WO2014193163A1

WO2014193163A1 - An injectable composition for bone defects and a preparation method thereof

Info

Publication number: WO2014193163A1
Application number: PCT/KR2014/004769
Authority: WO
Inventors: Hyun Seung Ryu; Kyung Dan Min
Original assignee: Bioalpha Corporation
Priority date: 2013-05-28
Filing date: 2014-05-28
Publication date: 2014-12-04
Also published as: KR20140140204A; KR101472046B1

Abstract

The present invention relates to a bone graft composition and a preparation method thereof, and more particularly to a bone graft composition, which is in the form of a porous scaffold comprising calcium phosphate compound particles, silk fibroin and poloxamer in a certain mixed ratio, and a preparation method thereof. Since the bone graft composition of the present invention can be changed into various shapes in a wet state and has an excellent ability to restore its original shape, it can be conveniently implanted and densely filled in bone defects.

Description

AN INJECTABLE COMPOSITION FOR BONE DEFECTS AND A PREPARATION METHOD THEREOF

The present invention relates to a bone graft composition and a preparation method thereof, and more particularly to a bone graft composition, which is in the form of a porous scaffold comprising calcium phosphate compound particles, silk fibroin and poloxamer, can be changed into various shapes in a wet state, has an excellent ability to restore its original shape, and thus can be conveniently implanted and densely filled in bone defects, and to a preparation method.

Bone graft biomaterials developed initially in the art had characteristics in that they are inert in vivo, but the applications thereof were significantly limited due to infection and inflammatory reactions which can occur in the surrounding tissue after implantation. Since then, with the rapid development of biomaterial technologies based on ceramic and polymer materials, materials that are biocompatible rather than bioinert were designed and developed, leading to the development of bioactive scaffolds for bone tissue regeneration which vary depending on the site and purpose of use. It is required that such bioactive scaffolds for bone tissue regeneration have different physical properties depending on the location of graft placement, are not be toxic to the surrounding tissue, and have relatively high mechanical properties compared to other artificial organs. Such bioactive scaffolds for bone tissue regeneration have been marketed and developed as various biomaterials depending on the properties of the raw materials and the intended use thereof.

All materials that are to be grafted into the human body, particularly polymer materials for bone tissue regeneration, should have good processability and moldability or have good in-situ polymerization properties so as to be suited to wounds. These materials are required to provide a suitable environment for the adhesion, growth and differentiation of cells, and degradation products thereof are also required to be biocompatible. Particularly, if the elongation of a bone graft material is too low, the bone graft material will have low utility because it is difficult to freely change the shape of the bone graft material upon injection or dense filling of the bone graft material. Also, if the tensile strength of a bone graft material is too low, it will be difficult to maintain the abilities of the bone graft material to fix its location and keep its external shape in the closure or implant placement stage after injection or dense filling of the bone graft material.

Accordingly, there is a need for the development of a bone graft composition that has biocompatibility and physical properties suitable for grafting in bone defects and that has the property of maintaining the formulation during a specific period after implantation.

Under this background, the present inventors have prepared a bone graft composition as a porous scaffold comprising calcium phosphate compound particles, silk fibroin and poloxamer at a specific mixing ratio, and have found that the composition can be changed into various shapes and has an excellent ability to restore its original shape, thereby completing the present invention.

It is an object of the present invention to provide a bone graft composition having excellent physical properties.

Another object of the present invention is to provide a method for preparing a bone graft composition having excellent physical properties.

To achieve the above objects, the present invention provides a bone graft composition comprising 35-65 parts by weight of calcium phosphate compound particles, 25-40 parts by weight of silk fibroin and 10-25 parts by weight of poloxamer.

The present invention is characterized in that a bone graft composition in the form of a porous scaffold comprising calcium phosphate compound particles, silk fibroin and poloxamer at a specific mixing ratio can be provided, which can be changed into various shapes in a wet state and has an excellent ability to restore its original shape.

In other words, the bone graft composition according to the present invention can be biodegraded with the passage of time after bone grafting, because it comprises the biodegradable polymers silk fibroin and poloxamer. Also, the bone graft composition according to the present invention can be freely changed into various shapes in a wet state by adding water thereto immediately before use, and thus can be conveniently implanted and densely filled in irregular bone defects. In addition, the bone graft composition comprises calcium phosphate compound particles, silk fibroin and poloxamer at a suitable mixing ratio, and thus the bone graft composition is in the form of a porous scaffold comprising calcium phosphate compound particles, silk fibroin and poloxamer and can be more freely changed into various shapes in a wet state. When a bone morphogenic protein together with water is added to the bone graft composition, the composition can further promote bone formation.

The present invention provides a bone graft composition, which is in the form of a porous scaffold comprising calcium phosphate compound particles, silk fibroin and poloxamer, can be changed into various shapes in a wet state, has an excellent ability to restore its original shape, and thus can be conveniently implanted and densely filled in bone defects.

FIG. 1 is a set of scanning electron microscope (SEM) photographs showing scaffolds prepared using various concentrations of silk fibroin solution.

FIG. 2 shows the results of examining the shape and restoration ability of scaffolds, prepared using various concentrations of silk fibroin solution, in a wet state. In FIG. 2, a): 4 wt%; b): 8 wt%; and c: 12 wt%.

FIG. 3 shows the results of examining the shape and restoration ability of scaffolds, prepared using various mixing ratios of hydroxyapatite fine particles and silk fibroin, in a wet state. In FIG. 3, a): 75/25; b): 50/50; and c): 25/75.

FIG. 4 shows the results of examining the shape and restoration ability of scaffolds, prepared using various mixing ratios of silk fibroin and poloxamer 407, in a wet state. In FIG. 4, a): 20/80; b): 50/50; and c): 80/20.

FIG. 5 shows the results of examining the shape and restoration ability of scaffolds, prepared using various mixing ratios of hydroxyapatite fine particles, silk fibroin and poloxamer 407, in a wet state. In FIG. 5, a): 50/10/40; b): 50/25/25; and c): 50/40/10.

FIG. 6 is a set of photographs showing the external shape (6A) and cross-sectional shape (FIG. 6B) of a bone graft composition according to the present invention.

FIG. 7 shows the results of a cytotoxicity test for bone graft compositions of Comparative Examples 1 and 2 and Examples 1 and 2.

As used herein, the term "bone graft composition" refers to a composition for use as bone defect replacement that is grafted in bone defects to fill the bone defects. Specifically, the term "bone graft composition" as used herein means an alloplastic synthetic bone graft material composition based on a calcium phosphate compound.

The bone graft composition of the present invention is mainly composed of calcium phosphate compound particles, silk fibroin and poloxamer.

As the first component of the bone graft composition of the present invention, calcium phosphate compound particles are similar to natural bone and functions to induce osteoconduction and bone growth.

As used herein, the term "calcium phosphate compound" refers to a compound comprising phosphoric acid and calcium.

In the present invention, the calcium phosphate compound may be selected from the group consisting of hydroxy apatite, tricalcium phosphate (TCP), monocalcium phosphate, tetracalcium phosphate, dicalcium phosphate, and a combination of two or more thereof, but is not limited thereto.

Preferably, the tricalcium phosphate may be β-tricalcium phosphate (β-TCP; Ca3(PO4)2).

In an example of the present invention, hydroxyapatite was used as the calcium phosphate compound in view of that it has excellent biocompatibility and bioactivity.

In the present invention, the hydroxyapatite may preferably be in the form of porous fine particles.

In the present invention, the hydroxyapatite can be obtained by drying hydroxyapatite powder, and calcining the dried hydroxyapatite powder at a temperature of preferably 1000~1400 ℃, more preferably 1200 ℃.

Silk fibroin and poloxamer, which are the remaining two components of the bone graft composition according to the present invention, serve as a hydrogel matrix that is gelled by addition of water. These components function to physically bond the calcium phosphate compound particles to each other and form the framework of the scaffold that is capable of freely changing its shape, thereby forming a formulation suitable for bone grafting.

As used herein, the term "silk fibroin" refers to the fibroin component of silk. In the present invention, silk fibroin functions to form the framework of a porous scaffold having a specific tensile strength and elongation. In a dry state, this scaffold framework is maintained in a specific shape, and in a wet state, it forms a hydrogel to physically bond the calcium phosphate compound particles to each other and can be freely changed into various shapes suitable for bone grafting. Accordingly, the bone graft composition according to the present invention can be present as a porous scaffold during distribution and storage, but can be gelled by addition of water immediately before use, and thus the deformable gel formed from the porous scaffold can be applied to bone defects.

Silk fibroin that is used in the present invention is commercially available or can be separated directly from silk by a degumming process. The silk fibroin may have a molecular weight ranging from 300,000 to 400,000 g/mole.

As used herein, the term "poloxamer" refers to a triblock copolymer (PEO-PPO-PEO) having two polyethylene glycol (PEG) chains bonded to a central chain of polypropylene glycol (PPG). Generally, the ratio of PEG/PPG in poloxamer may vary in the range from 1:9 to 8:2. The molecular weight of poloxamer may be in a wide range from 1,100 to 14,000 g/mole. Poloxamer is a temperature-sensitive polymer. In the present invention, poloxamer functions to impart injectability and moldability to the bone graft composition and to enable the bone graft material to be degraded rapidly after filling in bone defects so as to allow only the calcium phosphate-based bone graft material component to remain. In order to maintain the ease of injection and moldability in the room temperature range and formulation stability during storage and transport at room temperature, high-molecular-weight a poloxamer having a relatively low sol-gel transition temperature and high viscosity is preferably used. More preferably, a poloxamer that has a sol-gel transition temperature of 4~35 ℃ so as to be able to maintain the gel state at about 37 ℃ (body temperature) may be used in the present invention. Most preferably, poloxamer 407 having an excellent ability to maintain the gel state at about 37 ℃ (body temperature) may be used in the present invention.

In the present invention, the bone graft composition in the form of the porous scaffold can be made into a deformable gel by adding water thereto before use.

In the present invention, the bone graft composition may further comprise bone morphogenetic protein (BMP).

In the present invention, the bone morphogenetic protein may be selected from the group consisting of BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and a combination of two or more thereof, but is not limited thereto.

As described above, according to the present invention, the use of a combination of specific amounts of silk fibroin and poloxamer can provide a bone graft composition having increased elongation compared to a composition comprising silk fibroin alone or silk fibroin and poloxamer in amounts not covered by the range specified in the present invention. As the elongation of the scaffold increases, the shape deformability of the scaffold increases, and thus the scaffold can be easily applied to bone defects.

Particularly, the bone graft composition according to the present invention may have an elongation ranging from 35 to 45%. As the composition according to the present invention has an elongation in the above-specified range, it can be freely changed into various shapes in a wet state so that it can be easily filled in bone defects during implantation. Thus, it shows physical properties suitable for use as a bone graft material.

As used herein, the term "elongation" means the percentage of the difference between the length of the bone graft composition elongated until the composition is broken by an external force and the original length of the composition relative to the original length. Specifically, the elongation can be indicated by the following equation 1:

Equation 1

Elongation = (L_f-L_o)/L_o

wherein L_fis the length until breakage occurs,and L_ois the original length.

As the elongation of a bone graft material increases, the shape defoamability thereof increases, and thus the bone graft material is more conveniently used upon injection or dense filling in a wet state.

As used herein, the term "tensile strength" refers to the maximum stress measured until a specimen is broken by a tensile load, and means the maximum load at breakage, divided by the original cross-sectional area of the specimen. Higher tensile strength means that a greater force is required until the scaffold breaks, and also means that the formation of the bone graft composition can maintain its shape well not only when it is applied to bone defects, but also during distribution and storage.

In the present invention, elongation and tensile strength can be measured using a universal material testing machine (DTU-900MH200kN, Dae Kyung Tech, Korea). Specifically, elongation and tensile strength can be measured using a universal material testing machine after forming a specimen into a dumbbell shape having a diameter of 6 mm and a length of 115 mm. Herein, the initial distance between grips is 80 mm, and a testing speed of 50 mm/min is maintained. The testing is repeated 10 times, and the measurements are averaged.

The bone graft composition according to the present invention as described above comprises the calcium phosphate compound particles physically bonded to each other in a close state in the porous scaffold formed of silk fibroin and poloxamer. The bone graft composition in the form of the porous scaffold can be made into a hydrogel by adding water thereto immediately before it is implanted in bone defects. After the freely deformable hydrogel formed from the bone graft composition is into bone defects, the hydrogel is degraded and released, the calcium phosphate compound particles are maintained in the close state, and bone grows into the space between the calcium phosphate compound particles after release of the hydrogel.

The present invention also provides a method for preparing the bone graft composition, the method comprising the steps of:

1) preparing an aqueous solution of silk fibroin;

2) preparing an aqueous solution of poloxamer;

3) mixing calcium phosphate compound particles with a mixed solution of the aqueous solution of silk fibroin and the aqueous solution of poloxamer in such a manner that the amounts of calcium phosphate compound particles, silk fibroin and poloxamer are 35-65 parts by weight, 25-40 parts by weight and 10-25 parts by weight, respectively; and

4) freeze-drying the mixture resulting from step 3).

Step 1) of the method is a step of an aqueous solution of silk fibroin. In this step, silk fibroin is dissolved in water to obtain an aqueous solution of silk fibroin.

First, the silk fibroin that is used in this step may be separated from silk by a degumming process.

Silk is composed of a composition comprising fibroin, sericin, wax and inorganic matter. In the present invention, a degumming process is performed to remove components other than fibroin from silk. The degumming process can be performed by heating silk together with one or more selected from the group consisting of Marseilles soap, sodium bicarbonate, sodium carbonate, sodium hydroxide, sodium silicate and papain enzyme at a temperature of 90~95 ℃ for 30 minutes to 2 hours, followed by washing and drying.

Preferably, step 1) of the method may comprise the steps of:

a) dissolving silk fibroin in water containing at least one salt selected from the group consisting of lithium bromide, calcium chloride, lithium chloride and zinc chloride to obtain an aqueous solution of silk fibroin; and

b) removing a salt from the aqueous solution of silk fibroin by a dialysis process using a cellulose dialysis membrane.

Preferably, dissolving silk fibroin in step a) can be performed by heating at a temperature of 80~90 ℃ for about 1-4 hours.

Preferably, step b) may be performed by adding the silk fibroin solution to a cellulose dialysis membrane having a molecular weight of 6,000-12,000, sealing the membrane, immersing the sealed membrane in purified water and PEG (polyethylene glycol), and then stirring the membrane with a magnetic bar for about 3 days while replacing purified water and PEG (polyethylene glycol) twice a day, thereby removing a salt from the silk fibroin solution.

Step 2) of the method is a step of preparing an aqueous solution of poloxamer. In this step, poloxamer is dissolved in water to prepare an aqueous solution of poloxamer.

In one embodiment, step 2) can be performed by adding 25 g of poloxamer to 75 g of purified water, and then stirring the mixture at 4 ℃ and a constant speed of 100 rpm, thereby preparing an aqueous solution of poloxamer.

Herein, the kind and characteristics of poloxamer are as described above with respect to the bone graft composition.

Step 3) of the method is a step of mixing calcium phosphate compound particles with a mixed solution of the aqueous solution of silk fibroin and the aqueous solution of poloxamer in such a manner that the amounts of calcium phosphate compound particles, silk fibroin and poloxamer are 35-65 parts by weight, 25-40 parts by weight and 10-25 parts by weight, respectively. In this step, a specific amount of calcium phosphate compound particles are added to a mixed solution obtained by mixing the aqueous solution of silk fibroin of step 1) with the aqueous solution of poloxamer of step 2).

Step 3) can be performed by mixing the aqueous solution of silk fibroin with the aqueous solution of poloxamer to obtain a mixed solution, and adding calcium phosphate compound particles to the mixed solution, followed by mixing.

Herein, the characteristics and preparation method of the calcium phosphate compound particles are as described above with respect to the bone graft composition.

Step 4) of the method is a step of freeze-drying the mixture. In this step, a mixture of the calcium phosphate compound particles, the aqueous solution of silk fibroin and the aqueous solution of poloxamer is freeze-dried to obtain a bone graft composition.

In the present invention, step 4) may be performed by pouring the mixture of the calcium phosphate compound particles, the aqueous solution of silk fibroin and the aqueous solution of poloxamer into a mold, and then freezing the mixture at a temperature of -10 ℃ to -40℃, more preferably -15 ℃ to -30 ℃, and most preferably -20 ℃, followed by freeze drying.

In the present invention, the bone graft composition in the form of the bone scaffold prepared as described above may be made into a deformable gel by adding water thereto before use. In addition, a bone morphogenetic protein together with water may be added to the composition for use. The kind of bone morphogenetic protein is as described above with respect to the bone graft composition.

The present invention also provides a kit for bone implantation comprising:

a bone graft composition comprising A) 35-65 wt% of calcium phosphate compound particles, 25-40 wt% of a silk fibroin, and 10-25 wt% of a poloxamer; and an injection tool.

In the present invention, the injection tool may be forceps, a syringe, a syringe needle or the like.

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes and are not intended to limit the scope of the present invention.

Experimental Example 1: Selection of concentration of silk fibroin solution

4 wt%, 8 wt% and 12 wt% silk fibroin solutions were prepared, and then porous scaffolds were prepared therefrom. Then, the tensile strength and elongation of the porous scaffolds were measured.

The results of the measurement are shown in Table 1 below.

Table 1

Silk fibroin solution concentration (wt %)	Load (N)	Tensile strength (N/mm²)	Elongation (%)
4	4.7072	0.0941	12.658
8	11.6895	0.2338	33.318
12	12.7585	0.2552	49.574

As can be seen in Table 1 above, the porous scaffold prepared using the 4 wt% silk fibroin solution had low tensile strength and elongation compared to the 8 wt% and 12 wt% silk fibroin solutions. It is expected that the porous scaffolds prepared using the 8 wt% and 12 wt% silk fibroin solutions will have good restoration ability because they have high elongation.

Also, each of the prepared scaffolds was observed with a scanning electron microscope (SEM), and the results of the observation are shown in FIG. 1.

As can be seen in FIG. 1, the porous scaffold prepared using the 4 wt% silk fibroin solution has a large pore size compared to that of the porous scaffolds prepared using the 8 wt% or 12 wt% silk fibroin solution. Thus, it is believed that the porous scaffold prepared using the 4 wt% silk fibroin solution has weak bonds compared to those of the porous scaffolds prepared using the 8 wt% or 12 wt% silk fibroin solution. These results are consistent with the results of measurement of tensile strength as described above.

Meanwhile, the present invention provides a scaffold, which can be freely deformed while having good restoration ability so that it can be easily applied to bone defects and can also be applied curved bone defects. Because the scaffold can be changed into various shapes in a wet state, the shape and restoration ability of the scaffold in a wet state were examined. The results of the examination are shown in FIG. 2.

As can be seen in FIG. 2, the porous scaffold prepared using the 4 wt% silk protein solution did not change its shape in a wet state and did not restore its original shape. Also, the porous scaffold prepared using the 8 wt% or 12 wt% silk fibroin solution did change its shape in a wet state and then restored its original shape.

Thus, it can be seen that the optimal concentration of the silk fibroin solution is in the range of 8-12 wt%.

Experimental Example 2: Selection of mixing ratio between hydroxyapatite fine particles and silk fibroin

Hydroxyapatite fine particles and silk fibroin were mixed with each other at mixing ratios of 75/25, 50/50, 25/75 and 0/100, and then porous scaffolds were prepared therefrom. The tensile strength and elongation of each of the scaffolds were measured. In addition, the shape and restoration ability of each scaffold in a wet state were examined. The results of the examination are shown in FIG. 3.

The results are shown in Table 2 below and FIG. 3.

Table 2

Mixing ratio between hydroxyapatite and silk fibroin	Load (N)	Tensile strength (N/mm²)	Elongation (%)
75/25	10.7285	0.2146	19.770
50/50	9.1300	0.1826	23.328
25/75	11.0200	0.2204	24.022
0/100	12.7585	0.2552	49.574

As can be seen in Table 2 above, the tensile strength of the scaffold did not significantly differ from that of the porous scaffold formed of silk fibroin, but the elongation thereof decreased due to the addition of hydroxyapatite fine particles.

However, as can be seen in FIG. 3, even when hydroxyapatite was present, the restoration ability and utility of the scaffold were maintained due to the presence of silk fibroin.

From these results, it can be seen that the optimal mixing ratio between hydroxyapatite fine particles and silk fibroin is in the range from 50/50 to 25/75.

Experimental Example 3: Selection of mixing ratio between silk fibroin/poloxamer 407

Silk fibroin and poloxamer were mixed with each other at mixing ratios of 20/80, 50/50 and 80/20, and then porous scaffolds were prepared therefrom. The tensile strength and elongation of each of the prepared porous scaffolds were measured. In addition, the shape and restoration ability of each scaffold in a wet state were examined. The results of the examination are shown in FIG. 4.

The results are shown in Table 3 below and FIG. 4.

Table 3

Mixing ratio between silk fibroin and poloxamer	Load (N)	Tensile strength (N/mm²)	Elongation (%)
50/50	13.2880	0.2658	71.454
80/20	13.3174	0.2663	53.008
100/0	12.7585	0.2552	49.574

As can be seen in Table 3 above, the addition of poloxamer 407 did not significantly change the tensile strength of the scaffold, but increased the elongation of the scaffold. Because the shape deformability of the scaffold increases as the elongation increases, the scaffold is easily applied to bone defects.

Meanwhile, as can be seen in FIG. 4, when the content of poloxamer in the scaffold is higher than that of silk fibroin, the scaffold does not maintain its shape due to weak bonding strength and does not maintain its shape due to the aqueous solubility of poloxamer 407 in a wet state. However, when the mixing ratio between silk fibroin and poloxamer is suitable, the elongation of the scaffold is improved while the tensile strength does not differ from that of the scaffold formed of silk fibroin alone.

Thus, it can be seen that the optimal mixing ratio between silk fibroin and poloxamer solution is in the range from 50/50 to 80/20.

Experimental Example 4: Selection of mixing ratio between hydroxyapatite fine particles, silk fibroin and poloxamer 407

Hydroxyapatite fine particles, silk fibroin and poloxamer 407 were mixed with each other at mixing ratios of 50/10/40, 50/25/25 and 50/40/10, and porous scaffolds were prepared therefrom. Then, the tensile strength and elongation of each scaffold were measured.

The results are shown in Table 4 below and FIG. 5.

Table 4

Mixing ratio between hydroxyapatite, silk fibroin and poloxamer	Load (N)	Tensile strength (N/mm²)	Elongation (%)
50/25/25	11.0031	0.2201	41.304
50/40/10	10.7383	0.2148	37.219
0/80/20	13.2880	0.2658	71.454
0/100/0	12.7585	0.2552	49.574

As can be seen in Table 4 above, the tensile strength and elongation of the scaffold decreased due to the addition of hydroxyapatite compared to those of the porous scaffold formed of silk fibroin and poloxamer 407. However, the tensile strength and elongation of the scaffold did not significantly differ from those of the porous scaffold formed of silk fibroin alone.

Meanwhile, as can be seen in FIG. 5, as the content of poloxamer 407 in the scaffold increases compared to the content of silk protein, the bond of the scaffold becomes weaker, and when the scaffold is wet, only hydroxyapatite remains. However, it can be seen that the scaffold prepared using a suitable mixing ratio of silk fibroin and poloxamer has a good bond with hydroxyapatite, and thus maintains the bond even in a wet state while it is easily deformed and has a good ability to restore to its original shape after deformation.

From these results, it can be seen that the optimal mixing ratio between hydroxyapatite fine particles, silk fibroin and poloxamer is in the range from 50/25/25 to 50/40/10.

Example 1: Preparation of porous scaffold comprising hydroxyapatite fine particles and silk fibroin

Based on the results of Experimental Examples 1 and 2, a scaffold composition for bone regeneration was prepared in the following manner.

First, a 12 wt% aqueous solution of silk fibroin was prepared. 417 g of the prepared 12 wt% aqueous solution of silk fibroin (silk fibroin: 50 g) was mixed with 50 g of hydroxyapatite fine particles, and the mixture was poured into a mold having a size of 10x10x50 ㎜. Next, the mixture was frozen at a temperature of -20 ℃, followed by freeze drying for 3 days. The prepared porous scaffold was sterilized with EO gas, thereby obtaining a bone scaffold for bone regeneration.

Example 2: Preparation of porous scaffold comprising hydroxyapatite, silk fibroin and poloxamer 407

Based on the results of Experimental Examples 3 and 4, a scaffold composition for bone regeneration was prepared in the following manner.

First, a 12 wt% aqueous solution of silk fibroin was prepared. Then, 25 g of poloxamer 407 was completely dissolved in 75 g of purified water at 4 ℃ to prepare a 25 wt% aqueous solution of poloxamer 407. Next, 50 g of hydroxyapatite fine particles, 333 g of the 12 wt% aqueous solution of silk fibroin (silk fibroin: 40 g) and 40 g of the 25 wt% aqueous solution of poloxamer 407 (poloxamer 407: 10 g) were mixed with each other, and the mixture was poured into a mold having a size of 10x10x50 ㎜, and then frozen at a temperature of -20 ℃, followed by freeze drying for 3 days. The prepared porous scaffold was sterilized with EO gas, thereby obtaining a bone scaffold for bone regeneration.

Comparative Example 1: Preparationof porous scaffold composed of silk fibroin

First, a 12 wt% aqueous solution of silk fibroin was prepared. The prepared silk fibroin solution was poured into a mold having a size of 10 10 50 ㎜, and then frozen at a temperature of -20 ℃, followed by freeze drying for 3 days. The prepared porous scaffold was sterilized with EO gas, thereby obtaining a bone scaffold for bone regeneration.

Comparative Example 2: Preparation of porous scaffold comprising silk fibroin and poloxamer 407

First, a 12 wt% aqueous solution of silk fibroin was prepared. Then, 25 g of poloxamer 407 was completely dissolved in 75 of purified water to prepare a 25 wt% solution of poloxamer. Then, 667 g of the 12 wt% aqueous solution of silk fibroin (silk fibroin: 80g) and 80 g of the 25 wt% solution of poloxamer 407 (poloxamer 407: 20 g) were mixed with each other, and the mixture was poured into a mold having a size of 10x10x50 ㎜, and then frozen at a temperature of -20 ℃, followed by freeze drying for 3 days. The prepared porous scaffold was sterilized with EO gas, thereby obtaining a bone scaffold for bone regeneration.

Experimental Example 5: Examination of shape of bone graft composition of the present invention

FIG. 6(A) shows the external shape of the bone graft composition prepared in Example 2 of the present invention.

Also, the cross-sectional shape of the bone graft composition prepared in Example 2 of the present invention was observed with a scanning electron microscope (SEM), and the result of the observation is shown in FIG. 6(B).

As can be seen in FIG. 6, the bone graft composition of the present invention has a specific scaffold shape. Particularly, it can be seen that calcium phosphate compound particles are physically bonded to each other in a close state in the porous scaffold formed of silk fibroin and poloxamer.

Experimental Example 6: Cytotoxicity test

The results of a cytotoxicity test for the scaffold compositions of Comparative Examples 1 and 2 and Examples 1 and 2 indicated that these scaffold compositions all show high cell viability.

In order to quantitatively analyze the effects of the scaffold compositions of Comparative Examples 1 and 2 and Examples 1 and 2 on cell proliferation, the growth of cells was examined by an MTT assay using L-929 mouse fibroblast cells and an eluate obtained from each of the scaffolds. With respect to elution conditions, according to ISO 10993-9, 4 g of each test material was eluted with 20 ㎖ of a solvent in serum medium (MEM) at 37℃ for 24 hours. Cells were seeded into each well of a 96-well plate at a concentration of 1x10⁵cells/㎖ and cultured for 24 hours, and when the confluency of the cells reached about 80%, the medium was removed from each well, and then each well was treated with 1 ml of each of the eluate of each scaffold, a negative control and a positive control and incubated for 48 hours. As the negative control, an HDPE eluate was used, and as the positive control, 10% DMSO-containing medium was used. After 48 hours of incubation, 5 mg/㎖ of MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each well, which was then incubated in a 5% CO₂incubator at 37 ℃ for 4 hours. When a purple crystal was produced, 1 ml of dimethylsulfoxide (DMSO) solution was added to each well, which was then agitated for 1 hour to dissolve the crystal. 100 ㎕ of the resulting solution was added to each well of the 96-well plate, and the absorbance at 570 ㎚ was measured using an ELISA plate reader.

The results of the measurement indicated that the scaffolds of Comparative Examples 1 and 2 and Examples 1 and 2 showed cell viabilities of 104.67±8.69%, 92.90±7.12%, 117.98±4.29%, and 95.5±3.53%, respectively, suggesting that the scaffolds are highly compatible with cells (see FIG. 7).

Claims

A bone graft composition comprising 35-65 parts by weight of calcium phosphate compound particles, 25-40 parts by weight of silk fibroin and 10-25 parts by weight of poloxamer.
The bone graft composition of claim 1, which is in the form of a porous scaffold.
The bone graft composition of claim 1, wherein the calcium phosphate compound is selected from the group consisting of hydroxyapatite, tricalcium phosphate (TCP), monocalcium phosphate, tetracalcium phosphate, dicalcium phosphate, and a combination thereof.
The bone graft composition of claim 3, wherein the hydroxyapatite is in the form of porous fine particles.
The bone graft composition of claim 1, wherein the poloxamer has a sol-gel transition temperature between 4 ℃ and 35 ℃.
The bone graft composition of claim 5, wherein the poloxamer is poloxamer 407.
The bone graft composition of claim 1, which is made into a deformable gel by adding water thereto before use.
The bone graft composition of claim 7, which is used after a bone morphogenic protein (BMP) is added thereto together with water.
The bone graft composition of claim 8, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and a combination thereof.
The bone graft composition of claim 1, which has an elongation of 35-45%.
A method for preparing the bone graft composition of claim 1, the method comprising the steps of:

1) preparing an aqueous solution of silk fibroin;

2) preparing an aqueous solution of poloxamer;

3) mixing calcium phosphate compound particles with a mixed solution of the aqueous solution of silk fibroin and the aqueous solution of poloxamer in such a manner that the amounts of calcium phosphate compound particles, silk fibroin and poloxamer are 35-65 parts by weight, 25-40 parts by weight and 10-25 parts by weight, respectively; and

4) freeze-drying the mixture resulting from step 3).
The method of claim 11, wherein step 1) comprises the steps of:

a) dissolving silk fibroin in water containing at least one salt which is selected from the group consisting of lithium bromide, calcium chloride, lithium chloride and zinc chloride to obtain an aqueous solution of silk fibroin; and

b) removing the salt from the aqueous solution of silk fibroin by a dialysis process using a cellulose dialysis membrane.
A kit for bone implantation comprising: the bone graft composition according to claim 1; and an injection tool.
The kit of claim 13, wherein the injection tool is forceps, a syringe or a syringe needle.