JPH0414584B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a kit for preparing a filling composition for filling bone defects and cavities and generating new bone at the filling sites.

[Prior Art] In the field of surgery or orthopedics, bone defects or voids occur due to bone fractures or bone tumor excision, and in the field of dentistry, loss of jaw bone due to alveolar pyorrhea occurs. This is often encountered when a prosthesis is required for the area. Conventionally, in many cases, a method has been used in which the patient's own iliac bone or the like is removed and filled into the bone defect site to fill the defect or void in the bone tissue and to hasten the recovery and healing of the tissue. However, using this method requires resection of normal bone tissue other than the damaged area, which causes great pain to the patient and requires a great deal of labor during the surgery. Furthermore, if the bone defect is large, it is not always possible to collect enough autologous bone to fill it, and some kind of substitute must be used to make up for the shortage. As a substitute for this, for example, allogeneic bone and xenogeneic bone are available, but there are still problems such as rejection reactions with the implanted living tissue, and the postoperative course is not always good. It has not yet reached the practical stage.

Various metal alloys and organic substances have been used as hard tissue substitutes for living organisms, but they are said to deteriorate due to dissolution in the living environment, to be toxic to living organisms, and to be accompanied by foreign body reactions. Ceramic materials, which have excellent compatibility with living organisms and do not have the above-mentioned drawbacks, are now being used. Among these ceramic materials, artificial bones and teeth made of sintered bodies or single crystals of alumina, carbon, tricalcium phosphate, or hydroxyapatite, which have excellent biocompatibility, are being developed and are attracting attention.

Attempts have been made to fill bone defects and cavities with these combustion bodies or single crystals, but the shape of bone defects that require actual treatment is not constant;
Moreover, they have a complex shape, and it is difficult to process these sintered bodies or single crystals to fit the shape. Furthermore, even if these sintered bodies or single crystals are filled, the surrounding area Because it is significantly harder than bone tissue, the stimulation causes bone resorption around the filling material, causing problems such as loosening, and it has not yet reached the level of practical use.

On the other hand, the sintered body is made into a porous body by mechanical methods or by adding combustible fiber to powder during molding and sintering it, and this is used as a filling material for bone defects and cavities. However, the porosity of the porous body created by these methods is poor, for example, when attempting to mechanically process a ceramic sintered body to make it porous. Moreover, it is impossible to introduce a large amount of pores because the material is brittle and easily damaged. Furthermore, even when flammable fiber is added to ceramic raw material powder and molded and sintered to obtain a porous body, a large amount of flammable fiber is added to the powder. It is difficult to mold the material with high porosity. For this reason, there are drawbacks such as insufficient infiltration of biological osteogenic components necessary for the generation of new bone into the filling material, and a long period of time required for the filling material and bone tissue to become integrated.

[Objective of the Invention] Therefore, one object of the present invention is to provide a material that has excellent biocompatibility, forms bone tissue in a particularly short period of time without any foreign body reaction, and allows the filling material itself to be absorbed and replaced by the living body, and in particular, to improve the filling composition. It is an object of the present invention to provide a kit for preparing a composition for filling bone defects and cavities that can generate new bone up to the center of the object.

Another object of the present invention is to promote bone formation in the filled area and to particularly quickly repair and restore the structure and function of the bone tissue defect area.
An object of the present invention is to provide a kit for preparing a composition for partial filling.

Still another object of the present invention is to provide a kit for preparing a composition for filling bone defects and cavities in which new bone is generated particularly quickly.

Still another object of the present invention is to provide a kit for preparing a composition for filling bone defects and cavities that is easy to mold into a shape that matches the shape of the filling site.

The above and other objects of the present invention will become more apparent from the following description.

[Configuration of the Invention] According to the present invention, there is provided a kit for preparing a composition for filling bone defects and cavities, which is composed of at least hydroxyapatite heat-treated at 500° C. or higher, fibrinogen, thrombin, and calcium chloride. provided.

[Description of the invention] The present invention will be explained in more detail below.

The hydroxyapatite used in the present invention is hydroxyapatite heat-treated at 500° C. or higher, and hydroxyapatite obtained by heat-treating at 700° C. or higher is particularly preferable because new bone is generated quickly. The upper limit temperature of the heat treatment is not particularly limited, but since hydroxyapatite starts to decompose, it should be lower than the decomposition temperature. In addition, the hydroxyapatite used in the present invention may be artificially synthesized by known manufacturing methods such as wet, dry, or hydrothermal methods, or it may be a natural product obtained from bones etc. Good too.

On the other hand, fibrinogen used in the present invention is obtained from human or animal plasma. From the viewpoint of biocompatibility, when the target is a human, it is preferable to use fibrinogen obtained from human plasma as the raw material, and when the target is an animal, it is preferable to use fibrinogen obtained from the plasma of that animal as the raw material.
Such fibrinogen is specified in the Biological Products Standards supervised by the Pharmaceutical Affairs Bureau of the Ministry of Health and Welfare (1979, pp. 201-203).
Dry fibrinogen for medical use produced according to the above method can be used, and a commercially available powder of this product is available under the trade name "Fibrinogen-Midori" [Midori Juji Co., Ltd.]. This is made by adding coagulant protein and stabilizers to dry fibrinogen, such as sodium citrate and monosaccharides such as glucose, fructose, and mannite. Before use, it is dissolved in distilled water for injection or a low-salt buffer solution with a pH of 6 to 7. let A 0.01 to 0.03 molar citrate buffer is suitable as this low salt concentration buffer. The dissolution temperature is 32 to 36°C, and it is preferable to maintain the inside of the bottle containing fibrinogen under reduced pressure during dissolution. Under such dissolution conditions, dry fibrinogen powder dissolves within a range of about 2 to 10 W/V%. Also,
Fibrinogen may be used in powder form.
As detailed below, fibrinogen is solidified by adding thrombin and calcium chloride as solidifying agents when preparing a composition for filling bone defects and cavities using the kit of the present invention, and is combined with fibrin. Become.

When preparing a composition for filling bone defects and cavities using the kit of the present invention, the above-mentioned hydroxyapatite powder or granules (hereinafter collectively referred to as granules) or powders are prepared. There are cases in which ivy granules are used and cases in which the above-mentioned porous body of hydroxyapatite is used.

When using hydroxyapatite powder, for example, in one of the wet methods, it is obtained as a precipitate, so it is separated from the solution by a method such as filtration or centrifugation, dried, and then pulverized. Hydroxyapatite powder is obtained. In addition, in the case of a dry method and a hydrothermal method, the material is pulverized as necessary. When using natural hydroxyapatite such as bone, pulverization treatment is similarly performed. In any case, in the case of a powder, the maximum size depends on the size of the defect, but it is best to use a powder with a size of up to 5 mm to form the filling composition using the kit of the present invention. preferred above.

The hydroxyapatite powder thus obtained can be used as it is as a kit for preparing the filling composition of the present invention, but it is possible to improve the crystallinity of the particles and improve biocompatibility, and prevent bacterial infection and organic matter. Hydroxyapatite obtained by wet synthesis should be sterilized by heating sufficiently to prevent rejection reactions caused by oxidation. Afterwards, if necessary, it is preferable to carry out appropriate pulverization treatment to obtain powder particles.

The powder and granules obtained by each of the above synthesis methods and the powder and granules obtained by further calcination are further added with a liquid such as water or physiological saline, and then processed using a rolling granulator or the like. It can also be used to form granules. The particle size of the granules is not particularly limited, but is preferably about 0.1 to 5 mm in order to manufacture the filling composition of the present invention and to prevent the granules from flowing out to other parts of the body.

Separately, a mixed solution is prepared in which throbin and calcium chloride are dissolved in physiological saline or citrate buffer as a solidifying agent for solidifying fibrinogen to form fibrin. Normal thrombin 1
~500 NIH units/ml and calcium chloride 10-100
It is preferable to add mmol/ml; if it is less than the lower limit, the solidification of fibrinogen may be insufficient; on the other hand, if it is added in excess of the upper limit, the solidifying effect will not change much in many cases. A protease inhibitor may be added to the mixed solution to prevent fibrin from being dissolved depending on the site to be filled with the composition for filling bone defects and cavities. Plasmin inhibitors can be preferably used as protease inhibitors, and examples thereof include aprotinin, epsilon aminocaproic acid, planexamic acid, and soybean trypsin inhibitors. The amount of protease inhibitor added is usually 100 to 5000 KIE units/
It is about ml.

Next, when the above-mentioned mixture of fibrinogen and hydroxyapatite and the above-mentioned mixed solution are mixed and stirred, thrombin and calcium chloride insolubilize the fibrinogen into fibrin in about 10 to 30 minutes, and granules of hydroxyapatite are formed in the colloidal fibrin. Alternatively, a composition for filling bone defects and cavities in which the granules are dispersed can be obtained.

Since it takes about 10 to 30 minutes for fibrinogen to solidify with thrombin and calcium chloride, a mixed solution of thrombin and calcium chloride is added to the fibrinogen solution or powder, and then hydroxyapatite powder or granules are added to the fibrinogen solution or powder. A composition for filling bone defects and cavities can also be obtained by adding and stirring and mixing.

The kit for preparing the composition for filling bone defects and cavities of the present invention may be used in any combination as long as thrombin and fibrinogen are not placed in a container. For example, the following combinations can be mentioned.

(a) A combination kit comprising a container containing hydroxyapatite and fibrinogen solution or powder and a mixed solution consisting of thrombin, calcium chloride and optionally a protease inhibitor.

(b) A combination kit consisting of a container containing hydroxyapatite, a fibrinogen solution or powder, and a mixed solution of thrombin, calcium chloride and optionally a protease inhibitor.

(c) A combination kit comprising a container containing hydroxyapatite, a fibrinogen solution or powder, a thrombin container, a calcium chloride container, and a protease inhibitor.

As mentioned above, a porous body of hydroxyapatite can also be used in the kit for preparing the composition for filling bone defects and cavities of the present invention. In this case, for example, the aforementioned hydroxyapatite powder is suspended in a liquid such as distilled water to form a slurry,
A sponge-like organic porous material having continuous pores in a three-dimensional network structure is impregnated with slurry-like hydroxyapatite, and then dried and heated to burn and eliminate the organic porous material, thereby forming a hydroxyapatite porous material. It can be prepared. Next, after immersing hydroxyapatite in the fibrinogen solution or powder described above, the mixed solution containing thrombin, calcium chloride, and optionally a protease inhibitor is added dropwise to solidify the fibrinogen, thereby forming continuous pores in the hydroxyapatite porous body. A composition for filling bone defects and cavities filled with fibrin is obtained. As mentioned above, the reaction of fibrinogen with thrombin and calcium chloride takes some time, so a mixed solution containing thrombin and calcium chloride is added to the fibrinogen solution or powder, and the hydroxyapatite porous material is immediately immersed or reacted with the mixture. A composition for filling bone defects and cavities can also be obtained by dropping it onto a porous body.

Regardless of whether hydroxyapatite powder, granules, or porous materials are used, the bone defect or cavity is filled with hydroxyapatite,
Next, a fibrinogen solution or powder added to the mixed solution may be poured, or conversely, a fibrinogen solution or powder added to the mixed solution is first poured into the bone defect or cavity, and then hydroxyapatite is added. You can fill it up.

The content of hydroxyapatite in the composition for filling bone defects and cavities prepared using the kit of the present invention is 5 to 5% in the final product composition.
Preferably, the amount is 95 vol%, with the remainder comprising fibrin. Hydroxyapatite is 5vol%
If the fibrin content is less than 95 vol%, the formation of new bone may be slowed down, while if it exceeds 95 vol%, the fibrin content will be low and new blood vessels will be less likely to form.

[Effect] Hydroxyapatite has an osteogenic effect, and when filled into bone defects or cavities, new bone is formed. On the other hand, fibrin is a hard protein obtained by the action of fibrinogen and thrombin, and has the function of adhering the wound surface. Due to this adhesion, fibroblasts are generated on the wound surface, which eventually become fibrous cells and fix the tissue. At the same time, blood vessels are generated within the tissue. Therefore, in the present invention, hydroxyapatite is held in a fixed state by the adhesive action of fibrin, and the osteogenic action of hydroxyapatite is promoted, as well as angiogenesis is promoted to form new bone, thereby repairing living tissue. It can be sped up. In particular, when the kit of the present invention is filled with the composition for filling bone defects and cavities, new blood vessels are formed and extended to the center of the composition, and new bone is rapidly formed.

The composition for filling bone defects and cavities prepared using the kit of the present invention is prepared under sterile conditions using physiological saline or a low salt concentration buffer solution with a pH of 6 to 7, e.g.
Store it in a 0.01-0.03M citrate buffer, cut it into pieces that match the shape of the bone defect, or cut it into sections of approximately 1 mm to 15 cm, if necessary. It is possible to fill the empty space.

The kit of the present invention is extremely convenient because a composition for filling bone defects and cavities can be easily prepared by simply removing the kit from the container and mixing and stirring.

[Example] Next, the present invention will be explained with reference to its embodiments.

Example 1 60 mg of fibrinogen extracted and purified from bovine blood using the kit of the present invention was added to a 9% solution of thrombin in citrate buffer (PH6).
4 NIH units/ml, aprotinin 3000 KIE units/ml
A composition (hereinafter abbreviated as fibrin composition) was prepared by adding 1 ml of a 40mM calcium chloride solution containing the following. Next, hydroxyapatite powder (calcined at 900°C) was mixed with the fibrin composition at a volume ratio of 1:1 and solidified to prepare a composition for filling bone defects and cavities of the present invention. cut out to size
in 0.01M citrate buffer. Additionally, tricalcium phosphate powder (1150°C) was added to the fibrin composition.
(calcined), tetracalcium phosphate powder (calcined at 1350℃)
were mixed at a volume ratio of 1:1, and in the same manner, 5×
It was cut into a size of 4 x 3 mm and placed in 0.01M citrate buffer.

Next, a hole of 5 x 4 x 3 mm was made in the rabbit femur, filled with the above composition of the present invention, and the progress was observed after 4 weeks. Separately, only the fibrin composition, only the hydroxyapatite powder, only the tricalcium phosphate powder, and only the tetracalcium phosphate powder were filled into the holes, and the progress after 4 weeks was observed. In this case, as a control, only a hole was made and nothing was filled, and the progress was also observed.

As a result, when the composition for filling bone defects and cavities, which is a combination of the fibrin composition prepared using the kit of the present invention and hydroxyapatite, was filled, new bone formation was observed up to the center, and fibrin The amount of new bone produced was the highest compared to the combination of the composition and tricalcium phosphate or tetracalcium phosphate.

On the other hand, when only the fibrin composition was filled or when only a hole was made, the defect was filled with soft tissue, and only a small amount of new bone was observed around the defect.

hydroxyapatite, tricalcium phosphate,
When only tetracalcium phosphate was used, although new bone formation was observed inside the filler, almost no new bone was observed in the center of the filler.

Example 2 Fibrin composition and hydroxyapatite powder (calcined at 1200°C) in volume ratios of 40:1, 20:1, 1:1,
Mix and solidify in the same manner as in Example 1 at a ratio of 1:20 and 1:40, cut this into 5 x 4 x 3 mm, and place it in the same solution as in Example 1 to prepare each sample. Fill the 5 x 4 x 3 mm hole drilled in the rabbit femur, and
I observed the situation after a week.

As a result, new bone formation was observed up to the center of the hole when the mixtures were mixed at volume ratios of 1:20, 1:1, and 20:1, but when the mixtures were mixed at volume ratios of 1:40 and 40:1. No new bone had been generated up to the center of the hole.

Example 3 Using human-derived fibrinogen, Example 1
A fibrin composition was prepared in the same manner as above, and hydroxyapatite (granules fired at 800°C, particle size 2.0~
1.0 mm) was added at a fibrin composition: hydroxyapatite = 1:10 (volume ratio) and solidified, cut into 5 x 5 x 5 mm to prepare a sample, and placed in the same solution as in Example 1. Ta.

A bone tumor occurring in a human femur was excised and filled with the sample, and the progress after filling was observed using X-rays and bone scintigraphy. As a result of X-ray observation, new bone formation was observed from 1 week after the surgery, and from 3 to 3 days after surgery.
After 4 weeks, the boundary between the filled sample and the living bone became unclear. On the other hand, bone scintigraphy shows that preoperative
The tumor area, which had been cold, became hot after the surgery, suggesting that active bone formation was occurring.

Example 4 A hydroxyapatite porous material (calcined at 1200°C, porosity 80%) was immersed in the fibrinogen-containing solution used in Example 1 and taken out, followed by calcium chloride containing thrombin and aprotinin used in Example 1. The solution was added dropwise to prepare a composition for filling bone defects and cavities of the present invention. A sample was prepared by cutting out this composition into a size of 5 x 4 x 3 mm, and the sample was placed in 0.01M citrate buffer.

Next, a hole of 5 x 4 x 3 mm was made in the rabbit femur and filled with the above sample. When the progress was observed after 4 weeks, new bone formation was observed up to the center of the filled sample.

Claims (1)

[Claims] 1. A kit for preparing a composition for filling bone defects and cavities, comprising at least hydroxyapatite heat-treated at 500°C or higher, fibrinogen, thrombin, and calcium chloride.