Title of Invention	METHOD FOR PREPARING A PRION-FREE BONE GRAFTING SUBSTITUTE
Abstract	ABSTRACT The present invention relates to a method for preparing a hone graft substitute using bovine bone, and more particularly to a method for preparing a sale hone graft substitute which does not have the risk of infection with bovine spongiform encephalopathy, the method comprising treating bovine hone with sodium hypochlorite and treating the treated bone at a high temperature of more than 600 ('. The bone graft substitute does not cause an immune respon.se. because it is prepared by effectively removing lipids and organic substances from bovine bone having a structure very similar to that of the human bone. Also, it has excellent osteoconduetivity. and is free of prion, and thus it does not have the risk of infection with bovine spongiform encephalopathy. According to the disclosed invention, the bone graft substitute having such advantages can be prepared in a simple manner.

Title of Invention

METHOD FOR PREPARING A PRION-FREE BONE GRAFTING SUBSTITUTE

Abstract

ABSTRACT The present invention relates to a method for preparing a hone graft substitute using bovine bone, and more particularly to a method for preparing a sale hone graft substitute which does not have the risk of infection with bovine spongiform encephalopathy, the method comprising treating bovine hone with sodium hypochlorite and treating the treated bone at a high temperature of more than 600 ('. The bone graft substitute does not cause an immune respon.se. because it is prepared by effectively removing lipids and organic substances from bovine bone having a structure very similar to that of the human bone. Also, it has excellent osteoconduetivity. and is free of prion, and thus it does not have the risk of infection with bovine spongiform encephalopathy. According to the disclosed invention, the bone graft substitute having such advantages can be prepared in a simple manner.

Full Text	METHOD FOR PREPARING A PRION-FREE BONE GRAFTING SUBSTITUTE TECHNICAL FIELD The present invention relates to a method for preparing it bone graft substitute using bovine hone, and more particularly to a method for preparing a safe bone graft substitute which does not have the risk of infection with bovine spongiform encephalopathy, the method comprising treating bovine bone with sodium hypochlorite and treating the treated hone al a high temperature of more than 600 C. BACKGROUNDART A bone graft substitute BGSi refers to a graft material that N used to substitute for bone tissue defects caused by various dental diseases or traumas, disease-related degeneration or other loss of tissue, so as to fill pore spaces in the hone tissue and to promote the formation of new bone. The best graft is generally known to be autogenous bone graft, but the autogenous bone graft has -problems in that it requires a secondary surgical operation, is difficult to obtain the required amount, is difficult to carry out at small-scale hospitals, and has a possibility that makes patient's pain and morbidity severe. For this reason, various substitutes, including donated human bones, artificial bones, and artificially synthesized materials made of bone hydroxyapatite. have been used for grafting. Commercially available bone substitutes are advantageous in thai they are available in various forms, including powder, gel. slurry/putty, tablets, chips, morsels, pellets, sticks, sheets and blocks, are homogeneous, have a low risk with respect to infection and disease, eliminate the risk of pains resulting from the collection of a patient's own bones for grafting, and have reduced In an attempt to solve these problems, bone minerals obtained by physicochemically treating animal bones having a structure similar to that of human bones so as to remove organic suhsumees have been processed such that they could be used in dental or orthopedic surgical operations. A typical example thereof may include Bio-Oss© commercially available from Geistlich Biomaterials. A method for preparing said bone graft substitute using animal bones comprises the steps of: treating the thighbone of a bovine animal in a solvent having a boiling point of K0-120V to remove lipids; adding ammonia or primary amine to the treated bone to remove proteins and organic substances, thus obtaining hone Although there was an example where a cartilage was treated with sodium hypochlorite to selectively remove the collagen phase in order to observe the remaining cartilage structure (Broz. J..I. ct a!.. J. Mater. Sci. Mater. Med. 8:395. 1997). it has not yet been reported that sodium hypochlorite K used to remove all proteins in the preparation of bone minerals. Among such animal bones, the most frequently used bone is bovine bone, and said Bio-Oss© product is also produced using the bovine bone as a raw material. However, as the onset of bovine spongiform encephalopathy has recently been frequent, the safety of the bovine bone as a raw material with respect to bovine spongiform encephalopathy is not ensured. For this reason, in a step of processing bovine bone into a bone graft material, a prion that causes bovine spongiform encephalopathy must be removed. Because the prion is not completely removed even at a high temperature of 600 ( \ it cannot be removed hv methods known so far. and thus the development of a novel method is required. Accordingly, the present inventors have prepared a bone graft substitute, which does not have the risk of bovine spongiform encephalopathy, using a method comprising the steps of inactivating prion protein with sodium hypochlorite in a process of preparing a bone graft substitute using bovine bone, and heating the resulting hone at a high temperature of 600 . thereby completing the present invention. SUMMARY OF INN^NTION In one aspect, the present invention relates to a method for preparing a bone graft substitute, which is completely free of prion protein that causes bovine spongiform encephalopathy, the method comprising treating bovine bone uith -.odium hypochlorite and heating the treated bone at a high temperature. In another aspect, the present invention relates to a bone graft substitute composition containing the bone graft substitute prepared according to said method. Other features and embodiments of the present invention will become apparent from the following detailed description and claims. BRIEF DESCRIPTION OF DRAWINGS FIG, 1 is a. scanning electron microscope photograph of bone powder thermally treated at 600T'. FK1 2 shows XRD measurement results tor bone powder thermally treated at 600 V. FIG. 3 shows FT-IR measurement results for bone powder thermally treated at 600'. FIG. 4 shows photographs of tissue samples taken at 2 weeks after each of bone powder prepared in Example I. Bio-Oss'Rl. and OsteoGrafR')/N has been implanted into the circular defects of New Zealand while rabhits. FfG. 5 shows photographs of tissue samples taken at 4 weeks after each of bone powder prepared in Example I. Bio-Oss'"'. and OsteoGraf[ DETAILED DESCRIPTION OF THE INVENTION. AND PREFERRED EMBODIMENTS The present invention provides a method for preparing a bone graft substitute from bovine hone, the method comprising the steps of: (a) boiling bovine bone, from which blood components have been removed, in deionized water to remo\e lipids and proteins, and drying the boiled bone: 'bi grinding the dried bone, and immersing and shaking the ground bone powder in an organic solvent: (O removing the organic solvent and drying the bone powder: idi treating the dried bone powder, from which the solvent has been removed, with a solution of 2-2()r/c sodium hypochlorite: (e) removing the sodium hypochlorite solution from the bone powder and drying the resulting bone powder: and (fl thermally treating the dried bone powder at 600-1000"C for 1-6 hours to completely remove lipids and proteins. In the inventive method, the step of immersing the bone powder in the organic solvent is a step of removing lipids remaining in the bovine bone powder, in which the organic solvent may preferably be a mixed solvent of chloroform and methanol. The ratio of chloroform: methanol in the mixed solvent ma\ be 2-S:K-2, and preferabh 1:1. In the inventhe method, the step of treating the bone powder with the sodium hypochlorite solution is a step of' removing proteins remaining in the ho\ine hone powder and inactivating a prion that causes bovine spongiform encephalopathy. The sodium hypochlorite solution used in this step may be a solution having a sodium hypochlorite concentration of 2-20% (w/v>. and most preferably about 4% (w/v). The step of treating the bone powder with the sodium hypochlorite must be conducted for at least 20 minutes in order to inactivate the prion, and is preferably conducted for at least 72 hours in order to remove the remaining proteins. In the inventive method, the step (d) may additionally comprise adding I-10N sodium hydroxide to the sodium hypochloride solution in order to increase efficiency of inactivating the prion. The concentration of the sodium hydroxide is preferahh about 2N. The inventive method mav additionally comprise, after the step if), the steps of: sieving the thermally treated bone powder through a sieve having a pore size of 212-425 urn: and washing the sieved bone powder. In another aspect, the present invention provides a composition for bone graft substitution, containing the bone graft substitute prepared according to said method. The inventive composition for bone graft substitution may additionally contain at least one biologically active substance selected from the group consisting of a bone growth-promoting factor, a fibrin, a bone morphogenic factor, a hone growth agent, a ehemotherapeutic agent, an antibiotic, an analgesic, a bisphosphonate. a strontium salt, a fluorine salt, a magnesium salt, and a sodium sait. Also, it may additionally contain at least one chemical compound selected from the group consisting ot hyaluronic acid, chondroitin sulfate, alginic acid, chitosan. collagen. human body and promotes bone growth. Examples of the bone growth agent, which can be used in the present invention, include peptides or nucleic acids that facilitate hone formation, and antagonists for substances that inhibit bone format km. Examples of chemical additives, which are used to form the bone graft substitute in the present invention, include hyaluronic acid, chondroitm sulfate, alginic acid, chitosan. collagen, hydroxyapatite. calcium carbonate, calcium phosphate, calcium sulfate, and ceramics. Depending on the kind of the additives, the bone graft substitute can be formed in the shape of gel. strips, granules, chips, pellets, tablets, paste, etc. Examples Hereinafter, the present invention will be described in further detail with reference to examples. Il is to be understood, however, that these examples are for illustrative purposes only and are not to he construed to limit the scope of the present invention. Example 1: Preparation of bone graft substitute [Pretreatment and grinding step] A bovine femoral bone was cut to a size of ? cm' 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 pieces washed with deioni/ed water were boiled for 72 hours while replacing the deionized water at 12-hr intervals, thus primarily removing lipids and proteins present in the bones. The bone pieces from which the lipids and proteins have primarily been removed were completely dried in an oven at 60 C for 24 hours, and then ground to a size of less than 0.7 mm using a mill. [Defatting step! To 1 g of the ground bone powder. 2(1 ml of a mixed solvent of chloroform and methanol (1:1 \/vt was added and (he solution was shaken a( a rotating speed of 120 rpm for 24 hours so as to defat the bone powder. In order to remo\e the solvent remaining in the defatted hone powder, deionized water was added to the bone powder in a weight ratio of 50:1. and then the solution v\ as shaken at 120 rpm for 12 hours, thus removing the solvent remaining in the powder. At this time, the deionized water was replaced with fresh deionized water at 2-hr intervals in order to increase washing efficiency. The washed bone powder was completely dried in an oven at 60V. [Deproteinizing step] To 1 g of the defatted bone powder. 25ml of a solution of 4r added to I g of the bone powder, and the solution was shaken at 120 rpm for 72 hours, thus removing the sodium hypochlorite remaining in the powder. At this time, the deionized water was replaced with fresh deionized water every two hours for the first I 2 hours, and then replaced with fresh deionized w ater every I 2 horns. The water-washed bone powder was completely dried in an o\ en at 60 i'. [Thermal treatment step] The defatted, deproteinized and dried bone powder was thermally treated at high temperature to remove lipids and proteins remaining therein. The temperature of an electric furnace used for the thermal treatment was elevated at a rale of 2 ' Vmin. and the bone powder was thermally treated at 600' for 3 hours, followed by furnace cooling. [Sieving step] The thermal!v treated bone powder was sieved through a sieve having a pore size ot 215-425 :>_m. and the sieved hone powder was washed a few limes with deionized water to remove fine particles remaining on the surface thereof, and then dried in an oven at 60 V for 24 hours. The dried hone powder was collected and used as a bone graft substitute. The bone powder subjected to the above-described steps was analyzed using a scanning electron microscope and. as a result, hydroxyapatite particles having a size of 50-H0 nm were observed in the bone powder tRG. H. Also, the bone powder was analyzed by XRD and. LIS a result, it could be observed that a pure, low-crystalline apatite phase was produced in the bone powder (FIG. 2), Also, from the results of FT-IR analysis, it was confirmed that the bone powder was a low crystalline carbonate apatite containing a carbonate group, similar to human bone (FIG. 3). Example 2: Preparation of composition for bone gruff substitution To 100 g of desalted water. 20 g of hyaluronic acid was added to make a viscous hyaluronic acid solution, to which 10 g of the bone powder prepared in Example 1 was then added to make an injectable paste. Example 3: Evaluation of osteoconductivitv of bone graft substitute In order to examine the osteoconductivity of the inventive bone graft substitute, the evaluation of osteoconductivity was conducted for the hone graft substitute prepared according to the method of Example 1. and commercially available bone substitutes Bio-Oss,R' and OsteoGraf^'/N as control groups. In this Example. New Zealand white rabbits were used and circular defects having a diameter of S mm were formed in the cranial bones of the animals and then implanted with each of the inventive bone graft substitute. Bio-Oss'Rl and OsteoGraf,K7N granules. At 2 weeks and 4 weeks after the implantation, tissue samples were prepared and comparatively analyzed for osteoconduetiviry nn the basis of the amount of hone produced around each of the hone graft substitutes and the production or non-produclion of connective tissues. As a result, in the test samples prepared at 2 weeks after implantation of the three bone substitutes, only connective tissues were mostly produced around the portions implanted with Bio-Oss'R,anti OsteoGrafk'/N. and the formation of new bones was hardly observed around the implanted portions (FIG. 4). On the other hand, as shown in FIG. 4(c). it could be observed that the sample implanted with the bone graft substitute prepared in Example I had a large amount of new bones produced therein (see arrow in FIG. 4(c)). FIG. 5 shows photographs of tissue samples prepared at 4 weeks after implantation with the three bone substitutes. As shown in FIG. 5. in the tissue samples were formed and mosfh surrounded b\ connective tissues, and on the other hand, in the tissue samples implanted with the bone graft substitute prepared in the Example 1. a large amount of new bones were produced and grown around the bone graft substitute. This suggests that the bone graft substitute prepared according to the present invention has very excellent osteoconductivin compared to those of the prior bone graft substitutes. INDUSTRIAL APPLICABILITY As described above, the present invention provides the method for preparing the prion-free bone graft substitute, comprising treating bovine bone with sodium hypochlorite solution and subjecting the treated bone to high-temperature treatment, as well as a composition containing said bone graft substitute. The inventive hone graft substitute does not cause an immune response, because it is prepared by effectively removing lipids and organic substances from bovine bone having a ; i structure ver\ similar to that of the human bone. Also, it has excellent osteoconductivity. and is free of the prion, and thus it does not have the risk of infection with bovine spongiform encephalopathy. According to the present invention, the hone graft substitute having such advantages can be prepared in a simple manner. 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 onlv for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will he defined by the appended claims and equivalents thereof. THE CLAIMS What is Claimed is: 1. A method tor preparing a bone graft substitute from bovine bone, the method comprising the steps of: la) boiling bovine bone, from which blood components ha\e been removed, in deionized water to remove lipids and proteins, and drying the boiled hone: (b) grinding the dried bone, and immersing and shaking the ground bone powder in an organic solvent: (c) removing the organic solvent and drying the bone powder: (d) treating the dried bone powder, from which the solvent has been removed, with a solution of 2-20% sodium hypochlorite: if) thermalh treating the dried bone powder a completely remove lipids and proteins. 2. The method for preparing a bone graft substitute from bovine hone according to claim 1. wherein the organic solvent is a mixed solvent of chloroform and methanol. 3. The method for preparing a bone graft substitute from bovine bone according to claim 1. wherein the sodium hypochlorite concentration is about 4. The method for preparing a bone graft substitute from box hie bone according to claim 1. wherein the step (d) additionally comprises adding 1-4 ON sodium hydroxide to the sodium hypochloride solution. 5. The method for preparing a bone graft substitute from bovine bone according to claim 4. wherein the concentration of the sodium hydroxide is about 2N. 6. The method for preparing a bone graft substitute from bovine bone according to claim 1. which further comprises the steps after the step of sieving the thermally treated bone powder through a sieve having a pore size of 212-425 nm; and washing the sieved bone powder. 7. A composition for bone graft substitution, containing the bone graft substitute prepared by the method of any one claim among claims 1-6. 8. The composition for hone graft substitution according to claim 7. which additionally contains at least one biologically active substance selected from the group consisting of a bone growth-promoting factor, a fibrin, a hone morphogenic factor, a bone growth agent, a chemotherapeutic agent, an antibiotic, an analgesic, a hisphosphonate. a strontium salt, a fluorine salt, a magnesium salt, and a sodium ■.alt. 9. The composition for bone graft substitution according to claim ~ or s. which additionally contains at least one chemical compound selected from the group consisting of hyaluronic acid, chondroitin sulfate, alginic acid, chitosan. collagen, hydroxyapatite. calcium carbonate, calcium phosphate, calcium sulfate, and ceramics. 10. The composition for bone graft substitution according to claim 9. wherein the chemical compound is hyaluronic acid, and the composition is gel-type.

Full Text

METHOD FOR PREPARING A PRION-FREE BONE GRAFTING SUBSTITUTE
TECHNICAL FIELD
The present invention relates to a method for preparing it bone graft substitute using bovine hone, and more particularly to a method for preparing a safe bone graft substitute which does not have the risk of infection with bovine spongiform encephalopathy, the method comprising treating bovine bone with sodium hypochlorite and treating the treated hone al a high temperature of more than 600 C.
BACKGROUNDART
A bone graft substitute BGSi refers to a graft material that N used to substitute for bone tissue defects caused by various dental diseases or traumas, disease-related degeneration or other loss of tissue, so as to fill pore spaces in the hone tissue and to promote the formation of new bone. The best graft is generally known to be autogenous bone graft, but the autogenous bone graft has -problems in that it requires a secondary surgical operation, is difficult to obtain the required amount, is difficult to carry out at small-scale hospitals, and has a possibility that makes patient's pain and morbidity severe.
For this reason, various substitutes, including donated human bones, artificial bones, and artificially synthesized materials made of bone hydroxyapatite. have been used for grafting. Commercially available bone substitutes are advantageous in thai they are available in various forms, including powder, gel. slurry/putty, tablets, chips, morsels, pellets, sticks, sheets and blocks, are homogeneous, have a low risk with respect to infection and disease, eliminate the risk of pains resulting from the collection of a patient's own bones for grafting, and have reduced

In an attempt to solve these problems, bone minerals obtained by physicochemically treating animal bones having a structure similar to that of human bones so as to remove organic suhsumees have been processed such that they could be used in dental or orthopedic surgical operations. A typical example thereof may include Bio-Oss© commercially available from Geistlich Biomaterials.
A method for preparing said bone graft substitute using animal bones comprises the steps of: treating the thighbone of a bovine animal in a solvent having a boiling point of K0-120V to remove lipids; adding ammonia or primary amine to the treated bone to remove proteins and organic substances, thus obtaining hone

Although there was an example where a cartilage was treated with sodium hypochlorite to selectively remove the collagen phase in order to observe the remaining cartilage structure (Broz. J..I. ct a!.. J. Mater. Sci. Mater. Med. 8:395. 1997). it has not yet been reported that sodium hypochlorite K used to remove all proteins in the preparation of bone minerals.
Among such animal bones, the most frequently used bone is bovine bone, and said Bio-Oss© product is also produced using the bovine bone as a raw material. However, as the onset of bovine spongiform encephalopathy has recently been frequent, the safety of the bovine bone as a raw material with respect to bovine spongiform encephalopathy is not ensured. For this reason, in a step of processing bovine bone into a bone graft material, a prion that causes bovine spongiform encephalopathy must be removed. Because the prion is not

completely removed even at a high temperature of 600 ( \ it cannot be removed hv methods known so far. and thus the development of a novel method is required.
Accordingly, the present inventors have prepared a bone graft substitute, which does not have the risk of bovine spongiform encephalopathy, using a method comprising the steps of inactivating prion protein with sodium hypochlorite in a process of preparing a bone graft substitute using bovine bone, and heating the resulting hone at a high temperature of 600 . thereby completing the present invention.
SUMMARY OF INN^NTION
In one aspect, the present invention relates to a method for preparing a bone graft substitute, which is completely free of prion protein that causes bovine spongiform encephalopathy, the method comprising treating bovine bone uith -.odium hypochlorite and heating the treated bone at a high temperature.
In another aspect, the present invention relates to a bone graft substitute composition containing the bone graft substitute prepared according to said method.
Other features and embodiments of the present invention will become apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG, 1 is a. scanning electron microscope photograph of bone powder thermally treated at 600T'.
FK1 2 shows XRD measurement results tor bone powder thermally treated at 600 V.

FIG. 3 shows FT-IR measurement results for bone powder thermally treated at
600'.
FIG. 4 shows photographs of tissue samples taken at 2 weeks after each of bone powder prepared in Example I. Bio-Oss'Rl. and OsteoGrafR')/N has been implanted into the circular defects of New Zealand while rabhits.
FfG. 5 shows photographs of tissue samples taken at 4 weeks after each of bone powder prepared in Example I. Bio-Oss'"'. and OsteoGraf[ DETAILED DESCRIPTION OF THE INVENTION. AND PREFERRED EMBODIMENTS
The present invention provides a method for preparing a bone graft substitute from bovine hone, the method comprising the steps of: (a) boiling bovine bone, from which blood components have been removed, in deionized water to remo\e lipids and proteins, and drying the boiled bone: 'bi grinding the dried bone, and immersing and shaking the ground bone powder in an organic solvent: (O removing the organic solvent and drying the bone powder: idi treating the dried bone powder, from which the solvent has been removed, with a solution of 2-2()r/c sodium hypochlorite: (e) removing the sodium hypochlorite solution from the bone powder and drying the resulting bone powder: and (fl thermally treating the dried bone powder at 600-1000"C for 1-6 hours to completely remove lipids and proteins.

In the inventive method, the step of immersing the bone powder in the organic
solvent is a step of removing lipids remaining in the bovine bone powder, in which the organic solvent may preferably be a mixed solvent of chloroform and methanol. The ratio of chloroform: methanol in the mixed solvent ma\ be 2-S:K-2, and

preferabh 1:1.
In the inventhe method, the step of treating the bone powder with the sodium hypochlorite solution is a step of' removing proteins remaining in the ho\ine hone powder and inactivating a prion that causes bovine spongiform encephalopathy. The sodium hypochlorite solution used in this step may be a solution having a sodium hypochlorite concentration of 2-20% (w/v>. and most preferably about 4% (w/v). The step of treating the bone powder with the sodium hypochlorite must be conducted for at least 20 minutes in order to inactivate the prion, and is preferably conducted for at least 72 hours in order to remove the remaining proteins.
In the inventive method, the step (d) may additionally comprise adding I-10N sodium hydroxide to the sodium hypochloride solution in order to increase efficiency of inactivating the prion. The concentration of the sodium hydroxide is preferahh about 2N.
The inventive method mav additionally comprise, after the step if), the steps of: sieving the thermally treated bone powder through a sieve having a pore size of 212-425 urn: and washing the sieved bone powder.
In another aspect, the present invention provides a composition for bone graft substitution, containing the bone graft substitute prepared according to said method.
The inventive composition for bone graft substitution may additionally contain at least one biologically active substance selected from the group consisting of a bone growth-promoting factor, a fibrin, a bone morphogenic factor, a hone growth agent, a ehemotherapeutic agent, an antibiotic, an analgesic, a bisphosphonate. a strontium salt, a fluorine salt, a magnesium salt, and a sodium sait. Also, it may additionally contain at least one chemical compound selected from the group consisting ot hyaluronic acid, chondroitin sulfate, alginic acid, chitosan. collagen.

human body and promotes bone growth. Examples of the bone growth agent, which can be used in the present invention, include peptides or nucleic acids that

facilitate hone formation, and antagonists for substances that inhibit bone format km.
Examples of chemical additives, which are used to form the bone graft substitute in the present invention, include hyaluronic acid, chondroitm sulfate, alginic acid, chitosan. collagen, hydroxyapatite. calcium carbonate, calcium phosphate, calcium sulfate, and ceramics. Depending on the kind of the additives, the bone graft substitute can be formed in the shape of gel. strips, granules, chips, pellets, tablets, paste, etc.
Examples
Hereinafter, the present invention will be described in further detail with reference to examples. Il is to be understood, however, that these examples are for illustrative purposes only and are not to he construed to limit the scope of the present invention.
Example 1: Preparation of bone graft substitute
[Pretreatment and grinding step]
A bovine femoral bone was cut to a size of ? cm' 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 pieces washed with deioni/ed water were boiled for 72 hours while replacing the deionized water at 12-hr intervals, thus primarily removing lipids and proteins present in the bones. The bone pieces from which the lipids and proteins have primarily been removed were completely dried in an oven at 60 C for 24 hours, and then ground to a size of less than 0.7 mm using a mill.
[Defatting step!
To 1 g of the ground bone powder. 2(1 ml of a mixed solvent of chloroform and

methanol (1:1 \/vt was added and (he solution was shaken a( a rotating speed of 120 rpm for 24 hours so as to defat the bone powder. In order to remo\e the solvent remaining in the defatted hone powder, deionized water was added to the bone powder in a weight ratio of 50:1. and then the solution v\ as shaken at 120 rpm for 12 hours, thus removing the solvent remaining in the powder. At this time, the deionized water was replaced with fresh deionized water at 2-hr intervals in order to increase washing efficiency. The washed bone powder was completely dried in an oven at 60V.
[Deproteinizing step]
To 1 g of the defatted bone powder. 25ml of a solution of 4r
added to I g of the bone powder, and the solution was shaken at 120 rpm for 72 hours, thus removing the sodium hypochlorite remaining in the powder. At this time, the deionized water was replaced with fresh deionized water every two hours for the first I 2 hours, and then replaced with fresh deionized w ater every I 2 horns. The water-washed bone powder was completely dried in an o\ en at 60 i'.
[Thermal treatment step]
The defatted, deproteinized and dried bone powder was thermally treated at high temperature to remove lipids and proteins remaining therein. The temperature of an electric furnace used for the thermal treatment was elevated at a rale of 2 ' Vmin. and the bone powder was thermally treated at 600' for 3 hours, followed by furnace cooling.
[Sieving step]
The thermal!v treated bone powder was sieved through a sieve having a pore size

ot 215-425 :>_m. and the sieved hone powder was washed a few limes with deionized water to remove fine particles remaining on the surface thereof, and then dried in an oven at 60 V for 24 hours. The dried hone powder was collected and used as a bone graft substitute.
The bone powder subjected to the above-described steps was analyzed using a scanning electron microscope and. as a result, hydroxyapatite particles having a size of 50-H0 nm were observed in the bone powder tRG. H. Also, the bone powder was analyzed by XRD and. LIS a result, it could be observed that a pure, low-crystalline apatite phase was produced in the bone powder (FIG. 2), Also, from the results of FT-IR analysis, it was confirmed that the bone powder was a low crystalline carbonate apatite containing a carbonate group, similar to human bone (FIG. 3).
Example 2: Preparation of composition for bone gruff substitution
To 100 g of desalted water. 20 g of hyaluronic acid was added to make a viscous hyaluronic acid solution, to which 10 g of the bone powder prepared in Example 1 was then added to make an injectable paste.
Example 3: Evaluation of osteoconductivitv of bone graft substitute
In order to examine the osteoconductivity of the inventive bone graft substitute, the evaluation of osteoconductivity was conducted for the hone graft substitute prepared according to the method of Example 1. and commercially available bone substitutes Bio-Oss,R' and OsteoGraf^'/N as control groups. In this Example. New Zealand white rabbits were used and circular defects having a diameter of S mm were formed in the cranial bones of the animals and then implanted with each of the inventive bone graft substitute. Bio-Oss'Rl and OsteoGraf,K7N granules. At 2 weeks and 4 weeks after the implantation, tissue samples were prepared and

comparatively analyzed for osteoconduetiviry nn the basis of the amount of hone produced around each of the hone graft substitutes and the production or non-produclion of connective tissues.
As a result, in the test samples prepared at 2 weeks after implantation of the three bone substitutes, only connective tissues were mostly produced around the portions implanted with Bio-Oss'R,anti OsteoGrafk'/N. and the formation of new bones was hardly observed around the implanted portions (FIG. 4). On the other hand, as shown in FIG. 4(c). it could be observed that the sample implanted with the bone graft substitute prepared in Example I had a large amount of new bones produced therein (see arrow in FIG. 4(c)).
FIG. 5 shows photographs of tissue samples prepared at 4 weeks after implantation with the three bone substitutes. As shown in FIG. 5. in the tissue samples

were formed and mosfh surrounded b\ connective tissues, and on the other hand, in the tissue samples implanted with the bone graft substitute prepared in the Example 1. a large amount of new bones were produced and grown around the bone graft substitute. This suggests that the bone graft substitute prepared according to the present invention has very excellent osteoconductivin compared to those of the prior bone graft substitutes.
INDUSTRIAL APPLICABILITY
As described above, the present invention provides the method for preparing the prion-free bone graft substitute, comprising treating bovine bone with sodium hypochlorite solution and subjecting the treated bone to high-temperature treatment, as well as a composition containing said bone graft substitute. The inventive hone graft substitute does not cause an immune response, because it is prepared by effectively removing lipids and organic substances from bovine bone having a
; i

structure ver\ similar to that of the human bone. Also, it has excellent osteoconductivity. and is free of the prion, and thus it does not have the risk of infection with bovine spongiform encephalopathy. According to the present invention, the hone graft substitute having such advantages can be prepared in a simple manner.
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 onlv for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will he defined by the appended claims and equivalents thereof.

THE CLAIMS What is Claimed is:
1. A method tor preparing a bone graft substitute from bovine bone, the method
comprising the steps of:
la) boiling bovine bone, from which blood components ha\e been removed, in deionized water to remove lipids and proteins, and drying the boiled hone:
(b) grinding the dried bone, and immersing and shaking the ground bone
powder in an organic solvent:
(c) removing the organic solvent and drying the bone powder:
(d) treating the dried bone powder, from which the solvent has been removed, with a solution of 2-20% sodium hypochlorite:
if) thermalh treating the dried bone powder a completely remove lipids and proteins.
2. The method for preparing a bone graft substitute from bovine hone according to
claim 1. wherein the organic solvent is a mixed solvent of chloroform and methanol.
3. The method for preparing a bone graft substitute from bovine bone according to
claim 1. wherein the sodium hypochlorite concentration is about
4. The method for preparing a bone graft substitute from box hie bone according to
claim 1. wherein the step (d) additionally comprises adding 1-4 ON sodium
hydroxide to the sodium hypochloride solution.
5. The method for preparing a bone graft substitute from bovine bone according to
claim 4. wherein the concentration of the sodium hydroxide is about 2N.

6. The method for preparing a bone graft substitute from bovine bone according to claim 1. which further comprises the steps after the step of sieving the thermally treated bone powder through a sieve having a pore size of 212-425 nm; and washing the sieved bone powder.
7. A composition for bone graft substitution, containing the bone graft substitute prepared by the method of any one claim among claims 1-6.
8. The composition for hone graft substitution according to claim 7. which
additionally contains at least one biologically active substance selected from the
group consisting of a bone growth-promoting factor, a fibrin, a hone morphogenic
factor, a bone growth agent, a chemotherapeutic agent, an antibiotic, an analgesic, a
hisphosphonate. a strontium salt, a fluorine salt, a magnesium salt, and a sodium
■.alt.
9. The composition for bone graft substitution according to claim ~ or s. which
additionally contains at least one chemical compound selected from the group
consisting of hyaluronic acid, chondroitin sulfate, alginic acid, chitosan. collagen,
hydroxyapatite. calcium carbonate, calcium phosphate, calcium sulfate, and
ceramics.
10. The composition for bone graft substitution according to claim 9. wherein the
chemical compound is hyaluronic acid, and the composition is gel-type.

Documents:

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Patent Number

270018

Indian Patent Application Number

5980/CHENP/2008

PG Journal Number

48/2015

Publication Date

27-Nov-2015

Grant Date

24-Nov-2015

Date of Filing

04-Nov-2008

Name of Patentee

SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION

Applicant Address

SAN 4-2, BONGCHEON-DONG, GWANAK-GU, SEOUL 151-818,

Inventors:

#	Inventor's Name	Inventor's Address
1	RHEE, SANG-HOON,	112-506, HANSHIN APT., DONAM-DONG, SUNGBOK-GU, SEOUL 136-600,
2	CHUNG, CHONG-PYOUNG,	2701 JAMSIL DAEWOO LAKEWORLD APT., 58-1, SONGPA-DONG, SONGPA-GU, SEOUL 138-170,
3	PARK, YOON, JEONG,	110-402 LOTTE APT., GURO-5-DONG, GURO-GU, SEOUL 152-770

PCT International Classification Number

A61K2/28

PCT International Application Number

PCT/KR06/01773

PCT International Filing date

2006-05-12

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA