KR20160034557A

KR20160034557A - To induce bone regeneration using PLGA-Silk hybrid structure method of manufacturing

Info

Publication number: KR20160034557A
Application number: KR1020140125486A
Authority: KR
Inventors: 박찬흠; 이옥주; 주형우; 이정민; 김정호; 문보미; 박현정; 김동욱; 이민채; 김수현; 정주연
Original assignee: 한림대학교 산학협력단
Priority date: 2014-09-22
Filing date: 2014-09-22
Publication date: 2016-03-30

Abstract

The present invention relates to a bone inductive artificial bone support including a silk protein, and a method of manufacturing the same. In addition, the present invention relates to an artificial bone support physically combined with a PLGA support by mixing a silk protein (or silk fibroin) extracted from cocoon or fibroin with hydroxyapatite nanoparticles. It is tried to replace a damaged bone tissue with a normal bone tissue or to regenerate a damaged bone tissue, and interests in the development of a bone tissue for implantation is increased. A method of implanting a bone of another person or an animal, or a method of implanting a tissue of a patient has been widely used, but the immunological rejection occurs or a damaged area is large by implanting a tissue of others. Therefore, a case in which a material which can be used in a body of a patient is not sufficient, might occur. Accordingly, the development of a safe artificial bone which can be easily supplied and manufactured in various forms, is required. To solve the problem of the present invention, advantages of mass-productivity, biodegradability, a bone regeneration function, excellent biocompatibility, and having no biological hazards of silk fibroin, and advantages of safety, and mechanical strength biodegradability of PLGA (polylactic-glycolic acid) are mixed. Therefore, provided is the artificial bone having a structure of two materials, morphologically, a physically mixed hybrid support structure in which a PLGA support surrounds a silk fibroin support, and having advantages of the two materials.

Description

[0001] The present invention relates to a PLGA-silk hybrid structure for inducing bone regeneration,

The present invention relates to a method for producing bone-induced artificial bones containing silk fibroin (or silk protein).

Bone plays a key physiological role in regulating the calcium ion concentration in the body besides the mechanical function of supporting the human body and performing the action and producing the red blood cells and the white blood cells necessary for the human body in the bone marrow. The bone consists of a cortical bone with dense structure with high mechanical strength and a supporting bone with a structure such as an inner sponge. As the age increases, the bone density is lowered and the fracture easily occurs even in the case of small impact, and it is damaged by osteoporosis and arthritis. If such fracture occurs due to such physiological or physical damage, bone regeneration becomes difficult.

Treatment of bone regeneration includes natural treatment, treatment by drug or physical therapy, orthopedic surgery, etc. In cases where bone damage is so severe that recovery is difficult, normal tissue is protected, Remove. In recent years, attempts have been made to replace or repair damaged bone tissue with normal bone tissue, and thus interest in the development of bone tissue for implantation is increasing. In order to complement the injured bone tissue, studies are under way to develop a biotissue artificial bone that uses bone cells such as bone marrow derived stem cells to increase the bone formation effect. In addition, a method of transplanting bone of another person or animal, There is also a method of transplanting a tissue, but the transplantation of the other tissue may result in immunological rejection or the site of injury may not be sufficient for the patient's body. Therefore, it is necessary to develop safe artificial bones that can be easily manufactured and manufactured in various forms.

In the late 18th century, the development of artificial bones began with iron wires and pins made of precious metal materials. Since then, the development and design of alloys and the development of surgical techniques have led to the development of various internal fixtures such as metal pins, . These biomaterials include stainless steel, cobalt-chromium (Co-Cr) alloy, titanium (Ti) metal, etc. Artificial bones made of these materials are subject to corrosion, abrasion, loosening or re- There is a disadvantage. Ceramic materials are excellent in biocompatibility, have no corrosion and have good compressive strength, but have a disadvantage of low cutting strength, difficulty in forming various types of fixing devices, and inability to be flexible. However, the use of metal and ceramics has been widely used in biomedical engineering such as orthopedics, plastic surgery, cardiovascular surgery, skin urology, ophthalmology, neurosurgery, dental materials, cell culture, (Plate, screw, washer, pin, etc.) for bone fixation, bone repair material, filler, substitute.

The purpose of regenerating bone tissue is to create tissue similar to the original bone tissue to replace or restore lost bone tissue. In other words, a support, which is a porous template capable of proliferating and dividing bone cells, can be produced, and osteogenic factors and osteocytes are put into a support and cultured to produce bone tissue. Currently, ceramics most commonly used for artificial bones are HAp (hydroxyapatite) and tricalcium phosphates (TCPs), which have good bone conduction and bone adhesion and are useful for bone marrow biopsy. However, when it is made into a porous body, the mechanical properties are remarkably reduced and there is a limitation in processing, so it is considered that the disadvantage can be overcome by mixing with other agent.

Silk is a fibrous protein material usually obtained from a silkworm. It has been widely used as a material for clothing. Recently, researches are being conducted to use it as a new natural polymer resource. The composition of the silk protein is very similar to the amino acid composition of the collagen protein that constitutes the skin of the human body. Therefore, it is used in foods and cosmetics based on the skin affinity. The material for biotechnology such as enzyme immobilization carrier, cell culture support, wound covering material, artificial blood vessel It is used as medical material.

Silk proteins have cholesterol and hypoglycemia, promote alcohol metabolism, prevent and treat dementia, and improve memory. These functions are known to be closely related to the pharmacological effects of amino acids such as glycine, alanine, serine and tyrosine. In addition, collagen and fibronectin are typical natural polymer substances used as cell culture supporters, and they have an Arg-Gly-Asp (RGD) tripeptide commonly known to have cell recognition function. It promotes cell adhesion and proliferation, and is also present in the silk fibroin structure.

The characteristics of these silk proteins show the possibility of application of bio-bones and artificial skin, and silk fibroin has proven positive effects on cell adhesion and differentiation. However, since silk protein does not have mechanical strength such as bone, it has a limitation in application as an artificial bone and has a limitation that it is difficult to process in a certain form.

In order to solve the above problems, the present invention has been made to solve the above problems by mixing the advantages of silk fibroin with mass productivity, biodegradability, bone regeneration, excellent biocompatibility and biological hazards, safety of polylactic-glycolic acid (PLGA) It is intended to provide an artificial bone having the advantages of the above two materials by fabricating a physically mixed hybrid support structure in which a PLGA support surrounds a silk fibroin support in terms of structure and morphology of the two materials. The artificial bone is fused with the surrounding bone tissue to induce the regeneration of the new bone tissue, and finally the artificial bone is biodegraded.

The present invention provides a silk fibroin artificial bone for bone regeneration wherein 4 wt% of silk fibroin and 2 wt% of hydroxyapatite solution are physically bonded to a PLGA support.

The present invention also provides a method for producing hydroxyapatite, comprising: a first step of forming a mixture with hydroxyapatite and a silk fibroin solution; A second step of forming a PLGA support comprising voids; A third step of impregnating the PLGA support with the mixture solution of the first step to increase hydrophilicity and cell affinity while binding with the hydrophobic PLGA support; And a fourth step of lyophilizing the supporter. The present invention also provides a method for manufacturing a silk fibroin artificial bone for bone regeneration.

The method of forming voids of the PLGA support containing the voids is preferably selected from the group consisting of a salt extraction method, a freeze drying method, a weaving method, a method using a foaming agent, and an electrospinning method.

Preferably, the present invention may further include at least one selected from the group consisting of BMP-2, BMP-12, and calcium phosphate involved in the bone induction process.

In addition, the present invention may further include a fifth step of insolubilizing the artificial bone of the silk fibroin for bone regeneration after the fourth step. The above-mentioned insolubilization treatment may be performed by selecting one or more of methanol, ethanol, and propanol, or by hydration or high-temperature treatment.

According to the present invention, the two materials can replace the physical function as an artificial bone by maintaining proper pores and mechanical strength, which are structural and morphological characteristics, and have a bone healing effect by inducing regeneration of surrounding bone tissue. Over time, artificial bones can be regenerated into normal bone tissue as they are biodegraded and eliminated in the body.

1 is a process for producing silk fibroin solution.
Fig. 2 is a method for producing artificial bones for bone regeneration in PLGA, PLGA-Silk, and PLGA-Silk-HAp 3 groups.
FIG. 3 is a cross-sectional view of the silk fibroin and PLGA scaffold prepared by freeze-drying and salt extraction (A), the cross-section of a silk support prepared by freeze-drying, (B) Section of a PLGA-Silk support prepared by salt extraction method, and (D) a section of PLGA-Silk-HAp support prepared by salt extraction method.
Figure 4 shows the EDS results of the mixed support measured by a transformed field emission scanning electron microscope (VP-FE-SEM). (A) Silk support, (B) PLGA support, (C) PLGA-Silk support, (D) PLGA-Silk-HAp support (notation S is silk part, notation P is PLGA part, notation HAp is hydroxyapatite nano (Scale bar 100 mm.)
5 shows the result of measurement of the swelling force of the support (A), the result of measurement of the water absorption rate of the support (B), the result of measurement of the porosity of the support (C), and the result of measurement of the water contact angle of the support (D).
6 is a Fourier transform infrared spectroscopy (FT-IR) analysis result of HAp NPs, Silk, PLGA, PLGA-Silk, PLGA-Silk-HAp and supports.
FIG. 7 shows the result of thermogravimetric analysis (TGA) of HAp NPs, Silk, PLGA, PLGA-Silk, PLGA-Silk-HAp and supporter.
Fig. 8 shows the results of compressive strength measurement of Silk, PLGA, PLGA-Silk and PLGA-Silk-HAp supports.
FIG. 9 shows the result of cell fitness observations by MTT analysis after osteoblast culturing of Silk, PLGA, PLGA-Silk and PLGA-Silk-HAp scaffolds. (1,7,14 days)
FIG. 10 shows fluorescence microscopic observation after 4 weeks of osteoblast culturing by DAPI staining. (A) Silk support, (B) PLGA support, (C) PLGA-Silk support, and (D) PLGA-Silk-HAp support.
11 shows the experimental method of the skull defect Rat model. (A) bone defect site formation with a diameter of 3 mm, (B) scaffold implantation at a bone defect site, (C) skin incision site sealant, (D) scapular incision site skin incision, and (E) scaffold implant site skull extraction.
(B) Silk scaffolds, (C) PLGA scaffolds, (D) PLGA-Silk scaffolds, (E) scaffolds, and PLGA-Silk-HAp support.
(B) Silk scaffolds, (C) PLGA scaffolds, (D) PLGA-Silk scaffolds, (E) scaffolds, and ) PLGA-Silk-HAp support.

The present invention provides a silk fibroin artificial bone composition for bone regeneration wherein 4 wt% of silk fibroin and 2 wt% of hydroxyapatite solution are physically bonded to a PLGA support.

In order to increase the mechanical strength of silk fibroin, the present invention physically mixes PLGA so as to have strength similar to that of bone, and silk fibroin is mixed with hydroxyapatite (Hap), which is a major component of bone mineral, A silk fibroin artificial bone composition for bone regeneration in which PLGA and hydroxyapatite are physically bonded to a silk fibroin capable of being used for bone regeneration, and a method for producing the same.

The artificial bone of the present invention can acquire the necessary physical strength as a support by mixing silk fibroin with PLGA having high mechanical strength. In addition, since hydroxyapatite, which is a component of bone, is contained, it is easy to differentiate and proliferate, induce and adhere bone cells. In addition, the present invention provides a method for producing a silk artificial bone for bone regeneration, wherein the artificial bone according to the present invention is biodegraded by itself in the body as the bone regeneration progresses.

Preferably, the hydroxyapatite, BMP-2, BMP-2, and BMP-3 involved in the bone induction process are provided between the first step of forming the mixture with the silk fibroin solution and the fourth step of lyophilizing the support, 12, and calcium phosphate. The bone regeneration factor may be contained to promote bone regeneration.

According to the present invention, it is possible to replace the physical function as an artificial bone by maintaining adequate pores and mechanical strength, and to induce regeneration of surrounding bone tissue, thereby achieving bone healing. Over time, artificial bones can be regenerated into normal bone tissue as they are biodegraded and eliminated in the body.

The artificial bone for bone regeneration according to the present invention provides sufficient mechanical strength and bone regeneration ability as a bone substitute, and thus can be effectively used as a bone regeneration artificial bone in a patient with fracture and bone defect.

[0035] To describe again, the bone repairing silk fibroin artificial bone composition according to the present invention comprises a step of preparing a stock solution of silk fibroin, a step of mixing the hydroxyapatite and the silk fibroin solution, a step of forming the void of the PLGA support, A process of further forming mixed and internal voids, and a crystallization process.

The present invention can produce a silk fibroin artificial bone having a bone regeneration inducing effect and an excellent mechanical strength by a process of filling the voids of the mixed and PLGA scaffold while forming internal voids and being biodegraded more easily in the human body .

In addition, in the method for producing a silk fibroin artificial bone composition for bone regeneration according to the present invention, a fifth step of insolubilizing the artificial bone for bone regeneration silk fibroin after the fourth step may be further included. The above-mentioned insolubilization treatment may be performed by selecting one or more of methanol, ethanol, and propanol, or by hydration or high-temperature treatment. By the above-mentioned insolubilization treatment, the support of the silk fibroin artificial bone is more advantageous to maintain the original structure.

The following examples illustrate the invention. However, this is for facilitating the understanding of the invention, and the invention should not be construed as being limited thereto.

Each step will be described in detail.

1. Materials and Methods

1.1 Reagents and Materials

Silk and silage were supplied at Uljin Plant, and Hap (Hydroxyapatite) was supplied by Bio Alpha. PLGA (poly (lactic-co-glycolic acid) molar ratio 75:25, Resomer RG 756, Boehringer Ingelheim Co., Germany) was used with an average molecular weight of 90-126 kD. NaCl (sodium chloride, Orient Chem. Co., Ltd.) was used as a porous material and crystals of 180-259 mm were used. (MT) (3- (4,5-dimethylthiazol-2-yl) -) -methanol was used as a reagent for the cell culture, and MEM (Welgene, Gibco) 2,5-diphenyltetrazolium bromide, Duchefabiochemie, Haarlem, The Netherlands). Tetamine / Zolazepam HCl (Zoletil, Virbac, France) and Xylazine HCl (Rompun, Bayer, Korea) (1: 2) were used for rat anesthesia.

1.2 Silk Fibroin Extract

Sericin protein and impurities were removed from Bombyx mori. For this purpose, it was finely cut and then put in 0.02 M Na2CO3 or basic aqueous solution, heated to 100 for 1 hour, and washed 2-3 times with distilled water. The dried silk fibroin was dissolved in CaCl2 / Ethanol / H2O (1: 2: 8, moleratio) solution at a concentration of 15wt% at 95 for 40 minutes. Next, it was filtered once more with miracloth (Calbiochem, San Diego, CA, USA) to remove impurities. The purified silk fibroin solution (SF) was prepared by dialyzing the filtered silk fibroin solution in distilled water for 3 days in a 12,000-14,000 Da (Spectra / Por, Rancho Dominguez, CA, USA) dialysis membrane. At this time, the final concentration of the produced silk fibroin was adjusted to 7-9 wt%.

1.3 Fabrication of PLGA-Silk Fibroin-Hydroxyapatite (PLGA-Silk-Hap) Support

A silk fibroin mixed support composed of PLGA, silk fibroin, and hydroxyapatite was prepared for the purpose of supplementing mechanical strength of the support and improving bone regeneration ability. 8 wt% silk fibroin solution and 4 wt% hydroxyapatite were prepared. Because hydroxyapatite is a nanoparticle, it was mixed with 4 wt% of distilled water to disperse it uniformly and Sonication (operating 20 kHz, amplitude 20%, 15 min) was performed. 8 wt% silk fibroin solution and 4 wt% hydroxyapatite were mixed at a ratio of 1: 1. Finally, a mixed solution of 4 wt% silk fibroin and 2 wt% hydroxyapatite was prepared. Since PLGA is hydrophobic and has excellent strength, it is prepared as a skeletal structure of a support. First, PLGA was dissolved in methyl chloride (MC) at 20wt% overnight at room temperature. The PLGA solution was mixed with NaCl particles (180-250 mm) at a ratio of 1: 9 and filled in silicon molds (diameter 7 mm, height 3 mm) prepared in advance. The support filled with the mold was pressurized with a force of 60 kgf / cm 2 at room temperature for 24 hours using Carver Press (MH-50Y, CAP 50 tons, Japan). It was kept at 25 in a vacuum oven for 1 week to remove the solvent remaining on the PLGA support. The completed PLGA support was immersed in a silk fibroin-hydroxyapatite (Silk-Hap) mixed solution. Since the PLGA support is hydrophobic, penetration of the hydrophilic silk fibroin-hydroxyapat (Silk-Hap) solution is difficult. Therefore, the pores in the PLGA support were filled with silk fibroin-hydroxyapatite (Silk-Hap) solution using a vacuum pump. The supporter thus prepared was frozen at -80 ° C and then freeze-dried to remove all water. During freeze-drying, silk fibroin has a random small pore structure.

The process of manufacturing this is as follows.

1.3.1 Manufacture of Silk Fibroin Support

The silk fibroin support was prepared by diluting a silk fibroin solution made with 8 wt% with distilled water to make a 4 wt% silk fibroin solution. A 4 wt% silk fibroin solution was placed in square dishes and frozen at -80 for 12 hours. The completely frozen sample was lyophilized for 24 hours to remove moisture in the support. After immersing in 100% Et-OH for 15 minutes to induce insolubilization of the water-removed silk fibroin support, a silk fibroin support with no Et-OH was prepared by substituting Et-OH for each concentration.

1.3.2 Manufacture of PLGA support

The PLGA support was prepared by NaCl leaching method. First, PLGA was dissolved in methyl chloride (MC) at 20wt% overnight at room temperature. The PLGA solution was mixed with NaCl particles (180-250 mm) at a ratio of 1: 9 and filled in silicon molds (diameter 7 mm, height 3 mm) prepared in advance. The support filled with the mold was pressurized with a force of 60 kgf / cm 2 at room temperature for 24 hours using Carver Press (MH-50Y, CAP 50 tons, Japan). The sample was kept at 25 in a vacuum oven for 1 week to remove the remaining solvent.

1.3.3 Fabrication of PLGA-silk fibroin support

To prepare a PLGA-silk fibroin support, a 4 wt% silk fibroin solution was prepared as above. The PLGA support was prepared in the same manner as above. The vacuum switch was turned on and off 4-6 times to add a hydrophilic 4 wt% silk fibroin solution to the hydrophobic PLGA support. After freezing at -80, it was lyophilized for 24 hours and crystallized by immersion in 100% Et-OH.

1.4 Insolubilization treatment of silk fibroin-containing bone support

In the prepared PLGA-Silk-HAp support, silk fibroin was hydrophilic and easily dissolved in water, and thus it was difficult to maintain the structure. Therefore, the silk fibroin was subjected to insolubilization in order to control the degradation degree and maintain the physical properties in vivo. The structure of the PLGA-silk fibroin-hydroxyapatite support was maintained by treating with methanol, ethanol, propane, hydration, or high-temperature treatment as the insolubilizing step. In order to analyze the bone regeneration effect, structural characteristics and physical properties of the PLGA-silk fibroin-hydroxyapatite scaffold, a comparison was made using a single silk fibroin scaffold, a PLGA scaffold and a PLGA-silk fibroin scaffold as control groups. .

2. Analysis of the support prepared by the above method

2.1 Analysis of Structure of Support by SEM, FT-IR and TGA

(VP-FE-SEM, S-3500N, Hitachi, Tokyo, Japan) was used. The sample was cut vertically and placed on a carbon tape. The surface of the sample was observed after coating gold / palladium with a thickness of 10 nm for 120 seconds. Also, the EDS was measured through the EVO LS10 mounted on the VP-FE-SEM. Spectral range 2250-500 cm-1 was measured using FT-IR (BIO-RAD Excalibur Series, FT-IR Spectrometer, Cambridge, USA) The measurement was 30 to 700, the heating rate was 10 C / min, nitrogen was 100 mL / min, and the thermal decomposition of the support was measured using a thermal analysis system (TA Instruments, New Castle, DE, USA) Respectively.

2.2 Structural analysis results of PLGA-silk fibroin-hydroxyapatite scaffold

In order to confirm the structure of the PLGA-silk fibroin-hydroxyapatite support produced by the above method, it was observed using a scanning electron microscope. Both have a pore structure suitable for use as a bone support and this pore structure is large enough to allow penetration of bone differentiation cells. The small pores made of silk fibroin and HAp mixed solution are distributed in the relatively large pores made of PLGA among the pores. The large pores of PLGA increase the mechanical strength and the small pores are connected to each other, And gas exchange. At the same time, small-sized pores are expected to facilitate the penetration of bone cells and the density of osteocytes.

As a result of SEM-EDS analysis, silk fibroin, PLGA and hydroxyapatite were separated and mixed and arranged in a constant structure. Each peak (PLGA has no N, silk fibroin has N, which is a representative characteristic of protein, Apatite was found to be Ca and P peaks). Thus, physical mixing of hydrophobic PLGA and hydrophilic silk fibroin-hydroxyapatite was found to be advantageous for adhesion, penetration and proliferation of hydrophilic cells.

As a result of FT-IR measurement, it was confirmed that each inherent peak of PLGA-silk fibroin-hydroxyapatite was mixed. The peaks of Amide (1624), Amide (1511), Amide (1228) and the peaks of 1022, 601 and 559 of hydroxyapatite and 1748 and 1179 of PLGA which are unique peaks of silk protein in PLGA silk fibroin- And it is confirmed that they are mixed.

As a result of the TGA analysis, the starting temperatures of the modifying of the support by heat were measured to be 274 for silk fibroin, 301 for PLGA, 298 for PLGA-silk fibroin and 317 for PLGA-silk fibroin-hydroxyapatite, And that the stability of the composition was increased. The change of the denaturation temperature showed that each formulation was physically mixed without chemical structural change.

2.3 Evaluation of Physical Properties of PLGA-Silk Fibroin-Hydroxyapatite Support

2.3.1 Swelling power, water absorption measurement

For the swelling power and water absorption test of the silk fibroin oozing retarder prepared in the above example, each support was prepared by punching with a diameter of 6 mm and a thickness of 2 mm and immersing in distilled water for 24 hours. At this time, the weight of the support sufficiently wetted with water was written in Ws, and the weight of the support after vacuum drying in all 60 ovens was written as Wd. Finally, the swelling power and the water absorption were calculated by substituting each of the numerical values into the following equation.

2.3.2 Measurement of porosity

Each support was punched in the same manner as above and the porosity was measured using Et-OH. Et-OH was used because silk fibroin is hydrophilic but PLGA is hydrophobic, so Et-OH was used to reduce the error. Prior to loading the support, the volume of the cylinder containing Et-OH was measured and recorded as V1. After the substrate was immersed for 5 minutes, the total volume of Et-OH was measured and recorded with V2. The support immersed in Et-OH was removed and the volume of the cylinder was measured and recorded as V3. Finally, the porosity of the support was calculated as follows.

2.3.3 Measurement of water contact angle

Contact angle microscope (LEICA, E24D, software LAS EZ) was used to measure the water contact angle on the support. Each support was punched in the same manner as above, then placed on a slide glass, and 15 ml of cell culture media was dropped on the support. The water contact angle was measured after 30 seconds.

2.3.4. Measurement results of support properties

The results of the swelling test showed that the silk fibroin supporter showed the highest value and PLGA showed the lowest value. As expected, PLGA-silk fibroin showed intermediate levels between silk fibroin and PLGA and increased with the inclusion of hydroxyapatite, but not significantly. The water uptake was also determined to be the same as the swelling power.

The numerical value of the increased swelling power and water uptake means that various growth factors expressing bone cells can be stored for a long time when inserted into the body, and since metabolism and gas exchange of cells and cells are easy, It can help you more.

Porosity measurements showed that the porosity of the silk fibroin scaffold was the highest and the porosity of PLGA silk fibroin-hydroxyapatite was the lowest. These results show that the porosity of the support made of the mixed material decreases rather than the support made of the single material. However, the above results were not significant because the porosity difference was about 5%.

The contact angle measurement results of water were also found to increase with addition of silk fibroin and further increase with addition of hydroxyapatite. Therefore, it was confirmed that the hydrophilic property was increased when the silk fibroin was added to the single PLGA support, and the hydrophilic property was further increased when the hydroxyapatite was added. As a result, the hydrophobic PLGA scaffold is mixed with silk fibroin and hydroxyapatite, so that the polarity of the entire scaffold becomes hydrophilic. This result is closely related to cell affinity and cell adhesion.

2.3.5 Mechanical compression strength measurement

Each sample was punched using a 6 mm Biopsy punch in a round disk configuration with a diameter of 6 mm and a thickness of 2 mm. The prepared sample was measured with a Universal Testing Machine (QM 100), and the jig head speed was set to 5 mm / min. As a result of compressive strength measurement, it was confirmed that the compressive strength of the PLGA-silk fibroin scaffold was increased as compared with that of the single silk fibroin and PLGA scaffold, and further increased with the addition of hydroxyapatite. Because the compressive strength is very important due to the function of the bone substitute, PLGA-silk fibroin-hydroxyapatite is considered to be the most desirable form to replace the physical function as a bone substitute.

2.3.6 Cell Survival and Toxicity Test

MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium Bromide) assay was performed for cell viability and cytotoxicity. Each support was cut to a diameter of 6 mm and a thickness of 2 mm, and then sterilized and placed in a 96-well plate. Cells were osteoblast extracted from the iliac bone of the rats and 2 x 105 cells / ml of cells were dispensed in 100 ml of each support (2 X 104 cells / 100 ml). The cells were incubated in a 37, 5% CO2 incubator for 30 minutes to facilitate cell attachment, and then 100 ml of fresh media was added. The cell culture medium was DMEM (10% FBS, 1% A / A), changed into fresh medium twice a week, and incubated at 37 ° C in a 5% CO2 incubator for 1, 7 and 14 days. At each observation time, cells were mixed with MTT and medium mixed at 1: 9, and incubated for 2 hours to induce cell responses. After completion of the reaction, the supernatant was added to DMSO to induce color development. 100 ml of each color-developed DMSO was added to a new 96-well plate and the plate was measured at 540 nm using ELISA.

The proliferation rate of the primary cells was similar without any significant difference. However, as the cell proliferation rate of PLGA-silk fibroin-hydroxyapatite supplemented with hydroxyapatite was confirmed, the cytotoxicity was not observed but the proliferation rate .

2.3.7 Penetration of support cells

Osteoblasts were cultured for 4 weeks in the same manner as described above, and then fixed in 4% paraformaldehyde (PFA) for 4 hours. The cross-sectional area of the supernatant was observed by performing a general paraffin section procedure and the nuclei of the cells were stained with 4, 6-diamidino-2-phenylindole (DAPI). Observations were made with DAPI filter and FITC filter. The nuclei of the cells were observed in blue, and silk fibroin was observed in green because it had autofluorescent. As a result, it was observed that PLGA-silk fibroin-hydroxyapatite deeply digested and proliferated in the number of cells and the number of cells, and therefore, artificial bones according to the present invention are considered to be excellent in bone regeneration ability in living beings.

3. Measurement of bone regeneration effect

In order to confirm the bone regeneration effect of the PLGA-silk fibroin-hydroxyapatite scaffold prepared in this study, the bone regeneration effect in an animal model was confirmed. As an experimental animal, rats were used and the skull with a diameter of 3 mm was removed after anesthesia. A sterilized support of 3 mm in diameter and 0.5 mm in height was inserted into the removed area and completely sealed. After incubation for 4 weeks, the cells were plated on micro-CT and stained with H & E stain.

In the H & E results, in the control group (Blank), the regeneration of the bone tissue was delayed by the proliferation of the fibroblasts that had been regenerated before the bone tissue grew. In the case of the silk fibroin, PLGA and PLGA silk fibroin scaffolds, And the new bone tissue was not completely regenerated. However, in the PLGA-silk fibroin-hydroxyapatite scaffold, a new bone tissue was regenerated and the scaffold was completely degraded.

In the micro-CT results, H & E staining was also observed, and PLGA-silk fibroin-hydroxyapatite was found to be superior in bone regeneration ability.

Therefore, the silk fibroin, the PLGA, and the hydroxyapatite support according to the present invention can be utilized not only as a support for tissue regeneration but also as an orthopedic artificial bone, a periodontal regeneration inducing material, and a bone graft material.

Claims

A silk fibroin artificial bone composition for bone regeneration characterized in that 4 wt% of silk fibroin and 2 wt% of hydroxyapatite solution are physically bound to a PLGA support

The artificial bone composition for bone regeneration according to claim 1, wherein the artificial bone composition further comprises at least one selected from the group consisting of BMP-2, BMP-12 and calcium phosphate.

A first step of forming a mixture with hydroxyapatite and a silk fibroin solution; A second step of forming a PLGA support comprising voids; A third step of impregnating the PLGA support with the mixture solution of the first step to increase hydrophilicity and cell affinity while binding with the hydrophobic PLGA support; And a fourth step of lyophilizing the supporter. The method for manufacturing artificial bone of silk fibroin for bone regeneration

The method of claim 3, wherein the inner cavity forming method is selected from the group consisting of a salt extraction method, a freeze drying method, a weaving method, a method using a foaming agent, and an electrospinning method. Way

4. The method of claim 3, wherein the step of forming the mixture of the hydroxyapatite with the silk fibroin solution and the fourth step of lyophilizing the support comprise BMP-2, BMP-12, calcium phosphate Wherein the method further comprises the step of selecting at least one member selected from the group consisting of

4. The method according to claim 3, further comprising a fifth step of insolubilizing the artificial bone for bone regeneration silk fibroin after the fourth step

[Claim 7] The method according to claim 6, wherein the insolubilization treatment is performed by selecting one or more of methanol, ethanol, and propanol, and treating the selected one or more silk fibroin

7. The method of manufacturing a silk fibroin artificial bone composition for bone regeneration according to claim 6, wherein the insolubilization treatment is performed by hydration or high-temperature treatment