KR20220056140A - Composition for bone tissue regeneration comprising recombinant silk protein and porous scaffold comprising the same - Google Patents

Abstract

The present invention relates to a composition for bone tissue regeneration comprising a recombinant silk protein and a porous scaffold comprising the same. The composition for bone tissue regeneration comprising a recombinant silk protein according to the present invention uses a biocompatible polymer to ensure suitable mechanical intensity and elasticity, and has excellent cell adhesion cell sticking (adhesion) ability, wound healing effect, and differentiation ability into bone tissues or bones.

Description

Composition for bone tissue regeneration comprising recombinant silk protein and porous scaffold comprising the same

The present invention relates to a composition for bone tissue regeneration comprising a recombinant silk protein and a biocompatible polymer, and a porous scaffold comprising the same.

Repetitive protein polymers composed of the repetition of various natural or non-natural amino acid sequences are attracting attention as important substances. Unlike polymers made by chemical synthesis, protein polymers are made based on genetic information (genes), so the properties of various polymers such as length and three-dimensional properties can be precisely controlled. In addition, the protein polymer can have desired mechanical, chemical, and biological properties along with biocompatibility and biodegradation, and thus can be usefully used in biomaterials engineering, tissue engineering, and the like.

Repeat sequences of representative repeat proteins having the properties of protein polymers include elastin (GVGVP, VPGG, APGVGV), silk fibroin (GAGAGS), byssus (GPGGG), flagelliform silk (GPGGx) ), dragline silk (GPGQQ, GPGGY, GGYGPGS), collagen (GAPGAPGSQGAPGLQ, GAPGTPGPQGLPGSP), keratin (AKLKLAEAKLELA), sericin (SSTGSSSNTDSNSNSVGSSTSGGSS), and artificial repetitive proteinYSSTSSGGSS ), etc. Among them, the dragline spider web, which is used as a spider's lifeline and used to make a radial spider web, has high strength and elasticity. Spider silk is five times stronger per unit mass than steel and three times harder than high-quality man-made Kepler fibers.

On the other hand, the scaffold (Scaffold) refers to a physical support and an adhesive substrate made to enable in vitro culture and in vivo transplantation of tissue cells. These scaffolds are used for cell transplantation for human tissue regeneration, and also include mass culture and Its importance in propagation is very high.

In the present invention, it was attempted to develop a scaffold with excellent biocompatibility by preparing a cryogel using spider silk protein.

Korean Patent Registration No. 10-1380786

The present inventors have completed the present invention by confirming that a composition for bone tissue regeneration comprising a recombinant silk protein and a biocompatible polymer has excellent cell adhesion (adhesion) ability, wound healing efficacy, and differentiation ability into bone tissue or bone. .

Accordingly, it is an object of the present invention to provide a composition for bone tissue regeneration comprising a recombinant silk protein and a biocompatible polymer, and a porous scaffold comprising the same.

The present invention provides a composition for bone tissue regeneration comprising a recombinant silk protein and a biocompatible polymer.

The present invention also provides a porous cryogel scaffold comprising the composition for bone tissue regeneration.

The present invention comprises the steps of mixing a recombinant silk protein and a biocompatible polymer; freezing the mixture; and freeze-drying the frozen mixture to form pores; It provides a method of manufacturing a porous scaffold for tissue regeneration, comprising a.

In addition, there is provided a porous scaffold for bone or wound regeneration prepared by the above manufacturing method.

The composition for bone tissue regeneration comprising the recombinant silk protein and biocompatible polymer of the present invention may have excellent cell adhesion and proliferation ability including the recombinant silk protein. In addition, it is suitable for the production of a porous scaffold with appropriate mechanical strength and elasticity, including recombinant silk protein and biocompatible polymer, and is effective for differentiation and regeneration into bone tissue or bone, and wound regeneration.

1 is a conceptual diagram schematically illustrating a method for manufacturing a porous scaffold according to an embodiment of the present invention.
2 is an SEM image (X120) of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5.
3 is a graph showing the expansion rate of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5.
4 shows the compressive strength of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5.
5 shows the Young's modulus of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5;
6 is a cytotoxicity test result for the cryogel scaffolds prepared in Comparative Example 1 and Examples 2 to 5.
7 is a graph showing the cytotoxicity test results for the cryogel scaffolds prepared in Comparative Example 1 and Examples 2 to 5.
8 is a cell adhesion experiment result of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5.
9 is a result of wound healing assay and confirmed using confocal microscopy in order to confirm the interaction between recombinant silk protein and growth factor and the effect on cell growth.
10 is a result of RT-PCR to confirm the interaction between recombinant silk protein and growth factor and the effect on cell growth.
11 is an Alizarin red S staining experiment result for the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5.

Hereinafter, embodiments and embodiments of the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein.

Throughout this specification, when a part 'includes' a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated. As used throughout this specification, the terms 'about', 'substantially', etc. are used in or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used in the understanding of the present invention. It is used to prevent unfair use by unconscionable infringers of the disclosure in which exact or absolute figures are mentioned to help As used throughout this specification, the term 'step for (to)' or 'step for' does not mean 'step for'.

As one aspect for achieving the above object, the present invention provides a composition for bone tissue regeneration comprising a recombinant silk protein and a biocompatible polymer.

The composition for bone tissue regeneration of the present invention has excellent adhesion to the matrix of cells and cell proliferation, including recombinant silk protein. It has excellent cell adhesion and proliferation ability, making it suitable for bone differentiation, bone regeneration and wound regeneration.

The composition for bone tissue regeneration of the present invention may include a recombinant silk protein. In addition, it may include a biocompatible polymer and a bioactive material.

As used herein, the term “recombinant silk protein” refers to a synthetic silk protein having a molecular and structural profile that is close to that of a natural silk protein and biosynthesized by a recombinant protein production method. Since a spider silk fiber having properties similar to that of a spider silk fiber can be produced using the “recombinant silk protein”, the recombinant silk protein is also called a recombinant spider silk protein.

In the present invention, the recombinant silk protein may have a molecular weight of 5 to 300 kDa, specifically, may have a molecular weight of 100 to 200 kDa. In addition, the recombinant silk protein may include a peptide having the amino acid of SEQ ID NO: 1 in units of 1 to 160 repeats, and may have a structure in which the peptide having the sequence of SEQ ID NO: 1 is repeated 8 to 128 times.

SEQ ID NO: 1: NH ₂ -SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT-COOH

In the present invention, the recombinant silk protein was prepared according to the method described in Korean Patent No. 10-1317420.

In the present invention, the recombinant silk protein may be included in the composition for bone regeneration at a concentration of 1 μg/mL to 1000 μg/mL. More specifically, it may be included at a concentration of 100 μg/mL to 1000 μg/mL.

The biocompatible polymer is polyethylene oxide (PEO), polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), collagen, fibronectin, keratin, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, poly It may be selected from the group consisting of lactic acid, polyglycolic acid, dextran, and polylactic acid-polyglycolic acid (PLA-PGA), specifically polyethylene glycol diacrylate (PEGDA).

The biocompatible polymer may be included in the composition for bone regeneration at a concentration of 5% (w/v) to 20% (w/v).

In addition, the composition for bone regeneration of the present invention may further include a bioactive material.

The bioactive substance is not limited thereto, but includes, for example, plasma or serum components, hydroxyapatite, laminin, drugs, therapeutic agents, anesthetics, cell growth factors, antibodies, antibody-like molecules, nucleic acids, and polysaccharides. , one or more of these may be mixed and used.

More specifically, the bioactive substance may be a growth factor, plasma or serum component, and the growth factor is one or more species from the group consisting of TGF-beta, SCF, FGF-alpha, FGF-beta, IGF, VEGF, and KGF. You can choose.

In the composition of the present invention, the bioactive material may include 0.1 to 200 ppm, specifically 50 to 150 ppm.

In addition, the composition for bone regeneration has an additional wound healing or regeneration effect.

In one embodiment of the present invention, through a wound healing assay, it was confirmed that the spider silk protein had a wound regeneration or healing effect like the growth factor (FIG. 9).

In another aspect, the present invention provides a porous cryogel scaffold comprising a composition for bone tissue regeneration.

In the present invention, the description of "composition for bone tissue regeneration", "recombinant silk protein", "biocompatible polymer", and "bioactive material" is the same as described above.

In the present invention, the term “scaffold” refers to a physical support and an adhesive substrate made to enable ex vivo culture and implantation of tissue cells, and the scaffold is used for cell transplantation for human tissue regeneration, and also It is used for mass culture and propagation.

Most biologically active cells have a basic step that must be passed to survive when they come into contact with a substance inside or outside the body, and the first step is cell adhesion. In particular, when looking at the survival stage of fibroblasts and tissue cells, the cells preferentially adhere to the substrate, and after adhesion, the metabolism of organelles in the cytoplasm becomes active, and in order to facilitate proliferation and supply of nutrients, they move to a new site. will move

Therefore, the surface that activates the deposition of cells is the most basic means for doubling the proliferation of cells. The ability of these cells to adhere to the matrix can be artificially controlled by the components of the matrix. Scaffolds are carriers that support the regeneration and growth of cells, that is, materials that are the basis of artificial substrates, and are recently used as a coating for mass culture and proliferation of cells or flasks.

The porous scaffold of the present invention may be a cryogel having pores formed by freezing and freeze-drying processes.

In the present invention, the term “cryogel” refers to a porous hydrogel prepared at 0° C. or less (subzero temperature), and has a porous structure because it has an interconnected pore structure.

The average pore size of the porous cryogel scaffold may be 30 to 300 μm, specifically 60 to 150 μm. Accordingly, it may be suitable for cell seating, and may have appropriate mechanical strength and elasticity.

The porous scaffold of the present invention may have an elastic modulus of 60 to 80 kPa, specifically 62 to 70 kPa. Accordingly, it can be used for bone and bone tissue regeneration as it is suitable for inducing bone differentiation.

In another aspect, the present invention comprises the steps of mixing a recombinant silk protein and a biocompatible polymer; freezing the mixture; and freeze-drying the frozen mixture to form pores.

In the present invention, the terms "porous scaffold", "composition for bone tissue regeneration", "recombinant silk protein", "biocompatible polymer", and "bioactive material" are the same as described above.

In the present invention, the tissue regeneration may be bone, bone tissue regeneration, or wound regeneration.

The porous scaffold may promote differentiation of adipose-derived stem cells into bone tissue, or may be used for wound healing, wound regeneration, or therapeutic purposes. In addition, since it has excellent differentiation ability into bone or bone tissue and has an effect of promoting bone tissue regeneration or bone regeneration, it can be used for bone or bone tissue regeneration.

1 is a conceptual diagram schematically illustrating a method for manufacturing a porous scaffold according to an embodiment of the present invention.

Referring to FIG. 1 , first, a recombinant silk protein and a biocompatible polymer may be mixed. The types and properties of the recombinant silk protein and biocompatible polymer are as described above.

In addition, ammonium persulfate and N, N, N', N'-tetramethylethylenediamine (N, N, N', N'-tetramethylethylenediamine) may be added in the mixture preparation step, and the mixture is It may be stored at 0 to 6 ℃, more specifically, it may be stored at 4 ℃.

The mixture may then be frozen.

According to an embodiment of the present invention, the freezing may be performed at -50 to -10 °C for 10 to 50 hours. Specifically, it may be carried out at -25 to -15 °C for 15 to 25 hours. If it is out of the above range, cross-linking and polymerization between the recombinant silk protein and the biocompatible polymer may not be successful.

Next, the frozen mixture may be freeze-dried to form pores.

Specifically, by thawing ice crystals by freeze-drying with a freeze dryer or removing ice crystals, pores can be formed inside the scaffold as the biocompatible polymer and the recombinant silk protein are cross-linked. The freeze drying may be performed for 15 to 30 hours. In addition, unreacted residues may be removed by washing with sterile PBS after pore formation, and UV sterilization may be performed.

The porous scaffold of the present invention may be understood as a cryogel in which pores are formed by a freezing and thawing process.

Hereinafter, the present invention will be described in more detail through Examples according to an embodiment of the present invention, but these Examples do not limit the scope of the present invention.

Experimental Example 1: Preparation of recombinant silk protein

A recombinant silk protein in which the peptide having the amino acid shown in SEQ ID NO: 1 is repeated 48 times was prepared.

SEQ ID NO: 1: NH ₂ -SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT-COOH

The recombinant spider silk protein was prepared using the method described in Korean Patent Registration No. 10-1317420.

Experimental Example 2: Preparation of porous cryogel scaffolds

Porous cryogel scaffolds of Examples 1 to 5 and Comparative Example 1 were prepared.

Example 1.

A 20% (w/v) solution of polyethylene glycol diacrylate (PEGDA) dissolved in phosphate buffered saline (PBS) and a recombinant silk protein solution at a concentration of 2 μg/mL were mixed in a volume ratio of 1:1, and the final concentration of PEGDA was 10% (w/v) and the final concentration of recombinant silk protein was 1 μg/mL. While the mixed solution was stored at 4°C, ammonium persulfate (10% (w/v), Ammonium persulfate, APS; Sigma-Aldrich, USA) and N, N, N', N'-tetramethylethylenediamine (TEMED; Sigma-Aldrich, USA) was added to a final concentration of 0.4% (w/v) and 0.25% (v/v), respectively. The mixed solution to which ammonium persulfate and TEMED was added was pipetted into a mold and frozen at -20°C for 20 hours to slowly proceed with crosslinking. By removing ice crystals from the scaffold with a freeze dryer, a cryogel scaffold having pores of a cross-linked structure was prepared. The obtained cryogel scaffold was washed several times with sterile PBS to remove unreacted residues, and then sterilized with UV light for 1 hour.

Example 2.

A 20% (w/v) solution of PEGDA dissolved in phosphate buffered saline (PBS) and a 20 μg/mL concentration of recombinant silk protein solution were mixed in a volume ratio of 1:1, resulting in a final concentration of PEGDA of 10% (w/v). , and a porous cryogel scaffold was prepared in the same manner as in Example 1, except that the final concentration of the recombinant silk protein was 10 μg/mL.

Example 3.

A 20% (w/v) solution of PEGDA dissolved in phosphate-buffered saline (PBS) and a 200 μg/mL concentration of recombinant silk protein solution were mixed in a volume ratio of 1:1, resulting in a final concentration of PEGDA of 10% (w/v). A porous cryogel scaffold was prepared in the same manner as in Example 1, except that the recombinant silk protein final concentration was 100 μg/mL.

Example 4.

A 20% (w/v) solution of PEGDA dissolved in phosphate-buffered saline (PBS) and a recombinant silk protein solution at a concentration of 1000 μg/mL were mixed in a volume ratio of 1:1, resulting in a final concentration of PEGDA of 10% (w/v). A porous cryogel scaffold was prepared in the same manner as in Example 1, except that the final concentration of the recombinant silk protein was 500 μg/mL.

Example 5.

A 20% (w/v) solution of PEGDA dissolved in phosphate-buffered saline (PBS) and a recombinant silk protein solution at a concentration of 2000 μg/mL were mixed in a volume ratio of 1: 1 to obtain a final concentration of PEGDA of 10% (w/v). A porous cryogel scaffold was prepared in the same manner as in Example 1, except that the recombinant silk protein concentration was 1000 μg/mL.

Comparative Example 1: Preparation of porous cryogel scaffolds

Phosphate-buffered saline (PBS) and a 20% (w/v) solution of PEGDA dissolved in phosphate-buffered saline (PBS) were mixed in a volume ratio of 1:1 so that the final concentration of PEGDA was 10% (w/v). . A porous cryogel scaffold was prepared in the same manner as in Example 1, except that the recombinant silk protein was mixed.

Experimental Example 3: Measurement of physical properties of porous cryogel scaffolds

1) pore measurement

The shape and pore size of the cryogel scaffold were analyzed using scanning electron microscopy (SEM). 2 is an SEM image (X120) of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5. The average pore size of the cryogel scaffold prepared in Comparative Example 1, which is widely used in the past, was 60 to 150 μm.

The average pore size of the cryogel scaffolds prepared in Examples 3 and 5 containing recombinant silk protein was also measured to be 60 to 150 μm, which was similar to Comparative Example 2. In addition, it was confirmed through the SEM image that numerous pores (pores) having a size of 60 to 150㎛ are formed.

Therefore, it was found that there was no difference in pore size and the scaffold using PEGDA even when the recombinant silk protein was included.

2) Measurement of swelling rate

The swelling rate was measured according to a conventionally known gravimetric analysis method, and the swelling (Q) was calculated by the following formula.

3 is a graph showing the expansion rate of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5. Referring to FIG. 3 , the highest expansion rate was exhibited when the recombinant silk protein concentration was 1000 μg/mL. As a result of calculating the expansion rate obtained by comparing the weights before and after water content, it was confirmed that PEGDA-cryogel of Comparative Example 1, which is widely used, exhibited high water content like that of a hydrogel by about 15, and PEGDA and When the recombinant spider silk protein was mixed, it was confirmed that the water content was increased, and in particular, when the silk protein concentration was 1000 μg/ml, it was confirmed that this property was further improved.

3) Compressive strength and elasticity measurement

The strain and the stress of the elastic material were measured in the deformation region through a tensile compression tester.

4 shows the compressive strength of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5. The cryogel scaffolds prepared in Comparative Example 1, Example 3, and Example 5 all showed a similar tendency of compressive strength for a load up to 18N.

Therefore, it was found that there is no difference in compressive strength and the scaffold using PEGDA even when the recombinant silk protein is included.

In addition, the young's modulus value was calculated by obtaining the slope value at the inflection point using the compressive strength. 5 shows the Young's modulus of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5; Young's modulus is a mechanical property that measures the stiffness of a solid material. It is an elastic modulus that defines the relationship between stress (force per unit area) and strain of a linear elastic material in a uniaxial deformation region.

As a result, as shown in FIG. 5 , it was confirmed that the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5 all had a similar Young's modulus of about 64 to 68 kPa. Therefore, it was confirmed that the scaffolds of Examples 3 and 5 also had suitable physical properties for inducing bone differentiation, as in Comparative Example 1.

Next, since there was a report that the young's modulus can promote bone differentiation when the modulus is 60~70 kPa, the scaffold was used to confirm the effect of improving osteodifferentiation of adipose-derived stem cells and to confirm their use as a bone graft material. .

Experimental Example 4: Measurement of Effect of Porous Cryogel Scaffolds with Bone Grafts

1) Cytotoxicity testing

A cytotoxicity test according to the concentration of recombinant spider silk protein was performed using the Live/Dead viability kit. About 500,000 adipose-derived stem cells (hADSCs, human Adipose-Derived Stem cells) were seeded on the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5, and survived 2 days after seeding. Live/dead assay was performed to make cells fluoresce green and dead cells red.

6 and 7 are cytotoxicity test results for the cryogel scaffolds prepared in Comparative Example 1, Examples 2 to 5. 6 and 7 , as in the scaffold of Comparative Example 1 not containing spider silk protein, the concentration of recombinant spider silk protein was 1 μg/ml (not shown), 10 μg/ml to 100 μg/ml. It was confirmed that there was no cytotoxicity with a survival rate of 98% or more. On the other hand, when the concentration of recombinant spider silk protein exceeded 500 μg/ml, the proportion of dead cells (red) was high. (urea solution) was confirmed to cause cytotoxicity.

2) Cell adhesion assay

After seeding GFP-tagged 293T cells at a constant concentration on the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5, as the scaffold expands, whether the cells adhere well or permeate evenly location was evaluated using confocal microscopy.

8 is a cell adhesion experiment result of the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5. As a result, it was confirmed through confocal imaging that cells were well adhered and evenly distributed in all of the scaffolds prepared in Comparative Examples 1, 3, and 5.

Therefore, it was found that the scaffolds of Examples 3 and 5 containing the recombinant silk protein were also effective for cell adhesion as in Comparative Example 1.

3) Wound healing assay

Wound healing assay and RT-PCR were performed to confirm the interaction with growth factors and their effect on cell growth.

9 is a result of wound healing assay and confirmed using confocal microscopy in order to confirm the interaction between recombinant silk protein and growth factor and the effect on cell growth.

The negative control group was cultured in DMEM (Dulbecco's modified Eagle's medium) medium under serum free conditions, and the positive control group (positive control_DMEM+FBS) was cultured in DMEM medium containing FBS, and the GF group (DMEM+growth factor) is cell culture in DMEM medium containing growth factor, and Spider silk+GF group (DMEM+silk protein+GF) is DMEM medium containing spider silk protein and growth factor. has been cultivated in Growth factors used in the experiment were TGF-beta, SCF, FGF-alpha, FGF-beta, IGF, VEGF, and KGF.

As can be seen in FIG. 9 , as a result of scratching the cells grown confluently to a certain area and confirming cell regeneration for 2 days, the Spider silk+GF group treated with recombinant spider silk protein and growth factors It was confirmed that cells were rapidly regenerated to a similar extent to that of a positive control in which cells were cultured in a normal growth medium. Therefore, it was found that the recombinant silk protein promotes wound healing or regeneration.

10 is a result of RT-PCR to confirm the interaction between recombinant silk protein and growth factor and the effect on cell growth.

The negative control group (negative control_serum free DMEM) was cultured in DMEM medium under serum free conditions, the GF group (DMEM+growth factor) was cultured in DMEM medium containing growth factors, and the Silk group (silk+) DMEM) is a culture of cells in a DMEM medium with spider silk protein, and the silk_GF group (DMEM+silk protein+GF) is a culture of cells in a DMEM medium containing spider silk protein and growth factors. Growth factors used in the experiment were TGF-beta, SCF, FGF-alpha, FGF-beta, IGF, VEGF, and KGF.

As shown in FIG. 10 , as a result of RT-PCR, it was confirmed that the expression of vinculin, NF-kB, etc. was increased in the silk+GF group treated with both recombinant spider silk protein and growth factors. Vinculin is known to be involved in cell adhesion such as integrin and the connection of actin cytoskeleton, and NF-kB is also known to be involved in cell survival.

From the results of FIGS. 9 and 10 , it was confirmed that the recombinant spider silk protein increased cell differentiation and growth (proliferation) and increased the expression of proteins involved in focal adhesion.

4) Bone differentiation capacity evaluation (Alizarin red S staining)

Finally, in order to confirm the improvement of bone differentiation ability in vitro, cells were seeded on the scaffolds prepared in Comparative Examples 1, 3, and 5, and Serum Free Medium (SF) and BMP2, an osteogenic factor ( Calcium deposition was confirmed by Alizarin reds S staining 2 weeks after treatment with osteogenic factor).

11 is an Alizarin red S staining experiment result for the cryogel scaffolds prepared in Comparative Examples 1, 3, and 5. As a result, it was confirmed that red agglomerated calcium deposition spots in the scaffold of Example 3 containing 100 μg/ml spider silk protein were greater than in the other group. Therefore, it was confirmed that when the concentration of recombinant spider silk protein was 100 μg/ml, the calcium deposition level was high, so that it was most effective for inducing bone differentiation.

Therefore, it was found that the scaffold of Example 3 including the recombinant silk protein promoted osteodifferentiation compared to the scaffold of Comparative Example 1 not including the recombinant silk protein.

Although described above with reference to the embodiments of the present invention, those of ordinary skill in the art can variously modify the present invention within the scope not departing from the spirit and scope of the present invention described in the claims below. and can be changed.

<110> Medicosbiotech Inc. <120> Composition for bone tissue regeneration comprising recombinant silk protein and porous scaffold comprising the same <130> APP-2021-0287 <150> KR 10-2020-0139279 <151> 2020-10-26 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> spider silk peptide <400> 1 Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly Met Ala Ala Ala 1 5 10 15 Ala Ala Met Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser 20 25 30 Gln Gly Thr 35

Claims (11)

A composition for bone tissue regeneration comprising a recombinant silk protein and a biocompatible polymer. The method of claim 1,
In the recombinant silk protein, the peptide having the amino acid of SEQ ID NO: 1 is repeated 8 to 128 times, the composition for bone tissue regeneration. The method of claim 1,
The biocompatible polymer is polyethylene oxide (PEO), polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), collagen, fibronectin, keratin, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, poly Lactic acid, polyglycolic acid, dextran, and polylactic acid-polyglycolic acid (PLA-PGA) will be selected from the group consisting of, a composition for bone tissue regeneration. The method of claim 1,
The recombinant silk protein is contained in the composition at a concentration of 1 μg/mL to 1000 μg/mL, a composition for bone tissue regeneration The method of claim 1,
The biocompatible polymer is contained in the composition in a concentration of 5% (w / v) to 20% (w / v), the composition for bone tissue regeneration A porous cryogel scaffold comprising the composition of claim 1 . 7. The method of claim 6,
The average pore size of the scaffold is 60 to 150 ㎛, the porous cryogel scaffold. mixing a recombinant silk protein and a biocompatible polymer;
freezing the mixture; and
forming pores by freeze-drying the frozen mixture;
A method of manufacturing a porous scaffold for tissue regeneration, comprising a. 9. The method of claim 8,
The freezing step is performed at -50 to -10 °C for 10 to 50 hours, the method of manufacturing a porous scaffold. 9. The method of claim 8,
The porous scaffold is for bone tissue or wound regeneration, the method of manufacturing a porous scaffold. A porous scaffold for bone or wound regeneration prepared by the method of any one of claims 8 to 10.

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