WO2019031772A2

WO2019031772A2 - Granule aggregate for substituting bone and manufacturing method thereof

Info

Publication number: WO2019031772A2
Application number: PCT/KR2018/008846
Authority: WO
Inventors: Ki Soo Kim; Seok Beom Song
Original assignee: Bioalpha Corporation
Priority date: 2017-08-11
Filing date: 2018-08-03
Publication date: 2019-02-14
Also published as: WO2019031772A3; KR20190017513A; TW201909933A

Abstract

Provided are granule aggregate for a substituting bone and a manufacturing method thereof. More particularly, provided are granule aggregate for a substituting bone which optimizes the composition of a calcium phosphate compound and collagen in order to prevent a substituting bone from being falling into small pieces even when constant shear force is applied, thereby facilitating the supply of the substituting bone to a bone defect portion or a tooth extraction portion both of which require filling, and a manufacturing method thereof.

Description

GRANULE AGGREGATE FOR SUBSTITUTING BONE AND MANUFACTURING METHOD THEREOF

The present invention relates to granule aggregate for a substituting bone and a manufacturing method thereof, and more particularly, to granule aggregate for a substituting bone and a manufacturing method thereof which facilitate the supply of a substituting bone to a bone defect portion or a tooth extraction portion, both of which require filling, by optimizing the composition of calcium phosphate compound and collagen so as not to be fallen into small pieces even when a constant shearing force is applied.

A substituting bone is a material which is to be implanted to reinforce or fill a fractured bone defect portion or a tooth extraction portion. So far, an autogenous bone, an allogeneic bone, a heterogeneous bone, and a synthetic bone have been introduced.

Among these, the autogenous bone is the most ideal material which has few immunological side effects and excellent bone regeneration properties. However, the autogenous bone has disadvantages in that a secondary surgery is required for harvesting and an amount which can be harvested is limited. The allogeneic bone has advantages in that it is is easily supplied and a second surgery of a donor is not required. However, the allogenic bone has disadvantages in that there is a possibility that a disease may be transferred to others and ethical problems may be caused. The heterogeneous bone may be easily supplied and manufactured. However, the heterogeneous bone has disadvantages in that an animal disease may be transferred to human, and bone inducing ability is considerably decreased. The synthetic bone has advantages in that it is easily supplied and various forms thereof may be manufactured and the possibility of inflammatory reaction is low. However, the synthetic bone has disadvantages in that in vivo absorption is slow and the bone inducing ability is considerably decreased.

Among the above materials, despite their disadvantages, research on the production of a new synthetic bone has been actively carried out in order to improve bone inducing ability by promoting in vivo absorption of a synthetic bone which is easily supplied in various forms.

A substituting bone made of a conventional synthetic bone has been supplied in the form of a powder or a fixed block for a bone defect portion or a tooth extraction portion. In the case of a substituting bone in the form of a powder, there is a disadvantage in that an additional measure is required so that the substituting bone is not separated after being supplied to the bone defect portion or the tooth extraction portion. In the case of a substituting bone in the form of a block, there is a disadvantage in that the substituting bone does not efficiently correspond to various forms of the bone defect portion or the tooth extraction portion.

In order to solve these problems, there have been attempts to add collagen to a conventional synthetic bone to provide flexibility. U.S. Patent No. 7189263 (registered on May 13, 2007) and European Patent Publication No. 0621044 (published on October 26, 1994) disclose inventions in which 11 to 43 parts by weight of collagen and 200 to 100 thousand by weight of collagen are respectively mixed based on 100 parts by weight of calcium phosphate compound.

However, in this case, the viscosity of the mixed solution excessively increases due to excess collagen, so that it is difficult to uniformly disperse the synthetic bone. Therefore, there is a great demand in the industry for a synthetic substituting bone having a variable shape.

Prior Art

(Patent Document 1) US Patent No. 7189263 (registered on May 13, 2007)

(Patent Document 2) European Patent Publication No. 0621044 (published on Oct. 26, 1994)

The present invention provides granule aggregate for a substituting bone having desired level of viscoelasticity by adding collagen to a synthetic bone composed of a calcium phosphate compound so as to efficiently correspond to various forms of bone defect portions and tooth extraction portions, and at the same time, so as not to be fallen into small pieces even when shear force is applied.

The present invention also provides a manufacturing method of granule aggregate for a substituting bone.

In accordance with an exemplary embodiment, granule aggregate for a substituting bone includes: 100 parts by weight of a calcium phosphate compound and 0.5 to 3 parts by weight, preferably 0.7 to 2.8 parts by weight, more preferably 0.9 parts by weight to 2.6 parts by weight, even more preferably 1.1 to 2.5 parts by weight of collagen.

In addition, the granules may be prepared by spray-drying fine particles of the calcium phosphate compound and then sintering the fine particles to aggregate in order to form macro-particles, and then coating the surfaces thereof with collagen.

In addition, the average particle diameter of the granules may be 0.05 to 2 mm, preferably 0.08 to 1.5 mm, more preferably 0.1 to 1 mm.

In addition, the average surface area to the average particle diameter of the granules, that is, the average surface area/average particle diameter of the granules may be 0.3 to 45 mm, preferably 0.6 to 25 mm, more preferably 1 to 15 mm.

In addition, the average particle diameter of the fine particles of the calcium phosphate compound may be 10 to 1000 nm, preferably 30 to 500 nm, and more preferably 50 to 100 nm.

In addition, the sintering may be performed at 500 to 800℃, preferably 550 to 750℃, more preferably 600 to 700℃ for 60 to 90 minutes.

In addition, the shear resistance index of the granule aggregate for a substituting bone of the present invention may be in the range of 50 × 10^-9 to 2000 × 10^-9, preferably 100 × 10^-9 to 1000 × 10^-9, more preferably 200 × 10^-9 to 600 × 10^-9.

In accordance with another exemplary embodiment, the granule aggregate of the present invention may further include an osteogenesis promoting factor.

In accordance with another exemplary embodiment, the calcium phosphate compound may be selected from the group consisting of tricalcium phosphate, β-tricalcium phosphate, monocalcium phosphate, biphasic calcium phosphate, hepta calcium phosphate, tetra calcium phosphate, octacalcium phosphate, calcium pyrophosphate, calcium metaphosphate, carbonated apatite (calcium deficient hydroxyapatite), hydroxyapatide, oxyapatide, and a combination thereof.

In addition, the collagen may be obtained from a mammal, preferably a cow, more preferably a cow's tendon.

In accordance with another exemplary embodiment, the osteogenesis promoting factor may be selected from the group consisting of a transforming growth factor-beta (TGF-β), a fibroblast growth factor (FGF), bone morphogenic protein (BMP), a vascular endothelial growth factor (VEGF), an epidermal growth factor (EGF), an insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), a nerve growth factor (NGF), a hepatocyte growth factor (HGF), a placental growth factor (PGF), a granulocyte colony stimulating factor (G-CSF), ascorbate 2-phosphate, activin, inhibin, and a combination thereof.

In accordance with another exemplary embodiment, an extrusion container of the present invention may receive the granule aggregate for a substituting bone.

In accordance with another exemplary embodiment, the extrusion container may be a syringe.

In accordance with another exemplary embodiment, a manufacturing method of granule aggregate for a substituting bone includes,

(A) preparing a collagen mixed solution by adding 0.7 to 2.5 parts by weight, preferably 0.8 to 2.3 parts by weight, more preferably 0.9 to 2.1 parts by weight of collagen, and 0.02 to 0.06 parts by weight, preferably 0.03 to 0.05 parts by weight, more preferably 0.035 to 0.045 parts by weight of hydrogen chloride to 100 parts by weight of water,

(B) preparing a collagen aqueous solution by dissolving collagen in the collagen mixed solution,

(C) preparing a mixture for a substituting bone by mixing 40 to 70 wt%, preferably 45 to 65 wt%, more preferably 50 to 60 wt% of the collagen aqueous solution, and 30 to 60 wt%, preferably 35 to 55 wt%, more preferably 40 to 50 wt% of calcium phosphate compound,

(D) freezing the mixture for a substituting bone, and

(E) manufacturing granule aggregate for a substituting bone by freeze-drying the frozen mixture for a substituting bone.

In accordance with another exemplary embodiment, the manufacturing method of granule aggregate for a substituting bone of the present invention may further include a step of filling the mixture for a substituting bone in the extrusion container after the step (C) and prior to the step (D).

In addition, the calcium phosphate compound of the step (C) may form macro-particles by spray-drying fine particles of the calcium phosphate compound, and then sintering the fine particles to aggregate.

In addition, the average particle diameter of the granules may be 0.05 to 2 mm, preferably 0.08 to 1.5 mm, and more preferably 0.1 to 1 mm.

In addition, the average surface area to the average particle diameter of the granules may be 0.3 to 45 mm, preferably 0.6 to 25 mm, more preferably 1 to 15 mm.

In addition, the average particle diameter of the calcium phosphate compound may be 10 to 1000 nm, preferably 30 to 500 nm, more preferably 50 to 100 nm.

In addition, the sintering may be performed at 500 to 800℃, preferably at 550 to 750℃, more preferably at 600 to 700℃ for 60 to 90 minutes.

In addition, the shear force resistance index of the granule aggregate for a substituting bone of the present invention may be in the range of 50 × 10^-9 to 2000 × 10^-9, preferably 100 × 10^-9 to 1000 × 10^-9, more preferably 200 × 10^-9 to 600 × 10^-9.

In addition, the manufacturing method of granule aggregate for a substituting bone of the present invention may further include an osteogenesis promoting factor.

In addition, the calcium phosphate compound may be selected from the group consisting of tricalcium phosphate, β-tricalcium phosphate, monocalcium phosphate, biphasic calcium phosphate, hepta calcium phosphate, tetra calcium phosphate, octacalcium phosphate, calcium pyrophosphate, calcium metaphosphate, carbonated apatite (calcium deficient hydroxyapatite), hydroxyapatide, oxyapatide, and a combination thereof.

In addition, the osteogenesis promoting factor may be selected from the group consisting of a transforming growth factor-beta (TGF-β), a fibroblast growth factor (FGF), bone morphogenic protein (BMP), a vascular endothelial growth factor (VEGF), an epidermal growth factor (EGF), an insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), a nerve growth factor (NGF), a hepatocyte growth factor (HGF), a placental growth factor (PGF), a granulocyte colony stimulating factor (G-CSF), ascorbate 2-phosphate, activin, inhibin, and a combination thereof.

In addition, the freeze-drying of the step (E) may be performed at -90 to -50℃, preferably -80 to -60℃, more preferably -75 to -65℃.

In addition, the freeze-drying of the step (E) may be performed for 24 to 96 hours, preferably 30 to 84 hours, more preferably 36 to 72 hours.

In addition, the manufacturing method of granule aggregate for a substituting bone of the present invention may further include a step of sterilizing after the step (E).

In addition, the sterilizing may be sterilization using a gamma ray.

Meanwhile, the granule aggregate for a substituting bone of the present invention is characterized by being manufactured by the above-mentioned manufacturing method.

The granule aggregate for a substituting bone according to the present invention is advantageous in that the supply thereof is facilitated, various forms thereof may be manufactured, and the possibility of inflammatory reaction is low since a synthetic bone made of raw materials such as a calcium phosphate compound and collagen is used rather than a bone of a living body. In addition, by optimizing the composition of a calcium phosphate compound and collagen, the problem of the viscosity of a mixed solution being excessively increased, thereby making it difficult for a synthetic bone to be uniformly dispersed is solved. Above all, through such optimization of the composition, even if constant shear force is applied, a substituting bone does not fall into small pieces, so that it is easy to supply the substituting bone to a bone defect portion or a tooth extraction portion both of which require filling.

FIG. 1 is a coupling diagram showing an embodiment of an extrusion container for receiving granule aggregate for a substituting bone of the present invention;

FIG. 2 is a perspective view showing a body constituting the extrusion container of FIG. 1;

FIG. 3 is a perspective view showing a push rod constituting the extrusion container of FIG. 1;

FIG. 4 is a perspective view showing a gasket constituting the extrusion container of FIG. 1;

FIG. 5 is a perspective view showing a lid constituting the extrusion container of FIG. 1;

FIG. 6 is a photograph of another embodiment of an extrusion container in which the granule aggregate for a substituting bone of the present invention is received;

FIG. 7 is a photograph showing a state in which a portion of the granule aggregate for a substituting bone of the present invention is discharged from the extrusion container and then separated from the container by opening the lid and performing extrusion in the embodiment of FIG. 6;

FIG. 8 is a photograph showing absorbency of the granule aggregate for a substituting bone of the present invention; and

FIG. 9 is a photograph showing absorbency of commercially available conventional calcium phosphate compound aggregate.

Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, numerous specific details, such as specific elements, are set forth in order to provide a more thorough understanding of the present invention. It is to be understood that these specific details are provided to facilitate a thorough understanding of the present invention, and it will be apparent to those skilled in the art that the present invention may be practiced without these specific details. In addition, in the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

In addition, unless otherwise specified throughout this specification, the term "including" or "containing" refers to including any element (or component) without limitation, and cannot be interpreted as excluding the addition of another element (or component).

In the present invention, 'shear resistance index' is defined by the following formula:

Shear force resistance index = (mass of collagen/mass of a calcium phosphate compound) x average particle diameter of fine particles of the calcium phosphate compound/ (average surface area of granules/average particle diameter of granules).

The granule aggregate for a substituting bone of the present invention is characterized by including 100 parts by weight of a calcium phosphate compound, and 0.5 to 3 parts by weight, preferably 0.7 to 2.8 parts by weight, more preferably 0.9 to 2.6 parts by weight, even more preferably 1.1 to 2.5 parts by weight of collagen.

Here, the granules may be prepared by spray-drying fine particles of the calcium phosphate compound and then sintering the fine particles to aggregate in order to form macro-particles, and then coating the surfaces thereof with collagen.

As such, since the calcium phosphate compound forms granule rather than being in a fine particle state, mixing with collagen is facilitated, and injecting through an extrusion container becomes much easier. Particularly, the present invention makes it possible to change the shape of a substituting bone by adding collagen to the calcium phosphate compound, thereby allowing a bone defect portion and a tooth extraction portion to be filled completely without an empty space resulting in improving bone regeneration rate.

However, if relative content of collagen to the calcium phosphate compound is less than the above range, the granule aggregate easily falls into small pieces when shear force is applied by kneading the same with hands. Therefore, the aggregate must be handled with care, thereby significantly lowering the workability of a practitioner. On the contrary, when the relative content exceeds the above range, the viscosity of a mixed solution of the calcium phosphate compound is excessively increased due to excess collagen as described above, which makes it difficult to uniformly disperse the substituting bone.

The average particle diameter of the granules in which the fine particles of the calcium phosphate compound are aggregated together into macro-particles may be 0.05 to 2 mm, preferably 0.08 to 1.5 mm, more preferably 0.1 to 1 mm. When the average particle diameter is within the above range, resistance against the shear force described above may be secured, so that the practitioner may easily change the shape of the substituting bone by a simple movement of fingers.

In addition, the average surface area to the average particle diameter of the granules, that is, the average surface area of the granules/average particle diameter may be 0.3 to 45 mm, preferably 0.6 to 25 mm, more preferably 1 to 15 mm. When the average surface area to the average particle diameter is within the above range, resistance against shear force described above may be secured likewise, so that the workability of the practitioner may be secured.

The average particle diameter of the fine particles of the calcium phosphate compound constituting the granules may be 10 to 1000 nm, preferably 30 to 500 nm, more preferably 50 to 100 nm. When the average particle diameter is within the above range, a desired level of porosity may be obtained while suppressing excessive economical deterioration.

Sintering temperature at which the fine particles of the calcium phosphate compound are spray-dried and sintered to form granules is 500 to 800℃, preferably 550 to 750℃, more preferably 600 to 700℃, and sintering time may be 60 to 90 minutes. When the sintering temperature and the sintering time are within the above range, granules having an average particle diameter range and an average surface area range to the average particle diameter described above may be obtained while suppressing excessive economic deterioration.

In addition, shear resistance index of the granule aggregate for a substituting bone of the present invention may be 50 × 10^-9 to 2000 × 10^-9, preferably 100 × 10^-9 to 1000 × 10^-9, more preferably 200 × 10^-9 to 600 × 10^-9.

The shear resistance index is defined as described above as a dimensionless variable indicating the degree to which the granule aggregate for a substituting bone of the present invention maintains an aggregation state without being fallen into small pieces when shear force is applied:

Considering the meaning of each variable, first, as the mass of the collagen to the mass of the calcium phosphate compound becomes larger, the resistance of the aggregate against shear force becomes stronger due to the viscoelasticity of the collagen itself. Therefore, the mass of the collagen to the mass of the calcium phosphate compound is proportional to the shear force resistance index.

Next, as the average particle diameter of the fine particles of the calcium phosphate compound becomes greater, the surface area of the granules in which the fine particles are aggregated becomes smaller, so that the collagen is more distributed on the surface of the granules than inside the granules, making the resistance against shear force become stronger. Therefore, the average particle diameter of the fine particles of the calcium phosphate compound is proportional to the shear force resistance index.

Finally, as the average surface area of the granules to the average particle diameter of the granules becomes greater, the collagen is less distributed on the surface of the granules than inside the granules, making the resistance against the shear force become weakened. Therefore, the average surface area of the granules is inversely proportional to the shear resistance index.

When the shear resistance index of the granule aggregate for a substituting bone of the present invention is within the above range, a procedure may be easily proceeded since the agglomerate does not fall into small pieces while completely filling a bone defect portion or a tooth extraction portion without creating an empty space when filled. Particularly, when the shear resistance index is within the above range, the procedure becomes much simpler since the practitioner can make a desired shape of the granule aggregate for a substituting bone just by a simple movement of fingers with moderate strength.

The calcium phosphate compound may be used without limitation as long as it is a compound which may be used as a substituting bone in the art of the present invention, and may be selected from the group consisting of, for example, tricalcium phosphate, β-tricalcium phosphate, monocalcium phosphate, biphasic calcium phosphate, hepta calcium phosphate, tetra calcium phosphate, octacalcium phosphate, calcium pyrophosphate, calcium metaphosphate, carbonated apatite (calcium deficient hydroxyapatite), hydroxyapatide, oxyapatide, and a combination thereof.

In addition, the collagen may be used without limitation as long as it is a material which may be used as a substituting bone in the art of the present invention, and may be obtained from, for example, a mammal, preferably a cow, more preferably a cow's tendon.

The granule aggregate for a substituting bone of the present invention may further include an osteogenesis promoting factor, thereby further increasing bone regeneration rate to reduce the inconvenience of a patient.

The osteogenesis promoting factor may be used without limitation as long as it is a factor which may be used in the art of the present invention, and may be selected from the group consisting of, for example, a transforming growth factor-beta (TGF-β), a fibroblast growth factor (FGF), bone morphogenic protein (BMP), a vascular endothelial growth factor (VEGF), an epidermal growth factor (EGF), an insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), a nerve growth factor (NGF), a hepatocyte growth factor (HGF), a placental growth factor (PGF), a granulocyte colony stimulating factor (G-CSF), ascorbate 2-phosphate, activin, inhibin, and a combination thereof.

Meanwhile, the extrusion container of the present invention is characterized by receiving the granule aggregate for a substituting bone. The practitioner extrudes all or a portion of granule aggregate for a substituting bone of the present invention which has been received in the extrusion container with fingers, kneads the extruded granule aggregate into a desired shape, and then pushes the granule aggregate in the desired shape into a bone defect portion or a tooth extraction portion area to fill the same by pressing so that there is no empty space.

In the present invention, the extrusion container is not limited as long as it may provide the granule aggregate for a substituting bone of the present invention by an extrusion method, and may be, for example, a syringe.

FIG. 1 is a coupling diagram showing an embodiment of an extrusion container for receiving granule aggregate for a substituting bone of the present invention, and FIG. 2 to FIG. 5 are perspective views showing a body (1), a push rod (2), a gasket (3), and a lid (4) constituting the extrusion container of FIG. 1, respectively.

In addition, FIG. 6 is a photograph of another embodiment of an extrusion container in which the granule aggregate for a substituting bone of the present invention is received, and FIG. 7 is a photograph showing a state in which a portion of the granule aggregate for a substituting bone of the present invention is discharged from the extrusion container and then separated from the container by opening the lid and performing extrusion in the embodiment of FIG. 6.

Meanwhile, a manufacturing method for granule aggregate for a substituting bone of the present invention starts from a step of adding 0.7 to 2.5 parts by weight, preferably 0.8 to 2.3 parts by weight, more preferably 0.9 to 2.1 parts by weight of collagen, and 0.02 to 0.06 parts by weight, preferably 0.03 to 0.05 parts by weight, more preferably 0.035 to 0.045 parts by weight of hydrogen chloride to 100 parts by weight of water to prepare a collagen mixed solution. If the amount of hydrogen chloride is less than the above range, solubility of collagen is not sufficient to dissolve all of the above-mentioned amount of collagen. On the contrary, if the amount exceeds the above range, it is harmful to a human body due to high acidity.

When the collagen mixed solution is prepared as described above, collagen in the mixed solution is dissolved to prepare a collagen aqueous solution. Subsequently, a mixture for a substituting bone is prepared by mixing 40 to 70 wt%, preferably 45 to 65 wt%, more preferably 50 to 60 wt% of the collagen aqueous solution, and 30 to 60 wt%, preferably 35 to 55 wt%, more preferably 40 to 50 wt% of calcium phosphate compound.

The calcium phosphate compound may form macro-particles by spray-drying fine particles of the calcium phosphate compound, and then sintering the fine particles to aggregate.

If relative content of collagen to the calcium phosphate compound is less than the above range, the granule aggregate easily falls into small pieces when shear force is applied by kneading the same with hands as described above. Therefore, the aggregate must be handled with care, thereby significantly lowering the workability of a practitioner. On the contrary, when the relative content exceeds the above range, the viscosity of a mixed solution of the calcium phosphate compound is excessively increased due to excess collagen as described above, which makes it difficult to uniformly disperse the substituting bone.

The mixture for a substituting bone, in which the calcium phosphate compound and collagen are mixed, is then frozen, and then freeze-dried to manufacture a granule aggregate for a substituting bone of the present invention. The freeze-drying may be performed at -90 to -50℃, preferably -80 to -60℃, more preferably -75 to -65℃. When the freeze-drying temperature is within the above range, it is possible to prevent drying time from being excessively increased while suppressing excessive economic deterioration. In addition, the freeze-drying may be performed for 24 to 96 hours, preferably 30 to 84 hours, more preferably 36 to 72 hours. When the freeze-drying time is within the above range, it is possible to completely dry the granule aggregate while suppressing excessive economic deterioration.

The manufacturing method of granule aggregate for a substituting bone of the present invention may further include a step of filling the mixture for a substituting bone in the extrusion container prior to the freezing step, and a step of sterilizing after the freeze-drying step. The sterilizing may be performed without limitation as long as it is a sterilization method which may be used in the art of the present invention, and for example, may be sterilization using gamma rays.

Hereinafter, examples of the present invention will be described.

Examples

Example 1: Granule aggregate for a substituting bone

10 g of collagen powder (COLLAGEN SOLUTIONS, the Netherlands) was added to 500 ml of distilled water to which 7 ml of 0.5 N HCl had been added, and then sufficiently dissolved to prepare a collagen aqueous solution. 5 g of the collagen aqueous solution was mixed with 5 g of β-tricalcium phosphate granules having an average particle diameter of 500 μm and an average surface area of 3.5 mm² which had been prepared by sintering fine particles of the β-tricalcium phosphate having an average particle diameter of 70 nm at 600℃ for 90 minutes, and then the mixture was filled in an extrusion container. The filler was frozen and then freeze-dried at -75℃ for 60 hours to obtain granule aggregate for a substituting bone of the present invention. The viscoelasticity of the granule aggregate measured with a viscoelasticity meter (Malvern, UK) and the shear resistance index calculated are shown in Table 1 below.

Example 2: Hydroxyapatite

The same procedure as in Example 1 was carried out except that hydroxyapatite (CGBio, Korea) was used instead of β-tricalcium phosphate. The viscoelasticity and shear resistance index of thus-manufactured granule aggregate are shown in Table 1 below.

Example 3: Addition of an osteogenesis promoting factor

The same procedure as in Example 1 was carried out except that 0.25 mg of BMP-2 (Daewoong Pharmaceutical Co., LTD, Korea) was added to the distilled water. The viscoelasticity and shear resistance index of thus-manufactured granule aggregate are shown in Table 1 below.

Comparative Example 1: Excessive use of collagen

The same procedure as in Example 1 was carried out except that 20 g of the collagen powder was used. The viscoelasticity and shear resistance index of thus-manufactured granule aggregate are shown in Table 1 below. As a result of including collagen in an amount exceeding the scope of the present invention, the viscoelasticity was excessively increased (the measured value was lowered), and the shear resistance index was also out of the scope of the present invention. In addition, since the viscosity of the mixed solution of the calcium phosphate compound was excessively increased due to excessive viscoelasticity, it was difficult to uniformly disperse a substituting bone.

Comparative Example 2: Trace use of collagen

The same procedure as in Example 1 was carried out except that 1 g of the collagen powder was used. The viscoelasticity and shear resistance index of thus-manufactured granule aggregate are shown in Table 1 below. As a result of including collagen in an amount which is less than the scope of the present invention, the viscoelasticity was too low (the measured value was increased), and the shear resistance index was also out of the scope of the present invention. In addition, due to the significantly low viscoelasticity, the granule aggregate easily fell into small pieces when shear force was applied by kneading the same with hands. Therefore, the aggregate had to be handled with care, thereby significantly lowering the workability of a practitioner.

Test Example 1 and 2: Absorbency

The absorbency of the granule aggregate for a substituting bone of Example 1 and commercially available conventional calcium phosphate compound aggregate (Hansbiomed, Korea) were measured and shown in FIG. 8 and FIG. 9. The absorbency was measured by photographing the degree of absorption of the aggregate to blue liquid. FIG. 8 is a photograph of the aggregate of Example 1, and FIG. 9 is a photograph a photograph of commercially available aggregate. It can be seen from FIG. 8 and FIG. 9 that the absorbency of the present invention is superior to that of the prior art, and that such high absorbency increases bone regeneration rate by activating delivery of materials through blood, thereby solving a problem in which the substituting bone is pushed away by blood.

Manufacturing Example 1: Granule aggregate for a substituting bone (1)

10 g of collagen powder (COLLAGEN SOLUTIONS, the Netherlands) was added to 500 ml of distilled water to which 7 ml of 0.5 N HCl had been added, and then sufficiently dissolved to prepare a collagen aqueous solution. 5 g of the collagen aqueous solution was mixed with 5 g of β-tricalcium phosphate granules having an average particle diameter of 500 μm and an average surface area of 32 mm² which had been prepared by sintering fine particles of the β-tricalcium phosphate having an average particle diameter of 70 nm at 600℃ for 90 minutes, and then the mixture was filled in an extrusion container. The filler was frozen and then freeze-dried at -75℃ for 60 hours to obtain granule aggregate for a substituting bone.

Manufacturing Example 2: Granule aggregate for a substituting bone (2)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that β-tricalcium phosphate granules having an average surface area of 0.03 mm² was used.

Manufacturing Example 3: Granule aggregate for a substituting bone (3)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that β-tricalcium phosphate granules having an average particle diameter of 0.01 mm was used.

Manufacturing Example 4: Granule aggregate for a substituting bone (4)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that β-tricalcium phosphate granules having an average particle diameter of 5 mm was used.

Manufacturing Example 5: Granule aggregate for a substituting bone (5)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that β-tricalcium phosphate fine particles having an average particle diameter of 3 nm was used.

Manufacturing Example 6: Granule aggregate for a substituting bone (6)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that β-tricalcium phosphate fine particles having an average particle diameter of 10 μm was used.

Manufacturing Example 7: Granule aggregate for a substituting bone (7)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that the sintering was performed at 400℃.

Manufacturing Example 8: Granule aggregate for a substituting bone (8)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that the sintering was performed at 1100℃.

Manufacturing Example 9: Granule aggregate for a substituting bone (9)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that the sintering was performed for 30 minutes.

Manufacturing Example 10: Granule aggregate for a substituting bone (10)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that the sintering was performed for 2 hours.

Manufacturing Example 11: Granule aggregate for a substituting bone (11)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that 0.1 N HCl was used.

Manufacturing Example 12: Granule aggregate for a substituting bone (12)

The same procedure as in Manufacturing Example 1 was carried out to manufacture granule aggregate for a substituting bone, except that 5 N HCl was used.

Test Examples 3 to 14: Viscoelasticity and shear resistance index

The viscoelasticity of the granule aggregate prepared in Manufacturing Examples 1 to 12 measured with a viscoelasticity meter (Malvern, UK), and shear resistance index calculated are shown in Table 2 below.

	Viscoelasticity (°)	Shear resistance index (Х10^-9)
Test Example 3	10.5	8
Test Example 4	2.3	2774
Test Example 5	1.6	3416
Test Example 6	12.7	5
Test Example 7	1.9	3196
Test Example 8	11.3	6
Test Example 9	8.8	21
Test Example 10	2.4	2657
Test Example 11	9.1	17
Test Example 12	2.2	2843
Test Example 13	8.6	32
Test Example 14	2.6	2481

Although preferred embodiments of the present invention have been described above, the present invention is not limited to the specific embodiments described above, and it is to be understood by those skilled in the art that various changes and modifications can be made without departing from the gist of the present invention. Accordingly, the scope of the present invention should not be construed as being limited to the above embodiments, but should be determined by equivalents to the appended claims as well as the following claims.

Claims

Granule aggregate for a substituting bone, comprising:

100 parts by weight of a calcium phosphate compound; and

0.5 to 3 parts by weight of collagen.
The granule aggregate of claim 1 further comprising an osteogenesis promoting factor.
The granule aggregate of claim 1, wherein

the calcium phosphate compound is selected from the group consisting of tricalcium phosphate, β-tricalcium phosphate, monocalcium phosphate, biphasic calcium phosphate, hepta calcium phosphate, tetra calcium phosphate, octacalcium phosphate, calcium pyrophosphate, calcium metaphosphate, carbonated apatite (calcium deficient hydroxyapatite), hydroxyapatide, oxyapatide, and a combination thereof.
The granule aggregate of claim 1, wherein

the osteogenesis promoting factor is selected from the group consisting of a transforming growth factor-beta (TGF-β), a fibroblast growth factor (FGF), bone morphogenic protein (BMP), a vascular endothelial growth factor (VEGF), an epidermal growth factor (EGF), an insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), a nerve growth factor (NGF), a hepatocyte growth factor (HGF), a placental growth factor (PGF), a granulocyte colony stimulating factor (G-CSF), ascorbate 2-phosphate, activin, inhibin, and a combination thereof.
An extrusion container receiving granule aggregate for a substituting bone of any one of claims 1 to 4.
The extrusion container of claim 5, wherein the extrusion container is a syringe.
A manufacturing method of granule aggregate for a substituting bone, comprising:

(A) preparing a collagen mixed solution by adding 0.7 to 2.5 parts by weight of collagen and 0.02 to 0.06 parts by weight of hydrogen chloride to 100 parts by weight of water;

(B) preparing a collagen aqueous solution by dissolving collagen in the collagen mixed solution;

(C) preparing a mixture for a substituting bone by mixing 40 to 70 wt% of the collagen aqueous solution and 30 to 60 wt% of calcium phosphate compound;

(D) freezing the mixture for a substituting bone; and

(E) manufacturing granule aggregate for a substituting bone by freeze-drying the frozen mixture for a substituting bone.
The manufacturing method of claim 7 further comprising filling the mixture for a substituting bone in the extrusion container after the step (C) and prior to the step (D).