KR20130009720A - Osteoconductive bone graft and use thereof - Google Patents

Abstract

PURPOSE: A manufacturing method of xenogeneic bone originated bone graft substitute is provided to manufacture a xenogeneic bone originated bone graft substitute comprising pure hydroxyapatite with the excellent biocompatibility, cell adhesive ability and osteoconductivity by completely removing protein and fat from the xenogeneic bone. CONSTITUTION: A manufacturing method of xenogeneic bone originated bone graft substitute comprises the following steps:(i) step of cutting xenogeneic bone;(b) step of removing the blood component of the xenogeneic bone;(c) step of first removing fat and protein from the xenogeneic bone;(d) step of second removing fat and protein from the xenogeneic bone using an organic solvent;(e) step of removing the organic solvent;(f) step of processing the xenogeneic bone with sodium hypochlorite solution;(g) step of removing sodium hypochlorite solution;(h) step of completely removing the lipid and protein by heat-treating the xenogeneic bone;(i) step of cutting the xenogeneic bone in a block form; and(j) step of adding a bioactive substance to the xenogeneic bone.

Description

Bone graft derived from heterogeneous bone and method for manufacturing the same {Osteoconductive Bone graft and use thereof}

The present invention relates to a bone graft material having excellent biocompatibility, cell adhesion, and bone conductivity and a method of manufacturing the same using heterogeneous bone, and specifically, to remove proteins and fats from heterogeneous bones such as bovine bone, horse bone, and pig bone, and It relates to a method of preparing a particle or block type bone graft material having excellent biocompatibility, cell adhesion, and bone conductivity by surface treatment with an amino acid or adding a physiologically active substance.

Bone grafting substitute (BGS) is a bone grafting substitute (BGS), when a defect in bone tissue occurs due to various dental diseases, trauma, deterioration due to disease, or loss of other tissue, it replaces it and fills the space in the bone tissue and prevents the formation of new bone. It refers to a graft material used to promote it. Bone grafting materials include bio-derived bone grafting materials and synthetic bone grafting materials. Bio-derived bone graft materials include autogenous bone, allogeneic bone and heterogeneous bone. In order to obtain an autogenous bone, there is the inconvenience of having to perform surgery, and it has the disadvantage of being absorbed too quickly. On the other hand, allogeneic bone transplantation has concerns about immune response and the possibility of infection with viruses and diseases. In the case of artificially produced synthetic bone, there is no possibility of infection of viruses and diseases, but there is a problem that bone regeneration ability is lower than that of biologically derived bone, and absorption is too fast. To solve these shortcomings, a heterogeneous bone graft material derived from animal bones having a structure similar to that of human bones is used, and a representative product is BioOss from Geistlich. As a requirement to be developed as a xenograft material, only pure apatite should be extracted by removing xenoproteins and other impurities. It is most important to remove heterogeneous proteins that can cause an immune response and to prepare them to have a chemical composition similar to that of the bone tissue components of the human body. The method of manufacturing a bone graft material using the bone of the animal is to remove fat by adding a solvent having a boiling point of 80°C to 120°C to the bone of the cow's femur, and then add ammonia or primary amine to remove protein and organic matter. After preparing the mineral, it consists of a step of drying it by heating it for several hours at a high temperature of 250 ~ 600 ℃ (US Patent No. 5,167,961, US Patent No. 5,417,975).

On the other hand, the conventional method of manufacturing a block-shaped bone graft material was a method of coating ceramic on a porous polymer, heat treatment to burn the polymer, and sinter the remaining ceramic. The ceramic used is an artificially synthesized bone graft material, which is different from the bone graft material derived from heterogeneous bone (Korea Patent Application No. 10-2000-0046973).

There is a method of cutting homogeneous or heterogeneous bone tissue into a block, treated with hydrogen peroxide, demineralized with hydrochloric acid, treated with RSC solution, and then vacuum freeze-dried, but there is a possibility of causing a problem due to the remaining protein (Korean Patent Application No. 10). -2008-0138644).

Accordingly, the present inventors obtained a patent for a method of manufacturing a bone graft material without risk of infection of pathogenic substances by treating horse bones with sodium hypochlorite, pressing and high temperature treatment to effectively remove fat and organic matter. -0842012), there is a demand for a method of manufacturing a bone graft material to further improve the cell adhesion ability and new bone conduction ability of the bone graft material.

Accordingly, the present inventors have made diligent efforts to produce a bone graft material derived from heterogeneous bone consisting of pure hydroxyapatite from which proteins and fats have been removed, and excellent in biocompatibility, cell adhesion, and bone conduction, as a result of using sodium hypochlorite. It is treated and heated to high temperature to completely remove proteins and fats, and in this bone grafting material manufacturing process, acidic amino acids are used on the surface of the bone grafting material to reduce the roughness of the surface, thereby significantly increasing the adhesion of cells and derived from extracellular matrix. By introducing a component and a physiologically active factor such as a bone regeneration functional peptide into a bone graft material, it was confirmed that the conduction of new bone can be remarkably improved, and the present invention was completed.

An object of the present invention is to provide a bone graft derived from heterogeneous bone in the form of particles or blocks with improved biocompatibility, cell adhesion and bone conductivity while being composed of pure hydroxyapatite by removing proteins and fats, and a method for producing the same.

In order to achieve the above object, the present invention provides a method for producing a bone graft derived from heterogeneous bone in the form of particles comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) crushing the bone from which the blood component has been removed; (d) first removing and drying fats and proteins from the crushed bone; (e) immersing the dried bone in an organic solvent and shaking to secondly remove fat and protein from the bone; (f) removing and drying the organic solvent; (g) treating the bone powder from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing and drying the sodium hypochlorite solution from the bone powder; (i) heat-treating the dried bone powder to completely remove lipids and proteins; (j) separating the heat-treated bone powder into a sieve having pores of 212 to 1000 μm and 1000 to 2000 μm, respectively; And (k) treating the surface of the bone powder with an acidic amino acid.

The present invention also provides a block-type xenograft-derived bone graft manufacturing method comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) first removing and drying fats and proteins from the bones from which the blood components have been removed; (d) immersing the dried bone in an organic solvent and shaking to secondly remove fat and protein from the bone; (e) removing and drying the organic solvent; (f) treating the bone from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (g) removing sodium hypochlorite solution from the bone and drying it; (h) heat-treating the dried bone to completely remove lipids and proteins; (i) cutting the bone into blocks of a desired size; And (j) treating the surface of the bone with an acidic amino acid.

The present invention also provides a method for producing a bone graft material derived from heterogeneous bone in the form of particles comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) crushing the bone from which the blood component has been removed; (d) first removing and drying fats and proteins from the crushed bone; (e) immersing the dried bone in an organic solvent and shaking to secondly remove fat and protein from the bone; (f) removing and drying the organic solvent; (g) treating the bone powder from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing and drying the sodium hypochlorite solution from the bone powder; (i) heat-treating the dried bone powder to completely remove lipids and proteins; (j) separating the heat-treated bone powder into a sieve having pores of 212 to 1000 μm and 1000 to 2000 μm, respectively; And (k) adding at least one physiologically active substance selected from the group consisting of an extracellular matrix-derived component and a bone regeneration functional peptide to the bone powder.

The present invention also provides a block-type xenograft-derived bone graft manufacturing method comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) first removing and drying fats and proteins from the bones from which the blood components have been removed; (d) immersing the dried bone in an organic solvent and shaking to secondly remove fat and protein from the bone; (e) removing and drying the organic solvent; (f) treating the bone from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (g) removing sodium hypochlorite solution from the bone and drying it; (h) heat-treating the dried bone to completely remove lipids and proteins; (i) cutting the bone into blocks of a desired size; And (j) adding at least one physiologically active substance selected from the group consisting of an extracellular matrix-derived component and a bone regeneration functional peptide to the bone.

The present invention also provides a bone graft derived from heterogeneous bone in the form of particles or blocks prepared by the above manufacturing method.

The bone graft manufacturing method according to the present invention provides a bone graft material in the form of particles or blocks composed of pure hydroxyapatite by completely removing proteins and fats from heterogeneous bones such as bovine bone, horse bone, and pork bone. Compared to the low protein content, it is excellent in biocompatibility, so it is possible to manufacture a bone graft material that can be well fused without an inflammatory reaction in the tissue of the transplant site, and by treating the surface with an acidic amino acid, it is possible to increase the adhesion of bone cells. , By introducing a physiologically active factor, the conduction of new bone can be improved. The bone graft material according to the present invention can be used in dentistry, orthopedic surgery, plastic surgery, etc. to fill the bone damage to conduct the regeneration of new bone, and can also be used as a tissue engineering scaffold capable of culturing cells.

1 is a photograph of the appearance and observation of the particle shape and block shape bone graft material prepared in Example 1 with a differential scanning electron microscope.
Figure 2 shows the XRD measurement results of the particle shape and block type bone grafting material prepared in Example 1.
FIG. 3 shows a differential scanning electron microscope photograph of the surface of the bone graft material before and after treatment with an acidic amino acid on the particle and block type bone graft material prepared in Example 1. FIG.
4 is a result of observing the adhesion of cells before and after treatment with acidic amino acids to the particle and block bone graft materials prepared in Example 1. FIG.
5 is a result of observing the bone regeneration ability in the skull of a rabbit after adding an extracellular matrix to the bone graft material in the form of particles prepared in Example 1.
6 is a result of observing the adhesion of cells after treatment with an acidic amino acid to the bone graft material in the form of particles and blocks prepared in Example 1 and then adding an extracellular matrix.
7 is a result of observing the bone regeneration ability after treating the bone graft material in the form of particles prepared in Example 1, and the results of observing the bone regeneration ability, and after treating the surface with an acidic amino acid, even after treatment with the bone regeneration functional peptide It shows one result.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In one aspect, the present invention relates to a method for producing a bone graft material derived from heterogeneous bone in the form of particles comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) crushing the bone from which the blood component has been removed; (d) first removing and drying fats and proteins from the crushed bone; (e) immersing the bone powder in an organic solvent and shaking to secondly remove fat and protein from the bone; (f) removing and drying the organic solvent; (g) treating the bone powder from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing and drying the sodium hypochlorite solution from the bone powder; (i) heat-treating the dried bone powder to completely remove lipids and proteins; (j) separating the heat-treated bone powder into a sieve having pores of 212 to 1000 μm and 1000 to 2000 μm, respectively; And (k) treating the surface of the bone powder with an acidic amino acid.

In the present invention, the step (c) of crushing the bone from which the blood components have been removed is performed before removing the fat and protein from the bone, thereby increasing the surface area of the bone to facilitate the removal of fat and protein.

In the present invention, the step (e) of immersing the bone powder in an organic solvent is a step of removing residual fat present in the bone powder, and the organic solvent may be characterized in that it is a mixed solvent of chloroform and methanol, The mixed solvent used may have a chloroform:methanol ratio of 2 to 8:8 to 2, and is preferably 1:1.

In the present invention, the step of treating with sodium hypochlorite solution in step (g) is a step of decomposing and removing residual protein of bone powder into a water-soluble state, and inactivating denatured prion protein that causes mad cow disease. Sodium hypochlorite to be used may be a solution having a concentration of 2 to 20% (w/v), and it is most preferable to use a sodium hypochlorite solution having a concentration of 4% (w/v). In addition, the treatment time for the sodium hypochlorite is preferably 72 hours or more in order to remove prion and residual protein.

In the present invention, the heat treatment temperature in step (i) may be 550°C to 650°C.

In the present invention, the step (k) of treating the surface of the bone powder with the acidic amino acid may increase cell adhesion by adjusting the surface roughness of the bone graft material. Glutamic acid and aspartic acid may be used as the acidic amino acid. The treatment concentration of the acidic amino acid may be 1-20% (w/v), more preferably 5-10% (w/v). The treatment time can be 5-18 hours, more preferably 8-12 hours.

In another aspect, the present invention provides a method for producing a block-type xenograft-derived bone graft comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) first removing and drying fats and proteins from the bones from which the blood components have been removed; (d) immersing the dried bone in an organic solvent and shaking to secondly remove fat and protein from the bone; (e) removing and drying the organic solvent; (f) treating the bone from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (g) removing sodium hypochlorite solution from the bone and drying it; (h) heat-treating the dried bone to completely remove lipids and proteins; (i) cutting the bone into blocks of a desired size; And (j) treating the surface of the bone with an acidic amino acid.

In the present invention, the method of manufacturing a bone graft derived from xenograft in the form of a block does not include the step (c) of crushing the bone, unlike the method for manufacturing a bone graft in the form of particles, and after removing lipids and proteins from the bone, It includes the step of cutting into sized blocks.

In the present invention, the step (j) of treating the surface of the bone with the acidic amino acid can be used for both particle-type and block-type bone grafting materials, and increases cell adhesion by adjusting the surface roughness of the block-type bone grafting material. I can make it. Glutamic acid and aspartic acid may be used as the acidic amino acid. The treatment concentration of the acidic amino acid may be 1-20% (w/v), more preferably 5-10% (w/v). The treatment time can be 5-18 hours, more preferably 8-12 hours.

In one aspect, the present invention relates to a method for producing a bone graft material derived from heterogeneous bone in the form of particles comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) crushing the bone from which the blood component has been removed; (d) first removing and drying fats and proteins from the crushed bone; (e) immersing the dried bone in an organic solvent and shaking to secondly remove fat and protein from the bone; (f) removing and drying the organic solvent; (g) treating the bone powder from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing and drying the sodium hypochlorite solution from the bone powder; (i) heat-treating the dried bone powder to completely remove lipids and proteins; (j) separating the heat-treated bone powder into a sieve having pores of 212 to 1000 μm and 1000 to 2000 μm, respectively; And (k) adding at least one physiologically active substance selected from the group consisting of an extracellular matrix-derived component and a bone regeneration functional peptide to the bone powder.

In the present invention, the method may further include treating the surface of the bone with an acidic amino acid between steps (j) and (k). If the surface of the bone is treated with acidic amino acids and at least one physiologically active substance selected from the group consisting of components derived from extracellular matrix and functional peptides for bone regeneration is added, cell adhesion is increased and bone regeneration ability is increased. .

In another aspect, the present invention provides a method for producing a block-type xenograft-derived bone graft comprising the following steps:

(a) cutting the bone; (b) removing blood components from the cut bone; (c) first removing and drying fats and proteins from the bones from which the blood components have been removed; (d) immersing the dried bone in an organic solvent and shaking to secondly remove fat and protein from the bone; (e) removing and drying the organic solvent; (f) treating the bone from which the solvent has been removed with a 4% sodium hypochlorite solution to inactivate prion and other proteins; (g) removing sodium hypochlorite solution from the bone and drying it; (h) heat-treating the dried bone to completely remove lipids and proteins; (i) cutting the bone into blocks of a desired size; And (j) adding at least one physiologically active substance selected from the group consisting of an extracellular matrix-derived component and a bone regeneration functional peptide to the bone.

In the present invention, the method may further include treating the surface of the bone with an acidic amino acid between steps (i) and (j). If the surface of the bone is treated with acidic amino acids and at least one physiologically active substance selected from the group consisting of components derived from extracellular matrix and functional peptides for bone regeneration is added, cell adhesion is increased and bone regeneration ability is increased. .

In the present invention, new bone conduction may be promoted by adding one or more physiologically active substances selected from the group consisting of the extracellular matrix-derived component and bone regeneration functional peptide.

The extracellular matrix-derived component may be selected from the group consisting of collagen, fibronectin, laminin, hyaluronic acid, and glycosaminoglycans. The extracellular matrix-derived component binds to the bone graft material through physical adsorption and hydrogen bonding, and a chemical crosslinking agent can be used to strengthen the bonding force.

The bone regeneration functional peptide may be characterized in that (i) a peptide having bone regeneration ability and (ii) a peptide having an apatite binding ability are bonded to each other. (i) The peptide having bone regeneration ability may be selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 35, and (ii) the peptide having an apatite binding ability is the group consisting of the amino acid sequence of SEQ ID NO: 36 to SEQ ID NO: 39. Can be chosen from.

Specifically, (i) peptides having the bone regeneration ability are (a) bone morphogenetic proteins (bone morphogenetic protein, BMP) -2 , 4 amino acid sequence and amino acid sequence of each of the 2-18 positions of the 6 [BMP-2 In the case of (SEQ ID NO: 1), in the case of BMP-4 (SEQ ID NO: 2) and in the case of BMP-6 (SEQ ID NO: 3)], the amino acid sequence at positions 16-34 of BMP-2 (SEQ ID NO: 4), 47-71 Amino acid sequence at positions (SEQ ID NO: 5), amino acid sequence at positions 73-92 (SEQ ID NO: 6), amino acid sequence at positions 88-105 (SEQ ID NO: 7), amino acid sequence at positions 83-302 (SEQ ID NO: 8), 335 The amino acid sequence at position -353 (SEQ ID NO: 9) and the amino acid sequence at position 370-396 (SEQ ID NO: 10); BMP-4 amino acid sequence at position 74-93 (SEQ ID NO: 11), amino acid sequence at position 293-313 (SEQ ID NO: 12), amino acid sequence at position 360-379 (SEQ ID NO: 13) and amino acid sequence at position 382-402 (SEQ ID NO: 14) Amino acid sequence at positions 91-110 of BMP-6 (SEQ ID NO: 15), amino acid sequence at positions 407-418 (SEQ ID NO: 16), amino acid sequence at positions 472-490 (SEQ ID NO: 17) and 487- Amino acid sequence at position 510 (SEQ ID NO: 18) and amino acid sequence at positions 98-117 of BMP-7 (SEQ ID NO: 19), amino acid sequence at positions 320-340 (SEQ ID NO: 20), amino acid sequence at positions 400-409 (SEQ ID NO: Number 21) and the amino acid sequence at positions 405-423 (SEQ ID NO: 22);

(b) the amino acid sequence at positions 62-69 (SEQ ID NO: 23) of the bone sialoprotein II (BSP II), the amino acid sequence at positions 140-148 (SEQ ID NO: 24), the amino acid sequence at positions 259-277 (SEQ ID NO: 25), Amino acid sequence at positions 199-204 (SEQ ID NO: 26), amino acid sequence at positions 151-158 (SEQ ID NO: 27), amino acid sequence at positions 275-291 (SEQ ID NO: 28), amino acids at positions 20-28 (SEQ ID NO: 29) , An amino acid sequence at positions 65-90 (SEQ ID NO: 30), an amino acid sequence at positions 150-170 (SEQ ID NO: 31) and an amino acid sequence at positions 280-290 (SEQ ID NO: 32);

(c) any one or more selected from the group consisting of the amino acid sequence YGLRSKS (SEQ ID NO: 33), KKFRRPDIQYPDAT (SEQ ID NO: 34) and YGLRSKSKKFRRPDIQYPDAT (SEQ ID NO: 35) at positions 149-169 of bone sialoprotein I (BSP I, osteopontin) It may be characterized as being a peptide.

(ii) The peptide binding to the apatite mineral can be selected from the group consisting of SEQ ID NO: 36 STLPIPHEFSRE), SEQ ID NO: 37 (VTKHLNQISQSY), SEQ ID NO: 38 (SVSVGMKPSPRP), and SEQ ID NO: 39 (NRVFEVLRCVFD), and has bone tissue regeneration ability. It is chemically added to the N-terminus of the peptide to increase the binding ability to apatite, a constituent of bone, so that it can be stably bonded to a bone graft material or an apatite-coated implant surface.

In the present invention, the bone regeneration functional peptide is a peptide in which (i) a peptide having bone regeneration ability and (ii) a peptide having an apatite binding ability are bound, and can exist in a stable state by binding to the apatite surface, so that it is used for dental or orthopedics. It can be applied to surgical bone substitutes and apatite-coated metals, natural polymers, and synthetic polymers, and promotes the migration, proliferation and differentiation of cells related to bone tissue regeneration, thereby maximizing bone tissue regeneration capacity. Since it can exist stably while maintaining the peptide activity, it is useful for the development of bone tissue regeneration treatment technology using this.

In the present invention, the bone regeneration functional peptide is stably immobilized on the apatite surface, thereby increasing the stability of the peptide and maintaining its activity for a long period of time. Therefore, when implanted into the body, it is stably maintained at the implanted area, so that the effect of bone regeneration by the peptide can be sustained, and this has characteristics suitable for the treatment of regeneration of bone tissue and periodontal tissue.

In the present invention, the bone regeneration functional peptide may bind to an apatite selected from the group consisting of biologically derived hydroxyapatite bone minerals, synthetic hydroxyapatite, carbonate apatite, tricalcium phosphate, and monocalcium phosphate.

In the present invention, the dose of the bone regeneration functional peptide is preferably to contain 1-100mg per unit weight of the bone graft material (1g), more preferably 20-80mg per unit weight of the bone graft material. have.

In an embodiment of the present invention, a peptide having an apatite binding ability of SEQ ID NO: 36 and a peptide having a bone tissue regeneration ability of SEQ ID NO: 35 are combined to produce a functional peptide for bone regeneration of SEQ ID NO: 40, and the produced peptide is stable in a bone graft material. It was confirmed that the binding was confirmed, and the bone regeneration ability was confirmed by transplanting a bone graft material having the peptide stably fixed on the apatite surface to the bone defect.

In the present invention, the heterogeneous bone may be characterized in that it is selected from the group consisting of bovine bone, horse bone, and pig bone.

In another aspect, the present invention relates to a bone grafting material in the form of particles or blocks prepared by the method for producing a bone grafting material derived from heterogeneous bone.

* In the present invention, the "bone graft substitute" is a material for filling the space in the bone tissue, and the bone graft material is putty , Paste, moldable strips, blocks, chips, etc. can be molded and used, and can be formulated and used in the form of gels, granules, pastes, tablets, pellets, etc. using chemical additives. It is also possible.

When a bone graft material is formulated and used as described above, growth factors that promote bone growth, fibrin, bone morphogenetic factors, bone growth agents, chemotherapeutic agents, antibiotics, analgesics, bisphosphonates, strontum salts, fluoride salts, magnesium salts And sodium salts. The growth factors include bone morphogenic protein (BMP), platelet-derived growth factor (PDGF), transgenic growth factor (TGF-beta), insulin-like growth factor (IGF-I), IGF-II, fibroblast growth factor (FGF). ) And BGDF-II (beta-2-microglobulin), etc. can be used. As the bone morphogenetic factor, osteocalcin, bonesialo protein, osteogenin, BMP, and the like may be used. The bone growth agent may be used without limitation as long as it is harmless to the human body and promotes bone growth, and peptides or nucleic acids that promote bone formation, antagonists of substances that inhibit bone formation, and the like may be used.

Chemical additives used to formulate the bone graft material in the present invention include hyaluronic acid, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate, ceramic, and the like. It can be formulated in the form of granules, chips, pellets, tablets, and pastes.

Collagen, gelatin, chitosan, alginate, carboxymethylcellulose, hydroxypropylmethylcellulose, polyethylene glycol, poloxamer, Biocompatible polymers including polylactic acid, polylactic glycolic acid, and polycaprolactone may be used.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example One : Bone graft Produce

Example 1-1: particle shape Bone graft Manufacturing method

[Pre-treatment and grinding process]

The bones obtained from the cow's femur were cut ^{into about 2 cm 3 size using a bone cutter.} The cut bone fragment was immersed in deionized water for 24 hours to remove blood components present in the bone. The bone from which the blood component was removed was crushed to a size of 0.7 mm or less using a grinder. The bone powder was boiled for 72 hours while exchanging deionized water every 12 hours to primarily remove fats and proteins present in the bone. The bone fragments from which the fat and protein were primarily removed were completely dried in an oven at 60° C. for 24 hours.

[Degreasing treatment process]

A solvent in which 20 ml of chloroform and methanol were mixed in a volume ratio of 1: 1 per 1 g of dried bone powder was added and shaken at a rotation speed of 120 rpm for 24 hours to perform degreasing treatment. Deionized water was added at a rate of 50 g per 1 g of bone powder to remove the solvent remaining in the bone powder after the degreasing treatment was completed, and then shaken at 120 rpm for 12 hours to remove the solvent remaining in the powder. At this time, the washing efficiency was improved by replacing with new deionized water every 2 hours. The bone powder after washing with water was completely dried in an oven at 60°C.

[Deproteinization treatment process]

Protein present in bone powder was removed by adding 25 ml of 4% (w/v) sodium hypochlorite solution per 1 g of degreasing-treated dry bone powder and shaking at a rotation speed of 120 rpm for 24 hours. . In order to remove the solvent remaining in the deproteinized bone powder, 50 g of deionized water per 1 g of the bone powder was added and shaken at 120 rpm for 72 hours to remove the remaining sodium hypochlorite. At this time, for the first 12 hours, new deionized water was exchanged every 2 hours, and after that, it was exchanged with new deionized water every 12 hours. The bone powder after washing with water was completely dried in an oven at 60°C.

[Heat treatment process]

The dried bone powder, which had been degreased and deproteinized, was heat-treated at high temperature to remove residual lipids and proteins. The electric furnace used for the heat treatment was heated to 2 degrees per minute, and the bone powder was heat-treated at 600° C. for 3 hours and then furnace cooled.

[Separation process]

After heat treatment is completed, the bone powder is filtered using a sieve of 212~1000㎛, 1000~2000㎛, and the filtered bone powder is washed several times with deionized water to remove fine dust remaining on the surface, and then dried in an oven at 60℃ for 24 hours. It was obtained and used as a bone graft material.

Example 1-2: block type Bone graft Manufacturing method

[Pre-treatment process]

^{8cm 3} of the bone obtained from the femur of the horse using a fracture cutter Cut to size. The cut bone fragment was immersed in purified water for 6 to 15 hours, then the purified water was changed and the blood component present in the bone fragment was removed by immersing again for 6 to 15 hours. The bone fragments from which blood was removed with the purified water were heated for 3 hours or more per day, and limited to a maximum of 6 hours. This method was repeated for a minimum of 72 hours to a maximum of 80 hours to primarily remove lipids and proteins present in bone fragments. The bone fragments from which the fat and protein were primarily removed were dried for 12 hours at 120°C. The dried bone fragments were classified into cavernous bones and dense bones, respectively.

[Degreasing treatment process]

A solvent in which 6 ml of chloroform and methanol were mixed in a 1:1 volume ratio per 1 g of dried bone fragments was added and immersed for 24 hours to perform degreasing treatment, and methanol was discarded. In order to remove the solvent remaining in the bone fragments after the degreasing treatment was completed, the methanol was discarded after immersion in 6 ml of methanol per 1 g of bone for 12 hours. In order to remove the methanol remaining in the bone fragments, it was washed 6 times with 10 ml of purified water per 1 g of bone fragments, and then washed 6 times each 1 hour by a pressure heating method. The bone fragments after washing were completely dried in an oven at 120° C. for 12 hours.

[Deproteinization process]

After immersing for about 6 hours by adding 8 ml of 4% (w/v) sodium hypochlorite solution per 1 g of the dried bone fragments that have been degreased, the foam subsides and the sodium hypochlorite becomes colorless. Sodium chlorate was replaced. This method was repeated 3 to 5 times. In order to remove the solvent remaining on the deproteinized bone fragments, the bone fragments were washed 10 times with purified water for 12 hours, and then washed with purified water once every hour with a Lab Stirrer for a total of 10 times. After washing 10 times, 10L of purified water was filled again, heated for 30 minutes, and discarded twice. After washing, the bone fragments were completely dried at 120° C. for 12 hours.

[Heat treatment process]

The dried bone fragments that had been degreased and deproteinized were heat-treated at high temperature to remove residual lipids and proteins. The electric furnace used for heat treatment was heated to 2 degrees per minute, and bone fragments were heat treated at 600°C for 3 hours and then furnace cooled. Heat-treated bone fragments are heated for 1 hour by adding purified water. Heat washing was repeated several times to remove fine dust, dried in an oven at 120° C. for 12 hours, and used as a bone graft material.

[Process of cutting into blocks]

The dried bone graft material was cut into blocks of the desired size.

Example 1-3: above Bone graft Observation of appearance and crystallinity

The appearance and interior of the particle and block bone graft materials prepared in Examples 1-1 and 1-2 were observed. Inside, a differential scanning electron microscope was used. In addition, XRD was measured to analyze the degree of crystallinity to determine whether the bone graft material prepared as described above was made of apatite crystals. FIG. 1A is an appearance photograph and a differential scanning microscope photograph of a particle-shaped bone grafting material, and FIG. 1B is an appearance photograph and a differential scanning microscope photograph of a block-shaped bone grafting material. 2 is an XRD graph, FIG. 2A is an XRD graph of a particle-type bone grafting material, and FIG. 2B is an XRD graph of a block-type bone grafting material. It was confirmed that all were made of pure apatite crystals.

Example 2: Treatment with acidic amino acid

Example 2-1: surface-treated Bone graft Manufacturing method

Each 10 g of the bone grafting material prepared in Example 1 was put in 50 mL of a 5% (w/v) aspartic acid solution, immersed, and left for 6 hours. In order to remove the aspartic acid solution, it was washed with purified water until pH 7.0±0.5, and dried in an oven.

Example 2-2: Observation of roughness after surface treatment

The bone graft material prepared in Example 2-1 was fixed with a 2% glutaraldehyde solution. The fixed bone graft material was treated with 1% osmium tetroxide solution, washed, dehydrated and dried. The surface of the bone graft prepared as described above was observed with a differential scanning electron microscope.

The observation results are shown in a differential scanning electron microscope photograph of the bone graft material in FIG. 3. 3A is a photograph of the surface of the bone graft material in the form of particles before and after surface treatment, and FIG. 3B shows the surface of the bone graft material in the form of blocks before and after surface treatment.

The block-shaped bone graft material seems to have pores connected to the inside, and has a structure suitable for use as a tissue engineering support. In addition, it can be seen that the roughness decreased after surface treatment with an acidic amino acid.

Example 2-3: by surface treatment Bone graft Cell adhesion observe

The particle-type and block-type bone graft material prepared in Example 1 was placed in a 4-well chamber slide, and the cells were inoculated and incubated for 4 hours each. The bone graft material in which the cells (human osteosarcoma cells, purchased from Korea Cell Line Bank) were cultured was fixed with a 2% glutaraldehyde solution. The fixed bone graft material was treated with 1% osmium tetroxide solution, washed, dehydrated and dried. The surface of the bone graft material in which the cells were cultured was observed with a differential scanning electron microscope (FIG. 4). The above results were compared with the observation results for cell adhesion ability after surface treatment with an acidic amino acid as in Example 2-1.

As a result, as shown in FIG. 4, both the particle-type bone graft material (FIG. 4a) and the block-type bone graft material (FIG. 4B) showed that cell adhesion of the bone graft material after surface treatment was compared to that of the bone graft material before surface treatment. It can be seen that it was significantly increased. In the case of the block-shaped bone graft material, it was confirmed that the cells were well attached into the pore. This is because, as the fine powder on the surface of the bone graft material is removed by acidic amino acid treatment, the surface of the bone graft material becomes suitable for attaching cells.

Example 3: Extracellular matrix Addition of derived ingredients

Example 3-1: Extracellular matrix Containing derived ingredients Bone graft Produce

100 mg of ribose (D-ribose) was added and dissolved in 50 ml of a 2% (w/v) collagen solution. After putting 10 g of the bone graft material in the form of particles or blocks prepared in Example 1, it was placed in a desiccator and held in a vacuum state for 1 hour to allow the collagen solution to soak into the bone graft material. It was left for 5 days in refrigeration (4℃), and only bone graft material was collected and lyophilized. It was left to stand for 48 hours under vacuum at 140° C. and dried.

Example 3-2: Extracellular matrix By adding derived ingredients Bone graft Bone regeneration ability observe

The bone graft material prepared in Example 3-1 was transplanted from the defect of the skull of a rabbit to confirm bone regeneration ability. A circular bone defect with a diameter of 8 mm was formed on the skull of an anesthetized rabbit (Newzealand White rabbit, species name: cuniculus), and a collagen shielding membrane was covered on the bone defect, and the periosteum and the skin were double-sealed. Four weeks after transplantation, the animals were sacrificed, and the collected samples were fixed in formalin solution, and the tissues were embedded to prepare a specimen having a thickness of 20 μm. The prepared specimens were stained with basic fuchsin and toluidine blue to prepare non-demineralized specimens. The prepared specimen was observed with an optical microscope and photographed.

The left column is a photo at 40 magnification, and the right column is an enlarged photo of the square part of the left column at 100 magnification. GB stands for bone graft material, and NB stands for new bone. It was confirmed that the bone regeneration ability was increased in the case of the bone graft material (FIG. 5B) treated with the extracellular matrix-derived component than the bone graft material (FIG. 5A) that was not treated with the extracellular matrix-derived component (FIG. 5). This is because the introduction of extracellular matrix-derived components into the bone graft material created a more suitable environment for new bone to be regenerated.

Example 3-3: Acidic amino acid treatment + observation of cell adhesion ability of bone grafts by addition of components derived from extracellular matrix

As in Example 2-1, when the surface of the bone graft material was treated with an acidic amino acid and then the extracellular matrix-derived component was added, it was observed whether the cell adhesion ability further increased. As a result, as shown in Fig. 6, it was confirmed that the cell adhesion ability was further increased compared to the case where only the acidic amino acid was treated (Fig. 4). This is because the roughness is reduced by acidic amino acids, and the introduction of components derived from extracellular matrix creates an environment similar to that of the living body, making it more suitable for cell adhesion.

Example 4 : Bone regeneration Functional Peptide adding

Example 4-1: Bone regeneration Functional Peptide Containing Bone graft Manufacturing method

F-moc solid phase chemical synthesis using a peptide synthesis device to contain YGLRSKSKKFRRPDIQYPDAT (SEQ ID NO: 35) as a sequence with bone regeneration effect derived from Osteopontin in order from the N-terminal, and STLPIPHEFSRE (SEQ ID NO: 36) as a peptide with apatite binding ability. It was synthesized by the method. That is, it was synthesized using Rink resin (0.075mmol/g, 100 ~ 200 mesh, 1% DVB crosslinking) with Fmoc-(9-Fluorenylmethoxycarbonyl) bonded as a blocking group, and 50 mg of Rink resin in a synthesizer After swelling the resin with DMF, 20% piperidine/DMF solution was used to remove the Fmoc-group. From the C end in sequence, add 0.5M amino acid solution (solvent: DMF), 1.0M DIPEA (solvent: DMF&NMP), 0.5M HBTU (solvent: DMF) in 5, 10, and 5 equivalents, respectively, in a nitrogen stream for 1 to 2 hours. Reacted for a while. At the end of the deprotection and coupling steps, washing was performed twice with DMF and NMP. Even after coupling the last amino acid, deprotection was performed to remove the Fmoc-group.

To confirm the synthesis, the ninhydrin test method was used, and the resin that was tested and synthesized was dried with THF or DCM, added TFA cleavage cocktail at a rate of 20 ml per 1 g of resin, shaken for 3 hours, and filtered. The cocktail in which the resin and peptide are dissolved was separated. After removing the filtered solution using a rotary evaporator, add cold ether or directly add an excessive amount of cold ether to the TFA cocktail solution in which the peptide is dissolved to crystallize the peptide into a solid phase and centrifuge it. And separated. At this time, the TFA cocktail was completely removed through washing and centrifugation several times with ether. The peptide thus obtained was dissolved in distilled water and lyophilized.

NH ₂ -STLPIPHEFSRE-YGLRSKSKKFRRPDIQYPDAT-COONH ₂ (SEQ ID NO: 40)

The synthesized peptide sequence was cut from resin, washed, lyophilized, and separated and purified by liquid chromatography. The purified peptide was confirmed for molecular weight using MALDI analysis.

In order to test the stability in the body, 10 equivalents of FITC (Fluorescein isothicyanate) at the N-terminus at the time of preparation of SEQ ID NO: 40 was bound using triethylamine (1 ml per 1 g of resin), and the molecular weight was measured using MALDI-TOF. Synthesis was confirmed.

1200 mg of the peptide thus prepared was dissolved in 1 mL of third distilled water, added to 4 g of the bone graft material prepared in Example 1, immersed for 24 hours, and then lyophilized.

Example 4-2: Bone regeneration Functional Peptide In addition Hand Bone regeneration ability observe

As in Example 4-1, the peptides of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 40 were each bonded to the bone graft material, and then transplanted from the cranial circular bone defect of the rabbit to confirm bone regeneration. A circular bone defect with a diameter of 8 mm was formed on the skull of an anesthetized rabbit (Newzealand White rabbit, species name: cuniculus), and a bone graft material and a bone graft material containing a peptide were transplanted into the bone defect by 50 mg per defect, The periosteum and skin were double sutured. Two weeks after transplantation, the animals were sacrificed, and the collected sample was fixed in formalin solution, and the tissue was embedded to prepare a specimen having a thickness of 20 μm. The prepared specimens were stained with basic fuchsin and toluidine blue to prepare non-demineralized specimens. The prepared specimen was observed with an optical microscope and photographed.

In addition, the bone grafting material in the form of particles or blocks surface-treated with the acidic amino acid prepared in Example 2 was also treated with a bone regeneration functional peptide in the same manner as described above to observe the bone regeneration effect.

Figure 7 shows the bone regeneration effect by the bone grafting material to which the bone regeneration functional peptide is added, Figure 7a is the bone regeneration effect by the bone grafting material in the form of particles before treatment, Figure 7b is the particles after treatment with the bone regeneration functional peptide Bone regeneration effect by the form of bone grafting material, Figure 7c is the bone regeneration effect by the particle-type bone grafting material to which the bone regeneration functional peptide is also added after surface treatment with acidic amino acids. The left column is a photo at 40x magnification, and the right column is a photo enlarged at 100x magnification of the square part of the left column. GB stands for bone graft material, and NB stands for new bone.

As a result, it can be seen that when the bone regeneration functional peptide is treated, the bone regeneration effect is increased, and when both the acidic amino acid and the + bone regeneration functional peptide are treated, the effect is further increased. Therefore, when the acidic amino acid treatment and bone regeneration functional peptide of the present invention is used for a bone graft material made of apatite or an apatite-coated implant, the effect of regenerating bone tissue is expected to be great.

<110> Seoul national university, NIBEC <120> Osteoconductive Bone graft and use therof <130> P11-B119 <160> 40 <170> KopatentIn 1.71 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 1 Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His 1 5 10 15 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 2 Cys Ser Pro Lys His His Pro Gln Arg Ser Arg Lys Lys Asn 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 3 Cys Ser Ser Arg Lys Lys Asn Lys Asn Cys Arg Arg His 1 5 10 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 4 Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr His 1 5 10 15 Ala <210> 5 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 5 Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile 1 5 10 15 Val Gln Thr Leu Val Asn Ser Val Asn 20 25 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 6 Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser 1 5 10 15 Met Leu Tyr Leu 20 <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 7 Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr 1 5 10 15 Gln Asp <210> 8 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 8 Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp His Arg 1 5 10 15 Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe His His 20 25 30 Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr Thr Arg 35 40 45 Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe Ile Thr 50 55 60 Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala Leu Gly 65 70 75 80 Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile Ile Lys 85 90 95 Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu Asp Thr 100 105 110 Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp Val Thr 115 120 125 Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His Gly Phe 130 135 140 Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser Lys Arg 145 150 155 160 His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser Trp Ser 165 170 175 Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys Gly His 180 185 190 Pro Leu His Lys Arg Glu Lys Arg Gln Ala Lys His Lys Gln Arg Lys 195 200 205 Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr 210 215 220 <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 9 Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser 1 5 10 15 Met Leu Tyr Leu 20 <210> 10 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 10 Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr 1 5 10 15 Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg 20 25 <210> 11 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 11 Ser Ser Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile 1 5 10 15 Ser Met Leu Tyr Leu 20 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 12 Pro Lys His His Ser Gln Arg Ala Arg Lys Lys Asn Lys Asn Cys Arg 1 5 10 15 Arg His Ser Leu Tyr 20 <210> 13 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 13 Ser Ser Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile 1 5 10 15 Ser Met Leu Tyr Leu 20 <210> 14 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 14 Ser Met Leu Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr 1 5 10 15 Gln Glu Met Val Val 20 <210> 15 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 15 Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser 1 5 10 15 Val Leu Tyr Phe 20 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 16 Glu Leu Lys Thr Ala Cys Arg Lys His Glu Leu Tyr 1 5 10 <210> 17 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 17 Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser 1 5 10 15 Val Leu Tyr Phe 20 <210> 18 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 18 Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr 1 5 10 15 Arg Asn Met Val Val Arg Ala Cys 20 <210> 19 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Partial sequence of BMP-7 <400> 19 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser 1 5 10 15 Val Leu Tyr Phe 20 <210> 20 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Partial peptide of BMP-7 <400> 20 Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu 1 5 10 15 Tyr Val Ser Phe Arg 20 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Partial peptide of BMP-7 <400> 21 Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe 1 5 10 <210> 22 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Partial peptide of BMP-7 <400> 22 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 1 5 10 15 Arg Asn Met <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 23 Glu Glu Glu Gly Glu Glu Glu Glu 1 5 <210> 24 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 24 Asn Thr Thr Leu Ser Ala Thr Thr Leu Gly Tyr Gly Glu Asp Ala Thr 1 5 10 15 Pro Gly Thr Gly Tyr Thr Gly Leu Ala Ala Ile Gln Leu Pro Lys Lys 20 25 30 Ala Gly Asp Ile Thr Asn Lys Ala Thr Lys Glu Lys Glu 35 40 45 <210> 25 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 25 Tyr Glu Thr Tyr Asp Glu Asn Asn Gly Glu Pro Arg Gly Asp Thr Tyr 1 5 10 15 Arg Ile <210> 26 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 26 Glu Glu Gly Glu Glu Glu 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 27 Glu Glu Glu Glu Glu Glu Glu Glu 1 5 <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 28 Tyr Glu Ile Tyr Glu Ser Glu Asn Gly Glu Pro Arg Gly Asp Asn Tyr 1 5 10 15 Arg <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 29 Lys Asn Leu His Arg Arg Val Lys Ile 1 5 <210> 30 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 30 Asp Ser Ser Glu Glu Asn Gly Asp Asp Ser Ser Glu Glu Glu Glu Glu 1 5 10 15 Glu Glu Glu Thr Ser Asn Glu Gly Glu Asn 20 25 <210> 31 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 31 Asp Glu Glu Glu Glu Glu Glu Glu Glu Gly Asn Glu Asn Glu Glu Ser 1 5 10 15 Glu Ala Glu Val Asp 20 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 32 Ser Glu Asn Gly Glu Pro Arg Gly Asp Asn Tyr 1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 33 Tyr Gly Leu Arg Ser Lys Ser 1 5 <210> 34 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 34 Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr 1 5 10 <210> 35 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 35 Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln 1 5 10 15 Tyr Pro Asp Ala Thr 20 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> apatite binding peptide <400> 36 Ser Thr Leu Pro Ile Pro His Glu Phe Ser Arg Glu 1 5 10 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> aptite binding peptide <400> 37 Val Thr Lys His Leu Asn Gln Ile Ser Gln Ser Tyr 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> aptite binding peptide <400> 38 Ser Val Ser Val Gly Met Lys Pro Ser Pro Arg Pro 1 5 10 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> apatite binding peptide <400> 39 Asn Arg Val Phe Glu Val Leu Arg Cys Val Phe Asp 1 5 10 <210> 40 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> apatite binding smart peptide <400> 40 Ser Thr Leu Pro Ile Pro His Glu Phe Ser Arg Glu Tyr Gly Leu Arg 1 5 10 15 Ser Lys Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala 20 25 30 Thr

Claims (7)

Method for producing a bone-shaped xenograft-derived bone graft material comprising the following steps:
(a) cutting the xenograft; (b) removing blood components of the cut xenograft; (c) first removing fat and protein from the xenograft from which the blood component is removed and drying; (d) immersing the dried xenograft in an organic solvent and shaking the secondary to remove fat and protein from xenograft; (e) removing and drying the organic solvent; (f) treating the dislodged degenerated bone with a sodium hypochlorite solution at a concentration of 2-20% to inactivate prion and other proteins; (g) removing sodium hypochlorite solution from the xylem and drying; (h) heat treating the dried xenograft to completely remove lipids and proteins; (i) cutting the xylem into blocks of a desired size; And (j) adding at least one bioactive substance selected from the group consisting of extracellular matrix-derived components and bone regeneration functional peptides to the xenograft.
According to claim 1, Between (i) and (j), characterized in that it further comprises the step of treating the surface of the heterogenous bone with acidic amino acid, xenograft-derived bone graft material manufacturing method.
The method of claim 1, wherein the extracellular matrix-derived component is selected from the group consisting of collagen, fibronectin, laminin, hyaluronic acid and glycosaminoglycans. Method of producing bone graft derived bone xylem.
According to claim 1, wherein the bone regeneration functional peptide is at least one peptide selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 35 and one peptide selected from the group consisting of the amino acid sequence of SEQ ID NO: 36 to SEQ ID NO: 39 Method for producing a bone bone-derived bone graft material, characterized in that the combined peptide.
The method of claim 1, wherein the bone regeneration functional peptide is a bone bone-derived bone graft manufacturing method, characterized in that the peptide represented by the amino acid sequence of SEQ ID NO: 40.
The method of claim 1, wherein the hetero bone is bone bone-derived bone graft material manufacturing method, characterized in that selected from the group consisting of a bone bone.
In the group consisting of bone graft material prepared by the method of claim 1 and collagen, gelatin, chitosan, alginate, carboxymethyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol, poloxamer, polylactic acid, polylactic glycolic acid and polycaprolactone Composition for bone graft in the form of a gel or paste containing at least one selected polymer.

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