KR20130031870A - Peptide having apatite binding affinity and bone regeneration activity - Google Patents

Abstract

PURPOSE: A peptide is provided to ensure excellent binding affinity to an apatite inorganic material and to promote cell migration, proliferation, and differentiation. CONSTITUTION: A peptide with bone regeneration, which bind to apatite binds with one or more peptides selected from the group consisting of an amino acid sequences of sequence numbers 1-35 and peptides selected from the group consisting of amino acid sequences of sequence numbers 36-39. A bone graft material contains the peptide which is fixed on an apatite surface.

Description

Peptide having apatite binding affinity and bone regeneration activity}

The present invention relates to a peptide having bone tissue regeneration ability that specifically binds to the apatite inorganic surface, and more specifically, by combining an amino acid sequence having bone tissue regeneration ability and an amino acid sequence having an apatite binding ability, By providing a peptide that has binding ability to the apatite at the same time, it is stably fixed to the apatite surface and maintains effective activity for a long period of time, while a peptide having bone tissue regeneration ability that specifically binds to the apatite mineral surface exhibiting a bone regeneration effect, and for bone tissue regeneration containing the same It relates to the composition.

Bone or teeth are called hard tissues in the human body, and bones are composed of about 45% of bone minerals, 35% of organic matter, and 20% of moisture.The content of bone minerals in teeth is about 97% of enamel, 70% of dentin, and cement. It shows a content rate of about 50%, and the composition of inorganic substances is somewhat different depending on the type, part, and age of the animal.

Among the constituent materials, most of the organic materials are collagen, which is involved in the production of bones, maintaining toughness and elasticity, and exists as a matrix that selectively induces adhesion of bone cells to orient bone mineral particles. The main component of bone in nature, that is, bone minerals of vertebrates, is apatite mineral and hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), hydroxyapatite (Hydroxylapatite), carbonated apatite. hydroxy apatite, CHAp, A-type: Ca ₁₀ (PO ₄ ) ₆ [(OH) _2-2x (CO ₃ ) _x ], B-type: Ca _10-x [(PO ₄ ) _6-2x (CO ₃ ) it is known as _2x] (OH) ₂₎ and Ca ^{2 +} instead and Mg ^{2 +} and Na ⁺ is contained K ⁺ small amount of OH ^- instead of Cl ^-, F ^-. it is known that contains a small amount according to heal bone defects parts For dental materials and bone grafting materials for the purpose, research on making apatite inorganic material or coating the surface was continued.

The purpose of bone graft is to restore the morphological and physiological functions of the bone that maintains the biomechanical role of the bone, so the apatite bone graft material used at this time can be used immediately, does not cause an immune response, and is fast bone. It should be able to satisfy basic conditions such as promoting production and revascularization and maintaining bone support and continuity. However, although apatite itself can play a role as a medium having bone conduction, it does not have bone-inducing power for initial bone formation, which is essential for shortening the treatment period. In order to compensate for these shortcomings, studies in which apatite with a bioactive substance having chemotactic properties such as extracellular matrix proteins, tissue growth factors and bone morphogenetic proteins was used together with apatite were conducted, and INFUSE (containing BMP-2), GEM21S ( PDGF-containing) and other products have been developed. However, since these proteins are not stably fixed on the apatite surface, they are released from the apatite, and are then decomposed by exposure to systemic blood, it is difficult to maintain the activity on the apatite surface for a bone regeneration effect. Therefore, it is stably fixed on the apatite surface, and it is necessary to increase the bone regeneration effect to maintain effective activity for a long period of time.

Accordingly, the present inventors have made diligent efforts to solve the problems of the prior art as described above, and as a result of providing a peptide having bone formation ability and binding ability to apatite minerals at the same time, it can bind to the apatite mineral surface and exist in a stable state, and bone tissue By promoting the migration, proliferation, and differentiation of cells involved in regeneration, it is possible to finally maximize bone tissue regeneration ability, and it has been confirmed that the present invention has a high therapeutic effect on bone tissue regeneration.

An object of the present invention is to provide a peptide having a bone tissue regeneration ability that binds to apatite, stably immobilizing on the apatite inorganic surface and maintaining the activity of the peptide.

Another object of the present invention is to provide a bone graft material and a biomaterial for bone regeneration in which the peptide is fixed to the apatite surface.

In order to achieve the above object, the present invention provides a peptide having bone regeneration ability to bind to apatite, in which a peptide having bone regeneration ability and a peptide having apatite binding ability are combined.

The present invention also provides a bone graft material and a biomaterial for bone regeneration in which the peptide is fixed to the apatite surface.

The peptide having binding ability to apatite minerals and bone tissue regeneration ability according to the present invention can be present in a stable state by binding to the apatite surface, so that it can be used as a bone substitute for dental or orthopedic surgery and apatite-coated metal, natural polymer, and synthetic polymer. It can be applied and promotes the migration, proliferation, and differentiation of cells related to bone tissue regeneration, thereby maximizing bone tissue regeneration capacity in the end, and can exist stably while maintaining the peptide activity when transplanted into a living body, so bone tissue regeneration treatment technology using this It is useful for the development of

1 is a photograph of FITC-labeled bone minerals implanted in a rabbit skull circular bone defect, and after 4 weeks, tissues were collected and observed with a confocal microscope (FIG. 1A is a bone mineral to which a peptide is not bound, and FIG. 1B is FITC. Bone mineral to which the peptide of SEQ ID NO: 40 is labeled).
FIG. 2 shows FITC-labeled bone minerals were transplanted into a rabbit skull circular bone defect, and blood was collected before and after 1, 3, 7, 14, 21, and 28 days after transplantation. This is the value measured using a fluorometer for the released peptide (RFI: Relative fluorescence index, Blue line: Plasma negative control, measured using plasma without a fluorescent substance).
Figure 3 is a photograph of the bone minerals to which the peptide is bound is transplanted into a rabbit skull circular bone defect and the degree of bone regeneration is observed 2 weeks after the implantation (Figure 3A is a bone graft material to which the peptide of SEQ ID NO: 36 is bound, Figure 3B is SEQ ID NO: Bone grafting material to which the peptide of 35 is bound, Figure 3C is a bone grafting material to which the peptide of SEQ ID NO: 40 is bound).

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 order to develop the peptide according to the present invention, the amino acid sequence of the active site is separated and extracted from the osteogenic protein and the extracellular matrix, and the active structure is maintained through chemical modification after extraction.

In one aspect, the present invention relates to apatite in which 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 are bound It relates to a peptide having a binding bone tissue regeneration ability.

In the present invention, the peptide binding to the apatite mineral may 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), bone tissue regeneration ability It is chemically added to the N-terminus of the peptide having the to increase the binding ability to apatite, which is a constituent of bone, so that it can be stably bonded to the surface of a bone graft material or an apatite-coated implant.

In the present invention, the peptide having bone tissue regeneration ability may be characterized in that it is selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 to 35.

Specifically, the peptide having bone tissue regeneration ability is (a) an amino acid sequence at positions 2-18 among the amino acid sequences of (a) bone morphogenetic protein (BMP)-2, 4 and 6 [In the case of BMP-2 (SEQ ID NO: Number 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), amino acids at positions 47-71 Sequence (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), positions 335-353 The amino acid sequence of (SEQ ID NO: 9) and the amino acid sequence at positions 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.

In another aspect, the present invention relates to a bone graft material and a biomaterial for bone regeneration in which a peptide having bone tissue regeneration ability that binds to the apatite is fixed to the apatite surface.

In the present invention, the biomaterial for bone regeneration may be characterized in that it is selected from the group consisting of metals, natural polymers, and synthetic polymers.

In the present invention, in order to bind a peptide having bone tissue regeneration ability that binds to apatite to the apatite surface of a bone grafting material or a biomaterial for bone regeneration, a bone grafting material composed of apatite or a metal having a surface coated with apatite, natural polymer, synthetic Biomaterials such as polymers can be bonded by immersing them in the peptide solution, and a chemical crosslinking agent is not required to form bonds.

The peptide having bone tissue regeneration ability that binds to apatite according to the present invention is stably fixed 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.

The peptide having bone tissue regeneration ability that binds to apatite according to the present invention can 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 peptide having the ability to regenerate bone tissue that binds to the apatite is preferably to contain 1-100 mg per unit weight of the bone graft material, more preferably 20 per unit weight of the bone graft material. It may contain -80mg.

In an embodiment of the present invention, a peptide having bone tissue regeneration ability binding to the apatite of SEQ ID NO: 40 is prepared by combining the peptide having the apatite binding ability of SEQ ID NO: 36 with the peptide having the bone tissue regeneration ability of SEQ ID NO: 35. It was confirmed whether the peptide binds to the bone graft material stably, and the bone graft material having the peptide stably fixed on the surface of the apatite was transplanted to the bone defect to confirm the bone regeneration ability.

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.

Bone tissue that specifically binds to the apatite mineral surface Regeneration Have a smart Peptide Produce

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.

Bone tissue that specifically binds to the apatite mineral surface Regeneration Have a smart Peptide in vitro Stability check in

1200 mg of the prepared peptide was dissolved in 1 mL of tertiary distilled water, added to 4 g of bovine bone-derived minerals (OCS-B, Naivec), immersed for 24 hours, and then lyophilized.

Peptide-bound bone graft material 4g was put in 20 mL PBS, and a peptide release test was performed at 37°C. After 7 days, after removing all of the first 20 ml of the elution solvent, 20 ml of the new elution solvent is added again to elute at 37°C. Peptide elution experiments were performed for 14day, 28day, 56day, 84day, and 100day in the same manner as above. After the elution was completed, the bone material was collected and the peptide content was measured.

Test solution manufacturing method

3 g of the peptide-bound bone graft material was precisely weighed into powder, and about (2 g) equivalent to 160 mg of the peptide was precisely weighed, and 40 mL of the mobile phase A solvent was added, and sonication was performed for 1 hour. Thereafter, extraction was performed while stirring at 37±2° C. for 24 hours, and the supernatant of the extraction solvent was centrifuged at 3000 rpm for 10 minutes, and then filtered through a 0.22 μm millipore filter. Among them, 1 mL was taken, 3 mL of solvent A was mixed, and then filtered through a 0.22 μm millipore filter and used as a sample solution.

Standard liquid manufacturing method

The peptide standard was dried for 5 hours in a desiccator (silica gel), weighed about 10 mg precisely, dissolved in the mobile phase A solvent, and made exactly 10 mL, and used as a standard solution.

Test Methods

10 μL of the sample solution and the standard solution were tested under the operating conditions of the liquid chromatography method as follows to obtain the peak areas A _T and A _S of the sample solution and the standard solution.

Amount of peptide (mg) = Amount of peptide standard (mg) X

Operating conditions

Instrument: analytical HPLC (Shimadzu, Japan),

Column: Filled with 5um silica gel bound with C18 (length 250mm, inner diameter 4.6mm)

Mobile phase: 0.1% trifluoroacetic acid/DDW (solvent A) 0.098% trifluoroacetic acid/acetonitrile (solvent B)

Detector: Ultraviolet absorption photometer (measurement wavelength 230nm)

Flow rate: 1ml/min

Column temperature: constant temperature around 40℃

gradient condition: Time (min) B solvent composition (%) One 5 35 100 45 100 50 5 60 5

As a result, a dissolution experiment was conducted for 100 days to measure the peak of the peptide in the eluate (Table 2), Lot No. In 1, 2, and 3, no peak of the peptide was observed at retention time of 14.196 min. After the elution was completed, as a result of quantifying the amount of peptide, it was determined that 9.2mg, 9.18mg, and 9.78mg were included (Table 3). This proves that the peptide bound to the bone graft material is not released as an eluate, and is stably bound to the bone graft material.

Release result from bone mineral of the peptide of SEQ ID NO: 40 Lot No. day Name Ret. Time Area Height mg One 7 RT14.196 0.000 0 0 0 14 RT14.196 0.000 0 0 0 28 RT14.196 0.000 0 0 0 56 RT14.196 0.000 0 0 0 84 RT14.196 0.000 0 0 0 100 RT14.196 0.000 0 0 0 2 7 RT14.196 0.000 0 0 0 14 RT14.196 0.000 0 0 0 28 RT14.196 0.000 0 0 0 56 RT14.196 0.000 0 0 0 84 RT14.196 0.000 0 0 0 100 RT14.196 0.000 0 0 0 3 7 RT14.196 0.000 0 0 0 14 RT14.196 0.000 0 0 0 28 RT14.196 0.000 0 0 0 56 RT14.196 0.000 0 0 0 84 RT14.196 0.000 0 0 0 100 RT14.196 0.000 0 0 0

Content of peptide remaining in bone mineral of SEQ ID NO: 40 peptide Lot No. Name Ret. Time Area Height mg One RT14.196 14.244 1176751 51132 9.2 2 RT14.196 14.235 1174499 51711 9.19 3 RT14.196 14.229 124963 56018 9.78

Bone tissue that specifically binds to the apatite mineral surface Regeneration Have a smart Peptide in vivo Stability check in

40 peptides labeled with FITC were deposited on bone minerals derived from bovine bone (OCS-B, Naivec). After transplantation to the skull defect of rabbits, after 4 weeks of sacrifice, the bone minerals transplanted to the skull defect were observed with a confocal microscope (Confocal microscopy, Olympus, Japan). FITC-labeled SEQ ID No. 40 peptide was implanted with 10 mg and 20 mg of bone minerals bound to the cranial defect of rabbits, and before transplantation, at 1, 3, 7, 14, 21, and 28 days after transplantation Blood was collected and measured using a Fluorometer to determine whether the peptide was released into systemic blood.

As a result, it was confirmed that the peptide was well fixed on the surface of the bone graft material implanted in the skull, and did not show any spreading out to the surrounding tissues (FIG. 1B). The RFI values did not show any difference compared to the value before transplantation, confirming that the peptide contained in the bone mineral was not released into the blood, which proved that the peptide was well fixed to the bone mineral surface (FIG. 2).

Bone tissue that specifically binds to the apatite mineral surface Regeneration Have a smart Peptide in vivo in Bone regeneration ability Confirm

The peptides of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 40 prepared in Example 1 were each bonded to the bone graft material by the method of Example 2, and then transplanted from the skull circular bone defect of the 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 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.

Figure 3 shows the bone regeneration effect by a bone graft material containing a smart peptide having a bone tissue regeneration ability that specifically binds to the apatite mineral surface, the bone graft material (A, apatite-binding peptide) to which the SEQ ID NO: 36 peptide is bound The bone regeneration effect was small. Bone grafting material (B, bone regeneration effect peptide) to which SEQ ID NO: 35 peptide is bound has increased bone regeneration effect compared to A, and bone grafting material (C, apatite mineral surface) to which SEQ ID NO: 40 peptide is bound In the case of a smart peptide with bone tissue regeneration ability), it was observed that the bone regeneration ability was significantly increased compared to A and B. This proves that the apatite-binding peptide itself does not have a bone regeneration effect, and since a peptide with a bone regeneration effect cannot stably bind to a bone graft material, the bone regeneration effect is more effective than the case of a smart peptide that has both functions at the same time. It seems to be less. Therefore, when a smart peptide with bone tissue regeneration ability that specifically binds to the apatite mineral surface is used for a bone graft material made of apatite or an apatite-coated implant, it is expected that the effect of bone tissue regeneration will be great.

As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

<110> Seoul National University <120> Peptide having apatite binding affinity and bone regeneration activity <130> P10-B238 <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 (8)

At least one peptide selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to 35 and one peptide selected from the group consisting of the amino acid sequences of SEQ ID NO: 36 to SEQ ID NO: 39 are combined, provided that the peptides of the amino acid sequence of SEQ ID NO: 35 Peptide having a bone tissue regeneration ability to bind to apatite, except for the peptide to which the peptide of the amino acid sequence of SEQ ID NO: 36 is bound.
A bone graft material, characterized in that the peptide of claim 1 is fixed to the surface of the apatite.
The bone graft material of claim 2, wherein the apatite is selected from the group consisting of bio-derived hydroxyapatite bone mineral, synthetic apatite hydroxide, apatite carbonate, tricalcium phosphate and monocalcium phosphate.
The bone graft material according to claim 2, wherein the dose of the peptide contains 1-100 mg per unit weight of bone graft material (1 g).
Biomaterial for bone regeneration, characterized in that the peptide of claim 1 is fixed to the surface of the apatite.
The biomaterial for bone regeneration according to claim 5, wherein the biomaterial is selected from the group consisting of metal, natural polymer and synthetic polymer.
6. The biomaterial for bone regeneration according to claim 5, wherein the apatite is selected from the group consisting of bio-derived hydroxyapatite bone mineral, synthetic apatite hydroxide, apatite carbonate, tricalcium phosphate and monocalcium phosphate.
The biomaterial for bone regeneration according to claim 5, wherein the dose of the peptide contains 1-100 mg per unit weight (1 g) of the biomaterial for bone regeneration.

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