Control of biodegradability of composite biomaterial

[0001] [Technical field to which the invention belongs] The present invention relates to a method for controlling the biodegradability of a composite biomaterial containing a calcium salt (particularly hydroxyapatite) and collagen, and an improved composite biomaterial provided by the method. [0002] [Conventional technology] Conventionally, reimplantation of autologous tissue is often performed to repair a bone defect in the field of orthopedics. However, the use of self-tissue is a heavy burden on the patient, and the amount collected is limited, so supplementation with an artificial implant is indispensable. In addition to the mechanical properties similar to living bone, these artificial bones have biocompatibility and osteoconductivity-that is, they are gradually absorbed after application to the living body, taken into the bone regeneration cycle, and replaced with their own bone. -Is required. [0003] Originally, vertebrate bone is a complex consisting of the inorganic hydroxyapatite (HAp) and the organic collagen. In these, HAp forms a unique nanocomposite structure (self-organization) in which HAp is oriented along the collagen fiber in the c-axis direction in the living bone, and this structure gives the mechanical property peculiar to the bone. That is, it is not possible to obtain the same structure and characteristics as living bone simply by combining HAp and collagen. [0004] Therefore, various studies have been conducted to develop a composite biomaterial closer to living bone using HAp and collagen. For example, Mehlisch et al. Synthesized a mixture of HAp particles and collagen (Mehlisch, AS et al, Oral Surg Oral Med Oral Pathol, 70 (6) (1990), 685-692), and Miyamoto et al. Reinforced with HAp cement. Collagen was prepared and its biocompatibility was evaluated (KS Ten Huisen, et al, J. et al. Biomed Mater Res, 29 (7) (1995), 803-810). In addition, Ten Huisen et al. Produced HAp / Col nanocomposites by growing HAp crystals on collagen (Col) fibers using calcium hydrogen phosphate as a precursor of HAp (Y. Miyamoto et al., Biomaterials, 19 (1998), 707-715). However, none of them could reproduce the nanostructure similar to that of living bone. [0005] On the other hand, the present inventors utilize the self-organization of HAp and Col to HAp having a nanocomposite structure similar to that of living bone under biomimetic conditions (conditions similar to the in vivo environment where osteogenesis occurs). Succeeded in synthesizing the / Col complex (Japanese Patent Laid-Open No. 7-101708, JP-A-11-199209, JP-A-2000-5298, etc.). It was confirmed that this complex has excellent biocompatibility, is absorbed by osteoclasts, and promotes osteogenesis. However, since the complex is quickly absorbed and decomposed after transplantation, there is a problem that it lacks practicality as an artificial aggregate or the like. [0006] [Problems to be Solved by the Invention] In the present invention, in a complex containing a calcium salt (particularly hydroxyapatite) having a structure similar to that of a living bone and collagen, the complex is suitable for practical use by controlling the in vivo decomposition rate while maintaining its mechanical strength. The purpose is to provide biomaterials. [0007] [Means for solving problems] As a result of diligent studies to solve such a problem, the present inventors have found that the mechanical strength and the in vivo decomposition rate can be controlled by introducing a crosslink into the collagen fiber constituting the complex, and complete the present invention. I let you. [0008] That is, the present invention provides the following (1) to (9). (1) A method for controlling the in vivo decomposition rate of a composite biomaterial containing a calcium salt and collagen by introducing a crosslink into the collagen. (2) The method according to (1) above, wherein the calcium salt is hydroxyapatite. (3) The method according to (2) above, wherein the composite biomaterial has a microporous structure in which the c-axis of hydroxyapatite is oriented along the collagen fibers. (4) The method according to any one of (1) to (3) above, wherein the cross-linking is introduced into at least 1% or more of reactive functional groups in collagen. (5) The method according to any one of (1) to (4) above, wherein the cross-linking is introduced by a cross-linking reaction using glutaraldehyde. [0009] (6) The method according to (5) above, wherein the glutaraldehyde is used in an amount of 10 μmol to 10 mmol per 1 g of collagen in the composite biomaterial. (7) A composite biomaterial containing hydroxyapatite and collagen, wherein cross-linking is introduced into 1 g of collagen in the composite biomaterial using 10 μmol to 10 mmol of glutaraldehyde. (8) The composite biomaterial according to (7) above, wherein the cross-linking is introduced into at least 5% or more of the reactive ε-amino groups in collagen. (9) The composite biomaterial according to (7) or (8) above, wherein the composite biomaterial has a microporous structure in which the c-axis of hydroxyapatite is oriented along collagen fibers. [0010] BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the contents of the present invention will be described in detail. 1. Composite biomaterial of the present invention The composite biomaterial of the present invention has realized mechanical strength and in-vivo decomposition rate suitable for biomaterials by introducing crosslinks into collagen in a complex containing a calcium salt (particularly hydroxyapatite) and collagen. It is characterized by. [0011] As the calcium salt contained in the composite biomaterial of the present invention, calcium phosphate or calcium carbonate is preferable, and hydroxyapatite is most preferable. In this composite biomaterial containing collagen and hydroxyapatite, it is preferable that hydroxyapatite and collagen are oriented in a self-organizing manner to form a complex similar to living bone. In addition, "self-organization" generally means "the same or different kinds of atoms, molecules, fine particles, etc. gather by non-covalent interaction to form a specific structure (Tokyo Kagaku Dojin". From "Biochemistry Dictionary") ", especially in the present invention, calcium phosphate (hydroxyapatite: HAp) having an apatite structure is oriented peculiar to living bone, that is, the c-axis of HAp is collagen. It is meant to form a microporous structure oriented along the fibers. [0012] Hydroxyapatite has a general composition of Ca₅(PO₄)₃It is a compound called OH, and CaHPO depends on the non-stoichiometry of the reaction.₄, Ca₃(PO₄)₂, Ca₄O (PO₄)₂, Ca₁₀(PO₄)₆(OH)₂, CaP₄O₁₁, Ca (PO₃)₂, Ca₂P₂O₇, Ca (H₂PO₄)₂ H₂Contains a group of compounds called calcium phosphate such as O. Hydroxyapatite is Ca₅(PO₄)₃OH or Ca₁₀(PO₄)₆(OH)₂The basic component is the compound represented by the composition formula of, and a part of the Ca component is replaced with one or more selected from Sr, Ba, MG, Fe, Al, Y, La, Na, K, H and the like. You may. Also, (PO₄) Part of the component is VO₄, BO₃, SO₄, CO₃, SiO₄It may be replaced with one or more selected from the above. Furthermore, a part of the (OH) component is F, Cl, O, CO.₃It may be replaced with one or more selected from the above. Moreover, a part of each of these components may be defective. PO of apatite in living bone₄And some of the OH components are usually CO₃CO from the atmosphere during the manufacture of this composite biomaterial because it has been replaced by₃May be mixed and partially replaced with each component (about 0 to 10% by mass). [0013] In addition to ordinary microcrystals / amorphous and crystals, hydroxyapatite may be an isomorphic solid solution, a substituted solid solution, an intrusive solid solution, or may contain non-quantologic defects. .. Further, in this "hydroxyapatite", the atomic ratio (Ca / P) of calcium and phosphorus is preferably in the range of 1.3 to 1.8, and more preferably 1.5 to 1.7. When the atomic ratio is in the range of 1.3 to 1.8, the composition and crystal structure of apatite (calcium phosphate compound) in the product can have a composition and structure similar to those of apatite present in vertebrate bone. This is because the biocompatibility and bioabsorbability are increased. [0014] Currently, it is known that about 20 different molecular species of collagen exist in the living tissues of a wide range of animals including fish, not limited to mammals, and are collectively called "collagens". .. The collagen used in the present invention is not particularly limited in terms of animal species, tissue site, age, etc. as a starting material thereof, and any collagen can be used. Generally, collagen obtained from the skin, bones, cartilage, tendons, organs, etc. of mammals (eg, cows, pigs, horses, rabbits, rats, etc.) and birds (eg, chickens, etc.) is used. In addition, collagen-like proteins obtained from the skin, bones, cartilage, fins, scales, organs, etc. of fish (for example, cod, flatfish, flatfish, salmon, trout, tuna, mackerel, Thailand, sardines, sharks, etc.) are used as starting materials. You may use it. Alternatively, collagen obtained by a gene recombination technique may be used instead of extraction from animal tissue. [0015] Here, type I collagen is the most abundant and well-studied among the molecular species of collagen, and usually, when simply referred to as collagen, it often refers to type I collagen. The molecular species of collagen used in the present invention is not particularly limited, but it is preferable that type I collagen is the main component. Further, collagen may be used by appropriately chemically modifying the amino acid residue of the collagen protein, such as acetylation, saccharification, maleylation, phthalation, benzoylation, esterification, amidation, and guanidinization. [0016] Examples of the method for preparing collagen include a method of extracting collagen from the above-mentioned starting material (excluding gene recombination technology) with a neutral buffer solution or a dilute acid such as hydrochloric acid, acetic acid or citric acid. The former is called neutral salt-soluble collagen and the latter is called acid-soluble collagen. However, the amount of collagen extracted in each case is small, and most of them remain as insoluble collagen. As a method for solubilizing this insoluble collagen, an enzyme solubilization method and an alkali solubilization method are known. The former is called enzyme-solubilized collagen and the latter is called alkali-solubilized collagen, both of which can be solubilized as molecular collagen in a yield of almost 100%. [0017] The method for preparing collagen (extraction type) used in the present invention is not particularly limited, but if the molecular weight when collagen is solubilized is large, the strength of the complex becomes insufficient due to steric hindrance. It is preferable to use monomeric collagen. In particular, enzyme-solubilized collagen and alkali-solubilized collagen have a large amount of monomeric content, and in addition, the non-spiral portion (telopeptide) having most of the antigenicity of collagen is selectively decomposed at the preparation stage. -Since it is removed, it is suitable for the organic-inorganic composite biomaterial of the present invention. Collagen in which this non-spiral portion is decomposed and removed is called atelocollagen. [0018] Here, there is a difference in the isoionic point between the enzyme-solubilized collagen and the alkali-solubilized collagen. The isoionic point is the pH at which both positive and negative charges derived from the dissociation group peculiar to the protein molecule cancel each other out. In the case of collagen, it was solubilized when approaching the pH range of the isoionic point. It is known that things become fibrotic. Generally, the isoionic point of enzyme-solubilized collagen is pH 8-9, and the isoionic point of alkali-solubilized collagen is pH 4-5. In the present invention, it is more preferable to use enzyme-solubilized collagen in which collagen fibrosis progresses in a reaction vessel maintained at a pH of 7 to 11 and self-assembling is easy. Examples of the enzyme for solubilization include pepsin, trypsin, chymotrypsin, papain, and pronase. Pepsin and pronase are preferably used because of the ease of treatment after the enzyme reaction. [0019] 2. Method for producing a complex containing hydroxyapatite and collagen As a preferable example of the complex containing calcium salt and collagen, which is the base of the composite biomaterial of the present invention, a method for producing the complex containing hydroxyapatite and collagen will be described. [0020] Complexes containing hydroxyapatite and collagen are described, for example, by the method of Kikuchi et al. (Kikuchi, S. et al, J., Biomater., 22 (13) (2001), 1705-1711, S. Itoh et al, J. Biomed Mater Res, (2001), It can be manufactured according to 445-453). The complex is produced using at least three components, collagen, phosphate and calcium salt, as starting materials. Strictly speaking, it does not correspond to "salt", but in the present invention, the phosphate contains phosphoric acid and the calcium salt contains calcium hydroxide. [0021] [0021] Examples of the phosphoric acid source of the aqueous phosphate solution used include disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and phosphoric acid. The aqueous phosphate solution dissolves the above-mentioned collagen and is subjected to the reaction. [0022] Moreover, as a calcium source of the calcium salt aqueous solution used, for example, calcium carbonate, calcium acetate, calcium hydroxide and the like can be mentioned. The calcium salt aqueous solution may be a suspension as long as it is in a uniform state. For example, calcium carbonate is obtained by firing calcium carbonate and crushing it in a dairy pot or the like to obtain calcium hydroxide, and adding water to the calcium hydroxide. Suspension of can be preferably used. [0023] In the method for producing the complex, the aqueous calcium salt solution and the aqueous phosphate solution containing collagen are simultaneously added dropwise to the reaction vessel. Here, "simultaneous" does not mean only the form of dropping exactly at the same time, but also includes the form of dropping small amounts (about 0.01 to 5 ml) alternately. Both solutions may be added dropwise continuously as long as they are simultaneous, or may be added dropwise intermittently. An appropriate amount of pure water is put in the reaction vessel in advance. The amount of the pure water is not particularly limited, but it is preferably about the same as the amount of the calcium salt aqueous solution used. [0024] In the above-mentioned production method, it is important that the calcium ion concentration in the reaction vessel is maintained at 3.75 mM or less and the phosphate ion concentration is maintained at 2.25 mM or less. This is because if the concentration of calcium ion or phosphate ion exceeds the above range, suitable self-assembly of the complex is hindered. It is considered that this is because when the concentration of the above-mentioned ions convected in the reaction vessel exceeds those concentrations in the body fluid, spontaneous nucleation occurs. If the calcium ion concentration is maintained at 2.5 mM and the phosphate ion concentration is maintained at 1.5 mM or less, a complex having an average fiber length of 1 mm or more can be obtained, which is more preferable. [0025] In the above-mentioned production method, hydroxyapatite and collagen produced in the reaction vessel are preferably present in a weight ratio of 3: 2 to 9: 1, preferably 70:30 to 85:15. This is because it is important for self-organization that the weight ratio of hydroxyapatite to collagen when the ideal reaction occurs is closer to the composition of living bone (75:25). [0026] The ratio of the aqueous phosphoric acid solution containing collagen to the aqueous calcium salt solution is preferably in the range of 3: 1 to 1: 3. When the amount of the aqueous solution of phosphoric acid containing collagen is small, the composition becomes excessive in calcium and the strength is lowered, and when the amount of the aqueous solution containing calcium salt is small, calcium deficiency occurs and Young's modulus occurs. This is because the calcium may decrease, which may lead to a decrease in strength (see JP-A-11-199209). [0027] In the present invention, it is desirable to drop the reaction solution so that the pH of the reaction solution is in the range of 7 to 11 and the range of change is within 1. More preferably, the pH is in the range of 7 to 9, and the range of change is within 0.5. This is because native collagen causes precipitation at the isoelectric point in the pH range of 7 to 11 and the fibers regenerate, and calcium phosphate also tends to cause precipitation in this pH range. This is because the self-organization of collagen is promoted. When the pH exceeds 11, the water molecules are hydrated around the collagen molecules and it becomes difficult for the water molecules to separate in the subsequent pressure molding step, so that the water content of the complex becomes high and self-assembly is hindered. The strength may also decrease. On the other hand, if the pH is less than 7, both calcium phosphate and collagen are less likely to precipitate. In addition, when the range of change exceeds 1, the nucleation of calcium phosphate on collagen is disturbed and self-organization deteriorates (Kikuchi et.al., Biomaterials 22, (2000) p1705-1711)). [0028] In the above-mentioned production method, it is convenient to use a pH controller in order to perform suitable pH control. The pH controller is provided with a means for measuring the pH of the reaction solution and a means for adjusting the dropping amount of both solutions to be dropped, and has a certain range with respect to the pH (for example, 10) set as the desired value. The dropping amount of both solutions is adjusted based on the pH value of both solutions so as to maintain (for example, ± 0.3). Examples of the pH controller include those manufactured by NISSIN. It is preferable to carry out the reaction while constantly stirring both solutions and the reaction solution so that the pH of the reaction solution is not biased. [0029] In the production method, the temperature of the reaction solution is preferably maintained at 35 ° C to 40 ° C. This is because it is expected that complex formation will be performed under the same conditions as in vivo if the temperature is within this range. When the precipitate generated from the reaction solution is filtered, dried, and then pressure-molded, a complex in which hydroxyapatite and collagen are oriented and bonded in a self-organizing manner can be obtained. [0030] 3. Introduction of cross-linking In the complex containing the calcium salt (particularly hydroxyapatite) and collagen obtained as described above, crosslinks are introduced into the collagen fibers constituting the complex. Cross-linking is preferably carried out directly without isolating the complex from the reaction solution. In addition, a small amount of collagen or polysaccharide (1 to 100 mol% with respect to the amount of collagen in the complex) may be added to increase the number of cross-linking points. [0031] The cross-linking may be carried out by any method such as chemical cross-linking using a cross-linking agent or a condensing agent, physical cross-linking using γ-rays, ultraviolet rays, thermal dehydration, electron beams and the like. Examples of the cross-linking agent include aldehyde-based cross-linking agents such as glutal aldehyde and formaldehyde; isocyanate-based cross-linking agents such as hexamethylene diisocyanate; and carbodide-based cross-linking agents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. Agents; Polyepoxy-based cross-linking agents such as ethylene glycol diethyl ether; Transglutaminase and the like. The amount of these cross-linking agents used is preferably about 10 μmol to 10 mmol per 1 g of collagen in the complex. [0032] The cross-linking may be any portion of collagen, but it is particularly preferable to cross-link the carboxyl group and the hydroxyl group, the carboxyl group and the ε-amino group, or the ε-amino group. Further, it is preferable that at least 1% or more of the reactive functional groups are crosslinked, and more preferably 5% or more. This is because if the cross-linking is insufficient, the bone is decomposed quickly in the living body, and a sufficient filling effect of the bone defect cannot be expected. However, note that the use of excess cross-linking agent increases the water content of the complex and inhibits the binding between particles, resulting in the introduction of cross-linking between the fibers forming the complex, which reduces the strength of the complex. is required. [0033] Among the above-mentioned cross-linking methods, chemical cross-linking using a cross-linking agent such as glutaraldehyde is particularly preferable from the viewpoint of easy control of the degree of cross-linking and biocompatibility of the obtained complex. Hereinafter, as a preferred embodiment of the present invention, glutaraldehyde is used. a crosslinking method using will be described . [0034] The reaction solution of the complex containing hydroxyapatite and collagen obtained in the previous section is immediately after the complex synthesis or after aging for up to 3 hours, and glutaraldehyde is added with vigorous stirring to react for 10 minutes. After the cross-linking reaction, the complex is immediately filtered and washed 3 times with pure water to remove excess glutaraldehyde. Here, glutaraldehyde is preferably added in an amount of 10 μmol to 10 mmol, particularly 10 μmol to 1 mmol, based on 1 g of collagen in the composite biomaterial. Further, the temperature of the reaction solution is preferably maintained at 0 ° C to 40 ° C. [0035] 4. Improvement of physical properties (mechanical strength, in-vivo decomposition rate) by cross-linking The obtained crosslinked composite biomaterial has higher mechanical strength and a slower in vivo decomposition rate than the uncrosslinked composite biomaterial, and therefore has the in vivo retention required for artificial aggregates and the like. That is, the present invention provides a method for controlling the in vivo decomposition rate of a composite biomaterial while maintaining mechanical strength by introducing a crosslink between hydroxyapatite and collagen. [0036] The in vivo decomposition rate can be evaluated, for example, by transplanting the composite biomaterial into the bone of a mouse, rat, rabbit or the like and observing its in vivo retention. Further, the mechanical strength can be evaluated by, for example, the three-point bending strength and the Young's modulus obtained from the value thereof. [0037] Specifically, the organic-inorganic composite biomaterial obtained by adding 10 μmol to 10 mmol of glutaraldehyde to 1 g of collagen and introducing cross-linking has a mechanical strength of 7 MPa (uncross-linked) to 15 MPa or more (after cross-linking). Improved. The uncrosslinked sample was almost (90% or more) absorbed in the living bone in 4 weeks, whereas about 50% or more of the crosslinked composite biomaterial remained in the living bone even after 4 weeks. [0038] 5. How to use crosslinked composite biomaterials The crosslinked composite biomaterial obtained by the above method can be appropriately pressure-molded and used as an implant such as an artificial aggregate. The pressure molding is preferably performed in a temperature range of 0 ° C. or higher and 110 ° C. or lower, and in a pressure range of 10Mpa to 5Gpa. This is because when pressure molding is performed in this temperature range, most of the water contained in the precipitate is rapidly released. The temperature is preferably in the range of 25 ° C or more and 60 ° C or less, which releases a large amount of water, and particularly preferably in the range of 35 ° C or more and 45 ° C or less. Further, it is preferable to carry out while applying ultrasonic waves because self-organization can be further promoted. Examples of the pressure processing apparatus that can be used for pressure molding in the present invention include CIP manufactured by Kobe Steel, Ltd. [0039] The form and shape of the composite biomaterial of the present invention are not particularly limited, and can be molded into any form and shape according to the application, such as block shape, paste shape, film shape, granular shape, and sponge shape. The composite biomaterial of the present invention has elasticity like a sponge when it absorbs water, and has excellent biocompatibility, bone inducing ability or bone conduction ability. Therefore, when the composite biomaterial is used as an implant, it may be used after being once immersed in a suitable liquid such as physiological saline. The composite biomaterial thus implanted can rapidly bind to bone tissue and integrate with hard tissue on the donor side. [0040] In addition to the essential components calcium salt, phosphate, and collagen, the composite biomaterial of the present invention may further contain other components as long as the object and effect of the present invention are not impaired. Such components include, for example, St, Mg and CO.₃Examples thereof include inorganic salts such as, organic substances such as citric acid and phospholipids, bone morphogenetic proteins, and agents such as anticancer agents. [0041] The composite biomaterial of the present invention has a strength and composition close to that of living bone, and since both collagen and calcium phosphate, which are constituents, are biosoluble, it has a drug sustained-release effect, bone inducing ability, or bone conduction ability. Moreover, it has excellent mechanical strength and in-vivo retention (appropriate in-vivo decomposition rate) due to cross-linking. [0042] Further, by incorporating a cytokine having high physiological activity into the composite biomaterial of the present invention and culturing the tissue in a biologically similar environment or in vivo to which dynamics and electricity are added as a substrate, tissues such as bone marrow and liver are observed. The effect of reconstruction is also expected. For example, by using a composite material obtained by the present invention impregnated with an anticancer agent for reconstruction of excised bone such as osteosarcoma, it is possible to prevent cancer recurrence and induce living hard tissue. [0043] Therefore, as the use of the composite biomaterial obtained by the present invention, it is used as a living bone replacement type bone reconstruction material having bone induction and bone conductivity, and a living body used for tissue engineering containing amino acids, sugars and cytokines. Examples of the use as an active base material and a biocompatible drug such as an anticancer agent as a sustained-release base material can be mentioned. Specifically, artificial bone, artificial joint, joint material between tendon and bone, dental implant material, etc. , Percutaneous terminal for catheter, drug sustained-release base material, bone marrow induction chamber, tissue reconstruction chamber / base material, and the like. [0044] [Example] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.<u style="single">Example 1: Cross-linking</u><u style="single">HAp / Col</u><u style="single">Fabrication of complex</u>(1) Preparation of HAp / Col complex The Hap / Col complex was prepared according to the method of Kikuchi et al. (M. Kikuchi, et al., Biomater., 22 (13) (2001), 1705-1711). First, calcium carbonate (for alkali analysis, Wako Pure Chemical Industries, Ltd.), phosphoric acid (special grade, Wako Pure Chemical Industries, Ltd.) and atelocollagen derived from porcine skin (Nitta Gelatin) were prepared as starting materials. Calcium carbonate was calcined at 1050 ° C and then hydrodigested to form calcium hydroxide single phase. 40 mM calcium hydroxide suspension 2 dm³2 dm of 24 mM phosphoric acid aqueous solution containing 2 g of collagen³Was introduced into the reaction vessel via a tube pump. The pH in the reaction vessel was controlled to pH 9 by a controller, and the temperature was controlled to 40 ° C by a hot water bath. [0045] (2) Cross-linking reaction The reaction mixture was allowed to stand for 3 hours while suspended, and the cross-linking agent: glutaraldehyde was added with vigorous stirring to react for 10 minutes. After the cross-linking reaction, the complex was immediately filtered and washed with pure water three times. For comparison, a cross-linking reaction was carried out in the same manner using water-soluble carbodiimide and transglutaminase (both are condensing agents). [0046] The cross-linking reaction varies with respect to 1 g of collagen in the complex in the range of glutaraldehyde: 0.0191-13.5 mmol / g, water-soluble carbodiimide: 0.0191-8.8 mmol / g, and transglutaminase: 19.1-1910 mg / g, respectively. I let you go. In the case of glutaraldehyde, theoretically all ε-amino groups in the collagen molecule can be crosslinked at 0.191 mmol / g. [0047] (3) Measurement of characteristic values The characteristics of the obtained crosslinked complex were measured as follows. 1 Complex structure (particle size): The crosslinked complex was dispersed in pure water and observed using a transmission electron microscope Rapid-VueR (manufactured by Beckman-Colter). 2 Three-point bending strength: The crosslinked complex was dehydrated by uniaxially pressurizing at 20 MPa for 24 hours, and the three-point bending strength was measured by a universal testing machine (Autograph AGS-1kN, manufactured by Shimadzu). The measurement was performed using a crosslinked complex piece of 5 × 3 × 20 mm at a crosshead speed of 500 μm and a span of 15 mm. [0048] 3 HAp / Col / H₂O ratio: HAp / Col / H of the above pressure-molded crosslinked complex₂The O ratio was measured using a carbon determiner (LECO, RC-412). 4 Swelling degree: The above pressure-molded complex was immersed in phosphorus buffer (pH = 7.4, 37 ° C) for 4 weeks and weighed to determine the degree of swelling (formula below). [0049] [Number 1] Swelling degree (%) = [(Wx-Wo) / Wo] x 100 Wx: Initial weight, Wo: Weight after immersion [0050] 5 Amount of cross-linking: Using the crosslinked complex used for the three-point bending strength measurement, the amount of ε-amino groups was measured by the sulfo-SDTB method, and the amount of crosslinked was determined. [0051] (4) Result (1) As a result of transmission electron microscope observation, the fiber length of the glutaraldehyde crosslinked complex was 44.8 μm on average. In addition, it was found that the cross-linked hydroxyapatite and collagen did not show macro-orientation, and the cross-linking occurred randomly. The nanoscopic structure similar to that of living bone (orientation of collagen monofibrous HAp) was substantially maintained. As the glutaraldehyde concentration increased, the color of the complex changed from dark yellow to brown. This was thought to be due to the excess glutaraldehyde cross-linking between the self-assembled fibers and increasing the complex fiber length. [0052] 2 In the case of glutaraldehyde cross-linked complex, the three-point bending strength increases according to the glutaraldehyde content, 1.35 mmol / g. It reached the highest value in collagen (Fig. 1). The result is that excess glutaraldehyde cross-linking agent (1.35 mmol / g or more) introduces cross-linking between each fiber forming the complex, increasing the water content of the complex and thus inhibiting the binding between particles. It was suggested that the strength of the complex was reduced. In the cross-linked product by water-soluble carbodiimide or transglutaminase, the concentration did not necessarily change. [0053] 3 The HAp / Col ratio of the glutaraldehyde cross-linked complex was almost constant, but the water content increased with the amount of glutaraldehyde. This is because the crosslinks that occur within the self-assembled fibers do not affect the water retention of the complex, but the crosslinks that occur between the self-assembled fibers increase the water retention of the complex. Similar to glutaraldehyde, the collagen and water contents of water-soluble carbodiimide and transglutaminase increased according to the concentration of the reactant. [0054] 4 The degree of swelling mainly depends on the amount of collagen. Therefore, the degree of swelling was normalized by the amount of collagen to reflect the amount of cross-linking (Fig. 2). As a result, it was suggested that the degree of swelling decreased with the concentration of glutaraldehyde, and that cross-linking could control the biodegradability of the complex in living tissues. On the other hand, no increase in swelling was clearly observed with water-soluble carbodiimide or transglutaminase. This was thought to be because water-soluble carbodiimides and transglutaminase are condensing agents, and cross-linking makes the complex dense and prevents swelling. 5 As a result of sulfo-SDTB measurement, no free ε-amino group was detected at a concentration of 1.35 mmol / g of glutaraldehyde. This concentration is about 70 times the amount of glutaraldehyde required to crosslink the crosslinkable functional groups in collagen. [0055] (5) Conclusion In the case of a crosslinked glutaraldehyde, if the concentration of glutaraldehyde exceeds 1.35 mmol / g · col, the mechanical strength of the crosslinked product decreases, and 10 mmol / g to maintain the mechanical strength suitable for artificial aggregate. It seemed preferable to crosslink at a concentration of g · col or less. On the other hand, assuming that the decomposition in the living body is proportional to the degree of swelling, it was predicted that the larger the amount of cross-linking, the more the decomposition would be suppressed. In addition, the nanoscopic structure (collagen monofilament-like HAp orientation) similar to that of living bone was substantially maintained by cross-linking. [0056]<u style="single">Example 2: With a rabbit</u><u style="single">HAp / Col</u><u style="single">Biodegradability test of complex crosslinked products</u>(1) Test method The biodegradability of the HAp / Col complex crosslinked product was examined by implanting the crosslinked product (2 × 2 × 2 mm) obtained in Example 1 with various glutaraldehyde concentrations in the rabbit tibia. Evaluation was performed by gross findings (Fig. 3) and histological examination (hematoxylin-eosin staining) after 1, 2 and 4 weeks. [0057] (2) Result As a result of histological examination, no toxic reaction due to glutaraldehyde cross-linked product such as inflammatory reaction was observed. In addition, in all the crosslinked bodies, bone formation and bone conductivity similar to those of the uncrosslinked complex were observed around the crosslinked bodies. .. [0058] [0058] The absorption / decomposition rate of the HAp / Col complex cross-linked product decreased with the glutaraldehyde concentration, and 70-80% remained in the bone even after 4 weeks in the high-density cross-linking (191 μmol or more). About 50% of the collagen cross-linked with 19.1 μmol of glutaraldehyde remained, and about 85% or more of the collagen cross-linked with 675 μmol remained. Furthermore, in the case of cross-linked with 1.35 mmol of glutaraldehyde per 1 g of collagen, only the surface was absorbed, and 95% or more remained. The residual amount of ε-amino group in each crosslinked sample was 80-95%, 0-10%, and 0%. In particular, in the case of cross-linking with 1.35 mmol of glutaraldehyde, it was considered that excess glutaraldehyde formed a cross-linking network in the complex, further reducing the absorbability of the complex. [0059] (3) Conclusion From the above, it was confirmed that the complex crosslinked at a concentration of 19.1 μmol to 1.35 mmol of glutaraldehyde per 1 g of collagen has the mechanical strength required for artificial aggregate and the in vivo decomposition rate. From the above results and the results of Example 1, if cross-linking is introduced into at least 1% (preferably 5% or more) of the reactive ε-amino groups of collagen in the complex containing hydroxyapatite and collagen, it is mechanical. It was considered possible to achieve the in-vivo decomposition rate required for artificial aggregate while maintaining strength. Then, it was considered that at least about 10 μmol to 10 mmol of glutaraldehyde per 1 g of collagen should be used for the introduction of such a crosslink. [0060] [Effect of the invention] According to the present invention, in a complex containing a calcium salt (particularly hydroxyapatite) having a structure similar to that of living bone and collagen, the in vivo decomposition rate can be controlled while maintaining its mechanical strength. [Simple explanation of drawings] FIG. 1 shows the relationship between the cross-linking agent concentration and the three-point bending strength of the cross-linked complex. FIG. 2 shows the concentration of the cross-linking agent and the degree of swelling of the cross-linked complex (normalized by the amount of collagen). FIG. 3 is a photograph of each glutaraldehyde cross-linked complex 2 weeks after intratibial rabbit implantation.