KR101570832B1

KR101570832B1 - Bone graft substitute using cuttlefish bone and method for preparing thereof

Info

Publication number: KR101570832B1
Application number: KR1020130108124A
Authority: KR
Inventors: 이준; 김범수
Original assignee: 주식회사 본셀바이오텍
Priority date: 2013-09-09
Filing date: 2013-09-09
Publication date: 2015-11-20
Also published as: WO2015034307A1; KR20150029235A

Abstract

The present invention relates to a bone graft material using squid bone and a method for producing the bone graft material, and is characterized by forming a coating layer on a calcium phosphate-based porous material. The bone graft material according to the present invention includes a coating layer containing a biodegradable polymer in a calcium phosphate-based porous material, thereby further improving the compressive strength, which is a disadvantage of oozing retardation derived from the cuttlefish bone, It is more effective as bone graft material.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bone graft substitute using cuttlefish bone and a method for preparing the same,

The present invention relates to a bone graft material using squid bone and a method for producing the same, and more particularly, to a bone graft material including a coating layer containing a biodegradable polymer in a calcium phosphate based porous material and a method for producing the same.

In addition to its mechanical function to support and support the human body, bones also act as a reservoir of calcium to regulate calcium ion concentration in the body and possess important physiological functions of producing red blood cells and white blood cells for human body in bone marrow. Bones can be damaged by aging and other physiological reasons, or can be damaged by various accidents. Representative examples of physiological damage include osteoporosis and osteoarthritis (osteoarthritis), and osteonecrosis caused by interruption of blood supply to the bone. Currently, bone damage is treated primarily by mechanical and physical methods. Bone regeneration and treatment for bone loss is an important goal in oral surgery or orthopedic surgery caused by accidents, illnesses, infections, and the like. One way to do this is to implant the bone. Bone grafting involves the transplantation of bones from other people or animals, the implantation of the patient's own tissues, etc. However, when transplanted with other tissues, an immunological rejection occurs, or when the damaged area is large, There is a disadvantage that there is not enough material available. To overcome these drawbacks, research on bone graft materials is actively under way. Bone graft substitute (BGS) is a bone graft substitute (BGS), which can replace the bone graft deficient part due to various dental diseases, trauma, diseases, or other tissue loss, The implantable material used to make it. The calcium phosphate (Ca3 (PO4) 2, TCP) and hydroxyapatite (Ca10 (PO4) 6 (CaO) OH) 2, HAp) is known to have chemical components similar to natural bone or tooth tissues in terms of physical properties and biocompatibility, and is thus very useful as a biomaterial. TCP and HAp can be artificially synthesized by chemical methods, and recent studies have reported that biomaterial HAp or TCP can be produced from natural materials such as eggshells and corals. In particular, it is known that cuttlefish bones (CB) can be converted into HAp (CB-HAp), TCP (CB-TCP) or Biphasic calcium phosphate (CB-BCP)

The ideal condition for bone grafting is that blood vessels should be easily grown and blood circulation should be improved, and bone cells should be connected three-dimensionally for easy proliferation. Pore size of about 200-400 um is known to be adequate for hematogenous supply and bone cell infiltration. Since the inner structure of the squid bones has proper porosity characteristics satisfying the above porous conditions and most of the constituents of the squid bones of the squid are composed of calcium carbonate, many studies are being conducted to use them as a scaffold for bone regeneration . Nevertheless, squid bones are weak in strength and can not be utilized as materials for bone regeneration.

As a result of intensive efforts to improve the physical properties of the ophiolite, the present inventors have found that introduction of a coating layer containing a biodegradable polymer into a calcium phosphate-based porous body improves compressive strength and other performance as a bone graft material. .

Korean Registered Patent No. 10-1060251 (registered on August 23, 2011)

Ivankovic H. et al., Preparation of highly porous hydroxyapatite from cuttlefish bonel H Mater Sci Mater Med 2009; 20; 1039-1046 Sivakumar M, Kumar TS, Shantha KL, Rao KP. Development of hydroxyapatite derived from Indian coral. Biomaterials 1996; 17: 1709-1714. Manoli F, Dalas E. Calcium carbonate crystallization on xiphoid of the cuttlefish. J Cryst Growth 2000; 217: 422428. Birchall JD, Thomas NL. On the architecture and function of cuttlefish bone. J Mater Sci 1983; 18: 20812086.

It is an object of the present invention to provide a bone graft material having improved physical strength.

Another object of the present invention is to provide a method of manufacturing a bone graft material capable of improving physical strength.

In order to solve one object of the present invention, the present invention provides a bone graft material comprising a calcium phosphate-based porous body and a coating layer containing a polymer on the surface of the porous body.

In the present invention, a bone graft material is a bone graft material that replaces a bone defect in a bone tissue due to various dental diseases, trauma, disease, or loss of other tissues to fill the space in the bone tissue and promote the formation of new bone Refers to a graft material used, which includes a goal system, a bone filler, and an osteoporosis.

The polymer included in the coating layer of the present invention may be a biodegradable polymer and preferably the polymer is selected from the group consisting of polycaprolactone, polylactic acid (PLA), polyglycolic acid (PGA), polyphosphazene, polyanhydride, poly (Polyglycolic acid) (PLGA), collagen, chitosan, gelatin, fibrin, fibrinogen, chitin, hyaluronic acid, alginate, dextran, poly Copolymers, and mixtures thereof, and more preferably polycaprolactone, but is not limited thereto. Polycaprolactone has a higher tensile strength than other polymers and can impart appropriate physical strength to the calcium phosphate-based porous body of the present invention. Also, the polymers and other biopolymers that can be easily obtained by a person skilled in the art, such as the above-mentioned modified polymers, modified polymers, etc., are all included in the scope of the present invention.

The coating layer is preferably formed by immersing the polymer in a solution of 2 to 9 wt% of the polymer, but is not limited thereto. If the amount of the polymer in the solution is less than 2% by weight, the mechanical properties of the calcium phosphate-based porous body are not affected. If it exceeds 9% by weight, the porosity of the support is greatly reduced. Impregnation is difficult.

More preferably, the weight of the polymer contained in the solution may be 4 to 6% by weight. When a coating layer is formed by dissolving the polymer in an amount of 4 to 6% by weight, it has an ideal porosity as a bone support, is effective in improving physical properties such as compressive strength of a calcium phosphate-based porous body, effective.

The calcium phosphate-based porous body contained in the bone graft material of the present invention may be hydroxyapatite (HAp),? -Tricotinate (? -TCP) or biphasic calcium phosphate (BCP), preferably hydroxyapatite Lt; / RTI > The anhydrous calcium phosphate (BCP) is a mixture of hydroxyapatite (HAp) and calcium triphosphate (TCP), and may be formed in various ratios. In addition, the calcium phosphate-based porous body is preferably prepared from the cuttlefish bone, but not limited thereto, and natural materials capable of producing calcium phosphate-based porous bodies such as bones and corals of animals are all possible. When a calcium phosphate-based porous article is produced using a natural substance, particularly squid bone, it can be easily obtained at a low cost. In addition, it has an appropriate porosity as an ophiolite due to inherent porous structure of cuttlefish bone, and its porosity is maintained even when it is converted into? -Tricantinoate, hydroxyapatite, BCP or the like, and is particularly preferable as a bone graft material . However, the calcium phosphate-based porous body of the present invention is not limited to those produced from natural materials, and includes all of the chemically synthesized powders of hydroxyapatite, calcium triphosphate and the like produced through sintering and extrusion.

When the calcium phosphate-based porous body is manufactured using the cuttlefish bone, the calcium phosphate-based porous body can be manufactured through hot water treatment of the cuttlefish bone. Specifically, after removing the thick outer wall of the cuttlefish bone, the inner lamella matrix is cut to an appropriate size and hydrothermally treated. In the hydrothermal treatment, it is preferable that the cut squid bone is treated with a solution containing phosphate ions and reacted at 100-300 ° C for 6-48 hours, but it is not limited thereto. The solution containing the phosphoric acid may preferably be a solution containing ammonium phosphate, H ₃ PO ₄ (phosphoric acid), KH ₂ PO ₄ , but not limited thereto, all of the materials capable of supplying phosphoric acid Available. Also, conversion into a calcium phosphate-based porous body using natural materials such as bones and corals of an animal can be accomplished through the hydrothermal treatment described above.

In order to solve the above-mentioned problems, the present invention provides a method for producing a bone graft material, which comprises forming a coating layer from a calcium phosphate-based porous body.

Another aspect of the present invention relates to a method for manufacturing a bone graft material, which comprises the step of forming a coating layer on a calcium phosphate-based porous body.

The step of forming the coating layer preferably comprises immersing the calcium phosphate-based porous body in a solution in which the polymer is dissolved in a vacuum state, but is not limited thereto.

Also, the component contained in the coating layer formed on the surface of the calcium phosphate-based porous body may be a biodegradable polymer, and the polymer is preferably selected from the group consisting of polycaprolactone, polylactic acid (PLA), polyglycolic acid (PGA) (Polyglycolic acid) (PLGA), collagen, chitosan, gelatin, fibrin, fibrinogen, chitin, hyaluronic acid, alginate (polyglycolic acid) , Dextran, copolymers of these polymers, and mixtures thereof, but is not limited thereto. Also, all of the above-mentioned polymers, which can be easily obtained by a person skilled in the art such as modified substances, modified polymers, etc., are included in the scope of the present invention. The component of the coating layer is more preferably polycaprolactone.

The coating layer is preferably formed by immersing the polymer in a solution of 2 to 9 wt% of the polymer, but is not limited thereto. If the amount of the polymer in the solution is less than 2% by weight, the mechanical properties of the calcium phosphate-based porous body are not affected. If the amount exceeds 9% by weight, the porosity of the support is greatly reduced. It is difficult to impregnate the coating layer into the inside of the support.

More preferably, the weight of the polymer contained in the solution may be 4 to 6% by weight based on the total weight of the coating layer. When a coating layer is formed by dissolving the polymer in an amount of 4 to 6% by weight, it has an ideal porosity as a bone support, is effective in improving physical properties such as compressive strength of a calcium phosphate-based porous body, effective.

In the method for producing a bone graft material according to the present invention, the calcium phosphate-based porous body may be a composite of calcium phosphate (BCP) or calcium carbonate The porous calcium phosphate-based porous body is preferably made using the cuttlefish bone, but it is not limited thereto, and the calcium phosphate-based porous body such as bones and corals of an animal can be produced All natural substances are possible. In addition, the calcium phosphate-based porous body of the present invention is not limited to the one produced from a natural substance, but may be a metal such as calcium phosphate (HAp), calcium triphosphate (TCP, ideal calcium phosphate (BCP) It also includes all of the manufacturing methods.

When a calcium phosphate-based porous body is manufactured using the cuttlefish bone, the calcium phosphate-based porous body can be produced by subjecting the cuttlefish bone to hydrothermal treatment. Specifically, after removing the thick outer wall of the cuttlefish bone, the inner lamella matrix is cut to an appropriate size and hydrothermally treated. The hydrothermal treatment is preferably performed by treating the cut squid bone with a solution containing phosphate ions and reacting at 100-300 ° C for 6-48 hours, but is not limited thereto. The solution containing phosphoric acid may preferably be a solution containing ammonium phosphate, H ₃ PO ₄ (phosphoric acid), KH ₂ PO ₄ , but not limited thereto, and all of the substances capable of supplying phosphoric acid Available.

In addition, the conversion into the calcium phosphate type porous body using bones, corals, etc. of animals other than the cuttlefish bone can also be accomplished through the hydrothermal treatment described above.

According to the bone graft material and the bone graft material according to the present invention, the calcium phosphate-based porous body composed of the most similar components to the human bone, particularly the calcium phosphate-based porous body derived from the deep- It is possible to improve the compressive strength without significantly affecting the porosity which is an ideal condition of the bone graft material. It further improves the degree of cell adhesion and differentiation into osteoblasts, and does not affect toxicity, and is also excellent in biocompatibility. Therefore, the bone graft material according to the present invention can be utilized variously as a bone substitute because of its strong physical strength and porous structure.

In Fig. 1, A is an SEM image of an untreated cuttlefish bone (CB), and B is an SEM image of a cuttlefish bone (CB-HAp) converted to hydroxyapatite by hydrothermal action. C represents the XRD pattern of CB-HAp.
2, A shows the XRD pattern of the cuttlefish bone (CB-HAp) converted to hydroxyapatite coated with 1, 5, and 10% (w / v) PCL, SEM image of PC-coated CB-HAp support is shown. In B, the white arrow indicates the PCL coated on the CB-HAp.
In FIG. 3, A and B show MTT assay (A) and DNA content analysis (B) results of MG-63 cells cultured on CB-HAp support and PCL coated CB-HAp support, C is a fluorescence image showing the survival / toxicity level after 3 days of cell seeding.
Figure 4 shows an SEM image of CB-HAp coated with 1, 5, and 10% (w / v) PCL after 6 days of MG-63 cell culture.
Figure 5 is a graph showing alkaline phosphatase (ALP) activity of MG-63 cells cultured on CB-HAP and CB-HAp coated with 1, 5, and 10% (w / v) PCL. The data represent the mean (± SD) of the three samples, indicating that there is a significant difference from the CB-HAp uncoated with * p <0.05 and ** p <0.01.
6 is a graph showing quantitative analysis results of real-time PCR. The expression of each osteoblast marker gene was based on 18S and relative expression levels were based on cells cultured on uncoated scaffolds. Data indicate the mean (± SD) of the three samples, * p <0.05 and ** p <0.01 indicate significant differences from uncoated CB-HAp.

Hereinafter, the present invention will be described in more detail by way of examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Also, terms, techniques, and the like used in the present specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

< Manufacturing example > 1. Cuttlefish bone ( CB ) HAp ( Hydroxyapatite ) Support ( scaffold ) Produce

Cuttlefish bone (CB) extracted from Sepia esculenta (Korean Yellow Sea) was washed with water and dried. For hydrothermal reaction, the internal CB matrix (lamellae portion) was cut into pieces of cylindrical shape with a diameter of about 10 mm and a length of 10 mm using a trephine bar. A known hydrothermal reaction method (Rocha JH et al., J Biomed Mater. Res. 2006; 77: 160-168) was used to transform hydroxyapatite (HAp). After treating the CB pieces with an aqueous solution of 0.6M NH4H2PO4, the CB pieces were sealed in Teflon-lined stainless pressure vessels. The pressure vessel was placed in an electric furnace and heat treated at 200 ° C for 24 hours. The resulting HAp was washed with distilled water for further experiments and then dried at 100 ° C.

< Manufacturing example > 2. Hydroxyapatite On the support Of polycaprolactone (PCL) Coating treatment

PCL pellets (molecular weight 10,000; Sigma-Aldrich St. Louis, Mo.) were dissolved in dichloromethane to 1, 5, and 10% (w / v), respectively. The prepared CB-HAp support was immersed in the PCL solution for 5 minutes under vacuum to allow complete penetration into the porous support structure. The excess solution was then removed by centrifugation at 300 rpm for 30 seconds. The PCL coated samples were dried in an oven at 40 占 폚 for 6 hours and then dried again at room temperature for 7 days to volatilize the organic solvent.

< Test Example 1> CB / RTI > HAp Observation of the surface characteristics of the support and the degree of porosity

XRD (X-ray diffraction) analysis was performed with CuKa radiation at a scan rate of 0.02 / min (2 h), 50 kV, and 20-50 ° at 30 mA to characterize the composition of the converted components. The microstructure and morphology of the native CB, CB-HAp, and PCL-coated CB-HAp were analyzed by scanning electron microscopy (JEM-6360; JEOL, Tokyo, Japan).

1. Natural (unprocessed) CB

Figure 1 (A) is an SEM image of the microstructure of natural CB lamella. This structure was made up of parallel sheets and was supported by numerous pillars. The average spacing between lamellas was about 500 μm and the spacing between pillars was about 150 μm. The thickness of the lamella was slightly thicker than that of the pillars. In the high-resolution SEM images, wavy patterns were observed on the surface of the column, and some smooth and thin layers were observed in the lamellar structure.

2. CB Lt; RTI ID = 0.0 > HAp Support

Figure 1 (B) shows the microstructure of CB converted to HAp by hydrothermal treatment. The porous structure of CB-HAp was maintained during HAp conversion. Higher resolution images showed that the characteristic wavy pattern of the columnar surface of natural CB disappeared upon hydrothermal treatment and instead had rough surface characteristics. Fig. 1 (C) shows the results of XRD analysis for conversion of HAp. The XRD pickies of CB-HAp were almost identical to those of standard HAp. These results indicate that CB is completely converted to HAp by hydrothermal reaction.

The XRD pattern of the PCL coated CB-HAp support is shown in Figure 2 (A). After coating with PCL, typical PCL peaks in addition to the HAp peak on the pattern of the HAp-PCL complex were observed in the 20-25 ° region. No peak shifts appeared in the complex. These results indicate that the PCL polymer was successfully introduced into CB-HAp and no chemical reaction occurred. The increase of PCL concentration increased the peak intensity of PCL. However, there was no significant change in the peak of HAp. The PCL complex structure was further confirmed by SEM.

Figure 2 (B) shows the SEM shape of the porous CB-HAp after being coated with porous CB-HAp and PCL. When CB-HAp was coated with 1% PCL solution, PCL layer was hardly observed in the surface of the structure and in the sunken area between the column and parallel sheet structure. The roughness of the surface was not significantly different from that of the uncoated CB-HAp. In the support coated with 5% PCL solution, a thin layer covered the entire surface. As the concentration increased (10% PCL), the thick layer covered the surface with a large thickness, and many pores were clogged. The thicker the layer due to the PCL coating, the more the result of the polycrystalline structure of the surface was observed.

< Test Example 2> Compressive strength ( compressive strength ) _ And Porosity ( porosity ) Measure

A compressive strength test of a porous CB-HAp support (8 mm diameter x 5 mm height) was performed with an Instron test machine (Model: 4505, UK) at atmospheric conditions at a cross-head speed of 0.5 mm per minute. To measure the porosity, anhydrous ethanol was used as the liquid medium. Prior to the experiment, each sample, pycnometer, and liquid medium was preheated to 25 占 폚. Porosity was calculated using the following equation.

Porosity (%) = (Wp - Ws) / Wp - We X 100

In the above porous formulas, Ws indicates the weight of the support, and Wp indicates the weight of the ethanol-filled support in the pore. Four samples were tested.

As a result, the compressive strength of natural CB was 1.63 ± 0.13 MPa, and its strength decreased slightly to 1.11 ± 0.26 MPa during the conversion by hydrothermal reaction with HAp. 1% PCL coating showed little change in compressive strength. In contrast, the 5 and 10% PCL coatings increased significantly to compressive strengths of 2.32 ± 0.44 and 3.67 ± 0.46 MPa, respectively (see Table 1).

The total porosity of natural CB was 89.2 ± 2.3% and there was almost no change by hydrothermal reaction. When the CB-HAp support was coated with 1 and 5% PCL, there was no significant change in total porosity. In contrast, the total porosity of the CB-HAp support coated with 10% PCL was significantly reduced by about 20% (see Table 1).

Compressive strength and porosity of HAp deformed by hydrothermal action coated with natural raw squid bone (Raw CB) and PCL polymer Raw CB CB-HAp CB-HAp / 1% PCL CB-HAp / 5% PCL CB-HAp / 10% PCL Compressive strength (MPa) 1.63 + 0.13 1.11 ± 0.26 1.25 ± 0.56 2.32 + 0.44 ^a 3.67 ± 0.46 ^a Porosity (%) 89.2 ± 2.3 91.3 ± 1.9 89.6 ± 2.7 88.6 ± 1.3 76.8 ± 3.2 ^a

The data are for the mean ± standard deviation (SD) for four samples.

^a indicates an important difference from uncoated CB-HAp (p < 0.05).

< Test Example 3> PCL - Coated CB - HAp Biocompatibility of Biocompatibility )

1. Cell culture ( Cell culture )

MG-63 cells were used to assess the biocompatibility of PCL coated CB-HAp scaffolds. MG-63 cells were purchased from Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM Dulbecco's Modified Eagle Medium medium (Gibco-BRL, Gaithersbug, MD) containing 10% fetal bovine serum (Gibco-BRL) and 1% penicillin / streptomycin in a 5% Respectively. Osteoblast differentiation of MG-63 cells was induced by osteoblast stimulant (OS; 10 mM b-glycerophosphate, 50

mg / mL ascorbic acid, and 100 nM dexamethasone; Sigma-Aldrich). The badge and OS were changed every 2 days. For 3-dimensional culture on CB-HAp, 5 mm diameter and 2 mm high supports were used. Before cell inoculation, the support was sterilized with 70% alcohol for 30 minutes and washed 3 times (1 minute each) with 1X phosphate-buffered saline (PBS). Then, the cells were inoculated on a support at a density of 5 X 10 ⁴ for each support. After inoculation, the cells were incubated for 2 hours to allow for initial cell attachment and then rinsed with media. At various time intervals, the cell constructs were used for the analysis of cell proliferation, DNA content, viability / toxicity, cell adhesion, and osteoblast differentiation.

2. The degree of cell proliferation and DNA Content analysis

MTS and DNA content analyzes were performed at 1, 6, 12, and 18 days intervals to determine the extent of cell growth on uncoated CB-HAp and PCL-coated CB-HAp scaffolds.

To measure cell proliferation, CellTiter 96VR Aqueous One solution (Invitrogen, Carlsbad, Calif.) Was used. MG-63 cells were inoculated on CB-HAp and PCL-coated CB-HAP scaffolds and then cultured. 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS ) Reagents 100 ml and 500 ml of medium were mixed and added to each well. After 4 hours of incubation, the absorbance of the supernatant was measured at 490 nm using a microplate reader (SpectraMAX M3; Molecular Devices, Sunnyvale, Calif.).

Analysis of the DNA content using the PicoGreen dsDNA Quantitation kit (Molecular Probes, Eugene, OR) determined the number of cells attached and grown on the support. Cells were inoculated on CB-HAp and PCL-treated CB-HAp supports and then cultured. At standard working times (1, 6, 12, and 18 days), the support was washed with PBS and trimmed appropriately. To release the total DNA content, the pieces were allowed to soak for 30 minutes in 500 ml of lysis buffer (0.2% Triton X-100 and 5 mM MgCl2) and then centrifuged at 12,000 rpm at 4 ° C for 10 minutes . 10 ml of the supernatant was mixed with a DNA-binding fluorescent dye solution, and the fluorescence was measured with a plate reader (SpectraMAX M3) at an excitation wavelength of 480 nm and an emission wavelength of 528 nm.

The results of the MTS analysis are shown in Fig. 3 (A). Cells grew well during the entire incubation period and there was no significant difference in the degree of proliferation between uncoated CB-HAp and CB-HAp coated with 1% PCL. However, in contrast, 5% and 10% PCL were found to have higher visual density values than the other groups. When cells were cultured on CB-HAp coated with 10% PCL, cell growth rapidly increased until day 6, and the rate slowed down with increasing incubation period.

In addition, since the MTS assay is based on mitochondrial activity, we used DNA content analysis to more accurately measure cell proliferation. The results of the DNA content analysis are shown in FIG. 3 (B), which is consistent with the MTS analysis of FIG. 3 (A).

3. Survival / Toxicity Analysis

To determine cell viability and toxicity, Live / Dead Survival / toxicity kit (INvitrogen) was used as protocol. Cells were inoculated, incubated for 3 days, and phenol red and serum were removed by washing with OBS for 30 min. Live / Dead Survival / Toxic solution was added followed by CO ₂ After incubation for 30 min in an incubator, samples were observed using an inverted fluorescence microscope (DM IL LED Fluo; Leica Microsystems, Wetzlar, Germany).

To determine the toxicity of the PCL coating, a live / dead staining assay was performed. Although some dead cells (red) were observed on the PCL-coated CB-HAp, most of the MG-63 cells survived (green) (see Fig. 3 (C)). These results indicate that the coating of the CB-HAp support is not related to toxicity.

4. Observation of cell adhesion

SEM analysis was performed to observe the attachment of NG-63 cells to CB-HAp. Cells were seeded onto each support to observe the adhesion and growth of cells on the support, and cultured in growth medium for 6 days. The support was washed with PBS and fixed with 2.5% glutaraldehyde solution for 4 hours at 4 ° C. Samples were postfixed with 0.1% osmium tetroxide solution and dehydrated with increasingly high alcohol concentrations (25, 50, 75, 95, and 100). The dehydrated samples were sputter-coated with platinum and observed by SEM (JOM-6360).

4 is a SEM micrograph showing the attachment pattern of MG-63 cells. These cells were attached to the surface of the C-support and spread to the internal structure of CB-HAp and CB-HAp coated with 1 and 5% PCL. When cultured in CB-HAp coated with 10% PCL, almost all cells were grown only on the surface of the scaffold.

< Test Example 4> osteoblast osteoblast ) differentiation ) Measurement of degree

To determine whether MG-63 cells cultured on CB-HAp coated with uncoated CB-HAp and PCL can differentiate into osteoblasts, cells were treated with OS. Then, ALP activity was measured and qRT-PCR was performed to detect osteoblast marker expression.

Specifically, the degree of osteoblast differentiation was evaluated by alkaline phosphatase (ALP) activity assay using p-nitrophenylphosphate as a substrate. Activity was averaged by determining the amount of total protein with BCA protein assay kit (Pierce, Rockford, Ill.). The cells were inoculated and then cultured for 5 days in the medium containing the OS. The cultured support was washed with PBS and cut into small pieces. The attached cells were removed from the supporter and the pieces were sonicated in 1% Triton X-100 / PBS for 10 minutes in an icebox to obtain proteins. The samples were then centrifuged at 12,000 rpm to remove debris from the support, and the supernatant was used for ALP activity analysis.

In order to measure the degree of differentiation of osteoblasts in cells cultured on CB-HAp and PCL-coated CB-HAp supports, quantitative real-time (qRT) -PCR was performed to analyze the mRNA expression level of several osteoblast marker genes Respectively. After inoculation, the cells were cultured in the medium containing OS for 5 days, and the whole mRNA was extracted using RNA isolation kit (Ribospin, GeneAll, Seoul, Korea) and reverse transcribed with cDNA. PCR was performed using TaqMan Universal PCR Master Mix, TaqMan primer rtpting ALP (Hs01029144_m1), Runx2 (Hs00231692_m1), Col1aI (Hs00164004_m1), and 18S (Hs99999901_s1; Applied Biosystems, Carlsbad, CA) Respectively. All Taqman PCR assays were performed using the StepOne Plus Real-Time PCR System (Applied Biosystems). The 18S ribosomal RNA gene was amplified together, and the relative expression level was based on the CB-HAp scaffold.

As a result, when cultured on CB-HAp coated with 1% PCL, the pattern and level of activity were not significantly different from uncoated CB-HAp. In contrast, ALP activity was significantly increased in cells cultured on 5% and 10% PCL treated CB-HAp (see FIG. 5). The expression of marker genes of osteoblasts was consistent with the results of ALP activity analysis (see FIG. 6). On the other hand, when the cells were cultured on CB-HAp treated with 5% and 10% PCL, the expression of ALP, Runx2 and Collα increased significantly compared to the cells cultured on uncoated CB-HAp.

Claims

A bone graft material wherein a polycaprolactone solution is infiltrated into a calcium phosphate-based porous body converted into hydroxyapatite and then dried to form a polycaprolactone layer on the porous body.

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A method for producing a bone graft material, comprising the steps of: hydrothermally processing a bone of a cuttlefish, infiltrating the calcium phosphate-based porous body converted into hydroxyapatite by the cuttlefish bone, and drying the resultant, thereby forming a polycaprolactone layer on the porous body.

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[Claim 7] The method according to claim 6, wherein the hydrothermal treatment comprises treating the cut ground squid bone with a solution containing phosphate ions and reacting at 100 - 300 DEG C for 6-48 hours.