JPH08117323A - Apatite-coated base material and method for manufacturing the same - Google Patents

Abstract

PURPOSE: To manufacture an apatite-coated base material similar to the bio- apatite in physicochemical property and to use the material for the artificial bone material or culture base material, etc. CONSTITUTION: A surface of an organic polymer in a desired shape is coated with a calcium phosphate compound. This organic polymer is a protein or polyamino acid, etc., and the calcium phosphate compound is substantially bonded chemically to the organic polymer.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention is useful as a synthetic bone material such as a bone filling material and a bone replacement material, a container for tissue culture, a membrane, sponge, beads, and a culture substrate such as a freeze-dried product. The present invention relates to an apatite-coated substrate and a method for producing the same.

[0002]

2. Description of the Related Art Apatite is an inorganic calcium phosphate compound generally existing in the living body. The biological apatite of teeth and bones has a composition close to that of hydroxyapatite,
The usefulness of synthetic apatite as an artificial tooth root, artificial bone and bone substitute material is widely recognized. Synthetic apatite is molded into a dense body, a granular body, a porous body having an arbitrary porosity, and the like for clinical application. Synthetic apatite does not have a self-hardening property by itself, and thus needs to be sintered to be solidified.

Sintered apatite has significantly improved physicochemical properties and biocompatibility by the sintering treatment for forming, but is almost not absorbed by cells in vivo. Such a property is suitable because, when the sintered apatite is applied to an artificial dental root or the like, it is not absorbed into the tissue but is firmly adhered semipermanently. On the other hand, sintered apatite has an in-vivo activity of inducing bone formation by melting its surface in vivo and forming a chemical bond with bone even if it is not absorbed by cells in vivo. Have. Sintered apatite is also used as a bone substitute material because it has in vivo activity.

[0004] Heretofore, many studies have been reported to improve apatite by compounding it with various polymers. For example, it has been proposed to mix sintered apatite with an organic polymer such as gel-like collagen, fibrin, chitin and sodium alginate and simply dry the mixture or process it into a sheet or fiber and then dry. There is. This mixture is clinically applied as a biomaterial, and actually uses granular apatite as a bone filling material for periodontal bone defect or ridge preparation. In this bone filling material, the gel-like collagen and the fibrin glue function as an auxiliary agent for the purpose of initial fixing after the insertion of the filling material and after the operation.

In the above-mentioned bone prosthetic material, there is no bonding relationship between the sintered apatite and the polymer, and these components are merely mixed, and the mechanical properties required for the organic polymer as a base material. Will be responsible for the physical strength. Therefore, this bone prosthetic material is likely to collapse when wetted with physiological saline or blood, leaving a problem regarding the stability of the prosthetic material during insertion and initial fixation after surgery. Since the sintered apatite in the bone filling material is highly crystalline, it is not absorbed in the living body and is not a bone filling material or a bone replacement material in the original meaning.

In addition, a study has been reported that apatite is deposited in a biological tissue fluid on a polymer obtained by graft-polymerizing a phosphate group onto a high-density polyethylene film with respect to apatite (eg, Yoshihiro Yoshika, ON Tretinn).
i, Naohide Tomiyama, Yoshito Raito, Deposition of apatite on polymer surface, 9th Apatite Study Group, Proceedings 29, 199
3 years). In this research report, it takes about one month or more for the deposition of apatite, and the high molecular weight polyethylene must be ion-sputtered before the graft polymerization.
For this study, it remains unclear whether apatite is chemically bound to the phosphate groups attached to polyethylene. The reason for this is that the present inventors have confirmed that the apatite deposits almost in the same manner even in a polyethylene film which has been subjected to ion sputtering without graft polymerization of phosphate groups. In any case, the composite of the apatite and the polymer thus obtained is not used as a biomaterial, but is mainly applied as an industrial material. If deposition of apatite requires more than one month, it will be put into practical use. Is practically difficult. Further, the shape of polyethylene to be used needs to be relatively simple in order to carry out the ion sputtering process, so that even if this polymer is used as a biomaterial, its application range is extremely limited.

[0007]

When synthetic apatite is used as a bone filling material or bone replacement material, it does not disintegrate even when wetted with physiological saline or blood, and when the bone filling material is inserted and after the initial operation. It is desirable to be stable when fixing. Since synthetic apatite needs to be similar in physical and chemical properties to biological apatite in order to be absorbed in the living body, it is essential to be a bone substitute material or a bone replacement material in the original sense, and moreover, the production of apatite. It is also necessary for practical use to achieve the above in the shortest possible time. Further, the shape of the effective molecule to be used needs to be arbitrarily selected from a dense body, a granular body, a porous body, a sheet-like body, a fibrous body and the like.

[0008] Generally, the deposition of apatite on the surface of an inorganic substance or an organic substance in a solution containing calcium and phosphate ions is commonly experienced on a daily basis because the apatite adheres to a glass or plastic reaction vessel after an apatite deposition experiment. ing. At this time, the supply sources of calcium and phosphate ions may be reagents or other soluble calcium phosphate compounds or bioglass. When apatite adheres to a container, glass and plastics rarely act as nuclei of apatite, and it has been found that a crystal growth reaction mainly using apatite as a seed precedes.

When apatite is precipitated from a solution containing calcium and phosphate ions, organic substances such as proteins usually have an inhibitory effect on precipitation. That is, the organic substance easily forms a complex with calcium and phosphate ions, which causes a substantial decrease in the concentration of calcium and phosphate ions in the solution, the degree of supersaturation with respect to apatite is reduced, and the precipitation of apatite is delayed. Further, the organic matter is easily adsorbed by the precipitated apatite and has an action of inhibiting the crystal growth of the apatite. These inhibitory effects of organic matter depend on how freely the organic matter can be translationally diffused in the solution, and it can be presumed that if translational diffusion of the organic matter is suppressed, precipitation of apatite is promoted.

On the other hand, in a study on the clarification of the calcification mechanism of organic matter, it was reported that phosphoprotein extracted from dentin inhibits the precipitation and growth of calcium phosphate even at a very small concentration of about μmol. ing. However, it has also been confirmed that when the phosphoprotein is cross-linked to sepharose to suppress translational diffusion, the inhibitory effect on the precipitation of calcium phosphate salt disappears and, conversely, the precipitation of calcium phosphate salt is promoted. It has been found that the amount of precipitated calcium phosphate salt increases in proportion to the amount of crosslinked phosphoprotein, and the calcium phosphate salt is epitaxially precipitated.

The present inventors have paid attention to the above research from the viewpoint of synthesizing apatite, and by improving the precipitation operation of calcium phosphate salt in this research, it is possible to precipitate apatite on an organic polymer. I found out. The hydroxyapatite coated on the surface of an organic polymer has physicochemical properties very similar to those of biological apatite, and is useful when used as a synthetic aggregate.

Further, the culture substrate for tissue culture comprises a membrane of protein such as gelatin and collagen, a sponge, beads, a lyophilized culture bed, and the like. The bottom surface is usually coated with gelatin or collagen. In this type of culture substrate, when proteins such as gelatin and collagen are coated with apatite, the affinity with cells can be further increased.

Accordingly, the present invention aims to provide an apatite-coated substrate whose physicochemical properties closely resemble those of biological apatite. Another object of the present invention is to
The purpose of the present invention is to provide a synthetic aggregate having a high practical value as a bone filling material or a bone replacement material. Still another object of the present invention is to provide a culture substrate that allows cell culture in a state closer to that of a living body. Another object of the present invention is to provide a method for producing an apatite-coated substrate that efficiently deposits apatite on the surface of an organic polymer.

[0014]

In order to achieve the above object, the apatite-coated substrate according to the present invention has a desired shape, in which the surface of an organic polymer is coated with a calcium phosphate compound, and the calcium phosphate compound has a high organic content. Substantially chemically bound to the molecule. As the calcium phosphate compound to be coated, hydroxyapatite having a composition similar to that of biological apatite is generally used, but calcium phosphate dihydrate or octacalcium phosphate can also be coated depending on the application. By coating the surface of the organic polymer as shown in the photographs of FIGS. 3 to 6, this calcium phosphate compound binds to the organic polymer much more strongly than the binding by Van der Waals force, which is unconfirmed. Can be presumed to be substantially a chemical bond.

In the present specification, the apatite-coated substrate means a substrate in which the surface of an organic polymer is coated with apatite, that is, various calcium phosphate compounds, and when the substrate itself has a desired shape, In some cases, the organic polymer is further coated on a glass container or beads. The apatite-coated substrate of the present invention is a synthetic aggregate,
Organic polymers that can be used as a culture substrate or other biomaterials and are applied include proteins and polyamino acids such as sepharose, collagen, fibrin, chitin, gelatin, poly-D-lysine or poly-L-lysine, Examples include synthetic polymers such as methylmethacrylate and natural polymers such as resins.

The apatite-coated substrate of the present invention can be formed into a desired shape such as a dense body, a granular body, a porous body, a sheet-like body, or a fibrous body. When this apatite-coated substrate is used as a culture substrate, even if the culture bed is a protein or polyamino acid membrane, sponge, beads, lyophilized product, etc., it is a culture vessel with an inner bottom surface coated with protein or polyamino acid. May be. This incubator consists of glass or plastic petri dishes, dishes, plates,
It may be a flask, tray, or cell desk.

In the present invention, the synthetic aggregate means that it has a chemical composition different from that of the conventional artificial bone material, and although the bone prosthetic material and the bone substitute material are mainly used, only the artificial bone material is used. It is not limited to. When this synthetic aggregate is absorbed in vivo, it is necessary to select a protein or polyamino acid that is absorbed in vivo as the organic polymer. Further, when the synthetic aggregate is used as a bone substitute or bone substitute, the organic polymer is required to have biocompatibility, and collagen is suitable in this sense. Since gelatin is a modified form of collagen and has the same chemical composition, hydroxyapatite can be precipitated by the same treatment as collagen.

In the method of the present invention, the hydroxyapatite is deposited on the surface of the organic polymer by immersing the organic polymer in a supersaturated solution of a calcium phosphate compound. The supersaturated solution of the calcium phosphate compound contains, for example, a calcium salt and a phosphate or a calcium monoester phosphate, and in the case of the calcium monoester calcium salt, a hydrolase is also added. It is necessary that the organic polymer used is preliminarily cross-linked so as not to form a complex in a supersaturated solution of a calcium phosphate compound, and its translational diffusion is suppressed to accelerate the precipitation of apatite from the solution. In order to suppress the translational diffusion of the protein which is an organic polymer, a cross-linking formulation is relatively easy and preferable, but in addition to this, a formulation in which an inhibiting substance is added to a solution containing calcium and phosphate ions can also be carried out.

To crosslink a protein with an organic polymer,
For example, sepharose, collagen,
A protein such as fibrin, chitin, gelatin or a polyamino acid is used, and a phosphoprotein is cross-linked to this. This phosphoprotein may be phosvitin, casein, vitelin derived from egg yolk, phosphoprotein derived from hard tissue, or the like.
Applicable cross-linking agents are dimethyl suberoimidate, diethyl malonimidate dichloride, hexamethylene diisocyanate, glutaraldehyde, hydrazine, diphenylphosphoriazide, N-ethyl-N '-(3-dimethyl-aminopropyl). ) Carbodiimide hydrochloric acid and the like. Since the cross-linking agent inhibits the precipitation and growth of apatite,
It is desirable to thoroughly wash in advance.

In order to prepare a supersaturated solution of a calcium phosphate compound, a calcium compound such as calcium nitrate and a phosphate compound such as potassium dihydrogen phosphate are separately dissolved in a buffer solution, and the pH is 6.5 to 10 at a liquid temperature of 37 ° C. It is preferable to adjust to. At this time, if the pH is about 6 or less,
The dicalcium phosphate or octacalcium phosphate is likely to precipitate. On the other hand, when the pH is 11 or more, calcium hydroxide becomes a stable phase and the apatite precipitation efficiency deteriorates. A known pH stat (automatic pH adjuster) may be used in place of the buffer such as Hepes or Tris, and in this case, the calcium compound and the phosphate compound may be dissolved in water.

As a supersaturated solution of another calcium phosphate compound, a calcium salt of a phosphate monoester such as β-glycerophosphate may be used in place of the calcium compound and the phosphate compound, in which case a phosphonate such as alkaline phosphatase is used. Monoesterase is also used. Phosphomonoesterase is a general term for enzymes that hydrolyze phosphate monoesters. The calcium salt of phosphoric acid monoester and alkaline phosphatase are adjusted to pH 5.5 to 10.5 at a liquid temperature of 37 ° C., and preferably adjusted to pH 8.5 to 9.5 suitable for the activity of alkaline phosphatase.

Further, in order to absorb the obtained synthetic aggregate faster in vivo, carbon dioxide is added to a supersaturated solution of a calcium phosphate compound so that about 6% by weight of carbonate ion is contained in the crystal lattice as in the case of bone apatite. A compound may be added to increase the amount of carbonate ions contained in the crystal lattice of precipitated apatite. The concentration of the carbonate compound to be added varies depending on the organic polymer used, and is generally in the range of 0 to 20 mmol / liter, preferably around 4 mmol / liter. The type of the carbonic acid compound is not particularly limited, but if the calcium salt or phosphate of the carbonic acid compound is used, the calcium salt and phosphate concentrations in the calcium phosphate compound will vary.

[0023]

In the apatite-coated substrate of the present invention, the coated apatite has a low crystallinity and is very similar to biological apatite. Even if the synthetic aggregate is ultrasonically cleaned, the hydroxyapatite is not detached, and the hydroxyapatite is extremely strongly bonded to the organic polymer. Further, it is presumed that the synthetic aggregate does not disintegrate even when it is wet with physiological saline or blood, and that hydroxyapatite is substantially chemically bonded.

In the method of the present invention, the amount of precipitated apatite is generally adjusted by the reaction time, but the precipitation reaction can be further promoted by adding appropriate calcium salts and phosphates to the buffer solution in advance. It is preferable that the amounts of calcium salt and phosphate in the solution of the calcium phosphate compound are usually determined before and after the saturation even if the hydroxyapatite is supersaturated. When the concentration of the additive such as calcium salt and phosphate is excessively increased, the precipitation of hydroxyapatite in the solution starts, and the hydroxyapatite does not precipitate efficiently on the protein.

In the method of the present invention, in the cross-linking reaction of phosphoprotein with an organic polymer, an amino group of a protein or polyamino acid,
Imino groups, carboxyl groups, etc. are involved, and it is possible that more functional groups may be involved if the cross-linking agent is changed.
For this reason, at present, it is difficult to completely specify the functional groups involved in the above-mentioned crosslinking reaction, and there is applicability to a wide range of organic polymers.

[0026]

EXAMPLES Next, the present invention will be explained based on examples. Example 1 As a buffer solution, 20 to 100 mmol of N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (trade name: Tris, manufactured by Maruishi Chemical Co., Ltd.) is dissolved in distilled water. Next, the concentration of calcium nitrate is 0.5 to 2.6 mmol /
Liter and potassium dihydrogen phosphate (Kishida Chemical)
Were separately dissolved in the above buffer solution so that the concentration became 0.3 to 1.6 mmol / liter, and the pH was 6.5 to 10 at 37 ° C.
Adjust to. Each of these solutions is stored in a refrigerator set at 4 ° C. for long-term storage.

On the other hand, phosvitin is sepharose (trade name: EHAH-Sepharose 4B, manufactured by Formacia).
Since N-ethyl-N '-(3-dimethyl-aminopropyl) carbodiimide hydrochloric acid (manufactured by Nacalai Tesque) is used as a cross-linking agent for cross-linking at 5 μg or less per unit mg,
Make an aqueous solution having a concentration of 3 mg with respect to 1 ml of water.
Phosvitin and Sepharose are left in an aqueous solution of cross-linking agent for 1 day. After completion of standing, the phosvitin-crosslinked sepharose is washed on the glass filter to remove unreacted phosvitin and reaction by-products. The washed phosvitin-crosslinked sepharose is stored in a refrigerator set at 4 ° C.

The calcium nitrate and potassium phosphate solutions described above are heated to 37 ° C., and phosvitin-crosslinked sepharose is immersed in a mixed solution of equal amounts. Depending on the degree of supersaturation of the solution, in the case of a suitable supersaturated solution, apatite precipitates on Sepharose in about half a day. In order to deposit apatite on Sepharose in a large amount, the above dipping operation may be repeated.

X-ray analysis and infrared resonance absorption spectrum analysis of hydroxyapatite-bound sepharose
Precipitated apatite has low crystallinity, which is very similar to the X-ray analysis of the femur of the bovine bone (about 4 years old) used for comparison. In this apatite-bonded sepharose, the hydroxyapatite is not detached even after ultrasonic cleaning, and the hydroxyapatite is extremely strongly bonded to the sepharose. Moreover, since it does not disintegrate even when it is wet with physiological saline or blood, it is effective when used as a synthetic aggregate.

Example 2 The hydroxyapatite-bound sepharose produced in Example 1 is in a granular form and can be used as it is as a culture substrate similar to that in a living body. Further, the hydroxyapatite-bonded sepharose produced in Example 1 is dissolved in hot water and coated on the inner bottom surface of an incubator such as a petri dish or plate. The obtained culture substrate can be used as an incubator for tissue culture.

Example 3 As a buffer, 20 to 200 mmol of tris (hydroxymethyl) aminomethane is dissolved in distilled water. This buffer solution is kept warm at 37 ° C. and adjusted to pH 5.5 to 10.5, preferably pH 8.5 to 9.5. First, β-glycerophosphate is dissolved in the above buffer solution to a concentration of 2 to 12 mmol / liter.

On the other hand, the concentration of phosphoprotein phosvitin was about 0.04%, the concentration of hydrolase alkaline phosphatase (phosphate monoesterase type I) (manufactured by SIGMA) was about 0.04%, and Dimethyl suberoimidate (manufactured by SIGMA) is separately dissolved in the above buffer solution so that the concentration becomes about 0.03%, and these are stored in a refrigerator set at 4 ° C for long-term storage.

Phosvitin and alkali to collagen
30 mg of fibrillated collagen gel (S in cross-linked wet state of phosphatase)
IGMA), a dried body of the gel and a collagen sheet (all atelocollagen type I) (manufactured by Meiji Seika) are placed in a glass container. The collagen sheet has a shape as shown in FIGS. 1 and 2 when enlarged. To this glass container, 10 ml of the phosvitin solution, the enzyme solution and the cross-linking agent solution are added. This 3
The mixture is allowed to stand at 7 ° C. for 6 days, after the completion of the standing, the supernatant liquid is drained off and lightly washed with the buffer solution.

Deposition of apatite on crosslinked collagen fibers
Against out crosslinking treatment collagen is added above Phosvitin solution and each 10 ml of the enzyme solution.
This is left for about 3 hours at room temperature, and after the standing is completed, the supernatant liquid is drained off to quickly remove the excess liquid. Next, 40 ml of the above β-glycerophosphate calcium solution is added, and the mixture is left at 37 ° C. for about 20 hours.

In order to sufficiently deposit the hydroxyapatite on the collagen fibers, the addition of the phosvitin solution and the enzyme solution and the addition of the β-glycerophosphate solution are repeated 14 to 28 times. The white hydroxyapatite can be visually confirmed by repeating the operation 4 to 5 times, which is 4 to 5 days after the start of the reaction. The collagen sheet after 2 weeks from the start of the reaction is shown in FIGS. 3 and 4 and the collagen sheet after 4 weeks is shown in FIGS. 5 and 6 in an enlarged manner. The precipitation of white hydroxyapatite is clear from FIGS. 3 to 6. Can be confirmed. The apatite-precipitated collagen is thoroughly washed with distilled water, freeze-dried and then stored in a refrigerator. When using this as a bone substitute material, etc., it is gas sterilized if necessary.

X-ray diffraction of apatite-bonded collagen showed that the precipitated apatite had almost the same crystallinity as bone apatite, and that the apatite contained a little less than 1% carbonate ion in the crystal lattice by infrared resonance absorption. ing. According to thermogravimetric analysis, the amount of apatite precipitated in the reaction for 2 weeks is about twice the weight of collagen, regardless of whether fibrillar collagen or collagen sheet is used. Then, in the reaction for 4 weeks, the amount of precipitated apatite increases by about 20 to 30%.

The bone prosthetic material produced using re-fibrogenic collagen has some elasticity and can be deformed by finger pressure or tweezers, and the bone prosthetic material can be peeled off or detached even under gradual deformation. do not do. This bone filling material can be easily moistened with physiological saline, blood, or the like, and can be easily filled into a bone defect portion having an arbitrary shape without collapsing at that time.

The bone prosthesis material produced in Example 3 is filled in the two-walled bone defect portion prepared in the canine lower molar as shown in FIG. The progress one month after this filling is shown in the micrograph of FIG. 8, and from FIG. 8 regeneration of the cementum, alveolar bone and periodontal ligament can be recognized in the two-walled bone defect.

Example 4 Collagen membrane having a uniform pore diameter of 30 mg in a wet state (equal mixture of type I and III) (manufactured by COSTAR)
Is placed in a glass container, and 10 ml of each of the phosvitin solution, the enzyme solution, and the crosslinking agent solution obtained in Example 3 are added. This is allowed to stand at 37 ° C. for 6 days, after completion of standing, the supernatant is poured off, and the buffer solution obtained in Example 3 is lightly washed to crosslink phosvitin and alkaline phosphatase with collagen.

The crosslinked collagen film is then treated in the same manner as in Example 3 to sufficiently deposit hydroxyapatite on the collagen fibers. The apatite-bound collagen obtained 2 weeks after the reaction was attached to the inner bottom surface of a membrane filter type incubator (trade name: Transwell) made of polycarbonate, and this incubator had higher affinity with cells, It is suitable for studies on the movement of metabolites during culture.

Example 5 A glass petri dish (trade name: Celtite C-, coated with collagen (type I, type IV) on the inner bottom surface thereof.
4, manufactured by Sumitomo Bakelite Co., Ltd.), and the appropriate amount of the phosvitin solution, the enzyme solution, and the crosslinking agent solution obtained in Example 3 are added to the dish. This is left at 37 ° C. for 6 days and further washed with the buffer solution obtained in Example 3 to crosslink phosvitin and alkaline phosphatase with the collagen coat.

The crosslinked collagen coat is then treated in the same manner as in Example 3 to sufficiently deposit hydroxyapatite on the collagen coat in the petri dish. The collagen-coated dish obtained 2 weeks after the reaction has a higher affinity for cells and is suitable for primary culture of epithelial cells.

Example 6 In order to deposit calcium phosphate dihydrate or octacalcium phosphate on collagen fibers by treating in the same manner as in Example 3, phosphate monoesterase type II or type IV (SIGMA) was used as an enzyme. Company) is used. PH of reaction solution
Is set to an acidic region, preferably pH 4.5 to 6.2.

The bone substitute obtained by precipitating calcium phosphate dihydrate or octacalcium phosphate on collagen fibers has a little elasticity like the bone substitute prepared in Example 3, and can be acupressure or It can be deformed by tweezers or the like, and the bone prosthetic material does not peel or come off even under gradual deformation. This bone filling material can be easily moistened with physiological saline, blood, or the like, and can be easily filled into a bone defect portion having an arbitrary shape without collapsing at that time.

[0045]

EFFECTS OF THE INVENTION The apatite-coated substrate of the present invention has physicochemical properties similar to those of biological apatite and can be selected in any shape, and has practical value as a synthetic aggregate in the original meaning, a culture substrate, etc. high. This apatite coated substrate is
Hydroxyapatite does not desorb even when ultrasonically washed when used as a synthetic bone material such as bone filling material or bone replacement material, and does not disintegrate even when wetted with physiological saline or blood,
It is extremely stable when inserting a bone substitute and during initial fixation after surgery. Further, when this apatite-coated substrate is used as a culture substrate for tissue culture, the affinity with cells becomes higher than that of the conventional culture substrate.

According to the method of the present invention, an apatite-coated substrate can be easily produced simply by immersing an organic polymer in a supersaturated solution of a calcium phosphate compound. In order to produce a desired synthetic aggregate or culture substrate, an organic high It is possible to select a protein or polyamino acid that is absorbed in vivo as a molecule. Further, the method of the present invention, when the mechanical strength and elasticity of the synthetic aggregate is required, by appropriately changing the quality and shape of the organic polymer or the coating source, an apatite-coated substrate having desired properties. Can be manufactured.

[Brief description of drawings]

FIG. 1 shows a collagen sheet used in Example 2.
It is a weak magnification electron micrograph of 00 times.

2 is a 600 times magnified electron microscope photograph of FIG. 1. FIG.

FIG. 3 is a 200 × weak electron microscope photograph showing a collagen sheet in which hydroxyapatite was deposited for 2 weeks in Example 2.

FIG. 4 is a 600 times magnified electron microscope photograph of FIG.

FIG. 5 is a 200 × weak magnification electron micrograph showing a collagen sheet in which hydroxyapatite was deposited for 4 weeks in Example 2.

FIG. 6 is a 600 × magnified electron microscope photograph of FIG.

FIG. 7 is a photograph showing a state in which a two-walled bone defect portion was prepared in a dog's lower molar region.

[Fig. 8] A two-walled bone defect portion was prepared in a dog's lower molar region,
1 is a photomicrograph showing the progress of one month after filling with the synthetic aggregate of the present invention.

[Procedure amendment]

[Submission date] July 5, 1995

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 7

[Correction method] Change

[Correction content]

[Figure 7]

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Correction target item name] Figure 8

[Correction method] Change

[Correction content]

[Figure 8]

Claims (8)

[Claims] 1. An apatite-coated substrate in which a surface of an organic polymer having a desired shape is coated with a calcium phosphate compound, and the calcium phosphate compound is substantially chemically bonded to the organic polymer. 2. A synthetic aggregate in which the organic polymer having a desired shape is a protein or a polyamino acid, the surface of which is coated with hydroxyapatite, and the hydroxyapatite is substantially chemically bonded to the protein or the polyamino acid. . 3. Gelatin, sepharose, collagen, fibrin, chitin as protein or polyamino acid,
The synthetic aggregate according to claim 2, wherein poly-D-lysine or poly-L-lysine is used to cross-link the phosphoprotein before being immersed in a supersaturated solution of calcium phosphate. 4. A culture substrate in which the culture bed is an organic polymer having a desired shape, the organic polymer is coated with hydroxyapatite, and the hydroxyapatite is substantially chemically bonded to a protein or polyamino acid. . 5. The culture bed comprises a glass or plastic incubator coated with an organic polymer, and the organic polymer is gelatin, sepharose, collagen, fibrin, chitin, poly-D-lysine or poly-L-lysine. The culture substrate according to claim 4, which is a protein or a polyamino acid. 6. A method for producing an apatite-coated substrate, which comprises immersing an organic polymer in a supersaturated solution of a calcium phosphate compound to deposit the calcium phosphate compound on the surface of the organic polymer. 7. A solution of a phosphoric acid compound and a calcium compound is adjusted to pH 6.5 to 10, while a phosphoprotein is crosslinked with sepharose by a crosslinking agent, and this is immersed in a supersaturated solution of a calcium phosphate compound to form hydroxyapatite. A method for producing an apatite-coated substrate that is deposited on the surface of sepharose. 8. The pH of the phosphoric acid monoester calcium salt is adjusted to pH.
The phosphoprotein and phosphomonoesterase were cross-linked with collagen or gelatin by a cross-linking agent, and the resulting mixture was immersed in a supersaturated solution of calcium phosphate monophosphate to convert hydroxyapatite to collagen. Alternatively, a method for producing an apatite-coated substrate which is deposited on the surface of gelatin.