KR102527814B1 - Bone block with demineralized bone matrix-derived carrier introduced, and a method for manufacturing the same - Google Patents

Abstract

The present invention relates to a method for manufacturing a bone graft material composed only of demineralized bone and a bone graft material manufactured thereby. (b) preparing a demineralized bone paste by mixing demineralized bone powder and water in a predetermined ratio; A manufacturing method comprising the step (c) of treating the demineralized bone dough with an autoclave to obtain a demineralized bone-derived carrier for manufacturing a bone graft material, and mixing the demineralized bone-derived carrier for manufacturing a bone graft material with demineralized bone powder to bone graft material. preparing a forming mixture (d); There is provided a method for manufacturing a bone graft material comprising the step (e) of freeze-drying the mixture for forming a bone graft material.
According to the manufacturing method of the present invention, it is possible to prepare a demineralized bone-derived carrier having a viscosity suitable for use as a carrier for producing a bone filler. A bone graft material having excellent biocompatibility and excellent shape retention is provided.

Description

Bone graft material introduced with demineralized bone-derived carrier and manufacturing method thereof

The present invention relates to a bone graft material containing a demineralized bone-derived carrier and a method for manufacturing the same, and more particularly, to a bone graft material containing a demineralized bone-derived carrier and a method for manufacturing the same, which are composed only of demineralized bone and have excellent shape retention. .

Naturally occurring bone is composed of organic and inorganic materials. Organic substances include growth factors, cartilage tissue, collagen, and other proteins, and inorganic substances include calcium phosphate.

Bone implants are used to augment the natural regeneration process when bone defects or injuries occur, and must be biocompatible. In addition, an ideal bone graft should be capable of bone formation, i.e. bone conduction and bone induction simultaneously.

Bone transplantation for the purpose of treatment is performed for the purpose of reconstruction of lost bone and the purpose of reconstruction of joint surfaces that secrete synovial fluid. For example, treatment of nonunion fractures, joint fixation, filling of bone cavity, replacement of lost bone and joint parts, strengthening of acetabulum and skull, fusion of growth plate cartilage, bone and muscle transplantation for ligament loss, etc. Bone grafting is being done in the field. Bone reconstruction through bone transplantation includes autologous transplantation in which a part of one's own bone is harvested and transplanted to a target body part, allograft using the bone of someone other than the patient himself, and xenograft using the bone of another animal. there is.

Although autologous transplantation is the most effective, there are disadvantages in that the donor site is limited and secondary surgery is involved, so although the recovery of the bone defect is relatively slow compared to such autologous transplantation, allogeneic or xenotransplantation is autologous. It is emerging as an alternative to transplantation. In particular, demineralized bone (demineralized bone particles, demineralized bone matrix (DBM)) is a transplant material that is safe from the risk of disease transmission from the donor site, and has excellent bone regeneration ability for orthopedic, dental, and neurological applications. It is used as a filling material in areas where bone regeneration is slow or impossible in the field of surgery, etc., and is also used for bone replacement, joint fixation and strengthening, bone formation, bone trauma, and fracture treatment. Bone morphogenetic protein (BMP) present in demineralized bone is a representative material that increases bone regeneration and growth.

However, since it is difficult to maintain the shape of demineralized bone when transplanted alone, it was necessary to link with other materials. Accordingly, graft materials formulated by mixing demineralized bone with glycerol, a chemical carrier, or poloxamer, an organic synthetic polymer, have been developed.

However, the graft material mixed with glycerol has a problem in that the content of demineralized bone is significantly lowered and the bone regeneration action is slow. There was a problem that the burden could go.

On the other hand, a method of kneading demineralized bone powder with sterilized purified water and molding it into a stick shape has also been devised (Patent Document 1). There was a problem.

Accordingly, there has been a demand for the development of a bone graft material having excellent shape retention without using the above chemical carrier and a carrier capable of producing such a bone graft material.

Registered Patent Publication No. 10-1139660

The present invention has been made in view of the above problems, and an object of the present invention is to provide a bone graft material composed of only demineralized bone, excellent in bone induction and biocompatibility, and excellent in shape retention, and a manufacturing method thereof.

In addition, another object of the present invention is to provide a demineralized bone-derived carrier and a method for manufacturing the same, which can be used to manufacture a bone graft material composed of only demineralized bone and having excellent bone induction and biocompatibility and excellent shape retention will be.

The inventors of the present invention conducted intensive research for the purpose of advantageously solving the above problems. As a result, if bone powder is demineralized under predetermined conditions to produce demineralized bone powder, the demineralized bone powder and water are mixed in a predetermined ratio to prepare a demineralized bone paste, and then the demineralized bone paste is treated with an autoclave. , found that demineralized bone powder can be suitably dissolved in water to obtain a demineralized bone-derived carrier having a viscosity suitable for use as a carrier for producing a bone graft material, thereby completing the present invention.

According to the present invention, the step (a) of preparing demineralized bone powder by demineralizing bone powder;

(b) preparing a demineralized bone paste by mixing the demineralized bone powder prepared in step (a) with water;

A method for producing a demineralized bone-derived carrier for producing a bone graft material, comprising the step (c) of treating the demineralized bone dough prepared in step (b) with an autoclave,

A method for producing a demineralized bone-derived carrier for manufacturing a bone graft material is provided, wherein the mixing ratio of the demineralized bone powder and water in step (b) is, in weight ratio, the demineralized bone powder : water = 17-31 : 69-83.

Further, according to the present invention, a demineralized bone-derived carrier for manufacturing a bone graft material prepared by dissolving demineralized bone in water and having a viscosity of 495 Pa·s or more and 2000 Pa·s or less is provided.

In addition, according to the present invention, there is provided a method for producing a bone graft material in which the above-described demineralized bone-derived carrier for producing a bone graft material and demineralized bone powder are mixed and freeze-dried.

In addition, according to the present invention, a bone graft material into which the demineralized bone-derived carrier for manufacturing the above-described bone graft material is introduced is provided.

According to the present invention, there is provided a demineralized bone-derived carrier and a method for manufacturing the same, which can be used to manufacture a bone graft material composed of only demineralized bone and having excellent bone induction ability and biocompatibility as well as excellent shape retention.

In addition, according to the present invention, by introducing the demineralized bone-derived carrier, a bone graft material composed of only demineralized bone and having excellent bone induction ability and biocompatibility as well as shape retention and a manufacturing method thereof is provided.

1 is a photograph of a bone graft material manufactured by the manufacturing method of the present invention.
Fig. 2 is a schematic view of the bone graft material shown in Fig. 1;
3 is a photograph showing a state after molding the mixture for forming a demineralized bone-derived carrier and a bone graft material prepared in Examples 1, 4, and 6 of the present invention with a mold.
4 is a photograph showing a state after molding the mixture for forming a demineralized bone-derived carrier and a bone graft material prepared in Comparative Examples 1 and 3 of the present invention with a mold.
5 is a photograph showing a state in which demineralized bone and water are mixed in preparation of a demineralized bone-derived carrier in Comparative Examples 23 and 24 of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described in detail.

<Method for producing demineralized bone-derived carrier>

The demineralized bone-derived carrier according to the present invention is used to manufacture a bone graft material composed only of demineralized bone, and comprises the steps of: (a) demineralizing bone powder to prepare demineralized bone powder; (b) preparing a demineralized bone paste by mixing the demineralized bone powder prepared in step (a) with water in a specific ratio; It is prepared by a manufacturing method comprising the; step (c) of treating the demineralized bone dough with an autoclave. Hereinafter, each step will be described in detail.

[Step (a) of preparing demineralized bone powder by demineralizing bone powder]

In step (a), bone powder is demineralized under specific conditions to produce demineralized bone powder. Specifically, the step (a) includes demineralizing the bone powder; neutralizing demineralized bone; Freeze-drying step; may include.

Here, bone powder is obtained by pre-processing and pulverizing bone tissue collected from a donor, and a method for producing bone powder will be described later.

(Demineralization step)

Demineralization is to facilitate the release of bone-inducing proteins such as BMP by removing calcium phosphate present in bone tissue. In the step (a), demineralization is performed using an acidic solution such as HCl, EDTA, formic acid, citric acid, acetic acid, nitric acid, nitrous acid, etc. Among these, it is preferable to perform demineralization using an aqueous HCl solution.

It is preferable that the concentration of the acidic solution used at the time of demineralization is 0.3N or more and 1.5N or less. Further, the demineralization time is preferably 2 hours or more, more preferably 2.5 hours or more, preferably 7 hours or less, and 6.5 hours or less, when the concentration of the acidic solution used is 0.3N or more and 0.8N or less. More preferably, when the concentration of the acidic solution used is greater than 0.8N and less than or equal to 1.5N, the demineralization time is preferably 0.1 hour or more, more preferably 0.3 hour or more, still more preferably 0.5 hour or more, and 7 hours or less. It is preferable, it is more preferable that it is 6.5 hours or less, and it is still more preferable that it is 3 hours or less. By demineralization under the above conditions, calcium phosphate present in bone tissue is sufficiently removed so that bone-inducing proteins such as BMP can be easily released, and at the same time, the residual amount of bone-inducing protein in demineralized bone is increased, so that the bone of the bone graft material finally obtained is obtained. It is possible to make the induction ability excellent, and also minimize the damage applied to the bone structure, thereby increasing the durability and compressive strength of the finally obtained bone graft material, thereby making it excellent in shape retention.

On the other hand, the temperature at the time of demineralization is not particularly limited, but can be carried out under conditions of, for example, 15°C or more and 25°C or less.

(neutralization step)

In the neutralization step, the demineralized bone obtained through the demineralization step is neutralized. As a neutralization method, for example, sodium hydroxide (NaOH) can be used, and also washing several times with phosphate buffer saline, commercially available physiological saline, 0.9% sodium chloride solution, sterile distilled water, etc. or demineralization reaction It can be neutralized by adding it to an acidic solution. Specifically, after washing several times using 10 ml or more and 30 ml or less of sterile distilled water per 1 g of demineralized bone obtained through the demineralization step, it is neutralized by washing several times in a vacuum using 10 ml or more and 30 ml or less of phosphate buffer solution per 1 g of demineralized bone. can

(Lyophilization step)

The demineralized bone that has undergone the neutralization step is freeze-dried. The freeze-drying method is not limited thereto, but for example, the demineralized bone is evenly spread on a square dish, the square dish is packaged with an air permeable pouch, and then placed in a freeze dryer and freeze-dried for 36 hours or more to determine the moisture content of the demineralized bone. A method to make this less than 6% can be used.

The demineralized bone obtained through the freeze-drying step may be sterilized and stored before being provided to the next step, and sterilization is E.O. It can be performed by gas sterilization, gamma ray sterilization, E-beam sterilization, etc.

[Method for manufacturing bone powder]

The bone powder used in step (a) of the present invention includes pre-processing the bone collected from the donor; It is prepared by a manufacturing method comprising; crushing and screening.

In addition, the step of pre-processing the bone collected from the donor may include removing and washing soft tissue from the bone collected from the donor; immersing the washed bones in an organic solvent and stirring to firstly remove lipids and immune antigens (hereinafter sometimes referred to as “first delipidation and immunoantigen step”); It includes; crushing and screening the bones from which lipids and immune antigens are removed.

(Collection and transportation of bone tissue)

Bone tissue is collected and transported from the donor. As the bone (bone) suitable for producing the demineralized bone powder used in the present invention, autologous, allogeneic, or xenogeneic bone may be used. Useful sources of xenograft may be pigs, horses, or cattle. The bone may be cortical, reticular, or reticular cortical. Preferred bones are cortical allograft bones such as the femur, tibia, radius, and ulna.

(step of removing and washing soft tissue)

In this step, bone marrow and blood inside the bone are removed, preferably using high-pressure sterilized purified water and a centrifugal separator to remove and wash the bone marrow and blood.

(cell inactivation)

Before or after performing the step of removing and washing the soft tissue, after treating the bone tissue with antibiotics, it is preferable to store it in a freezer at -40 ° C or less. Thereby, cells in the bone tissue capable of causing immune rejection can be inactivated.

At this time, the bone tissue may be frozen and then thawed using a low-speed freezing system. The slow freezing system can be programmed to descend at a rate of 1°C/min using liquid nitrogen, allowing freezing without forming crystals in the tissue. For example, starting at room temperature 4 ° C, dropping at a rate of 1 ° C / min to -4 ° C, dropping from -4 ° C to 20 ° C / min at a rate of -45 ° C, Rising at a rate of 15°C/min to -10°C, descending at a rate of 0.5°C/min at -10°C to -20°C, and descending at a rate of 1°C/min at -20°C to -80°C It is possible to inactivate cells in bone tissue without generating crystals in the tissue by performing an operation to drop the bone to 10°C. In the case of storage thereafter, it is stored in a cryogenic freezer (-70 ° C), and in case of use, it can be melted at 4 ° C for 2 hours and at room temperature for 2 hours to proceed to the next step.

(1st delipidation and deimmunization step)

In this step, lipids and immune antigens are removed from bone tissue using various organic solvents such as hot water or chloroform/methanol, 95% ethanol, 70% ethanol, and diethyl ether. Specifically, it is not limited thereto, but, for example, using 5 ml or more and 30 ml or less of diethyl ether per 1 g of bone tissue, at a temperature of 15 ° C. or more and 25 ° C. or less and in a vacuum, for about 3 hours or more and 24 hours or less. After stirring for 2 hours, it may be dried for 2 hours or more and 24 hours or less.

(crushing and sorting step)

In this step, the bone tissue after the first step of delipidation and deimmunization is pulverized into various sizes. After crushing, the crushed bone pieces are separated and sorted according to size using sieves of various sizes. The term bone fragments as used herein refers to relatively small bone fragments such as regular or irregular fibers, chips, splinters, powders, and the like.

On the other hand, the grinding and sorting step is not limited to being performed after the first delipidation and deimmunization step, and may optionally be further performed before or after other steps in order to make the particle size of the demineralized bone powder within a desired range.

(Second delipidation and deimmunization step)

After the milling and sorting step and before the demineralization step, a second delipidation and deimmunization step may be performed to remove lipids and immune antigens that have not yet been removed in the first delipidation and deimmunization step. The type and amount of the solvent used, the stirring time and temperature, and the drying time may be the same as those in the first delipidation and deimmunization step.

[Demineralized bone dough manufacturing step (b)]

In step (b), a demineralized bone paste is prepared by mixing the demineralized bone powder obtained in step (a) with water in a predetermined ratio. Here, "demineralized bone dough" means a state in which demineralized bone powder and water are mixed, and the mixing method is not particularly limited.

In addition, the mixing ratio of demineralized bone powder and water is preferably demineralized bone powder:water = 17 to 31:69 to 83, more preferably 21 to 28:72 to 79, and 24 to 26:74 to 76 is more preferable. When the mixing ratio of demineralized bone powder and water is within the above range, the demineralized bone powder is suitably dissolved in water when treated with an autoclave, even though a sufficient amount of demineralized bone is ensured in the demineralized bone-derived carrier obtained, for use in producing a bone graft material. Since it has a viscosity suitable for use as a carrier, a bone graft material manufactured using the resulting demineralized bone-derived carrier can have both excellent bone induction ability and shape retention ability.

In addition, the particle size of the demineralized bone powder used in step (b) is preferably 500 μm or less, more preferably 400 μm or less, more preferably 300 μm or less, and most preferably 200 μm or less. By setting the particle size of the demineralized bone powder to the above upper limit or less, the demineralized bone powder dissolves more favorably in water when treated with an autoclave and has a viscosity suitable for use as a carrier for producing a bone graft material. The durability and compressive strength of the bone graft material manufactured using the carrier can be improved.

In addition, the water used in the step (b) is not particularly limited, but for example, distilled water (DW), sterilized purified water, and the like may be used.

[Autoclave processing step (c)]

In step (c), a demineralized bone-derived carrier having a viscosity suitable for use as a carrier for preparing a bone graft material is prepared by treating the demineralized bone dough prepared in step (b) with an autoclave.

The autoclave treatment is performed under conditions of heating and pressurization, and may be performed at a temperature of 100° C. or more and 130° C. or less and a pressure of 1.9 kgf/cm ² or more and 2.2 kgf/cm ^{2 or} less. In addition, the autoclave treatment time can be set according to the demineralization conditions in step (a) (delimination step), and the concentration of the acidic solution used in step (a) (demineralization step) is 0.3N. In the case of more than 0.8N or less, it is preferable to autoclave treatment for more than 10 minutes and less than 65 minutes under the demineralized condition for 2 hours or more, and more preferably autoclave treatment for more than 15 minutes and less than 60 minutes. In addition, when the concentration of the acidic solution used in (desalination step) in step (a) is greater than 0.8N and less than or equal to 1.5N, autoclave treatment for more than 10 minutes and less than 65 minutes under the condition of demineralization for 0.1 hour or more It is preferable, and autoclave treatment is more preferable for a time of 15 minutes or more and 60 minutes or less.

By autoclave treatment under the above conditions, the demineralized bone powder is suitably dissolved in water and has a viscosity suitable for use as a carrier for producing a bone graft material, while minimizing damage to the bone structure to obtain the resulting demineralized bone-derived carrier The durability and compressive strength of the bone graft material manufactured using the material can be improved.

<Demineralized bone-derived carrier>

The demineralized bone-derived carrier according to the present invention is produced by the above-described method, and since demineralized bone is suitably dissolved in water, it has a viscosity suitable for use as a carrier for producing a bone graft material.

Specifically, the demineralized bone-derived carrier according to the present invention has a viscosity of 495 Pa·s or more and 2,000 Pa·s or less, preferably 700 Pa·s or more and 1500 Pa·s or less. Since the demineralized bone-derived carrier according to the present invention has such a predetermined viscosity, it can act to enable molding when manufacturing a bone graft material, so that even if the obtained bone graft material is composed of only demineralized bone, it has excellent durability and compressive strength. It is possible to make the bone graft material excellent in shape retention. On the other hand, the method for measuring the viscosity of the demineralized bone-derived carrier according to the present invention is not particularly limited, and can be measured at 1/s shear rate at 25° C., for example, using a rheometer.

In addition, when a bone graft material is manufactured using the demineralized bone-derived carrier according to the present invention, since the bone graft material can be composed only of demineralized bone, it can have excellent bone inducing ability and biocompatibility.

<Method of manufacturing bone graft material>

The bone graft material according to the present invention comprises the steps (d) of preparing a mixture for forming a bone graft material by mixing the demineralized bone-derived carrier and demineralized bone powder; and (e) preparing a bone graft material by freeze-drying the mixture for forming the bone graft material.

[Preparation step (d) of mixture for forming bone graft material]

In step (d), the above-mentioned demineralized bone-derived carrier for preparing a bone graft material and demineralized bone powder are mixed.

Here, the demineralized bone powder mixed in the step (d) is not limited by its manufacturing method, and may be, for example, produced by the above-described step (a) of the present invention.

In addition, the demineralized bone powder mixed in step (d) has a particle size different from that of the demineralized bone powder prepared in step (a) by appropriately adjusting the grinding and sorting conditions in step (a). it could be In this case, the particle size of the demineralized bone powder mixed in step (d) is preferably greater than 150 μm and less than or equal to 2,000 μm, more preferably greater than or equal to 170 μm and less than or equal to 1500 μm, and more preferably greater than or equal to 200 μm and less than or equal to 850 μm. When the particle size of the demineralized bone powder mixed in step (d) is within the above range, it can be uniformly mixed with the demineralized bone-derived carrier for preparing a bone graft material, and thus the finally obtained bone graft material can have excellent durability and compressive strength.

In addition, in the above step (d), the mixing ratio of the demineralized bone-derived carrier for bone graft material manufacturing and the demineralized bone powder is preferably a weight ratio of: demineralized bone-derived carrier for bone graft material manufacturing: And, it is more preferable that it is 72-74: 26-28. When the mixing ratio of the demineralized bone-derived carrier for bone graft material manufacturing and the demineralized bone powder is within the above range, the demineralized bone-derived bone-derived carrier for bone graft material manufacturing and the demineralized bone powder are uniformly mixed, resulting in excellent durability and compressive strength of the finally obtained bone graft material. can do.

[Lyophilization step (e)]

In step (e), the mixture for forming a bone graft material prepared in step (d) is freeze-dried to prepare a bone graft material composed of demineralized bone.

Specifically, a bone graft material composed of demineralized bone is manufactured by molding a mixture for forming a bone graft material into a desired shape using a mold and freeze-drying it. Here, the freeze-drying method is not particularly limited, and sterilization treatment may be performed after freeze-drying. As a sterilization method, for example, E.O. Methods such as gas sterilization, kiln sterilization, and E-Beam sterilization are exemplified.

<Bone Graft Material>

The bone graft material according to the present invention is manufactured by the above-described manufacturing method and is composed of only demineralized bone. Since the bone graft material according to the present invention is composed only of demineralized bone, it has excellent bone induction ability and biocompatibility.

In addition, since the bone graft material according to the present invention is composed of only demineralized bone and manufactured by introducing the above-described demineralized bone-derived carrier, it has high durability, high compressive strength, and excellent shape retention.

The bone graft material according to the present invention can be used as a bone filler, bone graft material, implant, and bone support, in particular, as in High Tibial Osteotomy (HTO), foot & ankle surgery, etc. , it can be suitably used as a support for bone angle adjustment and bone tissue regeneration.

The shape of the bone graft material according to the present invention is not particularly limited, but is preferably a block shape, and more preferably a wedge shape as shown in FIGS. 1 and 2 . When it is in the shape of a block, the size is not particularly limited. Referring to FIG. 2, for example, the width W is 10 mm or more and 50 mm or less, the length L is 10 mm or more and 50 mm or less, and the height 1 (H1 ) may be 5 mm or more and 20 mm or less, and the height 2 (H2) may be 2 mm or more and 10 mm or less. This size can be changed by modifying the mold used in manufacturing the bone graft material.

Example

Hereinafter, the present invention will be described in detail by showing examples. However, the present invention is not limited to the following examples, and can be arbitrarily changed and implemented within a range that does not deviate from the scope of the claims of the present invention and their equivalents.

In the case where specific experimental methods and conditions are not mentioned below, it can be carried out using conventional experimental methods and conditions.

The durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate of the bone graft material composed of demineralized bone manufactured by the manufacturing method according to the present invention were measured by the following methods.

[durability]

Block-type bone graft material 1g (W ₀ ) prepared in Examples and Comparative Examples was immersed in 100mL of physiological saline and shaken at 100 rpm in an incubator at 37°C. After 3 hours, the remaining bone graft material blocks were collected, freeze-dried, and weighed to determine W _t . Then, the durability of the bone graft material block was evaluated by the following formula.

Durability (%) = (W _t /W ₀ ) × 100

[compressive strength]

A block-type bone graft material with a size of 0.5 cm × 0.5 cm was prepared, compressed with a universal physical property measuring device (manufactured by Instron, load cell 1 kN, crosshead speed 2 mm/sec), and the load at the strain 10% point was measured.

[Residual Calcium Concentration]

The concentration of calcium remaining in the bone graft material blocks prepared in Examples and Comparative Examples was measured in ppm units using inductively coupled plasma analysis (ICP-OES), and the units were converted into %.

[Residual amount of BMP-2]

0.1 g of the block-type bone graft material prepared in Examples and Comparative Examples was dissolved in 1 mL of a 4M guanidine solution. This process was performed in an incubator at 4° C. at 100 rpm for 16 hours. The solution was centrifuged at 4000 rpm for 30 minutes and the supernatant was recovered. The recovered solution was dialyzed against distilled water at 4° C. in a Spectrapor 3 tube (Mr 3500 cutoff) and lyophilized. The recovered freeze-dried powder was quantified at UV 450 nm using BMP-2 ELISA kit.

[Cell proliferation rate]

4g of the block-type bone graft material prepared in Examples and Comparative Examples was immersed in 20mL of cell culture medium, and then eluted at 37°C for 24 hours in a 5% CO ₂ incubator. This solution was directly contacted with osteoblast progenitor cells (MC3T3) at a concentration of 2 × 10 ³ cells (N ₀ ) in a non-coated 96 well plate (PS), and the cells were cultured. On the 5th day, MTT assay was performed to measure the number of cells, and it was set as N _t . And, the cell proliferation rate was measured by the following formula.

Cell proliferation rate (%) = (N _t /N ₀ ) × 100

<Example 1>

[1-1. Preparation of demineralized bone powder]

(1-1-1. Soft tissue removal and washing)

Soft tissue was removed from the bone tissue of the cadaver obtained from domestic and foreign tissue banks. This process was performed aseptically using a trimming set in a clean room. Then, the blood and bone marrow were removed using autoclaved water.

(1-1-2. Primary delipidation and deimmunization antigen)

After putting the dried bones in diethyl ether and stirring for 6 hours, they were dried in a sterile workbench for 14 ± 2 hours to first remove lipids and immune antigens.

(1-1-3. Crushing and sorting)

Next, the dried bones were pulverized using a bone grinder. Here, the grinding and screening conditions were set so that the particle size of the finally obtained demineralized bone powder was 200 μm or less, and the other conditions were set so that the particle size of the finally obtained demineralized bone powder was 200 μm or more and 850 μm or less. In addition, the finally obtained demineralized bone powder having a particle size of 200 μm or less is described in [1-2. Production of demineralized bone-derived carrier], and the finally obtained demineralized bone powder having a particle size of 200 μm or more and 850 μm or less is described in [1-3. Manufacture of block-type bone graft material].

(1-1-4. Secondary delipidation and deimmunization antigen)

The pulverized bone powder was put in 70% ethanol, treated at 15-25°C for 3 hours, put in 3% hydrogen peroxide, treated for 1 hour, and then washed three times for 10 minutes each time using sterile purified water. After washing, the bone powder was put into 70% ethanol again, treated at 15 ~ 25 °C for 1 hour, and then dried for 2 hours or more in a sterile workbench.

(1-1-5. Withdrawal)

The dried bone powder was treated with a demineralization solution (acidic solution, 0.5N HCl) at 15 to 25° C. for 6 hours.

(1-1-6. Chinese)

The demineralized bone powder was washed three times with sterilized purified water for 10 minutes each time, and then treated with DPBS (phosphate buffer solution) until the pH reached 7.

(1-1-7. Freeze drying)

The neutralized demineralized bone powder was evenly spread on a square dish, packed in an air permeable pouch, put in a freeze dryer and freeze-dried for 36 hours or more to obtain demineralized bone powder having a moisture content of less than 6%.

[1-2. Preparation of demineralized bone-derived carrier]

Above [1-1. Preparation of demineralized bone powder] by mixing the demineralized bone powder having a particle size of 200 μm or less and distilled water to prepare a demineralized bone paste. This demineralized bone dough was treated with an autoclave to obtain a demineralized bone-derived carrier. At this time, the mixing ratio of demineralized bone powder and distilled water was set to a weight ratio of demineralized bone powder : distilled water = 17.5 : 82.5. In addition, the autoclave treatment was performed for 60 minutes under conditions of a temperature of 121°C and a pressure of 2 kgf/cm ² .

A photograph of the demineralized bone-derived carrier obtained in Example 1 is shown in FIG. 3 (a). As shown in Fig. 3(a), in the demineralized bone-derived carrier obtained in Example 1, the demineralized bone powder was sufficiently dissolved in water. As a result of measuring the viscosity at s shear rate, the viscosity of the demineralized bone-derived carrier obtained in Example 1 was 714.6 Pa·s.

[1-3. Manufacture of block-type bone graft material]

The obtained demineralized bone-derived carrier, and the above [1-1. Preparation of demineralized bone powder] The mixture for forming a bone graft material was prepared by mixing the demineralized bone powder having a particle size of 200 μm or more and 850 μm or less obtained through the step. At this time, the mixing ratio of the demineralized bone-derived carrier and the demineralized bone powder was 73:27 by weight. The mixture for forming the bone graft material was put into a mold and molded into a block shape as shown in FIG. 1, and then freeze-dried in a freeze dryer to obtain a block-type bone graft material. On the other hand, a photograph after molding the mixture for forming the bone graft material of Example 1 into a mold is shown in FIG. 3(d). With respect to the obtained block-type bone graft material, durability and compressive strength were measured by the method described above. The results are shown in Table 1 below.

<Example 2>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed to be demineralized bone powder:distilled water = 20:80, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 2 was 790.8 Pa·s.

<Example 3>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed to be demineralized bone powder : distilled water = 22.5 : 77.5, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 3 was 889.5 Pa·s.

<Example 4>

Above [1-2. Preparation of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed so that the ratio of demineralized bone powder : distilled water = 25 : 75, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below. In addition, for Example 4, the residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described method, and the residual calcium concentration was 0.014%, the BMP-2 residual amount was 1074.7 pg/g, and the cell proliferation rate was 0.014%. It was 694%.

On the other hand, Fig. 3(b) shows a photograph of the demineralized bone-derived carrier obtained in Example 4, and Fig. 3(e) shows a photograph after molding the mixture for forming a bone graft material in a mold. The viscosity of the bone-derived carrier was 986.4 Pa·s.

<Example 5>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed to be demineralized bone powder : distilled water = 27.5 : 72.5, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 5 was 1203.1 Pa·s.

<Example 6>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed so that the ratio of demineralized bone powder : distilled water = 30 : 70, in the same manner as in Example 1, Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below.

On the other hand, Fig. 3(c) shows a photograph of the demineralized bone-derived carrier obtained in Example 6, and Fig. 3(f) shows a photograph after molding the mixture for forming a bone graft material in a mold. The viscosity of the bone-derived carrier was 1501.9 Pa·s.

<Comparative Example 1>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed to be demineralized bone powder : distilled water = 12.5 : 87.5, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below.

On the other hand, a photograph of the demineralized bone-derived carrier obtained in Comparative Example 1 is shown in Fig. 4 (a), and the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 1 was 186.3 Pa·s. In addition, a photograph after molding the mixture for forming a bone graft material of Comparative Example 1 in a mold is shown in FIG. 4(c), and it can be seen that a lot of moisture remains and molding is not performed.

<Comparative Example 2>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed to be demineralized bone powder : distilled water = 15 : 85, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 2 was 266.7 Pa·s.

<Comparative Example 3>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed to be demineralized bone powder : distilled water = 33.3 : 66.7, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below.

On the other hand, a photograph of the demineralized bone-derived carrier obtained in Comparative Example 3 is shown in FIG. 4(b), and in the demineralized bone-derived carrier obtained in Comparative Example 3, undissolved demineralized bone powder remains, and the viscosity can be measured with a rheometer device. It was impossible. In addition, a photograph after molding the mixture for forming a bone graft material of Comparative Example 3 in a mold is shown in FIG.

<Comparative Example 4>

Above [1-2. Production of demineralized bone-derived carrier], except that the mixing ratio of demineralized bone powder and distilled water was changed to be demineralized bone powder : distilled water = 32.5 : 67.5, in the same manner as in Example 1, demineralized bone powder and demineralized bone Derived carriers and block-type bone graft materials were prepared, and durability and compressive strength were measured according to the method described above. The results are shown in Table 1 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 4 was measured, but undissolved demineralized bone powder remained, making it impossible to measure the viscosity with a rheometer.

As described above, when the mixing ratio of demineralized bone powder and distilled water is within the range of the present invention (17 to 31:69 to 83 in weight ratio) during the manufacture of the demineralized bone-derived carrier for the manufacture of bone graft material, the demineralized bone is dissolved in water. It was found that it was suitably dissolved and that the demineralized bone-derived carrier had a viscosity suitable for use as a carrier for producing a bone graft material.

In addition, according to Table 1, it can be seen that using such a demineralized bone-derived carrier having a predetermined viscosity acts to enable molding during the manufacture of a bone graft material, resulting in a bone graft material with high durability and high compressive strength and excellent shape retention. It was possible (Examples 1 to 6).

In addition, from the results of Example 4, the bone graft materials (Examples 1 to 6) according to the present invention have a low residual calcium concentration, a high residual amount of BMP-2, and a high cell proliferation rate, and thus have excellent bone induction ability and biocompatibility. All were found to be excellent.

On the other hand, when the demineralized bone powder is used in an amount less than the scope of the present invention in the manufacture of a demineralized bone-derived carrier for the manufacture of a bone graft material, sufficient viscosity for use as a carrier for the manufacture of a bone graft material cannot be obtained. After that, it was found that the shape could not be maintained, and the durability and compressive strength of the obtained bone graft material were remarkably deteriorated (Comparative Examples 1 and 2).

In addition, when the demineralized bone powder is used beyond the scope of the present invention in the manufacture of a demineralized bone-derived carrier for the manufacture of a bone graft material, the demineralized bone powder is not sufficiently soluble in water, and the obtained demineralized bone-derived carrier is too stiff. , Demineralized bone powder was not mixed uniformly, so when manufacturing a block-type bone graft material, it was not molded according to the shape of the mold, and the resulting bone graft material had poor durability and compressive strength, resulting in poor shape retention (Comparative Examples 3 and 4) .

<Comparative Example 5>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 0.1N HCl and the demineralization time was changed to 0.5 hour, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 5 was 0.06 Pa·s.

<Comparative Example 6>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 5, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 6 was 0.09 Pa·s.

<Comparative Example 7>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 5, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 7 was 0.10 Pa·s.

<Comparative Example 8>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 0.1 N HCl and the demineralization time was changed to 1 hour, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 8 was 0.08 Pa·s.

<Comparative Example 9>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 8, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 9 was 0.12 Pa·s.

<Comparative Example 10>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-shaped bone graft material were prepared in the same manner as in Comparative Example 8, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 10 was 0.22 Pa·s.

<Comparative Example 11>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 0.1 N HCl and the demineralization time was changed to 3 hours, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 11 was 0.12 Pa·s.

<Comparative Example 12>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 11, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 12 was 0.33 Pa·s.

<Comparative Example 13>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 11, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 13 was 0.57 Pa·s.

<Comparative Example 14>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 0.1N HCl, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 14 was 0.69 Pa·s.

<Comparative Example 15>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 14, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 15 was 0.97 Pa·s.

<Comparative Example 16>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 14, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 2 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 16 was 1.23 Pa·s.

According to Table 2, in the production of the demineralized bone-derived carrier, when the concentration of the acidic solution used in the production of demineralized bone powder is less than the lower limit (0.3N) specified in the present invention, demineralization is not sufficient. It can be seen that the obtained bone graft material has a high residual calcium concentration and a low cell proliferation rate, so the osteoinduction ability is low, and it does not have sufficient viscosity to be used as a demineralized bone-derived carrier, and the durability and compressive strength of the obtained bone graft material are low, so the shape retention ability is also poor. (Comparative Example 5 to Comparative Example 16).

<Comparative Example 17>

The demineralization time in the above (1-1-5. Demineralization) was changed to 0.5 hour, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 17 was 105.8 Pa·s.

Meanwhile, in Table 3 below, the results of Example 4 are also shown.

<Comparative Example 18>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 17, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 18 was 113.3 Pa·s.

<Comparative Example 19>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 17, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 19 was 122.0 Pa·s.

<Comparative Example 20>

The demineralization time in the above (1-1-5. Demineralization) was changed to 1 hour, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 20 was 180.3 Pa·s.

<Comparative Example 21>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 20, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 21 was 221.8 Pa·s.

<Comparative Example 22>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Comparative Example 20, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Comparative Example 22 was 386.3 Pa·s.

<Comparative Example 23>

Above [1-2. Production of demineralized bone-derived carrier], except that the autoclave treatment was not performed, in the same manner as in Example 4 to manufacture demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material, but FIG. 5 (a ), the demineralized bone powder was not sufficiently soluble in water and was not suitable for use as a carrier for manufacturing a bone graft material.

<Comparative Example 24>

Above [1-2. Production of demineralized bone-derived carrier], except that the autoclave treatment time was changed to 10 minutes, in the same manner as in Example 4, to manufacture demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material. As shown in FIG. 5(b), the demineralized bone powder was not sufficiently soluble in water and was not suitable for use as a carrier for manufacturing a bone graft material.

<Example 7>

The demineralization time in the above (1-1-5. Demineralization) was changed to 3 hours, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 7 was 495.8 Pa·s.

<Example 8>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 7, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 8 was 669.2 Pa·s.

<Example 9>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 7, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 9 was 844.5 Pa·s.

<Example 10>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 10 was 910.6 Pa·s.

<Example 11>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 3 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 11 was 967.7 Pa·s.

According to Table 3 and above, in the production of the demineralized bone-derived carrier, the demineralization conditions and autoclave treatment conditions during the production of demineralized bone powder are the conditions specified in the present invention (the concentration of the acid solution is 0.3N or more and 0.8 or less, When the demineralized bone-derived carrier satisfies the following criteria: demineralization time is 2 hours or more and 7 hours or less, autoclave treatment time is 10 minutes or more and 65 minutes or less), the prepared demineralized bone-derived carrier has a viscosity suitable for use as a carrier for preparing a bone graft material, It can be seen that the block-type bone graft material into which the demineralized bone-derived carrier was introduced had low residual calcium concentration, good residual amount of BMP-2, excellent bone induction ability due to high cell proliferation rate, and excellent shape retention ability due to high durability and compressive strength. (Examples 4, 7 to 11).

On the other hand, in the manufacture of demineralized bone-derived carrier, when the demineralization conditions during manufacture of demineralized bone powder do not satisfy the conditions specified in the present invention, any of the remaining calcium concentration, BMP-2, and cell proliferation rate of the obtained bone graft material It was found that the bone induction ability of the bone graft material was poor because one or more of them were missing, and the demineralized bone-derived carrier did not have a viscosity suitable for use as a carrier for manufacturing a bone graft material, and the shape retention ability of the bone graft material was also poor because sufficient durability and compressive strength were not obtained ( Comparative Examples 17 to 22).

In addition, when autoclave treatment is not performed during the production of the demineralized bone-derived carrier, or when the autoclave treatment conditions do not satisfy the conditions specified in the present invention, the demineralized bone powder is not sufficiently soluble in water, and the bone graft material It was found that carriers derived from demineralized bone suitable for use as carriers for production could not be obtained (Comparative Examples 23 and 24).

<Example 12>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 1.0 N HCl and the demineralization time was changed to 0.5 hour, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 12 was 773.9 Pa·s.

<Example 13>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 12, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 13 was 800.2 Pa·s.

<Example 14>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 12, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 14 was 927.1 Pa·s.

<Example 15>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 1.0 N HCl and the demineralization time was changed to 1 hour, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 15 was 1013.4 Pa·s.

<Example 16>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 15, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 16 was 1058.5 Pa·s.

<Example 17>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 15, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 17 was 1193.2 Pa·s.

<Example 18>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 1.0 N HCl and the demineralization time was changed to 3 hours, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 18 was 1306.0 Pa·s.

<Example 19>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 18, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 19 was 1415.4 Pa·s.

<Example 20>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 18, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 20 was 1516.3 Pa·s.

<Example 21>

The demineralization solution in the above (1-1-5. Demineralization) was changed to 1.0 N HCl, and the above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 4, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 15 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 21 was 1670.7 Pa·s.

<Example 22>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 21, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 30 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 22 was 1708.4 Pa·s.

<Example 23>

Above [1-2. Demineralized bone powder, demineralized bone-derived carrier, and block-type bone graft material were prepared in the same manner as in Example 21, except that the autoclave treatment time in [Production of demineralized bone-derived carrier] was changed to 60 minutes. With respect to the obtained block-type bone graft material, durability, compressive strength, residual calcium concentration, BMP-2 residual amount, and cell proliferation rate were measured according to the above-described methods. The results are shown in Table 4 below. On the other hand, the viscosity of the demineralized bone-derived carrier obtained in Example 23 was 1832.6 Pa·s.

According to Table 4 and above, in the production of the demineralized bone-derived carrier, the demineralization conditions and autoclave treatment conditions during the production of demineralized bone powder are the conditions specified in the present invention (the concentration of the acid solution is greater than 0.8N and less than or equal to 1.5N). , demineralization time is 0.1 hour or more and 7 hours or less, autoclave treatment time is more than 10 minutes and 65 minutes or less), the prepared demineralized bone-derived carrier has a viscosity suitable for use as a carrier for manufacturing a bone graft material, It was found that the block-type bone graft material into which the demineralized bone-derived carrier was introduced had low residual calcium concentration, good residual amount of BMP-2, excellent bone induction ability due to high cell proliferation rate, and excellent shape retention ability due to high durability and compressive strength. It was possible (Examples 12 to 23).

In addition, in the production of the demineralized bone-derived carrier, when an acidic solution having a concentration of more than 0.8 N and 1.5 N or less is used in the production of demineralized bone powder, demineralization proceeds sufficiently even in a relatively short time, and the resulting demineralized bone is produced. It was found that the bone graft material containing the derived carrier had sufficient durability and compressive strength.

Claims (13)

(a) preparing demineralized bone powder by demineralizing bone powder;
(b) preparing a demineralized bone paste by mixing the demineralized bone powder prepared in step (a) with water;
Manufacturing a demineralized bone-derived carrier for manufacturing a bone graft material having a viscosity of 700 Pa s or more and 2000 Pa s or less by a method for manufacturing a demineralized bone-derived carrier for the manufacture of a bone graft material, comprising the step (c) of autoclaving the demineralized bone kneading material. step (I),
(d) preparing a mixture for forming a bone graft material by mixing the demineralized bone-derived carrier obtained in step (I) with demineralized bone powder; and
A method for manufacturing a bone graft material comprising the step (e) of preparing a bone graft material by freeze-drying the mixture for forming the bone graft material,
The step (a) may include demineralizing the bone powder; neutralizing the demineralized bone powder; Freeze-drying the neutralized bone powder;
The step of demineralizing the bone powder may include treating the bone powder with an acidic solution of 0.3N or more and 0.8N or less for 2 hours or more and 7 hours or less, or treating the bone powder with an acidic solution of 0.8N or more and 1.5N or less for 0.1 hour or more and 7 hours or less. It is carried out by processing for the following time,
The particle size of the demineralized bone powder prepared in step (a) is 200 μm or less,
The mixing ratio of the demineralized bone powder and water in step (b) is, in weight ratio, the demineralized bone powder : water = 21 ~ 28 : 72 ~ 79,
In the step (c), the demineralized bone dough is autoclaved for a time of more than 10 minutes and less than 65 minutes at a temperature of 100 ° C. or more and 130 ° C. or less and a pressure of 1.9 kgf / cm ² or more and 2.2 kgf / cm ^{2 or} less. do by doing,
The particle size of the demineralized bone powder mixed in step (d) is 200 μm or more and 850 μm or less,
The mixing ratio of the demineralized bone-derived carrier for preparing the bone graft material and the demineralized bone powder in step (d) is, in weight ratio, the demineralized bone-derived carrier for preparing the bone graft material: demineralized bone powder = 71-75: 25-29 , Method for manufacturing a bone graft material. According to claim 1,
The bone powder, removing the soft tissue from the bone and washing; immersing the washed bones in an organic solvent and stirring to remove lipids and immune antigens; A method for producing a bone graft material produced through the steps of crushing and selecting the bone from which the lipid and immunological antigens are removed. A bone graft material manufactured by the method for manufacturing a bone graft material according to claim 1. According to claim 3,
The bone graft material is a wedge-shaped, bone graft material. According to claim 4,
The bone graft material is used as a support for bone angle adjustment and bone tissue regeneration. delete delete delete delete delete delete delete delete

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