JPS6221542B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for preparing bone suitable for bone transplantation, and more specifically to a method for preparing bone suitable for use as an aggregate for repairing damaged bone tissue by treating bone with an enzyme. Concerning the preparation method.

When the affected bone is cut or removed due to osteonecrosis, etc., or when the bone tissue is damaged due to fracture, etc., it is generally used to supplement the repair with bone material to complete and accelerate the repair. ing. Aggregates used in this case include biological aggregates such as bone, skin collagen, and non-biological aggregates such as polyurethane, bone cement, cast powder, etc.
In general, non-biological aggregates have advantages such as strong supporting capacity, good fixing ability, and easy use.
It remains permanently in the body as a foreign substance, which poses a risk of cancer, and the scope of its application is extremely limited because biological fusion cannot be achieved with the bone tissue surrounding the transplanted affected area. There are some difficulties. On the other hand, biological aggregates contain osteogenic factors and do not have the disadvantages of non-biological aggregates, making them far superior as aggregates for repairing damaged bone tissue. Bone is preferred. Regarding the bone, bones of the same genus are preferred from the viewpoint of immunity and compatibility with the bone tissue of the transplant recipient, and among these, it is optimal to use autologous bone.

However, using one's own bone, which is most desirable, requires damage to other parts of the body during harvesting, and there is a limit to the amount that can be harvested. Regarding bone of the same species, which is considered preferable, the use of human bone to repair damaged bone tissue in Japan is limited at present, as Japan relies on amputated limbs as its source of bone material. There are drawbacks such as limitations. Therefore, we have no choice but to use bones from different species and genera, which pose fewer problems as a supply source.

Bone from different species has issues such as antigenicity and compatibility with the transplanted surrounding tissue.
After treating the bone with hydrochloric acid to demineralize and simultaneously remove cell membrane components that are the source of antigenicity, chemical treatment with calcium chloride or lithium chloride, or deproteinization treatment with pepsin, etc. Development efforts have been made to reduce the It's hard to say.

Although much remains to be elucidated regarding the necessary and sufficient conditions for an aggregate used for bone tissue repair, that is, an aggregate for bone transplantation, as well as factors that interfere with these conditions, the present inventors have As a result of intensive research in order to obtain superior bone material from different genera to be used as bone material for human transplantation, we found that bone from different genera, that is, animal bone, was treated with hyaluronan degrading enzymes to remove antigens. The present inventors have discovered that an excellent bone material can be obtained that has extremely low elasticity and is easily compatible with the surrounding tissues of the transplanted affected area.

That is, the purpose of the present invention is to provide a method for obtaining aggregate excellent as an aggregate for bone grafting from animal bones, and the present purpose is to provide a method for obtaining aggregate excellent as an aggregate for bone grafting from animal bones. can be achieved.

Next, the present invention will be explained in detail.

The bone marrow and surrounding soft tissues are removed from the collected animal bones, and the bones are degreased using organic solvents such as acetone and chloroform, and demineralized using dilute hydrochloric acid or formic acid. By performing enzyme treatment to remove hyaluronic acid, aggregate for bone grafting can be obtained. Further, it is preferable to freeze-dry the aggregate thus obtained from the viewpoint of preservability.

The animal bones used in the present invention include land animals and aquatic animals, such as cows, horses, pigs, sheep, goats, dogs, rabbits, guinea pigs, whales,
The bones of any animal can be used, such as chicken, ostrich, turkey, etc. In addition, there are no particular restrictions on the sex, part, age, etc. of these animals, but it is preferable to use young animals as much as possible, and from the viewpoint of individual size and availability of raw materials, it is usually
Long bones, flat bones, etc. of calves under 10 months of age are more preferred. However, it is not limited to these. In addition, these bones collected from animals may undergo protein denaturation over time at room temperature, destroy bone morphogenetic factors, and may not be able to achieve their purpose, so it is preferable to use fresh bones. Process the bone immediately after harvesting, or
Otherwise, it is necessary to freeze it immediately to maintain its freshness.

The removal of fat from the raw material bone, that is, the degreasing process,
Considering the purpose of use, it is not preferable to use a removal method that affects other bone components, and a normal fat removal method using an organic solvent such as acetone, chloroform, methanol, ethanol, or a mixture of these is preferable.

Deashing treatment is carried out according to the usual method of immersion in dilute hydrochloric acid, formic acid, ethylenediaminetetraacetate (EDTA) solution, etc. at low temperature.
The degree of demineralization may be either partial demineralization (surface demineralization) or complete demineralization, and complete demineralization is not necessarily required.

As the hyaluronic acid degrading enzyme used in the present invention, any enzyme obtained from microorganisms or animals can be used as long as it decomposes hyaluronic acid. For example, microbial-derived hyaluronidase, chondroitinase ABC, chondroitinase AC, animal-derived hyaluronidase, etc.
Commercially available hyaluronic acid degrading enzymes are sufficient to achieve the purpose.

Treatment with these enzymes is carried out for 2 to 10 days at a temperature range of 10 to 60°C. Although it can be carried out at temperatures below 10°C, this not only takes a long time but also increases the risk of bacterial contamination.
Since there is a risk of deterioration of bone components due to the action of proteolytic enzymes in bone tissue, it is necessary to add enzyme inhibitors, preservatives, etc., and to carry out the treatment under sufficient control. Furthermore, temperatures exceeding 60°C are not preferred because they may cause denaturation of bone components (protein denaturation). Usually, the treatment is carried out at a temperature of 25 to 40°C for 2 to 7 days. The pH during treatment is in the range of 4.5 to 10.0.

The amount of enzyme used for treatment is determined by the treatment time, bone size, etc., but 1g of bone (dry weight)
50 to 500 units of hyaluronic acid degrading activity is used per sample. If the amount of enzyme is less than 50 units, the treatment will be insufficient and the desired aggregate cannot be obtained.
Even if an amount of enzyme exceeding 500 units is used, there is no difference in the treatment effect, so it is uneconomical.

The bone grafting aggregate obtained in this way may be stored frozen as it is, or placed in a sterile solution such as 70% alcohol and stored at low temperature, but this may be difficult due to long-term storage and transportation convenience. It is preferable to store it as a freeze-dried product.

Regarding the bone obtained by the preparation method of the present invention,
When the glycosaminoglycan obtained by alkali extraction after pronase digestion was analyzed by electrophoresis using a Sethrose acetate membrane as a support, no hyaluronic acid was found.

Bone obtained by the preparation method of the present invention and Keelbon, that is, bone obtained by demineralization and hydrogen peroxide treatment, were transplanted under the abdominal fascia of rats and the bone formation was compared. The bone obtained by the preparation method of the invention was far superior in osteogenic ability.

According to the preparation method of the present invention, the difficulties of conventional methods such as antigenicity and compatibility with the transplanted tissue surrounding the affected area can be solved, and a graft bone material suitable for repairing damaged bone tissue can be obtained. It will be done.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto unless it exceeds the gist thereof.

Example 1 A frozen femur was immediately thawed after being cut and separated from a 3-month-old calf, the bone marrow and surrounding soft tissues were removed, and the bone was immersed in an equal volume mixture of 20 times the volume of chloroform and methanol. , kept at room temperature for 12 hours, then immersed in 20 times the amount of 0.6N hydrochloric acid, and heated at 4℃.
It was kept for 20 days. The size of the bone thus obtained was approximately 3 x 10 x 0.5 cm, and 40 ml of 0.1 M acetate buffer (PH 5.0) containing 0.15 M sodium chloride and 0.05% sodium azide was added to 2 g of the bone. 300 units of hyaluronidase (manufactured by Miles, USA) prepared from Streptomyces hyalurolyticus was added, kept at 37°C for 3 days, washed with water and freeze-dried to obtain 1.9 g of aggregate. As a result of electrophoretic analysis, no hyaluronic acid was found in the glycosaminoglycan extracted from this aggregate.

Example 2 After cutting and separating a 5-month-old calf, a frozen tibia was immediately thawed, the bone marrow and surrounding soft tissues were removed, and the bone was immersed in 20 times the amount of acetone.
It was kept at 4°C for 24 hours, then immersed in 20 times the volume of 0.6N hydrochloric acid, and kept at 4°C for 14 days while exchanging the hydrochloric acid every two days. The bones obtained in this way are approximately 3 x 20
×0.8cm in size, 4g contains 0.15M sodium chloride and 0.06% sodium acid.
Add 80 ml of 0.2M acetate buffer (PH5.5), add 1400 units of hyaluronidase (manufactured by Amano Pharmaceutical Co., Ltd.) prepared from Streptomyces hyalurolyticus, keep at 30°C for 5 days, and after washing with water. freeze-dried aggregate
Obtained 3.9g. As a result of electrophoretic analysis, no hyaluronic acid was found in the glycosaminoglycans extracted from this aggregate.

Example 3 A fresh skull cut and separated from a one-month-old calf, with bone marrow and attached soft tissue removed, was immersed in a mixture of 20 times the volume of chloroform and methanol in equal volumes, and kept at 15°C for 20 hours. Next, 20 times the amount of 0.6N
Immerse in hydrochloric acid and keep at 4℃ while changing the hydrochloric acid every 2 days.
It was kept for 15 days. The size of the bone thus obtained was approximately 4 x 4 x 0.5 cm, and 100 ml of 0.1 M acetate buffer (PH 6.0) containing 0.15 M sodium chloride and 0.06% sodium azide was added to 5 g of the bone.
Hyaluronidase prepared from bovine intestinal mucosa (USA,
1,100 units (manufactured by Miles Inc.) were added, kept at 35°C for 4 days, washed with water, and freeze-dried to obtain 4.8 g of aggregate. As a result of electrophoretic analysis, no hyaluronic acid was found in the glycosaminoglycans extracted from this aggregate.

Example 4 Immediately after cutting and separating a frozen tibia from a 4-month-old calf, the frozen tibia was thawed, the bone marrow and surrounding soft tissues were removed, and the bone was immersed in an equal volume mixture of 20 times the volume of chloroform and methanol. It was kept at room temperature for 12 hours, then immersed in 20 times the volume of 0.6N hydrochloric acid, and kept at 4°C for 5 days while exchanging the hydrochloric acid every two days. The bone thus obtained was approximately 1.5 x 1.5 x 0.3 cm in size, and 3 g of it was mixed with 0.15 M sodium chloride and
Add 60 ml of 0.1M Tris-HCl buffer (PH8.0) containing 0.06% sodium azide, add 900 units of chondroitinase ABC (manufactured by Seikagaku Corporation) prepared from Proteus vulgaris, and incubate at 37°C. Keep for 2 days, wash with water and freeze-dry to make aggregate.
Obtained 2.9g. As a result of electrophoretic analysis, no hyaluronic acid was found in the glycosaminoglycans extracted from this aggregate.

Example 5 Immediately after cutting and separating the frozen femur from a 3-month-old calf, the frozen femur was thawed, the bone marrow and surrounding soft tissues were removed, and the bone was immersed in a mixture of 20 times the volume of chloroform and ethanol in equal volumes. The sample was kept at 4°C for 20 hours, then immersed in 20 times the volume of 0.6N hydrochloric acid, and kept at 4°C for 30 days while exchanging the hydrochloric acid every two days. The size of the bone thus obtained was approximately 4 x 15 x 0.6 cm, and 60 ml of 0.1 M Tris-HCl buffer (PH7.3) containing 0.15 M sodium chloride and 0.06% sodium azide was added to 3 g of the bone. Arthrobacter aurescens
400 units of chondroitinase AC (manufactured by Seikagaku Kogyo Co., Ltd.) prepared from above were added, kept at 37°C for 3 days, washed with water, and freeze-dried to obtain 2.8 g of aggregate. As a result of analysis by electrophoresis, no hyaluronic acid was found in the glycosaminoglycan extracted from this aggregate.

Example 6 The aggregates obtained in Examples 1 to 5 were
A small piece of 1.5 mm weighs approximately 250 g, anesthetized with ether.
It was transplanted under the abdominal fascia of SD rats, sacrificed at one-week intervals after surgery, and the status of bone formation by the transplanted bone was observed until the 8th week. On the other hand, as a control, aggregates (same size) treated with hyaluronidase in Example 1 without the addition of hyaluronidase were similarly implanted under the abdominal fascia of rats, and bone formation was observed.

As a result, in the examples using the aggregates obtained in Examples 1 to 5, cartilage formation was observed in the 1st to 2nd week, calcification and formation of immature bone were observed in the 4th week, and thereafter, over time, cartilage formation was observed. After the 5th week, lamellar bone, bone marrow,
Bone cells appeared, and almost normal bone formation was observed by the 8th week. On the other hand, in the control bone, cartilage appeared in the 1st to 2nd week, which was no different from the aggregate treated with the treatment method of the present invention, but the subsequent formation of normal bone was significantly delayed, and cartilage appeared in the 8th week. Only the same degree of lime deposition as in the case of using the aggregate of the present invention was observed.

Claims (1)

[Claims] 1 After degreasing and decalcifying the animal bones,
A method for preparing bone for transplantation, characterized by removing hyaluronic acid using a hyaluronic acid degrading enzyme.