NUM> of Mg ion, <NUM> of Cl ion, and <NUM> of HCO<NUM> ion, and having a pH of <NUM>, in which calcium phosphate would be crystallized by spontaneous nucleation in about <NUM> to <NUM> hours if the solution was left at <NUM> as it was, was used (this unstable supersaturated calcium phosphate solution was different from the liquid of Patent Document <NUM>). Fibroblast growth factor-<NUM> (FGF-<NUM>) was added at each of concentrations of <NUM>µg/ml and <NUM>µg/ml to the unstable supersaturated calcium phosphate solution. A titanium pin for intracorporeal fixation (DePuy Synthes, cell drill <NUM>/<NUM> Ti, <NUM>-<NUM>) was immersed therein at <NUM> for <NUM> hours, and coated with FGF-<NUM> by co-precipitated with apatite, to thereby produce each of <NUM> or <NUM> co-precipitated apatite FGF-<NUM> (co-precipitated ApFGF) pins. Similarly, FGF-<NUM>-free unstable supersaturated calcium phosphate solution was used to produce each of <NUM> or <NUM> Ap pins. The co-precipitated ApFGF pins produced were each loaded into a tube and vacuum-dried at <NUM> Pa at room temperature for <NUM> hours. The lid of the tube receiving each of the pins was closed immediately after the drying, and each of the pins was packaged by using an anaerobic-dry storage system (I. ) composed of a gas-shielding storage bag, an oxygen absorbing agent, and a synthetic zeolite drying agent.

Half these ApFGF pins were subjected to γ-ray irradiation at a dose of <NUM> ± <NUM> kGy with <NUM>Co as a radiation source. The irradiation was performed at ordinary temperature, and the pins were stored at <NUM> also over a transport period. The co-precipitated ApFGF pins not subjected to γ-ray irradiation were stored at <NUM>. In order to evaluate the cell proliferation activity of FGF-<NUM> supported on each of the co-precipitated ApFGF pins including the pins subjected to γ-ray irradiation and the pins not subjected to γ-ray irradiation, these pins were each immersed and dissolved in a <NUM> sodium citrate solution for <NUM> minutes. An FGF-<NUM>-free Ap pin as a control was also immersed in a <NUM> sodium citrate solution for <NUM> minutes to thereby dissolve a coating layer. Since calcium in the dissolution liquid could promote cell proliferation, the samples were uniformed in terms of the calcium concentration after ICP emission spectrometric analysis, and each added to mouse fibroblast strains NIH3T3, and the proliferation rate was measured using Cell Counting Kit-<NUM>. The proliferation rate of the FGF-<NUM>-free Ap pin as a control was defined as <NUM>, and any co-precipitated ApFGF pin showing statistically significantly high proliferation rate was rated as "having activity". An operation including coating by co-precipitation, vacuum-drying, γ-ray irradiation or no γ-ray irradiation, and measurement of the proliferation rate was repeatedly trialed four times.

Table <NUM> shows the number of co-precipitated ApFGF pins rated as "having activity" in the four repeated trials. <FIG> illustrates the values of the proliferation rates measured.

As shown in Table <NUM>, <NUM> pins/<NUM> pins in the γ-ray irradiation group of the co-precipitated ApFGF pins, and <NUM> pins/<NUM> pins in the no irradiation group thereof were rated as "having cell proliferation activity", and the number of such pins each having cell proliferation activity was the same between the γ-ray irradiation group and the no irradiation group, in the four repeated trials. As illustrated in <FIG>, the co-precipitated ApFGF pins not only in the γ-ray irradiation group, but also in the no irradiation group each exhibited a proliferation rate about <NUM> times higher than that of the Ap pin, the proliferation rate in the no γ-ray irradiation group was statistically significantly higher than the proliferation rate (proliferation rate = <NUM>) of the Ap pin (p = <NUM>), and the difference between the proliferation rate in the γ-ray irradiation group and the proliferation rate (proliferation rate <NUM>) of the Ap pin was extremely close to a significant level (p = <NUM>). No significant difference (p = <NUM>) was confirmed between the proliferation rate in the irradiation group and that in the no irradiation group. In other words, it was revealed that, in a case where the intracorporeal fixation pin made of titanium as a metal for transplantation was coated with the composition where FGF-<NUM> was embedded into apatite, the protein attained ionizing radiation sterilization resistance by embedding, namely, embedding into apatite exhibited radioprotective effect on the bioactive protein. Herein, because embedding into apatite exhibited radioprotective effect on the bioactive protein in a condition of gamma-ray irradiation at <NUM> ± <NUM> kGy, such radioprotective effect would be exhibited also against gamma-ray irradiation at less than <NUM> ± <NUM> kGy.

Titanium pins for intracorporeal fixation, which may be used for fracture fixation, were coated with an inorganic salt solid to which FGF-<NUM> having cell proliferation activity was adsorbed, the resultants were entirely subjected to ionizing radiation sterilization, and thereafter whether or not FGF-<NUM> had cell proliferation activity was examined.

An FGF-<NUM>-free supersaturated calcium phosphate solution was prepared using the same unstable supersaturated calcium phosphate solution as in Example <NUM>, and <NUM> or <NUM> titanium pins for intracorporeal fixation (DePuy Synthes, cell drill <NUM>/<NUM> Ti, <NUM>-<NUM>) were immersed therein at <NUM> for <NUM> hours, to thereby produce respective Ap pins whose surfaces were coated with apatite. The Ap pins were immersed in a supersaturated calcium phosphate solution containing <NUM>µg/ml of FGF-<NUM> for several seconds, and frozen at -<NUM>, to thereby produce adsorbed apatite FGF-<NUM> (adsorbed ApFGF) pins each coated with apatite to which FGF-<NUM> was adsorbed. The pins were vacuum-dried at room temperature, subjected or not subjected to γ-ray irradiation, stored, and evaluated with respect to cell proliferation activity in the completely same conditions as in Example <NUM>. An operation including coating with apatite to which FGF-<NUM> was adsorbed, γ-ray irradiation or no γ-ray irradiation, and measurement of the proliferation activity was repeatedly trialed five times.

Table <NUM> shows the number of adsorbed ApFGF pins rated as "having activity" in the five repeated trials. <FIG> illustrates the values of the proliferation rates measured.

As shown in Table <NUM>, <NUM> pins/<NUM> pins in the γ-ray irradiation group of the adsorbed ApFGF pins, and <NUM> pins/<NUM> pins in the no irradiation group thereof were rated as "having cell proliferation activity", and the number of pins having cell proliferation activity in the γ-ray irradiation group was about one-fifth of the no irradiation group, in the five trials. Both the groups were subjected to the chi-square test, and a significant difference (p = <NUM>) was recognized between the γ-ray irradiation group and the no irradiation group and thus it was revealed that the adsorbed ApFGF pin lost bioactivity of FGF-<NUM> by γ-ray irradiation sterilization. As illustrated in <FIG>, while the adsorbed ApFGF pin in the no irradiation group exhibited a proliferation rate about <NUM> times (p = <NUM>) higher than that exhibited by the Ap pin, the proliferation rate exhibited by the adsorbed ApFGF pin in the γ-ray irradiation group was decreased to <NUM> times higher than that exhibited by the Ap pin and no significant difference from that exhibited by the Ap pin was recognized (p = <NUM>), and it was revealed that the cell proliferation rate of FGF-<NUM> significantly (p = <NUM>) disappeared by about <NUM>% by γ-ray irradiation. In contrast, as illustrated in <FIG> described above, the co-precipitated ApFGF pins in both the γ-ray irradiation group and the no irradiation group each exhibited a proliferation rate about <NUM> times higher than that exhibited by the Ap pin, and no significant difference (p = <NUM>) was recognized between the irradiation group and the no irradiation group. In other words, while Patent Documents <NUM>, <NUM> and <NUM> describe radiation sterilization of a composition of a combination of a protein having bioactivity and an inorganic salt, modes of such a combination include "mixing", "adsorption", "contact", and "embedding", and it has been found that the difference in combination mode leads to a large difference in protein bioactivity after terminal sterilization and a mode "embedding" allows such protein bioactivity to be maintained at a high efficiency.

The titanium pins for intracorporeal fixation, as in Example <NUM>, were immersed in a <NUM>% gelatin solution containing <NUM>µg/ml of FGF-<NUM> for several seconds, and frozen at -<NUM>, to thereby produce pins (gelatin FGF) coated with gelatin into which FGF-<NUM> was embedded. The pins were vacuum-dried at room temperature, subjected to or not subjected to γ-ray irradiation, stored, and evaluated with respect to cell proliferation activity, in the completely same conditions as in Example <NUM>. An operation including coating with gelatin into which FGF-<NUM> was embedded, γ-ray irradiation or no γ-ray irradiation, and measurement of the proliferation rate was repeatedly trialed four times.

Table <NUM> shows the number of gelatin FGF pins rated as "having activity" in the four repeated trials. <FIG> illustrates the values of the proliferation rates measured.

As shown in Table <NUM>, <NUM> pins/<NUM> pins in the γ-ray irradiation group of the gelatin FGF pins each coated with gelatin into which FGF-<NUM> was embedded, and <NUM> pins/<NUM> pins in the no irradiation group thereof were rated as "having cell proliferation activity", and the number of such pins each having cell proliferation activity was the same between the γ-ray irradiation group and the no irradiation group, in the four repeated trials. However, as illustrated in <FIG>, while the gelatin FGF pin in the no irradiation group exhibited a proliferation rate about <NUM> times higher than that exhibited by the Ap pin, the proliferation rate exhibited by the gelatin FGF pin in the γ-ray irradiation group was decreased to about <NUM> times higher than that exhibited by the Ap pin, and it was revealed that the cell proliferation rate of FGF-<NUM> statistically significantly (p = <NUM>) disappeared by about <NUM>% by γ-ray irradiation. In other words, it was found that, in a case where a matrix into which the protein having bioactivity was embedded was an organic substance such as gelatin, radioprotective effect was low unlike cases of Example <NUM> and <FIG> where a matrix for embedding was the inorganic salt solid. Patent Document <NUM> describes a carrier sterilized after synthesis, in which bioactivity is incorporated into a carrier matrix including an inorganic, organic, or organic and inorganic substance, but does not indicate which combination or material of the carrier matrix including an inorganic, organic, or organic and inorganic substance allows bioactivity to be maintained even after sterilization, or which sterilization method allows bioactivity to be maintained even after sterilization. It is revealed in Examples of the present disclosure that a case where the protein having bioactivity is embedded into a matrix of the inorganic salt solid exhibits much excellent radioprotective effect as compared with a case where the protein is embedded into an organic matrix. The reason is considered that the organic substance may generate many radicals by radiation irradiation unlike the inorganic salt solid.

A hydroxyapatite powder containing <NUM>% of polyvinyl alcohol and having a size of <NUM> microns or less was press-molded and sintered at <NUM> for <NUM> hour, to thereby produce dense discs made of apatite (ellipse of <NUM> diameter × <NUM> width × <NUM> thickness). The production method was essentially the same as the method of producing the apatite ceramic for artificial bone. The discs made of apatite were coated with co-precipitated ApFGF and the resultants were vacuum-dried and subjected to γ-ray irradiation according to the same method as in Example <NUM>, and thereafter NIH3T3 cells were cultured on the disc and the cell proliferation rate was measured. In a case where NIH3T3 cells were cultured directly on the disc, the adhesiveness of the cells changes by the influence of the protein, therefore a disc made of apatite in a no irradiation group, as a control, was immersed in an unstable supersaturated calcium phosphate solution to which bovine serum albumin (BSA) was added instead of FGF-<NUM>, and coated with ApBSA. An operation including coating, γ-ray irradiation or no γ-ray irradiation, and measurement of the proliferation rate was repeatedly trialed three times.

Table <NUM> shows the number of discs made of co-precipitated ApFGF apatite, rated as "having activity" in the three repeated trials. <FIG> illustrates the values of the proliferation rates measured.

As shown in Table <NUM>, <NUM> discs/<NUM> discs in the γ-ray irradiation group of the discs made of co-precipitated ApFGF apatite, and <NUM> discs/<NUM> discs in the no irradiation group thereof were rated as "having cell proliferation activity", and the number of such discs made of apatite, each having cell proliferation activity, was the same between the γ-ray irradiation group and the no irradiation group, in the three trials. As illustrated in <FIG>, the discs made of co-precipitated ApFGF apatite in both the γ-ray irradiation group and the no irradiation group each exhibited a proliferation rate about <NUM> times higher than that exhibited by the Ap pin, and no significant difference (p = <NUM>) was recognized between the irradiation group and the no irradiation group. In other words, it was revealed that, in a case where a ceramic for transplantation was coated with the composition where FGF-<NUM> was embedded into apatite, the protein attained ionizing radiation sterilization resistance by embedding, namely, embedding into apatite exhibits radioprotective effect on the bioactive protein, like a case where a metal for transplantation was coated.

Round bars made of polyether ether ketone (PEEK) (<NUM> diameter × <NUM> length) as a polymer were used, and the surfaces thereof were coated with co-precipitated ApFGF and the resultants were vacuum-dried and subjected to γ-ray irradiation or no irradiation, stored, and measured with respect to the cell proliferation rate, in the same conditions as in Example <NUM>. Polyether ether ketone is a polymer which may be used in transplantation.

Table <NUM> shows the number of round bars made of co-precipitated ApFGF-PEEK, rated as "having activity", in the three repeated trials.

As shown in Table <NUM>, <NUM> bars/<NUM> bars in the γ-ray irradiation group of the co-precipitated ApFGF-PEEK round bars, and <NUM> bars/<NUM> bars in the no irradiation group thereof were rated as "having cell proliferation activity", and the number of pins having cell proliferation activity in the γ-ray irradiation group was two-thirds of the no irradiation group, in the three trials. It was revealed that bioactivity of FGF-<NUM> in the co-precipitated ApFGF-PEEK round bar disappeared by γ-ray irradiation sterilization. Patent Document <NUM> describes a method of obtaining a substrate coated, by contacting a substrate with an acidified composition including a brine mixture including calcium, magnesium, phosphoric acid, hydrogen carbonate ion and a bioactive substance, to result in an increase in pH and thereby generate co-precipitation of a salt and the bioactive substance. Although there is no mention in the invention recited in the claims of Patent Document <NUM>, the specification thereof describes that gamma-ray irradiation can also be performed after the last step. However, it is revealed in Examples of the present disclosure that, in a case where the protein having bioactivity is co-precipitated on a polymer for transplantation to thereby coat the polymer therewith, no sufficient radioprotective effect can be achieved and a case of co-precipitation on and coating of a metal or ceramic for transplantation exhibits much excellent radioprotective effect. The reason is considered that the polymer may generate many radicals by radiation irradiation unlike the metal or ceramic.

Titanium pins for external fixation, as a metal for transplantation, were coated with apatite into which FGF-<NUM> was embedded, in the same conditions as in Example <NUM> and the resultant was subjected to γ-ray irradiation at a dose of <NUM> ± <NUM> kGy, and how the atmosphere in the γ-ray irradiation influences on FGF-<NUM> having reaction activity with an anti-FGF-<NUM> antibody was examined.

Co-precipitated ApFGF pins were produced using titanium pins for intracorporeal fixation in the same manner as in Example <NUM>, and sealed and packaged. Here, three atmosphere conditions of (i) the same anaerobic-dry packaging as in Example <NUM>, (ii) degassing packaging, and (iii) nitrogen packing packaging were applied. The degassing was performed as a vacuum degassing treatment at <NUM> kPa for <NUM> seconds. Nitrogen replacement was performed by allowing a high-purity nitrogen gas to flow in. Thereafter, γ-ray irradiation was performed at a dose of <NUM> ± <NUM> kGy. A co-precipitated ApFGF pin not subjected to γ-ray irradiation (no irradiation group) in the same anaerobic-dry packaging as in Example <NUM> was adopted as a control. The co-precipitated ApFGF pin not subjected to γ-ray irradiation was immersed in a <NUM> sodium citrate solution for <NUM> minutes to thereby dissolve a coating layer, and FGF-<NUM> supported on the pin was detected by western blotting using an anti-FGF-<NUM> antibody. A dissolution liquid was concentrated to <NUM>-fold by freeze-drying and then subjected to western blotting. The antibody here used was a human FGF-<NUM> mouse monoclonal antibody (Thermo Fisher Scientific) involving in bioactivity of FGF-<NUM>. Image data acquired was used for quantitative determinination and comparison of a signal intensity detected at a position of <NUM> kDa (molecular weight of FGF-<NUM>: <NUM>,<NUM>) with Imge Lab (Bio-Rad Laboratories, Inc.

A band signal was clearly detected at a position of <NUM> kDa in the no irradiation group. When the signal intensity in the no irradiation group as a control was <NUM>%, the signal intensities in the irradiation group were (i) <NUM>% in irradiation in the same anaerobic-dry packaging as in Example <NUM>, (ii) <NUM>% in irradiation in degassing packaging, and (iii) <NUM>% in irradiation in nitrogen packing packaging (<FIG>). Accordingly, it was revealed that γ-ray irradiation in a degassing state or in a nitrogen atmosphere suppressed a decrease of FGF-<NUM> having reaction activity with an anti-FGF-<NUM> antibody and in particular a degassing state showed high protective effect on FGF-<NUM> in γ-ray irradiation as compared with that in a nitrogen atmosphere. Even in a case where FGF-<NUM> having reaction activity with an anti-FGF-<NUM> antibody was decreased to <NUM>% in irradiation in anaerobic-dry packaging, the cell proliferation rate of FGF-<NUM> was comparable with that in the no irradiation group, as described in Example <NUM>.

Titanium pins for external fixation, as a metal for transplantation, were coated with apatite into which FGF-<NUM> was embedded, in the same conditions as in Example <NUM> and the resultants were subjected to γ-ray irradiation at a dose of <NUM> ± <NUM> kGy at room temperature and at a low temperature, and how the temperature in the γ-ray irradiation influences on reaction activity with an anti-FGF-<NUM> antibody of FGF-<NUM> was examined.

Co-precipitated ApFGF pins were produced using titanium pins for intracorporeal fixation in the same manner as in Example <NUM>, and sealed and packaged by the same degassing packaging as in Example <NUM>. The pin to be subjected to γ-ray irradiation at a low temperature was subjected to γ-ray irradiation in the coexistence with about <NUM> of dry ice, and the pin to be subjected to γ-ray irradiation at room temperature was subjected to γ-ray irradiation without dry ice. The sublimation temperature of dry ice is -<NUM> at atmospheric air pressure. Accordingly, the temperature of dry ice by itself was a temperature of -<NUM> or less. The dose of irradiation was the same as in Example <NUM>. Thereafter, FGF-<NUM> supported on the pin after the γ-ray irradiation was detected by western blotting according to the same method as in Example <NUM>.

<FIG> illustrates the influence of the temperature in radiation sterilization on FGF-<NUM> of <NUM> kDa, having reaction activity with an anti-FGF-<NUM> antibody embedded into apatite. Bands were detected at a position of <NUM> kDa in both groups. It was further revealed that irradiation at a low temperature of around -<NUM> by dry ice led to a signal intensity increased twofold as compared with that in irradiation at room temperature (<FIG>). It was revealed therefrom that γ-ray irradiation at a low temperature in the coexistence of dry ice in addition to degassing packaging suppressed a decrease of FGF-<NUM> having reaction activity with an anti-FGF-<NUM> antibody, and exhibited high protective effect on FGF-<NUM> as compared with γ-ray irradiation at room temperature.

FGF-<NUM> in Example <NUM> was changed to rhBMP-<NUM>, a dense disc made of apatite was coated with co-precipitated ApBMP and the resultant was subjected to γ-ray irradiation at a dose of <NUM>±<NUM> kGy, and the presence of protective effect of BMP-<NUM> against the γ-ray irradiation was examined.

A dense disc made of apatite, produced in the same manner as in Example <NUM>, was used, FGF-<NUM> of Example <NUM> was changed to human recombinant BMP-<NUM> (rhBMP-<NUM>) as an osteogenic protein, and coating with co-precipitated ApBMP was performed. In a control, coating with co-precipitated ApBSA was performed using bovine serum albumin (BSA) instead of rhBMP-<NUM>. After the respective apatite discs coated with co-precipitated ApBMP and co-precipitated ApBSA were vacuum-dried and subjected to γ-ray irradiation, rat bone marrow-derived mesenchymal stem cells were seeded on each of the discs, and a bone differentiation marker was measured after <NUM> days from bone differentiation induction. The mesenchymal stem cells here used were primary mesenchymal stem cells isolated from the bone marrow of a <NUM>-week-old F344/NSlc rat, and the mesenchymal stem cells seeded on each of the discs were cultured in a bone differentiation induction medium to which <NUM> mMβ glycerophosphate and <NUM> ascorbic acid were added, for <NUM> days soon after seeding, in a condition of no addition or addition of <NUM> dexamethasone (Dex). A half amount of the medium was exchanged every two days. After cultured for <NUM> days, the cells were frozen and lysed in <NUM>% Triron-X-containing PBS, and alkaline phosphatase (ALP) activity as a bone differentiation marker was quantitatively determined using LabAssay ALP (FUJIFILM Wako Pure Chemical Corporation). The amount of DNA in a cell lysate was quantitatively determined using Quant-iT (registered trademark) PicoGreen (registered trademark) dsDNA Reagent and Kits (Thermo Fisher Scientific) in order to evaluate activity per the number of cells.

<FIG> illustrates the ALP activity per the amount of DNA after differentiation induction in the absence or presence of Dex on a disc made of apatite coated with apatite into which BMP-<NUM> was embedded and subjected to γ-ray irradiation. As illustrated in <FIG>, the ALP activity per the amount of DNA after differentiation induction in the absence of Dex on a disc made of co-precipitated ApBMP apatite was significantly higher than that of ApBSA in the no γ-ray irradiation group (p = <NUM>), and comparable with that of ApBSA in the irradiation group (p = <NUM>). On the other hand, the ALP activity per the amount of DNA after differentiation induction in in the presence of Dex was significantly higher than those of ApBSA in both the γ-ray irradiation group and the no irradiation group (p = <NUM>, <NUM>). In other words, it was shown that embedding BMP-<NUM> as an osteogenic protein in apatite allows bone differentiation promotion action in the presence of Dex, as one bioactivity of BMP-<NUM>, to be maintained even after γ-ray sterilization. In other words, it was revealed that, in a case where a ceramic for transplantation was coated with a composition where BMP-<NUM> was embedded into apatite, ionizing radiation sterilization resistance can be attained by embedding and specific bioactivity of protein can be maintained, as in a case where FGF-<NUM> was embedded into apatite, namely, that embedding into apatite exhibited radioprotective effect on specific bioactivity of protein.

Titanium pins for external fixation, as a metal for transplantation, were coated with apatite into which FGF-<NUM> was embedded, in the same conditions as in Example <NUM>, and immersed in a solution (AsMg solution) of L-ascorbic acid phosphate magnesium salt n-hydrate for several seconds and vacuum-dried. The co-precipitated ApFGF pins produced were subjected to γ-ray irradiation at a dose of <NUM> ± <NUM> kGy, and how the coexistence of AsMg in the γ-ray irradiation influences on FGF-<NUM> having reaction activity with an anti-FGF-<NUM> antibody was examined.

After titanium pins for intracorporeal fixation were coated with apatite into which FGF-<NUM> was embedded, in the same manner as in Example <NUM>, the resultants were immersed in a <NUM> AsMg solution for several seconds twice, and vacuum-dried, to thereby add AsMg to the apatite into which FGF-<NUM> was embedded. The co-precipitated ApFGF pins produced were sealed and packaged in the same degassing packaging as in Example <NUM>, and subjected to γ-ray irradiation at a dose of <NUM> ± <NUM> kGy. Thereafter, FGF-<NUM> supported on the pin after the γ-ray irradiation was detected by western blotting according to the same method as in Example <NUM>.

<FIG> illustrates the influence of addition of AsMg to apatite into which FGF-<NUM> is embedded, in radiation sterilization, on reaction activity with an anti-FGF-<NUM> antibody of FGF-<NUM> embedded. Bands were detected at a position of <NUM> kDa in all the groups of no γ-ray irradiation, γ-ray irradiation in the presence of AsMg, and γ-ray irradiation in the absence of AsMg. In a case where the signal intensity in the no irradiation group was <NUM>%, the signal intensity in the γ-ray irradiation group in the presence of AsMg was <NUM>%. However, the signal intensity in the γ-ray irradiation group in the absence of AsMg was <NUM>%, and was a signal intensity about one-fourth of the γ-ray irradiation group in the presence of AsMg. Accordingly, it was revealed that γ-ray irradiation in a condition of addition of AsMg to apatite into which FGF-<NUM> was embedded suppressed a decrease of FGF-<NUM> having reaction activity with an anti-FGF-<NUM> antibody and that, in this condition, protective effect of FGF-<NUM> in γ-ray irradiation was high as compared with that in the absence of AsMg. It is considered that AsMg is one of ascorbic acid compounds having anti-oxidation action and thus excellent protective effect suppresses generation of radical due to radiation irradiation.

Example <NUM>: cell proliferation activity after radiation sterilization of external fixation titanium pin coated with apatite into or to which not only FGF-<NUM>, but also heparin was embedded or adsorbed.

A external fixation titanium pin coated with apatite into which both FGF-<NUM> and heparin were embedded, and a external fixation titanium pin coated with apatite to which both FGF-<NUM> and heparin were adsorbed were produced, these pins were subjected to γ-ray irradiation at a dose of <NUM> ± <NUM> kGy, and thereafter whether or not FGF-<NUM> had cell proliferation activity was examined.

Sodium heparin was added at a concentration of <NUM> units/ml to an unstable supersaturated calcium phosphate solution to which FGF-<NUM> was added at each of concentrations of <NUM>µg/ml and <NUM>µg/ml as in Example <NUM>. External fixation titanium pins (DePuy Synthes, cell drill <NUM>/<NUM> Ti, <NUM>-<NUM>) were immersed in these unstable supersaturated calcium phosphate solution in the same conditions as in Example <NUM>, and coated with FGF-<NUM> and heparin co-precipitated together with apatite (co-precipitated ApFGF heparin pin). On the other hand, sodium heparin was added at a concentration of <NUM> units/ml to the supersaturated calcium phosphate solution containing <NUM>µg/ml of FGF-<NUM> of Example <NUM>. The external fixation titanium pin was immersed in the unstable supersaturated calcium phosphate solution for several seconds in the same conditions as in Example <NUM>, and coated with apatite to which FGF-<NUM> and heparin were adsorbed (adsorbed ApFGF heparin pin). The resultants were vacuum-dried, subjected to γ-ray irradiation or no irradiation, stored, and evaluated with respect to the cell proliferation activity in the same conditions as in Example <NUM>.

Table <NUM> shows the number of co-precipitated or adsorbed ApFGF heparin pins rated as "having activity" in three repeated trials.

As shown in Table <NUM>, <NUM> pins/<NUM> pins in the γ-ray irradiation group of the co-precipitated ApFGF heparin pins, and <NUM> pins/<NUM> pins in the no irradiation group thereof were rated as "having cell proliferation activity", and the number of pins having cell proliferation activity was the same between the γ-ray irradiation group and the no irradiation group, in the three trials. On the other hand, <NUM> pins/<NUM> pins in the γ-ray irradiation group of the adsorbed ApFGF heparin pins, and <NUM> pins/<NUM> pins in the no irradiation group thereof were rated as "having cell activity", and the number of pins having cell proliferation activity in the γ-ray irradiation group was about one-third of the no irradiation group. Both the groups were subjected to the chi-square test, and a significant difference (p = <NUM>) was recognized between the γ-ray irradiation group and the no irradiation group. In other words, it was revealed that the adsorbed ApFGF heparin pin more strongly tended to lose bioactivity of FGF-<NUM> by γ-ray irradiation sterilization.

As illustrated in <FIG>, the cell proliferation rate in the γ-ray irradiation group, normalized under the assumption that the cell proliferation rate in the no γ-ray irradiation group was <NUM>%, was about <NUM>% with respect to the co-precipitated ApFGF heparin pin, but was as slight as <NUM>% with respect to the adsorbed ApFGF heparin pin, and there was a statistically significant difference (p = <NUM>) between both the values. In other words, it was revealed that the intracorporeal fixation pin made of titanium as a metal for transplantation, coated with the composition in which both FGF-<NUM> and heparin were embedded into apatite, had higher ionizing radiation sterilization resistance than that coated with apatite to which both FGF-<NUM> and heparin were adsorbed, namely, radioprotective effect was exhibited in a case where not only the protein having bioactivity, but also heparin having no bioactivity was further embedded into apatite.

Example <NUM>: comparison of cell proliferation activity after radiation sterilization between external fixation titanium pin coated with apatite into which FGF-<NUM> was embedded and external fixation titanium pin coated with apatite into which both FGF-<NUM> and heparin were embedded.

The cell proliferation rate of the co-precipitated ApFGF pin of (Reference) Example <NUM> and the cell proliferation rate after radiation sterilization of the co-precipitated ApFGF heparin pin of Example <NUM> were compared to examine the effect of embedding of heparin in addition to FGF-<NUM>.

In (Reference) Example <NUM>, a dissolution liquid produced by dissolving a coating layer on the co-precipitated ApFGF pin after radiation sterilization, in a <NUM> sodium citrate solution, was added to mouse fibroblast strain NIH3T3, and the cell proliferation rate was quantitatively evaluated. As a result, the value of the cell proliferation rate was <NUM> ± <NUM> (<FIG>). On the contrary, in a case where a dissolution liquid produced by dissolving a coating layer on the co-precipitated ApFGF heparin pin after radiation sterilization, in a <NUM> sodium citrate solution, was added to mouse fibroblast strain NIH3T3, the cell proliferation rate was high to such an extent as to exceed the quantitation limit. The dissolution liquid was here diluted by <NUM>-fold and added to mouse fibroblast strain NIH3T3, the cell proliferation rate was quantitatively evaluated, and thereby <NUM> ± <NUM> was obtained as a value of the cell proliferation rate. In other words, it was indicated that when polysaccharide sach as heparin which was derived from an extracellular matrix and which had, by itself, no direct cell proliferation/differentiation activity was embedded together with a bioactive protein, higher bioactivity after radiation sterilization can be maintained at a higher level.

Example <NUM>: cell proliferation activity after radiation sterilization of zirconia for artificial joint-artificial bone, coated with apatite into or to which not only FGF-<NUM>, but also heparin was embedded or adsorbed.

Zirconias for artificial joint-artificial bone, coated with apatite into which both FGF-<NUM> and heparin were embedded, and zirconias for artificial joint-artificial bone, coated with apatite to which both FGF-<NUM> and heparin were adsorbed were produced, and these were subjected to γ-ray irradiation at a dose of <NUM> ± <NUM> kGy, and thereafter whether or not FGF-<NUM> had cell proliferation activity was examined.

A zirconia square bar (<NUM> × <NUM> × <NUM>) was coated, vacuum-dried, subjected to γ-ray irradiation or no irradiation, stored, and measured with respect to the cell proliferation rate in the same conditions as in Example <NUM>.

Table <NUM> shows the number of zirconia bars with co-precipitated ApFGF/heparin or adsorbed, rated as "having activity", in two repeated trials.

As shown in Table <NUM>, <NUM> bars/<NUM> bars in the γ-ray irradiation group of the zirconia bars with co-precipitated ApFGF/heparin, and <NUM> bars/<NUM> bars in the no irradiation group thereof were rated as "having cell activity", and the number of such zirconia bars having cell proliferation activity was the same between the γ-ray irradiation group and the no irradiation group, in the two trials. On the other hand, <NUM> bars/<NUM> bars in the γ-ray irradiation group of the zirconia bars with adsorbed ApFGF/heparin, and <NUM> bars/<NUM> bars in the no irradiation group thereof were rated as "having cell activity", and the number of such pins each having cell proliferation activity in the γ-ray irradiation group was about one-third of the no irradiation group. Both the groups were subjected to the chi-square test, and a significant difference (p = <NUM>) was recognized between the γ-ray irradiation group and the no irradiation group. In other words, it was revealed that the zirconia with adsorbed ApFGF/heparin more strongly tended to lose bioactivity of FGF-<NUM> by γ-ray irradiation sterilization.

As illustrated in <FIG>, the cell proliferation rate in the γ-ray irradiation group, normalized under the assumption that the cell proliferation rate in the no γ-ray irradiation group was <NUM>%, was about <NUM>% with respect to the zirconia with co-precipitated ApFGF/heparin, but was as slight as <NUM>% with respect to the zirconia with adsorbed ApFGF/heparin, and there was a statistically significant difference (p = <NUM>) between both the values. In other words, it was revealed that the zirconia as a ceramic for transplantation, coated with the composition in which both FGF-<NUM> and heparin were embedded into apatite, had higher ionizing radiation sterilization resistance than that coated with apatite to which both FGF-<NUM> and heparin were adsorbed, namely, radioprotective effect was exhibited in a case where not only the protein having bioactivity, but also heparin having no bioactivity was further embedded into apatite.

The co-precipitated ApFGF pins in γ-ray irradiation and no irradiation, produced in (Reference) Example <NUM>, and the adsorbed ApFGF pins in γ-ray irradiation and no irradiation, produced in (Reference) Example <NUM>, were each immersed in a <NUM> sodium citrate solution for <NUM> minutes, and co-precipitated ApFGF and adsorbed ApFGF each serving as the coating layer were each lysed. Each lysate was chemically analyzed by an ICP emission spectrometric analysis method, and each amount of calcium and phosphorus in the co-precipitated ApFGF and the adsorbed ApFGF was quantitatively determined. The results are shown in Table <NUM>.

It was indicated from Table <NUM> that the co-precipitated ApFGF serving as the coating layer included calcium phosphate as a main component. The Ca/P molar ratio (<NUM> to <NUM>) in the co-precipitated ApFGF was a value close to the theoretic Ca/P molar ratio (<NUM>) of apatite (Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>) including no impurity element. It is known that in a case where a phosphate group of apatite is replaced with any of impurities, a Ca/P molar ratio will be more than <NUM>, and representative impurities with which the phosphate group is replaced include a carbonate group. Since the co-precipitated ApFGF of Example <NUM> and Example <NUM> were produced in a solution containing carbonate ion, it was considered that apatite containing a carbonate group was co-precipitated together with FGF-<NUM> to embed FGF-<NUM> thereinto or apatite and calcium carbonate were co-precipitated together with FGF-<NUM> to embed FGF-<NUM> thereinto.

An unstable supersaturated calcium phosphate solution was used which included <NUM> of Ca ion, <NUM> of phosphate ion, <NUM> of K ion, <NUM> of Na ion, <NUM> of Cl ion, and <NUM> of HCO<NUM> ion, which had a pH of <NUM>, and in which calcium phosphate would be crystallized by spontaneous nucleation in about <NUM> to <NUM> hours if left at <NUM>. Zirconias with co-precipitated ApFGF/heparin were produced in the completely same conditions as in Example <NUM> except that the immersion time was <NUM> hours, and the resultants were vacuum-dried, subjected to γ-ray irradiation, stored, and measured with respect to the cell proliferation rate.

Table <NUM> shows the number of zirconias with co-precipitated ApFGF/heparin, rated as "having activity".

As shown in Table <NUM>, <NUM> zirconias/<NUM> zirconias in the γ-ray irradiation group of the zirconias with co-precipitated ApFGF/heparin, and <NUM> zirconias/<NUM> zirconias in the no irradiation group thereof were rated as "having cell activity", and the number of zirconias having cell proliferation activity was the same between the γ-ray irradiation group and the no irradiation group. In other words, it was revealed that, also in a case where a Ca-PO<NUM>-K-Na-Cl-based unstable supersaturated calcium phosphate solution was used to coat zirconia as a ceramic for transplantation with the composition in which both FGF-<NUM> and heparin were embedded into apatite, embedding into apatite exhibited the radioprotective effect.

The co-precipitated ApFGF heparin coating layers produced in Example <NUM> were subjected to compositional analysis by the same method as in (Reference) Example <NUM>. The co-precipitates in production of the zirconia with co-precipitated ApFGF/heparin in Example <NUM> were placed on a silicon non-reflective plate, and analyzed by a powder X-ray diffraction method. Powder X-ray diffraction was performed using CuKα rays in conditions of <NUM> kV and <NUM> mA. The results of compositional analysis are shown in Table <NUM>.

It was indicated from Table <NUM> that the co-precipitated ApFGF serving as a coating layer included calcium phosphate. The Ca/P molar ratio in the co-precipitated ApFGF was <NUM> to <NUM> in the no γ-ray irradiation group and was <NUM> to <NUM> in the irradiation group.

It was considered from the Ca/P ratio that FGF-<NUM> and heparin were embedded into carbonate group-containing apatite or FGF-<NUM> and heparin were embedded into apatite and calcium carbonate, as in Example <NUM>. Furthermore, because the Ca/P molar ratio of amorphous calcium phosphate is typically <NUM>, it was also indicated that amorphous calcium phosphate was to be deposited.

<FIG> represents the results of analysis by a powder X-ray diffraction method. It is known that amorphous calcium phosphate appears as a broad peak large in half-value width at around <NUM>°. As illustrated in <FIG>, a broad peak large in half-value width, corresponding to amorphous calcium phosphate, was confirmed at around <NUM>° in a powder X-ray diffraction pattern of the co-precipitate. Three strong diffraction lines ((<NUM>), (<NUM>), (<NUM>)) characteristic of crystalline apatite, appearing at <NUM>°, <NUM>°, and <NUM>°, were not separated, and formed one broad peak at around <NUM>°. Accordingly, the apatite as the co-precipitate was a low crystalline apatite. Additionally, a peak of calcium carbonate was observed at <NUM>°. In other words, the co-precipitated product contains amorphous calcium phosphate, low crystalline apatite, and calcium carbonate as main components. The co-precipitated product does not include crystalline apatite with low solubility, but includes amorphous calcium phosphate, low crystalline apatite, and calcium carbonate each having relatively high solubility, as main components, and is suitable for gradually releasing protein embedded.

Claim 1:
A method for producing a medical instrument for use in mammals, including human beings, comprising a step of coating a part or the entirety of a substrate with a crystalline or an amorphous form of an inorganic salt into which a protein having bioactivity and heparin are embedded and a step of exposing the coated substrate to ionizing radiation at a dose sufficient for sterilization, wherein
the substrate is a metal, a ceramic or both,
the inorganic salt is one or more inorganic salts selected from the group consisting of apatite, tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, and calcium carbonate,
the inorganic salt is provided in a step selected from the group consisting of controlled delay co-precipitation method in a neutral or weak alkaline unstable supersaturated calcium phosphate solution which generates spontaneous nucleation, a coverage sandwich method, and a drying method, and
the medical instrument is one or more medical instruments selected from the group consisting of an intracorporeal fixation pin, an intracorporeal fixation screw, an artificial bone, a bone prosthetic material, a dental endosseous implant, a spinal fixation device, an intramedullary nail, and a spinal cage.