WO2008032928A1

WO2008032928A1 - Method for preparing bone grafting substitute from horse bone

Info

Publication number: WO2008032928A1
Application number: PCT/KR2007/003686
Authority: WO
Inventors: Sang Hoon Rhee; Chong-Pyoung Chung; Yoon-Jeong Park; Sang Hyuk Han
Original assignee: Seoul National University Industry Foundation; Nano Intelligent Biomedical Engineering Corporation. Co. Ltd
Priority date: 2006-09-12
Filing date: 2007-07-31
Publication date: 2008-03-20
Also published as: KR20080024006A; KR100842012B1

Abstract

The present invention relates to a method for preparing a bone graft substitute using horse bone. More specifically, relates to the method for preparing a bone graft substitute, which does not cause a xenogenic immune response and has no risk of infection with mad cow disease, which can occur when bovine bone is used in xenogenic bone grafting, the method comprising treating horse bone with sodium hypochlorite, hydrothermally treating the treated bone under pressure at a temperature higher than 121 °C, and thermally treating the hydrothermally treated bone at higher than 500 °C. According to the disclosed method, a bone graft substitute, which, when transplanted into the human body, does not cause immune responses and has no risk of infection with organic pathogenic substances such as a denatured prion protein that causes mad cow disease, can be prepared in a simple manner by effectively removing fatty substances and organic substances from horse bone having a structure highly similar to that of human bone.

Description

Method for Preparing Bone Grafting Substitute from Horse Bone

TECHNICAL FIELD

The present invention relates to the method for preparing the bone graft substitute using horse bone, and more particularly to the method for preparing the bone r.graft substitute, which does not cause a xenogenic immune response and has no risk of infection with Creutzfeldt Jakob Disease, which has a potential to occur when bovine bone is used in xenogenic bone grafting. The method comprising treating horse bone with sodium hypochlorite, treating hydrothermally the treated bone at the temperature higher than 121 ^°C under a pressure, and then heat- treating the hydrothermally treated bone at the temperature higher than 500 ^°C .

BACKGROUND ART

As used herein, the term "bone graft substitute" (BGS) refers to a graft material, which can be used to fill spaces in bone tissues and to promote the formation of new bone, when bone defects are created by various dental diseases, trauma, degeneration caused by disease, or other loss of tissue. Generally, the best graft material is known to be autogenous bone which is taken from the patient's own bone. However, the autogenous bone graft has problems in that it requires secondary surgery, is difficult to obtain the required amount, can be absorbed into the body early, is difficult to perform in small-size hospitals and can make the patient's pain and disease conditions severe.

For this reason, various bone graft substitutes, including donated human bone, artificial bone, and synthetic materials comprising bone component (hydroxyapatite), have been used in bone grafting. Commercial bone graft substitutes are available in several forms, including powders, gels, slurry/putties, tablets, chips, morsels, pellets, sticks, sheets and blocks. Such bone graft substitutes have the following advantages: ensuring product consistency; less risk for infection and disease; no morbidity or pain caused by harvesting of the patient's own bone for grafting; and availability of the substitute in different volumes (that is, it is not limited by the harvest site in patients). However, they are greatly different from human bone, and thus have various problems, including slow tissue regeneration rate.

In an attempt to solve such problems, bone graft substitutes have been prepared by physically and chemically treating animal bone, having a structure similar to that of human bone, to remove organic materials and recover only inorganic components from the animal bone, and processing the inorganic components into graft materials, which can be used in dental or orthopedic surgery. Typical commercial products, prepared using this method, may include Bio-Oss^® (Geistlich Biomaterials).

The method for preparing bone graft materials using animal bone comprises the steps of: degreasing bovine femur by refluxing it in a solvent having a boiling point of 80-120^°C ; heating the degreased bovine bone with ammonia or primary amine to remove proteins and organic matter, thus producing bone mineral; heating the bone mineral at a high temperature of 250-600 °C for a few hours; and drying the heated material (US Patent Nos. 5,167,961 and 5,417,975).

An example of treating a cartilage with sodium hypochlorite to selectively remove the collagen phase in order to observe the remaining cartilage structure was reported (Broz, JJ. et ai, J. Mater. ScL Mater. Med., 8:395, 1997). However, an example of using sodium hypochlorite to remove all proteins in the preparation of bone mineral has not yet been reported. Among such animal bones, bovine bone is most frequently used, and the product Bio-Oss^® is also produced using bovine bone as a raw material. However, as the onset of mad cow disease (bovine spongiform encephalopathy or Creutzfeldt- Jacob disease) has recently become frequent, the safety of cattle as a raw material from mad cow disease cannot be secured. For this reason, in a step of processing bovine bone into bone graft substitutes, prion that causes mad cow disease should be removed. Because the prion is not completely removed, even at a high temperature of 600 "C , it cannot be removed by the methods for preparing bone graft substitute, known to date. Thus, there is a need to develop a method of preparing bone graft substitutes using animal bone safe from mad cow disease.

Accordingly, the present inventors have found that a bone graft substitute safe from mad cow disease can be prepared using the method comprising the steps of treating horse bone with sodium hypochlorite, which can degrade and inactivate prion and other proteins, treating hydrothermally the treated bone in purified water at the temperature higher than 121 ^°C under a pressure to inactivate proteins, and then heat-treated the hydrothermally treated bone at 600 ^°C , thereby completing the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide the method for preparing the bone graft substitute using horse bone, the method comprising treating horse bone with a sodium hypochlorite solution and further treating the treated bone at high temperature.

Another object of the present invention is to provide the bone graft substitute composition containing the bone graft substitute prepared according to said method. To achieve the above objects, according to one aspect of the present invention, there is provided the method for preparing the bone graft substitute using horse bone, the method comprising the steps of: (a) boiling horse bone, from which blood components have been removed, in purified water, to remove fatty substances and proteins from the bone, and then drying the boiled bone; (b) powdering the dried bone, and then soaking and shaking the bone powder in an organic solvent; (c) removing the organic solvent and then drying the remaining bone powder; (d) treating the dried bone powder with the 2-20% (w/v) solution of sodium hypochlorite; (e) removing the sodium hypochlorite solution and impurities from the bone powder, and then drying the remaining bone powder; (f) adding purified water to the bone powder, treating hydrothermally the bone powder solution at 120-125 °C for the time range from 30 minutes to 5 hours under a pressure, and then drying the hydrothermally treated powder; and (g) heat-treating the dried bone powder at 500-1000 ^°C for 1-24 hours to completely remove lipids and proteins from the bone powder.

According to another aspect of the present invention, there is provided the bone graft substitute composition containing, as an active ingredient, horse bone powder, which is prepared according to said method and has no risk of infection with mad cow disease and from which fatty substances, lipids and proteins have been removed so as not to cause a xenogenic immune response.

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 shows the results of XRD measurement of bone powder thermally treated at 600^°C . DETAILED DESCRIPTION OF THE INVENTION, AND PPRFERRED EMBODIMENTS

In one aspect, the present invention relates to the method for preparing a bone graft substitute using horse bone, the method comprising the steps of: (a) boiling horse bone, from which blood components have been removed, in purified water, to remove fatty substances and proteins from the bone, and drying the boiled bone; (b) crushing the dried bone, and then soaking and shaking the bone powder in an organic solvent; (c) removing the organic solvent and then drying the remaining bone powder; (d) treating the dried bone powder with a 2-20% (w/v) solution of sodium hypochlorite; (e) removing the sodium hypochlorite solution and impurities from the bone powder, and then drying the remaining bone powder; (f) adding purified water to the bone powder, treating hydrothermally the bone powder solution at 120-125 °C for 30 minutes to 5 hours under a pressure, and then drying the hydrothermally treated powder; and (g) heat-treating the dried bone powder at 500-1000 ^°C for 1-24 hours to completely remove lipids and proteins from the bone powder.

As used herein, the term "bone graft substitute" refers to a material for filling spaces in bone tissues. For use, the bone graft substitute can be shaped in the form of, for example, putties, pastes, strips, blocks or chips, using a method such as pressing, compression, contact under pressure, packing, solidification or hardening. Alternatively, it can be formulated in the form of, for example, gels, granules, pastes, tablets or pellets, using chemical additives. In addition, it may also be used in the form of powder itself.

When the bone graft substitute is formulated as described above to use, it is preferably used in combination with a biologically active substance. Examples of the biologically active substance include bone growth-promoting factors, fibrin, bone morphogenetic factors, bone growth-promoting agents, chemotherapeutic agents, antibiotic agents, analgesics, bisphosphonates, strontium salts, fluorine salts, magnesiukm salts, and sodium salts.

In the present invention, the step (b) of soaking the bone powder in the organic solvent is carried out to remove fatty substances remaining in the horse bone powder. The organic solvent is preferably the mixed solvent of chloroform and methanol. The ratio of chloroform: methanol in the mixed solvent may be 2-8: 8-2, and preferably 1 :1.

In the present invention, the step (d) of treating the bone powder with sodium hypochlorite solution is carried out to degrade and remove proteins remaining in the horse bone powder in a water-soluble state and to inactivate denatured prion proteins that cause mad cow disease. In the step (d), sodium hypochlorite may be used in the 2-20% (w/v) solution of sodium hypochlorite, and preferably the 4% (w/v) solution of sodium hypochlorite.

In the present invention, the heat-treatment temperature in the step (g) is preferably 550-650 °C . Also, steps (d) and (e) are carried out to remove proteins and organic substances. In the step (f), the bone powder is hydrothermally treated at 120-125 ^°C for more than 30 minutes under a pressure, and preferably 3 hours, to degrade and remove proteins from the bone powder.

Furthermore, the preparation method according to the present invention may additionally comprise, after the step (g), the step of sieving the heat-treated bone powder through the sieves having mesh size range of 212-425 μm, 425-1000 μm, 1000-2000 μm and 1000-3000 μm, respectively. In another aspect, the present invention relates to the bone graft substitute composition containing, as an active ingredient, horse bone powder, which is prepared according to the said method and has no risk of infection with mad cow disease and from which fatty substances, lipids and proteins have been removed so as not to cause a xenogenic immune response.

The bone graft substitute composition according to the present invention preferably additionally contains one or more biologically active substances selected from the group consisting of bone growth-promoting factors, fibrin, bone morphogenetic factors, bone growth agents, chemotherapeutic agents, antibiotic agents, analgesics, bisphosphonates, strontium salts, fluorine salts, magnesiukm salts, and sodium salts. Also, the bone graft substitute composition according to the present invention preferably additionally contains one or more chemical compounds selected from the group consisting of hyaluronic acid, chondroitin sulfate, alginate, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate and ceramics.

Examples of the growth factors include BMP (bone morphogenic protein), PDGF (platelet-derived growth factor), TGF-beta (transgenic growth factor), IGF-I (insulin-like growth factor), IGF-II, FGF (fibroblast growth factor) and BGDF-II (beta-2-microglobulin). Examples of the bone morphogenetic factors include osteocalcin, bonesialoprotein, osteogenin and BMP. As the bone growth- promoting agent, any substance may be used without limitation, as long as it is harmless to the human body and promote bone growth. Examples of the bone growth-promoting agents include peptides or nucleic acids, which promote bone formation, and antagonists against substances which inhibit bone formation.

In the present invention, the chemical additives that are used to formulate the bone graft substitute include hyaluronic acid, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate and ceramics. Depending on the kind of chemical additive, the bone graft substitute can be formulated in the form of gels, strips, granules, chips, tablets or pastes.

In the present invention, when the chemical compound is hyaluronic acid, the bone graft substitute can be formulated into a gel-type bone graft substitute composition.

In the present invention, the step of treating the bone powder with sodium hypochlorite is preferably carried out for more than 72 hours in order to remove prion and the remaining proteins.

Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1 : Method for preparing bone graft substitute

[Pretreatment and powdering process]

Bone taken from the equine femur was cut with a bone cutter into pieces having a size of about 5 cm³. The bone pieces were soaked in purified water for 24 hours to remove blood components from the bone. The bone pieces, washed with deionized water, were boiled in purified water for 72 hours while replacing purified water at the interval of 12 hours, thus primarily removing fatty substances and proteins from the bone. The bone pieces, from which fat and proteins have been primarily removed, were completely dried in an oven at 60 "C for 24 hours and crushed with a pulverizer to a size of less than 10 mm³. [Degreasing process]

1 g of the bone powder was added to 20 ml of the mixed solvent of chloroform and methanol (1 :1 v/v), and the solution was shaken at the revolution speed of

120 rpm for 24 hours to degrease the bone powder. To remove the solvent from the degreased bone powder, 1 g of the bone powder was added to 50 g of purified water and shaken at 120 rpm for 12 hours, thus removing the solvent from the powder. Herein, the purified water was replaced with fresh purified water at a 2-hr interval in order to increase water- washing efficiency. The water- washed bone powder was completely dried in the oven at 60 °C .

[Deproteinizing process]

1 g of the degreased dried bone powder was added to 20 ml of the 4% (w/v) solution of sodium hypochlorite and shaken at a revolution speed of 120 rpm for 72 hours to remove proteins from the bone powder and to inactivate a prion protein that causes mad cow disease. To remove the solvent from the deproteinized bone powder, 1 g of the bone powder was added to 50 g of purified water and shaken at 120 rpm for 72 hours, thus removing the remaining sodium hypochlorite. Herein, the purified water was replaced with fresh purified water at a 2-hr interval for 12 hours after the start of shaking, and then replaced with fresh purified water at a 12-hr interval. 1 g of the water- washed bone powder was added to 50 g of purified water and hydrothermally treated at 121 ^°C for 3 hours under a pressure. Finally, the bone powder was completely dried in an oven at 60 ^°C .

[Thermal treatment process] The degreased, deproteinized, dried bone powder was heat-treated at high temperature to remove the remaining lipids and proteins. An electric furnace used in the heat-treatment was heated at a rate of 2 ^°C/min, and the bone powder was thermally treated at 600 °C for 3 hours, and then cooled in the furnace.

[Sieving process]

The thermally treated bone powder was sieved through sieves having mesh sizes of 212-425 μm, 425-1000 μm, 1000-2000 μm and 1000-3000 μm, respectively, and the sieved bone powder was washed several times with purified water to remove fine dust remaining on the surface thereof. The water-washed powder was dried in an oven at 60 °C for 24 hours and then collected for use as a bone graft substitute.

The prepared bone graft substitute was analyzed by XRD and, as a result, it could be seen that the bone graft substitute consisted of pure apatite crystals (see FIG.

1).

Example 2: Method for preparing bone graft substitute composition

20 g of hyaluronic acid was added to 100 g of demineralized water to prepare a viscous hyaluronic acid solution. To this solution, 10 g of the bone powder prepared in Example 1 was added, thus preparing an injectable paste.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides a method for preparing a bone graft substitute, which comprises treating horse bone with a sodium hypochlorite solution, followed by hydrothermal treatment and high-temperature treatment. Also, the present invention provides the bone graft substitute composition containing the bone graft substitute prepared according to said method. According to the present invention, a bone graft substitute, which, when transplanted into the human body, does not cause immune responses and has no risk of infection with organic pathogenic substances such as a denatured prion protein that causes mad cow disease, can be prepared in a simple manner by effectively removing fatty substances and organic substances from horse bone having a structure highly similar to that of human bone.

While the present invention has been described in detail with reference to 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 method for preparing a bone graft substitute using horse bone, the method comprising the steps of:

(a) boiling horse bone, from which blood components have been removed, in purified water, to remove fatty substances and proteins from the bone, and drying the boiled bone; (b) crushing the dried bone, and soaking and shaking the bone powder in an organic solvent;

(c) removing the organic solvent and drying the remaining bone powder;

(d) treating the dried bone powder with a 2-20% (w/v) solution of sodium hypochlorite; (e) removing the sodium hypochlorite solution and impurities from the bone powder, and drying the remaining bone powder;

(f) adding purified water to the bone powder, treating hydrothermally the bone powder solution at 120-125 °C for 30 minutes to 5 hours under a pressure, and drying the hydrothermally treated powder; and (g) heat-treating the dried bone powder at 500-1000 ^°C for 1-24 hours to completely remove lipids and proteins from the bone powder.

2. The method for preparing a bone graft substitute using horse bone 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 using horse bone according to claim 1, wherein the concentration of sodium hypochlorite solution is 4% (w/v).

4. The method for preparing a bone graft substitute using horse bone according to claim 1, wherein the thermal treatment temperature in the step (g) is 550-650 °C .

5. The method for preparing a bone graft substitute using horse bone according to claim 1 , which additionally comprises a step of sieving the thermally treated bone powder through sieves having mesh sizes of 212-425 μm, 425-1000 μm, 1000- 2000 μm or 1000-3000 μm, after the step (g).

6. A bone graft substitute composition containing, as an active ingredient, horse bone powder, which is prepared by the method of any one claim among claims 1-

5, and has no risk of infection with mad cow disease and from which fatty substances, lipids and proteins have been removed so as not to cause a xenogenic immune response.

7. The bone graft substitute composition according to claim 6, which additionally contains one or more biologically active substances selected from the group consisting of bone growth-promoting factors, fibrin, bone morphogenetic factors, bone growth agents, chemotherapeutic agents, antibiotic agents, analgesics, bisphosphonates, strontium salts, fluorine salts, magnesiukm salts, and sodium salts.

8. The bone graft substitute composition according to claim 6, which additionally contains one or more chemical compounds selected from the group consisting of hyaluronic acid, chondroitin sulfate, alginate, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate and ceramics.

9. The bone graft substitute composition according to claim 8, wherein the chemical compound is hyaluronic acid.