RU2393819C1 - Method of preparing prion-free bone graft substitute - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention refers to medicine, more specifically to the method of preparing a prion-free bone graft substitute made of bovine bone, involving preparation of a bovine bone powder with sodium hypochlorhide and its heat treatment at 600 -1000 C. ^ EFFECT: invention provides the bone graft substitute not causing the immune response, having high bone conduction, and also eliminating risk of infection with a spongy bovine encephalopathy. ^ 10 cl, 3 ex, 5 dwg

Description

FIELD OF THE INVENTION

The present invention relates to a method for preparing a bone graft substitute using bovine bone and, in particular, to a method for preparing a safe bone graft substitute that eliminates the risk of cattle spongiform encephalopathy, comprising treating bovine bone with sodium hypochlorite at a high temperature of more than 600 ° C.

State of the art

Bone graft substitute (BGS) refers to a transplant material that is used to replace bone defects resulting from various dental diseases or injuries associated with degeneration disease or other tissue loss, which fills the pore space in the bone tissue and promotes the formation of new bone. An autologous bone graft is known to be the best graft, but an autogenous bone graft has problems associated with the need for repeated surgery, there is a difficulty in obtaining the required amount, there is a difficulty in performing the operation in small hospitals and there is a possibility that contributes to pain for patient and the severity of the incidence.

For this reason, various substitutes are used for transplantation, including human donor bones, artificial bones and artificially synthesized materials made from bone hydroxyapatite. Commercially available bone substitutes have the advantage that they are available in various forms, including powder, gel, suspension / putty, tablets, chips, pieces, pellets, slats, plates and blocks, are homogeneous, have a low risk for infection and disease, reduce the risk of pain associated with taking the patient’s own bones for transplantation, and have reduced size restrictions. However, these commercial bone substitutes have various problems. For example, since their structure is significantly different from the physical structure of human bones, they have a low rate of tissue regeneration.

In an attempt to solve these problems, bone minerals obtained by physicochemical treatment of animal bones with a similar structure to the structure of human bones, with the removal of organic substances, were processed in such a way that they could be used in dental and orthopedic surgery. A typical example may include Bio-Oss®, commercially available from Geistlich Biomaterials.

A method for preparing said bone graft substitute using animal bones involves the following steps: treating the ruminant femur with a solvent having a boiling point of 80-120 ° C to remove lipids; adding ammonia or a primary amine to the treated bone to remove proteins and organic substances to produce a bone mineral; heating the bovine mineral at a temperature of 250-600 ° C for several hours, followed by drying (US patent No. 5167961 and No. 5417975).

Although there was an example where cartilage was treated with sodium hypochlorite to selectively eliminate the collagen phase in order to study the remaining cartilage structure (Broz, JJ. Et al, J. Mater. Sci. Mater. Med., 8: 395, 1997), there was not yet It is noted that sodium hypochlorite is used to remove all proteins in the preparation of bone minerals.

Among these animal bones, the most commonly used bone is bovine bone, and said Bio-Oss® product is also obtained using bovine bone as a raw material. However, since until recently the manifestation of bovine spongiform encephalopathy was often observed, the safety of bovine bone as a raw material relative to bovine spongiform encephalopathy was not ensured. For this reason, in the stage of converting bovine bone into bone graft material, the prion that causes spongiform encephalopathy in cattle must be removed. Since the prion does not completely disappear even at a high temperature of 600 ° C, it cannot be removed by methods known from the prior art, which requires the development of a new method.

In this regard, the present inventors received a bone graft substitute that did not exclude the risk of spongiform cattle encephalopathy using a method comprising the steps of inactivating prion protein with sodium hypochlorite during the preparation of a bone graft substitute using bovine bone and heating the resulting bone at high temperature 600 ° C, thus completing the present invention.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for preparing a bone graft substitute that is completely free of prion protein that causes bovine spongiform encephalopathy, a method comprising treating bovine bone with sodium hypochlorite and heating the treated bone at high temperature.

In another aspect, the present invention relates to a bone graft substitute composition comprising a bone graft substitute prepared in accordance with said method.

Other features and embodiments of the present invention will appear from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 is a photograph taken by scanning electron microscopy of a bone powder thermally treated at 600 ° C;

figure 2 shows the results of the measurement of XRD (X-ray diffraction analysis) for bone powder, heat treated at 600 ° C;

figure 3 shows the measurement results of FT-IR (Fourier transform infrared spectral analysis) for bone powder, heat treated at 600 ° C;

4 shows photographs of tissue samples taken 2 weeks after implantation of each bone powder prepared in Example 1, Bio-Oss® and OsteoGrar® / N, in circular defects of New Zealand white rabbits;

5 shows photographs of tissue samples taken 4 weeks after implantation of each bone powder prepared in Example 1 into circular defects of New Zealand white rabbits.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for preparing a bone graft substitute from bovine bone, the method comprising the steps of: (a) boiling bovine bone from which blood components have been removed in deionized water to remove lipids and proteins and drying the cooked bone; (b) grinding the dried bone, immersing and mixing 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; (e) removing the sodium hypochlorite solution from the bone powder and drying the resulting bone powder; (f) heat treatment of dried bone powder at 600-1000 ° C for 1-6 hours to completely remove lipids and proteins.

In the method of the invention, the step of immersing the bone powder in an 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 may be 2-8: 8-2 and preferably 1: 1.

In the method of the invention, the step of treating the bone powder with a sodium hypochlorite solution is the step of removing the proteins remaining in the bovine bone powder and inactivating the prion, which causes bovine spongiform encephalopathy.

The sodium hypochlorite solution used in this step may be a solution containing sodium hypochlorite at a concentration of 2-20% (w / v) and most preferably about 4% (w / v). The step of treating the bone powder with sodium hypochlorite should be carried out for at least 20 minutes to inactivate the prion and preferably be carried out for at least 72 hours to remove the remaining proteins.

In the method of the invention, step (d) may further comprise adding 1-10N sodium hydroxide to the sodium hypochlorite solution to increase prion inactivation efficiency. The concentration of sodium hydroxide is preferably about 2N.

The method according to the invention may additionally include, following step (f), the following steps: filtering the heat-treated bone powder through a filter having a pore size of 212-425 μm; and washing the filtered bone powder.

In another aspect, the present invention provides a bone graft replacement composition comprising a bone graft substitute prepared in accordance with said method.

The bone graft replacement composition of the invention may further comprise at least one biologically active substance selected from the group consisting of bone growth promoting factor, fibrin, bone morphogenetic factor, bone growth agent, chemotherapeutic agent, antibiotic, analgesic, diphosphonate , strontium salts, fluorine salts, magnesium salts and sodium salts. It may also optionally contain 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.

In yet another aspect, the present invention provides a gel type composition for bone graft replacement, wherein said chemical compound is hyaluronic acid.

As used herein, the term “bone graft substitute” refers to a material for filling a space in bone tissue. The bone graft substitute can be used in the form of putty, paste, moldable plates, blocks, chips, etc., which are formed by pressing, compacting, press sealing, compressing, squeezing, or stuffing the bone powder in the desired shape. It can also be used in the form of a gel, granules, paste, tablets, pellets, etc. that are formed using chemical additives, and it can be used in powder form as it is.

If the bone graft substitute is used in the above forms, it is preferable to add biologically active substances to it. Examples of biologically active substances that can be used in the present invention include bone growth promoting factor, fibrin, bone morphogenetic factor, bone growth agent, chemotherapeutic agent, antibiotic, analgesic, diphosphonate, strontium salt, fluorine salt, magnesium salt and sodium salt .

Examples of growth factor that can be used in the present invention include BMP (bone morphogenetic protein), PDGF (platelet 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 a bone morphogenetic factor that can be used in the present invention include osteocalcin, bonesialo protein, osteogenin, BMP and the like. The bone growth agent can be used without any specific restrictions as long as it is harmless to the human body and stimulates bone growth. Examples of a bone growth agent that can be used in the present invention include peptides or nucleic acids that provide bone formation, and antagonists of substances that inhibit bone formation.

Examples of chemical additives that are used to form the bone graft substitute in the present invention include hyaluronic acid, chondroitin sulfate, alginic acid, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate and ceramics. Depending on the type of additives, the bone graft substitute can form in the form of a gel, strip, granules, pellets, tablets, pastes, etc.

Information confirming the possibility of carrying out the invention

Hereinafter, the present invention will be described in further detail with reference to examples. However, it should be understood that these examples are for illustrative purposes only and are not interpreted to limit the scope of the claims of the present invention.

Example 1: Preparation of a bone graft substitute

Stage pre-processing and grinding

The bovine femur was trimmed to a size of 5 cm ³ using a bone saw. The chopped bone pieces were immersed in deionized water for 24 hours to remove the blood components present in the bone. Pieces of bone washed with deionized water were cooked for 72 hours when the deionized water was changed at 12-hour intervals, thus initially removing lipids and proteins present in the bones. Pieces of bone, from which lipids and proteins were initially 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.

Degreasing Stage

To 1 g of ground bone powder was added 20 ml of a mixed solvent containing chloroform and methanol (1: 1 v / v) and the solution was shaken at a rotation speed of 120 rpm for 24 hours, so that the bone powder was degreased. To remove the solvent remaining in the defatted bone powder, deionized water was added to the bone powder in a mass ratio of 50: 1, and then the solution was shaken at 120 rpm for 12 hours, thereby removing the solvent remaining in the powder. At this time, deionized water was replaced with fresh deionized water at 2-hour intervals to increase the washing efficiency. The washed bone powder was completely dried in a thermostat at 60 ° C.

Deproteinization Stage

To 1 g of ground bone powder was added 25 ml of a solution of 4% (w / v) sodium hypochlorite and the powder solution was shaken at a rotation speed of 120 rpm for 24 hours to remove proteins present in the bone and to inactivate the prion, which causes spongiform encephalopathy cattle. To remove the solvent present in the deproteinized bone powder, 50 g of deionized water was added to 1 g of bone powder and the solution was shaken at 120 rpm for 72 hours, thereby freeing sodium hypochlorite remaining in the powder. At this time, deionized water was replaced with fresh deionized water every two hours in the first 12 hours and replaced with fresh deionized water every subsequent 12 hours. The bone powder washed with water was completely dried in a thermostat at 60 ° C.

Heat Treatment Stage

Fat-free, deproteinized and dried bone powder was thermally treated at high temperature to remove lipids and proteins remaining there. The temperature of the electric furnace used for the heat treatment was increased at a rate of 2 ° C / min, and the bone powder was thermally treated at 600 ° C for 3 hours, followed by cooling in the furnace.

Filtration stage

The thermally treated bone powder was filtered through a filter having a pore size of 215-425 μm, and the filtered bone powder was washed several times with deionized water to remove fine particles remaining on its surface, and then dried in an oven at 60 ° C for 24 hours. Dried bone powder was collected and used as a substitute for bone graft.

The bone powder obtained in the above steps was analyzed using a scanning electron microscope and, as a result, hydroxyapatite particles having a size of 50-80 nm were observed in the bone powder (FIG. 1). Also, bone powder was analyzed using XRD and, as a result, it was observed that a clean, low crystalline apatite phase formed in the bone powder (FIG. 2). It was also confirmed from FT-IR analysis that the bone powder was a low crystalline carbonatapatite containing a carbonate group similar to human bone (Fig. 3).

Example 2: Preparation of a bone graft replacement composition

To 100 g of demineralized water was added 20 g of hyaluronic acid to form a viscous solution of hyaluronic acid, to which was then added 10 g of bone powder prepared in Example 1 to obtain an injectable paste.

Example 3: Determination of bone conduction of a bone graft substitute

To evaluate the bone conductivity of the bone graft substitute of the invention, bone conductivity was determined for the bone graft substitute prepared according to the method of Example 1 and the commercially available bone substitutes Bio-Oss® and OsteoGraf® as control groups. In this example, New Zealand white rabbits were used and circular defects having a diameter of 8 mm were formed in the bones of the animal skulls and then implanted with each of the bone graft substitute, Bio-Oss® and OsteoGraf® / N pellets. At 2 weeks and 4 weeks after implantation, tissue samples were prepared and a comparative bone conductivity analysis was performed based on the volume of bone formed around each bone graft substitute and the formation or absence of connective tissue.

As a result, in test samples prepared 2 weeks after implantation of three bone substitutes, only connective tissue was formed around the areas implanted with Bio-Oss® and OsteoGraf® / N, and new bone formation was hardly observed around the implanted areas (Fig. 4) . On the other hand, as shown in FIG. 4 (c), it can be seen that the sample implanted with the bone graft substitute prepared in Example 1 had more new bones formed therein (see arrow in FIG. 4 (c) )

Figure 5 shows photographs of tissue samples formed at 4 weeks after implantation with three bone substitutes. As shown in FIG. 5, very few new bones were formed in tissue samples implanted with Bio-Oss® and OsteoGraf® / N, mainly surrounded by connective tissues, and, on the other hand, in tissue samples implanted with a bone graft substitute prepared in example 1, a large number of new bones were formed and grown around a bone graft substitute. This suggests that the bone graft substitute prepared in accordance with the present invention has excellent bone conductivity compared to previous bone graft substitutes.

Industrial applicability

As described above, the present invention provides a method for preparing a prion-free bone graft substitute, comprising treating bovine bone with sodium hypochlorite solution and presenting the treated bone to a high temperature treatment, as well as a composition containing said bone graft substitute. The bone graft substitute of the invention does not elicit an immune response because it is prepared by effectively removing lipids and organic solvents from bovine bone having a structure similar to that of a human bone. It also has excellent bone conduction, is free of prions and, therefore, has no risk of infection with bovine spongiform encephalopathy. According to the present invention, a bone graft substitute having such advantages can be prepared in a similar manner.

Although the present invention has been described in detail with reference to specific features, it is obvious to a person skilled in the art that this description is presented only for a preferred embodiment and does not limit the scope of the present invention. Thus, the true scope of the present invention will be determined by the claims and their equivalents.

Claims (10)

1. The method of preparation of prion-free bone graft substitute from bovine bone, comprising the following stages:
(a) boiling bovine bone, from which blood components have been removed, in deionized water to remove lipids and proteins, and drying the cooked bone;
(b) grinding the dried bone, immersing and mixing 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;
(e) removing the sodium hypochlorite solution from the bone powder and drying the resulting bone powder; and
(f) heat treatment of dried bone powder at 600-1000 ° C for 1-6 hours to completely remove lipids and proteins. 2. The method according to claim 1, in which the organic solvent is a mixed solvent of chloroform and methanol. 3. The method according to claim 1, in which the concentration of sodium hypochlorite is about 4% (mass / volume). 4. The method according to claim 1, in which stage (d) further comprises adding 1-10 N sodium hydroxide to the sodium hypochlorite solution. 5. The method according to claim 4, in which the concentration of sodium hydroxide is about 2N. 6. The method according to claim 1, which, after stage (f), further includes the step of sieving the heat-treated bone powder through a filter having a pore size of 212-425 μm; and washing the filtered bone powder. 7. A composition for replacing a bone graft containing a bone graft substitute prepared by the method according to any one of claims 1 to 6. 8. The composition according to claim 7, which further comprises at least one biologically active substance selected from the group consisting of a factor that stimulates bone growth, fibrin, bone morphogenetic factor, bone growth agent, chemotherapeutic agent, antibiotic, analgesic, diphosphonate, strontium salts, fluorine salts, magnesium salts and sodium salts. 9. The composition according to claim 7 or 8, which further comprises at least one chemical compound selected from the group consisting of hyaluronic acid, chondroitin sulfate, alginic acid, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, sulfate calcium and ceramics. 10. The composition according to claim 9, in which the chemical compound is hyaluronic acid, made in the form of a paste.

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