WO2008032929A1

WO2008032929A1 - Bone graft containing osteogenesis enhancing peptides

Info

Publication number: WO2008032929A1
Application number: PCT/KR2007/003687
Authority: WO
Inventors: Chong-Pyoung Chung; Yoon-Jeong Park; Jue-Yeon Lee
Original assignee: Seoul National University Industry Foundation; Nano Intelligent Biomedical Engineering Corporation. Co. Ltd
Priority date: 2006-09-13
Filing date: 2007-07-31
Publication date: 2008-03-20
Also published as: KR100757241B1

Abstract

The present invention relates to a physiologically active peptide for promoting osteogenesis, and a bone graft material for dental and orthopedic applications, which contains the peptide. More specifically, the bone graft material which contains a peptide having CEEEEEEEGG added to the N-terminus of a peptide derived from a bone morphogenetic protein and/or an extracellular matrix. The peptide for promoting bone tissue formation, which is contained in the bone graft material, can promote the growth, proliferation and differentiation of bone regeneration-associated cells to maximize tissue regeneration. Also, the peptide has a reduced risk of immune responses due to its low molecular weight when applied in vivo, and can be present in a stable form in vivo to show a long-lasting effect. Accordingly, the peptide makes it expedient to perform surgical operations for the regeneration of periodontal tissue, alveolar bone and other bone tissues, and is expected to show high therapeutic effects.

Description

BONE GRAFT CONTAINING OSTEOGENESIS ENHANCING

PEPTIDES

TECHNICAL FIELD

The present invention relates to a physiologically active peptide for promoting osteogenesis, and a bone graft material for dental and orthopedic applications, which contains said peptide, and more particularly to a bone graft material which contains a peptide having CEEEEEEEGG added to the N-terminus of a peptide derived from a bone morphogenetic protein and/or an extracellular matrix.

BACKGROUND ART

In the oral and maxillofacial region and the orthopedic region, bone defects occur due to various causes, including infection, functional degeneration resulting from aging, trauma, cysts and tumors. In order to promote the aesthetic and functional restoration, stabilization and healing of such bone defects, bone grafting has been attempted in various ways. Because the object of bone grafting is to restore the morphological and physiological functions of bone to maintain the biomechanical role of bone, bone graft materials should be capable of satisfying various basic requirements. That is, the bone graft materials should be ready to use, should not cause an immune response, should be capable of promoting rapid osteogenesis and revascularization, should be capable of supporting bone and should be capable of maintaining the continuity of bone. In particular, the bone graft materials are mostly used in combination with implants in bone regeneration, and in this case, it may be ideal to use implants having suitable viability, which can promote osseointegration and, at the same time, maintain the stability of fixtures. Bone graft materials, which have been most frequently used in bone regeneration up to date, include autogenous bone graft materials, allogenic bone graft materials, heterogeneous bone graft materials and synthetic bone graft materials. Among them, the autogenous bone graft materials have a problem in that they are difficult to harvest, because secondary surgery is required for bone harvesting. The allogenic bone graft materials have the possibility of infection with disease. For these reasons, the heterogeneous bone graft materials and the synthetic bone graft materials have been mainly used. For example, bone graft material products produced by treating bovine bone in several steps have been marketed, and bone graft materials synthesized from hydroxypapatite powder have also been marketed. Among them, heterogeneous bone graft materials produced from bovine bone have the largest share of the domestic market of bone graft materials, in which Bio-Oss (Geistlich Sohne, Swiss) forms more than 50% of the domestic market, and Capset (Lifecore, USA) and Bio-Gen (BioTek, Italy) have

the second-largest share. In addition, BBP ( Oscotec Inc., Korea) and the like

form the domestic market of bone graft materials.

However, although such existing products can serve as an osteoconductive scaffold, they have a limitation in that osteogenesis can be induced after a considerable period of time after surgical operations, because they have no osteoinductivity for initial osteogenesis, which is essential to shorten the treatment period. In order to complement this shortcoming, physiologically active substances inducing chemotaxis, for example, extracellular matrix proteins, tissue growth factors, or bone morphogenetic proteins, have been experimentally used in bone grafting procedures to provide rapid osteogenesis. However, the use of such proteins has problems in that the proteins are relatively expensive, cause immune responses or have a short in vivo half-life, and are exposed to systemic blood flow when applied in vivo, such that it is difficult to maintain their effective concentrations at local sites. For this reason, a novel technology capable of overcoming such problems is required.

Accordingly, the present inventors have made many efforts to solve the above- described problems occurring in the prior art and, as a result, found that a bone graft material which contains a peptide having CEEEEEEEGG added to the N- terminus of a peptide derived from the active site of a bone morphogenetic protein and/or an extracellular matrix, can increase the differentiation rate of regeneration-associated cells into bone tissue to maximize tissue regeneration, has a reduced risk of immune responses when applied in vivo, due to its low molecular weight, and can be present in a stable form in vivo to show a long- lasting effect, thereby completing the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a bone graft material for dental and orthopedic applications, which contains a peptide having CEEEEEEEGG added to the N-terminus of a peptide derived from a bone morphogenetic protein and/or an extracellular matrix.

To achieve the above object, the present invention provides a bone graft material which contains a peptide having an amino acid sequence of SEQ ID NO: 36 added to the N-terminus of an amino acid sequence selected from the group consisting of the following (a) and (b): (a) one or more amino acid sequences selected from among amino acid sequences of SEQ ID NOs: 1 to 35; and (b) two linked amino acid sequences selected from among amino acid sequences of SEQ ID NOs: 1 to 35.

Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates confocal laser scanning fluorescence microphotographs showing the pattern of cell adhesion to a peptide-containing bone graft material prepared in each of Examples 1 and 3. In FIG. 1, (a) shows the pattern of cell adhesion to a bone graft material containing no peptide, (b) shows the pattern of cell adhesion to a bone graft material containing a bone morphogenetic protein- derived peptide, and (c) shows the pattern of cell adhesion to a bone graft material containing an extracellular matrix-derived peptide.

FIG. 2 illustrates confocal laser scanning fluorescence microphotographs showing the deposition of calcium on a peptide-containing bone graft material, prepared in each of Examples 1 and 3 and into which cells C₂C₁₂ were inoculated to culture in a calcein-containing medium for hard tissue formation for 14 days. In FIG. 2, (a) shows the degree of calcification on a bone graft material containing no peptide, (b) shows the degree of calcification on a bone graft material containing a bone morphogenetic protein-derived peptide, and (c) shows the degree of calcification on a bone graft material containing an extracellular matrix-derived peptide.

FIG. 3 shows the bone regeneration effect of a peptide-containing bone graft material, prepared in each of Examples 1 and 3 and grafted into bone defects in the rabbit cranial bone. In FIG. 3, (a) shows a bone graft material containing no peptide, and (b) and (c) show bone graft materials containing a bone morphogenetic protein-derived peptide and an extracellular matrix-derived peptide, respectively.

DETAILED DESCRIPTION OF THE INVENTION, AND PPRTERRED EMBODIMENTS In one aspect, the present invention relates to a bone graft material which contains a peptide having an amino acid sequence of SEQ ID NO: 36 added to the N-terminus of an amino acid sequence selected from the group consisting of the following (a) and (b): (a) one or more amino acid sequences selected from among amino acid sequences of SEQ ID NOs: 1 to 35; and (b) two linked amino acid sequences selected from among amino acid sequences of SEQ ID NOs: 1 to 35.

In the present invention, the above-described bone graft material was tested for cell adhesion, calcification, and cranial bone regeneration effects. As a result, it could be seen that the bone graft material could increase the differentiation rate of regeneration-associated cells into bone tissue to maximize tissue regeneration, had a reduced risk of immune responses when applied in vivo, due to its low molecular weight, and could be present in a stable form in vivo to show a long- lasting effect.

To the bone graft material according to the present invention, cells can stably adhere. Also, the bone graft material according to the present invention promotes the calcification of cells and increases bone regeneration.

In the present invention, the bone graft material is preferably obtained by soaking a bone graft material in a solution of said peptide. As the bone graft material for soaking in the peptide solution, all kinds and types of bone graft materials known in the art may be used, and preferred examples thereof include organism-derived bone mineral powders and porous blocks, originated from autogeneous bone, bovine bone and porcine bone, synthetic hydroxyapatite powders and porous blocks, tricalcium phosphate powders and porous blocks, monocalcium phosphate powders and porous blocks, bone graft materials made of silicon dioxide (silica), and bone-packing graft materials made of a mixture of silica and polymer. Hereinafter, the inventive bone graft material containing the peptide derived from the bone morphogenetic protein and/or extracellular matrix will be described in detail.

In order to develop the peptide according to the present invention, active site amino acid sequence is isolated and extracted from a bone morphogenetic protein and/or an extracellular matrix, and then subjected to chemical modification so as to maintain the active structure thereof. The peptide comprises an amino acid sequence of SEQ ID NO: 36 (CEEEEEEEGGG consisting of one cystein, seven glutamic acids and two glycines) chemically added to the N-terminus of the bone morphogenetic protein- and/or extracellular matrix-derived peptide, and thus increases the affinity of calcium for the bone graft material to increase the content of calcium in the bone graft material.

Herein, the bone morphogenetic protein- and/or extracellular matrix-derived peptide may be either at least one amino acid sequence selected from among amino acid sequences of SEQ ID NOs: 1 to 35, or two linked amino acid sequences selected from among the amino acid sequences of SEQ ID NOs: 1 to 35.

Particularly, the bone morphogenetic protein-derived peptide essentially contains any one amino acid sequence selected from the group consisting of: an amino acid sequence of positions 2-18 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 1); an amino acid sequence of positions 2-18 of bone morphogenetic protein (BMP)-4 (SEQ ID NO: 2); an amino acid sequence of positions 2-18 of bone morphogenetic protein (BMP)-6 (SEQ ID NO: 3); an amino acid sequence of positions 16-34 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 4); an amino acid sequence of positions 47-71 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 5); an amino acid sequence of positions 73-92 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 6); an amino acid sequence of positions 88-105 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 7); an amino acid sequence of positions 83-302 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 8); an amino acid sequence of positions 335-353 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 9); an amino acid sequence of positions 370-390 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 10); an amino acid sequence of positions 74-93 of bone morphogenetic protein (BMP)-4 (SEQ ID NO: 11); an amino acid sequence of positions 293-313 of bone morphogenetic protein (BMP)-4 (SEQ ID NO: 12); an amino acid sequence of positions 360-379 of bone morphogenetic protein (BMP)-4 (SEQ ID NO: 13); an amino acid sequence of positions 382-402 of bone morphogenetic protein (BMP)- 4 (SEQ ID NO: 14); an amino acid sequence of positions 91-110 of bone morphogenetic protein (BMP)-6 (SEQ ID NO: 15); an amino acid sequence of positions 397-418 of bone morphogenetic protein (BMP)-6 (SEQ ID NO: 16); an amino acid sequence of positions 472-490 of bone morphogenetic protein (BMP)-

6 (SEQ ID NO: 17); an amino acid sequence of positions 487-510 of bone morphogenetic protein (BMP)-6 (SEQ ID NO: 18); an amino acid sequence of positions 98-117 of bone morphogenetic protein (BMP)-7 (SEQ ID NO: 19); an amino acid sequence of positions 320-340 of bone morphogenetic protein (BMP)-

7 (SEQ ID NO: 20); an amino acid sequence of positions 390-409 of bone morphogenetic protein (BMP)-7 (SEQ ID NO: 21); and an amino acid sequence of positions 405-423 of bone morphogenetic protein (BMP)-7 (SEQ ID NO: 22).

The extracellular matrix-derived peptide essentially contains any one amino acid sequence selected from the group consisting of: an amino acid sequence of positions 62-69 of bone sialoprotein II (BSP II) (SEQ ID NO: 23); an amino acid sequence of positions 139-148 of BSP II (SEQ ID NO: 24); an amino acid sequence of positions 259-277 of BSP II (SEQ ID NO: 25); an amino acid sequence of positions 199-204 of BSP II (SEQ ID NO: 26); an amino acid sequence of positions 151-158 of BSP II (SEQ ID NO: 27); an amino acid sequence of positions 275-291 of BSP II (SEQ ID NO: 28); an amino acid sequence of positions 20-28 of BSP II (SEQ ID NO: 29); an amino acid sequence of positions 65-90 of BSP II (SEQ ID NO: 30); an amino acid sequence of positions 150-170 of BSP II (SEQ ID NO: 31); an amino acid sequence of positions 280-290 of BSP II (SEQ ID NO: 32); an amino acid sequence (YGLRSKS: SEQ ID NO: 33) of positions 149-169 of bone sialoprotein I (BSP I, osteopontin); an amino acid sequence (KKFRRPDIQ YPD AT: SEQ ID NO: 34) of positions 149-169 of BSP I; and an amino acid sequence (YGLRSKSKKFRRPDIQYPDA: SEQ ID NO: 35) of positions 149-169 of BSP I.

The prepared peptide is physically contained in a bone graft material by soaking the bone graft material in a solution of the peptide, thus preparing the inventive bone graft material containing the bone morphogenetic protein- and/or extracellular matrix-derived peptide.

The peptide according to the present invention has a molecular weight lower than that of the bone morphogenetic protein or the extracellular matrix, and thus is not sensitive to in vivo enzymatic reactions, has low in vivo immunogenicity and is easy to handle due to its high stability. Thus, when the peptide contained in a bone graft material for tissue regeneration is used in bone graft procedures, the desired concentration of the active peptide can be locally present while showing activity, so that its therapeutic effects can be increased. Thus, the active peptide has characteristics suitable for the regeneration and repair of bone tissue and periodontal tissue.

In addition, the peptide is preferably contained in the bone graft material in an amount of 10-100 mg per g of the bone graft material. More preferably, the peptide consists of SEQ ID NO: 1 and SEQ ID NO: 2 and is contained in an amount of 20-80 mg per g of the bone graft material.

Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1 : Incorporation of bone morphogenetic protein-derived peptide into bovine bone-derived bone mineral particles

A bone morphogenetic protein-derived peptide used in this Example was a chemically synthesized peptide, obtained by adding an amino acid sequence of SEQ ID NO: 36 to the N-terminus of an amino acid sequence of SEQ ID NO: 4 that is a peptide containing the active domain of a bone morphogenetic protein (BMP-2). Bovine bone-derived bone mineral particles were washed with ethanol under reduced pressure, and then left to stand in a vacuum oven at 100^°C for 20 hours to remove impurities from the particle surface. 80 mg of the prepared peptide was dissolved in 1 ml of triple-distilled water, and 1 g of the washed bovine bone-derived mineral particles were soaked in the peptide solution for 24 hours, and then dried.

Example 2: Incorporation of bone morphogenetic protein-derived peptide into synthetic hydroxyapatite and tricalcium phosphate

Synthetic hydroxyapatite and tricalcium phosphate bone graft powder materials were washed with ethanol under reduced pressure, and then left to stand in a vacuum oven at 100 °C for 20 hours to remove impurities from the powder surface. 80 mg of the same peptide as described in Example 1 was dissolved in 1 ml of triple-distilled water, and 1 g of the washed synthetic hydroxyapatite and tricalcium phosphate powders were soaked in the peptide solution for 24 hours, and then dried.

Example 3: Incorporation of extracellular matrix-derived peptides into bovine bone-derived bone mineral particles

Extracellular matrix-derived peptides used in this Example were chemically synthesized peptides, obtained by chemically synthesizing a peptide of SEQ ID NO: 25, containing an active domain structure for the induction of calcification of bone sialoprotein II, and a peptide of SEQ ID NO: 27, containing a cell adhesion site, and adding an amino acid sequence of SEQ ID NO: 36 to the N- termini of the synthesized peptides. Bovine bone-derived bone mineral particles were washed with ethanol under reduced pressure, and then left to stand in a vacuum oven at 100°C for 20 hours to remove impurities from the particle surface. 80 mg of the prepared peptides were dissolved in 1 ml of triple- distilled water, and 1 g of the washed bovine bone-derived mineral particles were soaked in the peptide solution for 24 hours, and then dried.

Example 4: Incorporation of extracellular matrix-derived peptides into synthetic hvdroxyapatite and tricalcium phosphate

Synthetic hydroxyapatite and tricalcium phosphate bone graft material powders were washed with ethanol under reduced pressure, and then left to stand in a vacuum oven at 100 ^°C for 20 hours to remove impurities from the surface. 80 mg of the same peptides as described in Example 3 were dissolved in 1 ml of triple-distilled water 1, and 1 g of the washed synthetic hydroxyapatite and tricalcium phosphate powders were soaked in the peptide solution for 24 hours, and then dried.

Example 5: Cell adhesion to peptide-containing bone graft materials according to the present invention

Human osteosarcoma cells (Korean Cell Line Bank, KCLB No. 21543) were inoculated into the peptide-containing bone graft materials prepared in each of Examples 1 and 3, and then cultured for 2 hours. The bone graft materials, into which the osteoblasts were inoculated to culture, were fixed with 2% glutaraldehyde solution. The fixed bone graft materials were treated with 1% triton X-IOO, and then the attached cell nuclei were stained with DAPI. The cells attached to the bone graft materials were observed with a confocal laser scanning microscope (FIG. 1).

In FIG. 1, (a) shows the pattern of cell adhesion to a bone graft material containing no peptide, (b) shows the pattern of cell adhesion to the bone graft material containing the bone morphogenetic protein-derived peptide, and (c) shows the pattern of cell adhesion to the bone graft material containing the extracellular matrix-derived peptide. As can be seen in FIG. 1, it could be observed that the stained cell nuclei were observed more in the bone graft material (b), containing the peptide of SEQ ID NO: 4, and the bone graft material (c), containing the peptides of SEQ ID NOs: 25 and 27, than in the bone graft material (a) containing no peptide, suggesting that the cells stably adhered to the bone graft materials (b) and (c) (see portions indicated by circles in FIG. 1).

Example 6: Calcification of cells by peptide-containinfi bone graft materials according to the present invention

Mesodermal stem cells C₂C₁₂ (America Type Culture Collection, CRL- 1772) were inoculated on the peptide-containing bone graft materials, prepared in each of Examples 1 and 3, and were cultured for 14 days in a medium for hard tissue formation, containing 1 βglπJl of calcein (fluorescent substance for calcium staining). The cultured osteoblasts were fixed with 2% glutaraldehyde solution. The fixed bone graft materials were treated with 1% triton X-IOO, and then the cytoplasm attached to the bone graft materials was stained with a fluorescent- labeled phalloidin solution. After the staining, the samples were washed and fixed, and then calcium deposited on the surfaces of the fixed bone graft materials was observed with a confocal laser scanning microscope (FIG. 2).

In FIG. 2, (a) shows the degree of calcification on a bone graft material containing no peptide, (b) shows the degree of calcification on the bone graft material containing the bone morphogenetic protein-derived peptide, and (c) shows the degree of calcification on the bone graft material containing the extracellular matrix-derived peptide. As can be seen in FIG. 2, calcium fluorescence appeared more in the bone graft material (b), containing the peptide of SEQ ID NO: 4, and the bone graft material (c), containing the peptides of SEQ ID NOs: 25 and 27, than in the bone graft material (a) containing no peptide, suggesting that the bone morphogenetic protein and the extracellular matrix- derived peptides promoted the calcification of cells.

Example 7: Effect of inventive peptide-containing bone graft materials on regeneration of rabbit cranial bone

The peptide-containing bone graft materials, prepared in Examples 1 and 3, were inserted into a circular bone defect in the rabbit cranial bone in order to examine their bone regeneration ability. For this purpose, at the cranial sites of anesthetized New Zealand white rabbits (Cuniculus), a circular bone defect having a diameter of 8 mm was formed. A bone graft material and the peptide- containing bone graft materials were grafted into the bone defects in an amount of 50 mg/defect, and the bone membrane and the skin were double sutured to each other. At 2 weeks after the grafting, the animals were sacrificed, and samples collected from the animals were fixed in formalin solution. Then, the tissues were embedded so as to prepare samples having a thickness of 20 μm. The prepared samples were stained with basic fuchsine and toluidine blue, thus constructing non-decalcified samples. The constructed samples were photographed with an optical microscope.

FIG. 3 shows the bone regeneration effects of the bone graft materials, containing the bone morphogenetic protein-derived peptide or the extracellular matrix- derived peptide. As can be seen in FIG. 3, when the inventive bone graft materials, containing the bone morphogenetic protein- derived peptide or the extracellular matrix-derived peptide, were grafted into the rabbit cranial bone defects, they showed a remarkably improved bone regeneration ability within 2 weeks, compared to the bone graft material (a) containing no peptide (see (b) and (c) in FIG. 3).

INDUSTRIAL APPLICABILITY

As described above in detail, the present invention provides a bone graft material for dental and orthopedic applications, which contains a peptide having

CEEEEEEEGG added to the N-terminus of a peptide derived from a bone morphogenetic protein and/or an extracellular matrix for promoting bone tissue regeneration. The inventive bone graft material which contains the peptide having CEEEEEEEGG added to the N-terminus of the bone morphogenetic protein- and/or extracellular matrix-derived peptide, can increase the differentiation rate of regeneration-associated cells into bone tissue, thus maximizing tissue regeneration. Also, the peptide has a reduced risk of immune responses due to its low molecular weight when applied in vivo, and can be present in a stable form in vivo to show a long-lasting effect. Accordingly, the peptide makes it expedient to perform surgical operations for the regeneration of periodontal tissue, alveolar bone and other bone tissues, and is expected to show high therapeutic effects.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS

What is Claimed is:

L A bone graft material which contains a peptide having an amino acid sequence of SEQ ID NO: 36 added to the N-terminus of an amino acid sequence selected from the group consisting of the following (a) and (b):

(a) one or more amino acid sequences selected from among amino acid sequences of SEQ ID NOs: 1 to 35; and (b) two linked amino acid sequences selected from among amino acid sequences of SEQ ID NOs: 1 to 35.

2. The bone graft material which contains a peptide according to claim 1, which is obtained by soaking a bone graft material in a solution of said peptide.

3. The bone graft material which contains a peptide according to claim 1, wherein the bone graft material is selected from the group consisting of: organism-derived bone mineral powders and porous blocks, synthetic hydroxyapatite powders and porous blocks, tricalcium phosphate powders and porous blocks, monocalcium phosphate powders and porous blocks, bone graft materials made of silicon dioxide (silica), and bone-packing graft materials made of a mixture of silica and polymer.