KR101348335B1 - Osteoconductive Bone graft and use thereof - Google Patents

Abstract

The present invention relates to a bone graft material having excellent biocompatibility, cell adhesion ability and bone conductivity using xenografts and a method of manufacturing the same. The bone graft material manufacturing method according to the present invention can provide a bone graft material that can be fused well without an inflammatory reaction in the tissue of the transplant site due to excellent biocompatibility because the content of the protein is lower than the conventional xenograft-derived bone graft material By treating the surface with acidic amino acids, the adhesion of bone cells can be increased, and the conductivity of new bone can be improved by introducing bioactive factors.

Description

Bone graft derived from xenograft and its manufacturing method {Osteoconductive Bone graft and use

The present invention relates to a bone graft material having excellent biocompatibility, cell adhesion ability and bone conductivity and a method for producing the same using xenograft, specifically, to remove protein and fat from xenograft such as bovine bone, horse bone, pork bone and The present invention relates to a method for producing a bone or bone-shaped bone graft material having excellent biocompatibility, cell adhesion, and bone conductivity by surface treatment with amino acids or addition of a bioactive substance.

Bone grafting substitute (BGS) is a replacement for bone defects caused by various dental diseases or traumas, disease degeneration or other tissue loss, which fills the space in bone tissue and forms new bone. Refers to the implant used to promote it. Bone grafts include bioderived bone grafts and synthetic bone grafts. Bio-derived bone grafts include autologous bone, allogeneic bone and xenograft. In order to obtain autogenous bone, there is an inconvenience of having to perform surgery and it has the disadvantage of being absorbed too quickly. Allogeneic bone transplantation, on the other hand, has the potential for immune response and viral and disease infections. In the case of artificially manufactured synthetic bone, there is no possibility of infection of viruses and diseases, but the bone regeneration ability is lower than that of biologically derived bones, and the absorption is too fast. In order to solve this disadvantage, xenograft grafts derived from bones of animals having a structure similar to human bones are used, and a typical product is Geistlich's BioOss. The requirement for developing xenografts is to extract only pure apatite by removing heterologous proteins and other impurities. It is most important to remove heterologous proteins that can cause an immune response and to have a chemical composition similar to that of human bone tissue. Method for producing bone graft material using the bone of the animal is to remove the fat by adding a solvent having a boiling point of 80 ℃ to 120 ℃ bovine femoral bone, and then remove the protein and organic matter by adding ammonia or primary amine After preparing the mineral, it is composed of a step of drying by heating 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 for producing a bone-shaped bone graft material used a method of coating the ceramic on the porous polymer, heat treatment to burn the polymer and then sintering the remaining ceramic. The ceramic used is an artificially synthesized bone graft material, which is different from bone graft material derived from xenograft (Korean Patent Application No. 10-2000-0046973).

There is a method of cutting homologous or heterologous bone tissue in the form of a block, followed by hydrogen peroxide treatment, demineralization with hydrochloric acid, RSC solution treatment, and vacuum freeze drying, but there is a possibility of causing problems due to the remaining protein (Korean Patent Application No. 10 -2008-0138644).

Thus, the present inventors have obtained a patent for a method for producing bone graft material which is treated with sodium hypochlorite, pressurized and heated at high temperature to effectively remove fats and organics so that there is no risk of infection of pathogenic substances (Korea Patent Publication No. 10 -0842012), a method for producing a bone graft material for further improving the cell adhesion and new bone conduction ability of bone graft material.

Accordingly, the present inventors made up of pure hydroxyapatite from which proteins and fats were removed, and as a result of diligently trying to produce a bone-derived bone graft material excellent in biocompatibility, cell adhesion ability, and bone conduction, sodium hypochlorite as sodium hypochlorite Treatment and heating to a high temperature to completely remove proteins and fats, and in this bone graft manufacturing process using acidic amino acids on the surface of the bone graft material can significantly increase the adhesion of cells by reducing the surface roughness, derived from extracellular matrix By physiologically active factors such as components and bone regeneration functional peptides were introduced into the bone graft material was confirmed that can significantly improve the conduction of new bone has been completed the present invention.

Disclosure of the Invention An object of the present invention is to provide a bone grafting bone-derived bone graft material and a method for producing the same, which are composed of pure hydroxyapatite to remove proteins and fats, thereby improving biocompatibility, cell adhesion, and bone conductivity.

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

(a) cutting the bone; (b) removing blood components of the cut bone; (c) grinding the bone from which the blood component is removed; (d) first removing fat and protein from the ground bone and drying; (e) immersing the dried bone in an organic solvent and shaking to remove the lipid and protein from the bone secondly; (f) removing and drying the organic solvent; (g) treating the solvent-free bone powder with 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing sodium hypochlorite solution from the bone powder and drying it; (i) heat treating the dried bone powder to completely remove lipids and proteins; (j) fractionating the heat treated bone powder into sieves having a pore size of 212 to 1000 µm and 1000 to 2000 µm, respectively; And (k) treating the surface of the bone powder with acidic amino acids.

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

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

(a) cutting the bone; (b) removing blood components of the cut bone; (c) grinding the bone from which the blood component is removed; (d) first removing fat and protein from the ground bone and drying; (e) immersing the dried bone in an organic solvent and shaking to remove the lipid and protein from the bone secondly; (f) removing and drying the organic solvent; (g) treating the solvent-free bone powder with 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing sodium hypochlorite solution from the bone powder and drying it; (i) heat treating the dried bone powder to completely remove lipids and proteins; (j) fractionating the heat treated bone powder into sieves having a pore size of 212 to 1000 µm and 1000 to 2000 µm, respectively; And (k) adding at least one bioactive 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 of the cut bone; (c) first removing fat and protein from the bone from which the blood components have been removed and drying; (d) immersing the dried bone in an organic solvent and shaking to remove the lipid and protein from the bone secondly; (e) removing and drying the organic solvent; (f) treating the solvent-free bone with 4% sodium hypochlorite solution to inactivate prion and other proteins; (g) removing sodium hypochlorite solution from the bone and drying; (h) heat treating the dried bone to completely remove lipids and proteins; (i) cutting the bone into blocks of the desired size; And (j) adding at least one bioactive 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 xenograft-derived bone graft material in the form of a particle or block produced by the method.

The bone graft material manufacturing method according to the present invention completely removes proteins and fats from xenografts such as bovine bones, horse bones, pork bones, and provides a bone graft material composed of pure hydroxyapatite or a block-type bone graft material according to the conventional xenografts Compared with the low protein content, it has excellent biocompatibility and can produce bone graft material that can be fused well without inflammatory reaction in the tissue of the graft site. The surface treatment with acidic amino acid can increase the adhesion of bone cells. By introducing physiologically active factors, it is possible to improve the conduction of new bone. The bone graft material according to the present invention can be used to fill the bone damage in dentistry, orthopedics, plastic surgery, etc. to conduct regeneration of new bone, and can also be used as a tissue engineering support for culturing cells.

Figure 1 is a photograph of the appearance and differential scanning electron microscope of the particle and block-shaped bone graft material prepared in Example 1.
Figure 2 shows the XRD measurement results of the particle and block-shaped bone graft prepared in Example 1.
Figure 3 shows a differential scanning electron micrograph of the surface of the bone graft material before and after treatment with the acidic amino acid in the particle and block-shaped bone graft material prepared in Example 1.
Figure 4 is a result of observing the adhesion of the cells before and after treatment with the acidic amino acid to the particle and block-shaped bone graft prepared in Example 1.
5 is a result of observing bone regeneration in the skull of the rabbit after adding the extracellular matrix to the bone graft material in the form of particles prepared in Example 1.
Figure 6 is a result of observing the adhesion of the cells after the addition of the extracellular substrate after treatment with acidic amino acid to the bone and bone-shaped bone graft material prepared in Example 1.
FIG. 7 shows the results of observing bone regeneration after treating bone regeneration functional peptides in the bone-shaped bone graft material prepared in Example 1, and treating bone regeneration ability after treating the surface with acidic amino acid and then treating bone regeneration functional peptides. One result is shown.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to a method for producing bone xenograft-derived bone graft material in the form of particles comprising the following steps:

(a) cutting the bone; (b) removing blood components of the cut bone; (c) grinding the bone from which the blood component is removed; (d) first removing fat and protein from the ground bone and drying; (e) immersing the bone powder in an organic solvent and shaking the secondary to remove fat and protein from the bone; (f) removing and drying the organic solvent; (g) treating the solvent-free bone powder with 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing sodium hypochlorite solution from the bone powder and drying it; (i) heat treating the dried bone powder to completely remove lipids and proteins; (j) fractionating the heat treated bone powder into sieves having a pore size of 212 to 1000 µm and 1000 to 2000 µm, respectively; And (k) treating the surface of the bone powder with acidic amino acids.

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

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

In the present invention, the step of treating with the sodium hypochlorite solution of step (g) is used to decompose and remove the residual protein of bone powder in a water-soluble state, inactivating the denatured prion protein causing mad cow disease, Sodium hypochlorite to be used may be a solution of a concentration of 2 to 20% (w / v), preferably a sodium hypochlorite solution of 4% (w / v) concentration is most preferred. In addition, the time for treating the sodium hypochlorite is preferably treated for 72 hours or more to remove the prion and residual protein.

In the present invention, the heat treatment temperature of the step (i) may be 550 ℃ ~ 650 ℃.

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

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

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

In the present invention, the method for producing bone-derived bone graft material in the form of a block, unlike the method for producing bone graft material in the form of a particle, does not include the step of crushing the bone, and removes the lipid and protein from the bone. Cutting into blocks of size.

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 and block-type bone graft material, and increases the cell adhesion by adjusting the surface roughness of the block-type bone graft material. You can. As the acidic amino acid, glutamic acid and aspartic acid may be used. The treatment concentration of the acidic amino acid may be 1-20% (w / v), more preferably 5-10% (w / v). The treatment time may be 5-18 hours, more preferably 8-12 hours.

The present invention relates to a method for producing bone xenograft-derived bone graft material in the form of particles comprising the following steps:

(a) cutting the bone; (b) removing blood components of the cut bone; (c) grinding the bone from which the blood component is removed; (d) first removing fat and protein from the ground bone and drying; (e) immersing the dried bone in an organic solvent and shaking to remove the lipid and protein from the bone secondly; (f) removing and drying the organic solvent; (g) treating the solvent-free bone powder with 4% sodium hypochlorite solution to inactivate prion and other proteins; (h) removing sodium hypochlorite solution from the bone powder and drying it; (i) heat treating the dried bone powder to completely remove lipids and proteins; (j) fractionating the heat treated bone powder into sieves having a pore size of 212 to 1000 µm and 1000 to 2000 µm, respectively; And (k) adding at least one bioactive 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 comprise treating the surface of the bone with an acidic amino acid between steps (j) and (k). By treating the surface of bone with acidic amino acids and adding one or more physiologically active substances selected from the group consisting of extracellular matrix-derived components and bone regeneration functional peptides, it is possible to increase cell adhesion and increase bone regeneration. .

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

(a) cutting the bone; (b) removing blood components of the cut bone; (c) first removing fat and protein from the bone from which the blood components have been removed and drying; (d) immersing the dried bone in an organic solvent and shaking to remove the lipid and protein from the bone secondly; (e) removing and drying the organic solvent; (f) treating the solvent-free bone with 4% sodium hypochlorite solution to inactivate prion and other proteins; (g) removing sodium hypochlorite solution from the bone and drying; (h) heat treating the dried bone to completely remove lipids and proteins; (i) cutting the bone into blocks of the desired size; And (j) adding at least one bioactive 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 comprise treating the surface of the bone with an acidic amino acid between steps (i) and (j). By treating the surface of bone with acidic amino acids and adding one or more physiologically active substances selected from the group consisting of extracellular matrix-derived components and bone regeneration functional peptides, it is possible to increase cell adhesion and increase bone regeneration. .

In the present invention, new bone conduction can be promoted by adding one or more physiologically active substances selected from the group consisting of the extracellular matrix-derived component and the 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 is combined with bone graft material through physical adsorption and hydrogen bonding, and can use a chemical crosslinking agent to strengthen the binding 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. (i) the peptide having bone regeneration ability may be selected from the group consisting of amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 35, (ii) the peptide having apatite binding capacity is a group consisting of the amino acid sequence of SEQ ID NO: 36 to SEQ ID NO: 39 Can be selected 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 Case (SEQ ID NO: 1), BMP-4 (SEQ ID NO: 2) and BMP-6 (SEQ ID NO: 3)], the amino acid sequence of 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 of the position -353 (SEQ ID NO: 9) and the amino acid sequence of the positions 370-396 (SEQ ID NO: 10); Amino acid sequence at positions 74-93 (SEQ ID NO: 11), amino acid sequence at positions 293-313 (SEQ ID NO: 12), amino acid sequence at positions 360-379 (SEQ ID NO: 13), and amino acid sequence at positions 382-402; (SEQ ID NO: 14) Amino acid sequence of positions 91-110 (SEQ ID NO: 15), amino acid sequence of positions 407-418 (SEQ ID NO: 16), amino acid sequence of positions 472-490 (SEQ ID NO: 17), and 487- Amino acid sequence at position 510 (SEQ ID NO: 18) and amino acid sequence at position 98-117 (SEQ ID NO: 19) of BMP-7, amino acid sequence at position 320-340 (SEQ ID NO: 20), amino acid sequence at position 400-409 (SEQ ID NO: Number 21) and the amino acid sequence at positions 405-423 (SEQ ID NO: 22);

(b) amino acid sequence at positions 62-69 of the bone sialoprotein II (BSP II) (SEQ ID NO: 23), amino acid sequence at positions 140-148 (SEQ ID NO: 24), 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 acid at positions 20-28 (SEQ ID NO: 29) , Amino acid sequences 65-90 (SEQ ID NO: 30), amino acids 150-170 (SEQ ID NO: 31) and amino acid sequences 280-290 (SEQ ID NO: 32);

(c) at least one selected from the group consisting of amino acid sequences 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 in that the peptide.

(ii) The peptide that binds 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), and have bone tissue regeneration ability. It is chemically added to the N-terminus of the peptide to increase the binding ability to apatite, which is a constituent of bone, so that it can stably bind to bone graft material or apatite coated implant surface and the like.

In the present invention, the bone regeneration functional peptide is (i) a peptide having bone regeneration ability and (ii) a peptide having an apatite binding ability is combined, it can be present in a stable state by binding to the surface of the apatite dental or orthopedic It can be applied to surgical bone substitutes and apatite-coated metals, natural polymers, and synthetic polymers, and promotes the transfer, proliferation and differentiation of cells related to bone tissue regeneration, and finally maximizes bone tissue regeneration ability. It can exist stably while maintaining the peptide activity is useful for the development of bone tissue regeneration treatment technology using the same.

In the present invention, the bone regeneration functional peptide is stably fixed to the apatite surface, thereby increasing the stability of the peptide and can maintain activity for a long time. Thus, when implanted into the body, by maintaining a stable in the transplanted local, the bone regeneration effect by the peptide can be sustained, which has characteristics suitable for the treatment of bone tissue and periodontal tissue regeneration.

In the present invention, the bone regeneration functional peptide can bind to apatite selected from the group consisting of bio-derived hydroxyapatite bone mineral, synthetic apatite hydroxide, apatite carbonate, 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 (1g) of bone graft material, more preferably may contain 20-80mg per unit weight of bone graft material have.

In one embodiment of the present invention by combining the peptide having the apatite binding capacity of SEQ ID NO: 36 and the peptide having bone tissue regeneration ability of SEQ ID NO: 35 to prepare a bone regeneration functional peptide of SEQ ID NO: 40, the produced peptide is stable to bone graft material It was confirmed that the binding, and the bone graft material in which the peptide is stably fixed to the surface of the apatite was implanted into the bone defect to confirm the bone regeneration ability.

In the present invention, the xylem may be characterized in that selected from the group consisting of bovine bone, horse bone and pork bone.

In still another aspect, the present invention relates to a bone graft material in the form of a particle or block prepared by the method for producing a bone-derived bone graft.

In the present invention, the "bone graft substitute" is a material for filling the space in the bone tissue, the bone graft material using a method such as compression, compression, pressure contact, packing, pressing, hardening, putty, It can be used in the form of pastes, moldable strips, blocks, chips, etc., and can be formulated in the form of gels, granules, pastes, tablets, pellets, etc. using chemical additives. It is possible.

When the bone graft material is formulated as described above, growth factor, fibrin, bone morphogenesis factor, bone growth agent, chemotherapeutic agent, antibiotic, analgesic agent, bisphosphonate, strontum salt, fluoride salt, magnesium salt to promote bone growth 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, and fibroblast growth factor (FGF). ) And BGDF-II (beta-2-microglobulin) can be used. As the bone morphogenesis factor, osteocalcin, headquarter alloprotein, bonesialo protein, osteogenin, BMP, and the like can 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 may use peptides or nucleic acids that promote bone formation and antagonists of substances that inhibit bone formation.

Chemical additives used to formulate bone graft material in the present invention is hyaluronic acid (hyaluronic acid), collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate, ceramics, and the like, depending on the type of the additive, gel, strip, Formulation is possible in the form of granules, chips, pellets, tablets, pastes and the like.

Collagen, gelatin, chitosan, alginate, carboxymethylcellulose, hydroxypropylmethylcellulose, polyethyleneglycol, poloxamer, to prepare pharmaceutical compositions in the form of gels or pastes to increase the ease of use of the bone graft material of the present invention, Biocompatible polymers including polylactic acid, polylactic glycolic acid, polycaprolactone and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and 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 form Bone graft  Manufacturing method

[Pretreatment and Grinding Process]

The bone obtained from the femoral region of the cow was cut to a size of about 2 cm ³ using a bone cutter. The cut bone pieces were immersed in deionized water for 24 hours to remove blood components present in the bone. The bone from which blood components were removed was ground to a size of 0.7 mm or less using a grinder. The bone powder was boiled for 72 hours with deionized water exchanged every 12 hours to remove the fat and protein present in the bone. The bone fragments from which the fat and protein were first removed were completely dried in an oven at 60 ° C. for 24 hours.

[Degreasing process]

A solvent in which 20 ml of chloroform and methanol were mixed in a 1: 1 volume ratio was added per 1 g of the dried bone powder, and then degreased by shaking for 24 hours at a rotation speed of 120 rpm. Deionized water was added at a rate of 50 g per 1 g of bone powder in order to remove the remaining solvent in the bone powder completed degreasing and then shaken at 120 rpm for 12 hours to remove the solvent remaining in the powder. At this time, the flushing efficiency was increased by replacing with fresh deionized water every two hours. The washed bone powder was completely dried in an oven at 60 ℃.

Deproteinization Process

25 g of 4% (w / v) sodium hypochlorite solution was added per 1 g of dried bone powder degreased and shaken for 24 hours at a rotational speed of 120 rpm to remove proteins present in the bone powder. . To remove the remaining solvent in the deproteinized bone powder, 50 g of deionized water was added per 1 g of bone powder and shaken at 120 rpm for 72 hours to remove the remaining sodium hypochlorite. At this time, the first 12 hours were exchanged with fresh deionized water every 2 hours, after which every 12 hours was replaced with fresh deionized water. The washed bone powder was completely dried in an oven at 60 ℃.

[Heat treatment process]

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

[Separation process]

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

Example  1-2: Block Type Bone graft  Manufacturing method

[Pretreatment Process]

Bones from the femoral area of the horse using a bone cutter 8cm ³ Cut to size. The cut bone pieces were immersed in purified water for 6-15 hours, and then purified water was removed and the blood components present in the bone pieces were removed by immersing again for 6-15 hours. The bone fragments from which blood was removed with the purified water were limited to heating for at least 3 hours per day, up to 6 hours. This method was repeated for at least 72 to 80 hours to remove the fat and protein present in the bone fragments first. The bone fragments from which the fat and protein were first removed were dried at 120 ° C. for 12 hours. The dried bone fragments were classified into spongy bone and dense bone, respectively.

[Degreasing process]

6 g of chloroform and methanol mixed in a 1: 1 volume ratio were added per 1 g of the dried bone pieces, and the mixture was immersed for 24 hours to degrease, and then the methanol was discarded. To remove the remaining solvent in the degreasing bone fragments, the methanol was immersed in 6 ml of methanol per 1 g for 12 hours and then discarded. In order to remove the methanol remaining in the bone fragments 6 times with 10ml purified water per 1g bone fragments and then washed repeatedly for 1 hour 1 time 1 time by the pressure heating method. The washed bone pieces were completely dried in an oven at 120 ° C. for 12 hours.

Deproteinization Process

Sodium hypochlorite solution at 4% (w / v) concentration of 8 ml per 1 g of dried bone pieces degreased is added, and after 6 hours of soaking, the bubbles disappear and the sodium hypochlorite turns colorless. Sodium chlorate was replaced. This method was repeated 3 to 5 times. In order to remove the remaining solvent in the deproteinized bone fragments, the bone fragments were washed 10 times with purified water for 12 hours, and then washed with Lab Stirrer a total of 10 times once every hour with purified water. After washing 10 times, 10L of purified water was filled again, and then heated and discarded twice for 30 minutes. The washed bone pieces were completely dried at 120 ° C. for 12 hours.

[Heat treatment process]

After degreasing and deproteinization, the dried bone fragments were heat-treated to remove the remaining lipids and proteins. The electric furnace used for the heat treatment was heated to 2 degrees per minute, the bone pieces were heat-treated at 600 ℃ for 3 hours and then cooled. The heat treated bone pieces are put in purified water and heated for 1 hour. Heat washing was repeated several times to remove fine dust, dried in an oven at 120 ° C. for 12 hours, and used as bone graft material.

[Process to cut 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 inside of the particle and block bone grafts prepared in Examples 1-1 and 1-2 were observed. Internally, a differential scanning electron microscope was used. In addition, XRD was measured to determine whether the bone graft material prepared as described above was made of apatite crystals and analyzed the crystallinity. Figure 1a is an external photograph and a differential scanning microscope picture of the bone graft material in the form of particles, Figure 1b is an external view and a differential scanning microscope picture of the bone graft material in the form of a block. Figure 2 is an XRD graph, Figure 2a is an XRD graph of the granular bone graft material, Figure 2b shows an XRD graph of the block-shaped bone graft material. All were confirmed to be pure apatite crystals.

Example  2: treatment with acidic amino acid

Example  2-1: Surface Treatment Bone graft  Manufacturing method

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

Example  2-2: Roughness observation after surface treatment

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

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

The bone graft in the form of a block appears to have pores connected to the inside, and has a structure suitable for use as a support for tissue engineering. It can also be seen that the roughness was reduced after the surface treatment with an acidic amino acid.

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

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

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

Example  3: Extracellular matrix  Derived ingredient addition

Example  3-1: Extracellular matrix  Derived components Bone graft  Produce

100 mg of ribose (D-ribose) was dissolved in 50 ml of a 2% (w / v) collagen solution. 10 g of the graft material in the form of a particle or block prepared in Example 1 was added, and then placed in a desiccator and maintained in a vacuum state for 1 hour to infiltrate the collagen solution to the inside of 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 dry for 48 hours in a vacuum at 140 ℃.

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

The bone graft material prepared in Example 3-1 was implanted in the rabbit's skull bone defect to determine the bone regeneration ability. A circular bone defect with a diameter of 8 mm was formed in the cranial area of the anesthesia rabbit (Newzealand White rabbit, name: cuniculus), the collagen shield was covered with the bone defect, and the periosteum and the skin were double-sealed. Animals were sacrificed 4 weeks after transplantation. The specimens were fixed in formalin solution and embedding tissues were prepared with a thickness of 20 μm. The prepared specimens were stained with basic fuchsin and toluidine blue to prepare non-limeous specimens. The specimens were photographed with an optical microscope.

The column on the left is a 40-magnification photo, and the column on the right is a magnification of the square of the left column at 100 magnification. GB stands for bone graft and NB stands for new bone. In the case of bone graft material treated with extracellular matrix-derived components (FIG. 5B) than bone graft material not treated with extracellular matrix-derived components (FIG. 5A), bone regeneration was increased (FIG. 5). This is because the extracellular matrix-derived components were introduced into the bone graft material, thereby creating a more suitable environment for regeneration of new bone.

Example  3-3: Acid Amino Acid Treatment + Extracellular matrix  By adding derived ingredients Bone graft Cell adhesion  observe

After treating the surface of the bone graft material with an acidic amino acid as in Example 2-1, it was observed whether the cell adhesion ability is further increased when the extracellular matrix-derived components are added. As a result, as shown in Figure 6, it was confirmed that the cell adhesion ability was further increased than when only acidic amino acid treatment (Fig. 4). This is because the roughness is reduced to an acidic amino acid and at the same time, the introduction of an extracellular matrix-derived component creates an environment similar to that in vivo, thereby making it more suitable for cell adhesion.

Example  4 : Bone regeneration  Functional Peptides  adding

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

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

Synthesis was confirmed using the ninhydrin test method. After the test was completed, the synthesized resin was dried with THF or DCM, and TFA cleavage cocktail was added at a rate of 20 ml per 1g of resin. Through the cocktail was dissolved resin and peptide dissolved. The filtered solution was removed using a rotary evaporator, and then cold ether was added or an excessive amount of cold ether was directly added to the TFA cocktail solution in which the peptide was dissolved to crystallize the peptide into a solid phase. Separated. At this time, the TFA cocktail was completely removed by washing and centrifuging with ether several times. 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 cleaved from the resin, washed, lyophilized and separated and purified by liquid chromatography. The purified peptide was confirmed molecular weight using MALDI analysis.

In the preparation of SEQ ID NO: 40, 10 equivalents of Fluorescein isothicyanate (FITC) was bound to the N-terminus using triethylamine (1 ml per 1 g of resin) at the N-terminus, 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 tertiary distilled water, and then added to 4 g of the bone graft material prepared in Example 1, which was deposited for 24 hours, and then lyophilized.

Example  4-2: Bone regeneration  Functional Peptides  In addition Of Bone regeneration  observe

As in Example 4-1, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 peptides were respectively coupled to the bone graft material, and then transplanted in the skull cranial bone defect of the rabbit to confirm bone regeneration. A circular bone defect with a diameter of 8 mm was formed in the skull portion of the anesthesia rabbit (Newzealand White rabbit, namely cuniculus), and a bone graft material containing bone graft material and a peptide was transplanted by 50 mg per defect part, Periosteum and skin were double sealed. Two weeks after the implantation, the animals were sacrificed, and the collected specimens were fixed in formalin solution and embedded in tissue to prepare specimens having a thickness of 20 μm. The prepared specimens were stained with basic fuchsin and toluidine blue to prepare non-limeous specimens. The specimens were photographed with an optical microscope.

In addition, the bone regeneration function was observed by treating the bone regeneration functional peptide in the same manner as the above in the bone or bone-type bone graft material treated with the acidic amino acid prepared in Example 2.

Figure 7 shows the bone regeneration effect by the bone graft material added bone regeneration functional peptide, Figure 7a is a bone regeneration effect by the bone graft material in the form of particles before treatment, Figure 7b is a particle after treating the bone regeneration functional peptide Bone regeneration effect by the bone graft material of the form, Figure 7c is a bone regeneration effect by the granular bone graft material added bone regeneration functional peptide after surface treatment with acidic amino acid. The left column is a 40x photo, and the right column is a magnified 100x square. GB stands for bone graft and NB stands for new bone.

As a result, it can be seen that the bone regeneration effect is increased when the bone regeneration functional peptide is treated, and the effect is further increased when both the acidic amino acid and + bone regeneration functional peptide is treated. Therefore, when the acidic amino acid treatment and bone regeneration functional peptides of the present invention are used in apatite-based bone grafts or apatite-coated implants, bone tissue regeneration effect is expected to be great.

Attach an electronic file to a sequence list

Claims (13)

delete delete delete delete Method for producing bone bone-derived bone graft material in the form of particles comprising the following steps:
(a) cutting the xenograft; (b) removing blood components of the cut xenograft; (c) grinding the xenograft from which the blood component is removed; (d) first removing fat and protein from the ground xenograft and drying; (e) immersing the dried xenograft in an organic solvent and shaking the secondary to remove fat and protein from xenograft; (f) removing and drying the organic solvent; (g) treating the dissociated bone bone powder with sodium hypochlorite solution at a concentration of 2-20% to inactivate prion and other proteins; (h) removing sodium hypochlorite solution from the xenograft powder and drying the solution; (i) heat treating the dried xenograft powder to completely remove lipids and proteins; (j) fractionating the heat treated hetero bone powder into a sieve having a pore size of 212 to 1000 μm, and then fractionating the sieve bone having a pore size of 1000 to 2000 μm; And (k) one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 35, and a bone regeneration functional peptide to which the peptide consisting of amino acid sequences of SEQ ID NO: 36 is bound to the xenograft powder Adding more than one bioactive substance.
According to claim 5, Between (j) and (k), characterized in that it further comprises the step of treating the surface of the hetero bone powder with acidic amino acid, xenograft-derived bone graft material manufacturing method.
delete delete delete delete The method of claim 5, 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.
6. The method of claim 5, wherein the xenograft is selected from the group consisting of bovine bone, horse bone and pork bone.
In the group consisting of bone graft material prepared by the method of claim 5 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.

Images