KR102840628B1 - 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 and has appropriate mechanical strength and elasticity, and has excellent cell adhesion (adhesion) ability, wound healing efficacy, and bone tissue or bone differentiation ability.

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 a variety of naturally occurring or unnatural amino acid sequences, are attracting attention as important materials. Unlike polymers created through chemical synthesis, protein polymers are created based on genetic information (genes), allowing precise control of various polymer properties, such as length and three-dimensional properties. Furthermore, protein polymers can exhibit desirable mechanical, chemical, and biological properties, including biocompatibility and biodegradability, making them useful in fields such as biomaterials engineering and tissue engineering.

Representative repetitive protein repeat sequences with the characteristics 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 (SSTGSSSNTDSNSNSVGSSTSGGSSTYGYSSNSRDGSV), and synthetic repetitive proteins. Among these, dragline silk, which is used as the lifeline of spiders and when creating a radial web shape, has high strength and elasticity. Spider silk is five times stronger per unit mass than steel and three times stronger than high-quality man-made Kepler fibers.

Meanwhile, a scaffold refers to a physical support and adhesive substrate that is designed to enable in vitro culture and in vivo transplantation of tissue cells. Such scaffolds are used for cell transplantation for human tissue regeneration, and their importance is also very high in mass culture and proliferation of cells.

In the present invention, a cryogel was manufactured using spider silk protein to develop a scaffold with excellent biocompatibility.

Korean Patent No. 10-1380786

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

Accordingly, the present invention has as a specific problem to be solved 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 provides a method for manufacturing a porous scaffold for tissue regeneration, comprising 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 addition, a porous scaffold for bone or wound regeneration manufactured by the above manufacturing method is provided.

The composition for bone tissue regeneration comprising the recombinant silk protein and a biocompatible polymer of the present invention can exhibit excellent cell adhesion and proliferation capabilities due to the inclusion of the recombinant silk protein. Furthermore, the composition is suitable for producing a porous scaffold with appropriate mechanical strength and elasticity due to the inclusion of the recombinant silk protein and a biocompatible polymer, and is effective in bone tissue or differentiation and regeneration into bone, as well as wound healing.

FIG. 1 is a schematic diagram illustrating a method for manufacturing a porous scaffold according to one embodiment of the present invention.
Figure 2 is an SEM image (X120) of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5.
Figure 3 is a graph showing the expansion ratio of cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5.
Figure 4 shows the compressive strength of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5.
Figure 5 shows the Young's modulus of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5.
Figure 6 shows the results of a cytotoxicity test for cryogel scaffolds manufactured in Comparative Example 1 and Examples 2 to 5.
Figure 7 is a graph showing the results of cytotoxicity tests on cryogel scaffolds manufactured in Comparative Example 1 and Examples 2 to 5.
Figure 8 shows the results of cell attachment experiments on cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5.
Figure 9 shows the results of a wound healing assay performed using confocal microscopy to confirm the interaction between recombinant silk protein and growth factors and their effects on cell growth.
Figure 10 shows the results of RT-PCR performed to confirm the interaction between recombinant silk protein and growth factors and their effects on cell growth.
Figure 11 shows the results of an Alizarin red S staining experiment on cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5.

Hereinafter, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments and examples described herein.

Throughout this specification, when a part is said to "include" a certain component, this does not mean that other components may be included, but rather that other components may be excluded, unless otherwise specifically stated. The terms "about" and "substantially" used throughout this specification are used in a meaning close to or at the numerical value when manufacturing and material tolerances inherent to the referred meaning are presented, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure, which contains exact or absolute numerical values to aid understanding of the present invention. The terms "step of doing" or "step of" used throughout this specification do not mean "step for doing."

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 a cell substrate and cell proliferation by including a recombinant silk protein, and has appropriate mechanical strength and elasticity by mixing the recombinant silk protein and a biocompatible polymer in an appropriate ratio, and 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 substance.

The term "recombinant silk protein" in the present invention refers to a synthetic silk protein that has a molecular and structural profile that is nearly identical to that of natural silk protein, and is biosynthesized using a recombinant protein production method. Since the "recombinant silk protein" can be used to produce spider silk fibers with properties similar to spider silk fibers, the recombinant silk protein is also referred to as a recombinant spider silk protein.

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

Sequence number 1: NH ₂ -SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT-COOH

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

In the present invention, the recombinant silk protein may be included in the bone regeneration composition 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 may be selected from the group consisting of polyethylene oxide (PEO), polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), collagen, fibronectin, keratin, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, dextran, and polylactic acid-polyglycolic acid (PLA-PGA), and may be specifically polyethylene glycol diacrylate (PEGDA).

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

In addition, the bone regeneration composition of the present invention may additionally contain a bioactive substance.

The above-mentioned bioactive substances include, but are not limited to, plasma or serum components, hydroxyapatite, laminin, drugs, therapeutic agents, anesthetics, cell growth factors, antibodies, antibody-like molecules, nucleic acids, and polysaccharides, and 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 may be selected from the group consisting of TGF-beta, SCF, FGF-alpha, FGF-beta, IGF, VEGF, and KGF.

In the composition of the present invention, the bioactive substance may be included in an amount of 0.1 to 200 ppm, specifically 50 to 150 ppm.

In addition, the composition for bone regeneration additionally has a wound healing or regenerative effect.

In one embodiment of the present invention, it was confirmed through a wound healing assay that spider silk protein has a wound regeneration or healing effect similar to a 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 descriptions of the “composition for bone tissue regeneration,” “recombinant silk protein,” “biocompatible polymer,” and “bioactive substance” are as described above.

In the present invention, the term "scaffold" refers to a physical support and adhesive substrate that is made to enable in vitro culture and in vivo transplantation of tissue cells. The scaffold is used for cell transplantation for human tissue regeneration, and is also used for mass culture and proliferation of cells.

Most biologically active cells must undergo a fundamental process to survive when exposed to substances inside or outside the body. This first step is cell adhesion. Specifically, examining the survival stages of fibroblasts and tissue cells, cells first adhere to a substrate. After adhesion, metabolism within the cytoplasm of the cell's organelles becomes active, and they migrate to new sites to facilitate proliferation and nutrient supply.

Therefore, a surface that activates cell deposition is the most fundamental means of multiplying cell proliferation. This cell adhesion to the substrate can be artificially controlled by the composition of the substrate. A scaffold is a carrier that supports cell regeneration and growth, i.e., the basis of an artificial substrate. Recently, it has been used as a coating on vessels or flasks for mass cell culture and proliferation.

The porous scaffold of the present invention may be a cryogel in which pores are formed by a freezing and freeze-drying process.

In the present invention, the term "cryogel" means a porous hydrogel manufactured at a subzero temperature or lower than 0°C, and has a porous structure because it has an interconnected pore structure.

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

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

In another aspect, the present invention provides a method for producing a porous scaffold for tissue regeneration, comprising 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 as described above.

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

The porous scaffold described above can be used to promote differentiation of adipose-derived stem cells into bone tissue, or for wound healing, wound regeneration, or therapeutic purposes. Furthermore, it has excellent differentiation potential into bone or bone tissue and has the effect of promoting bone tissue regeneration, making it suitable for use in bone or bone tissue regeneration.

FIG. 1 is a schematic diagram illustrating a method for manufacturing a porous scaffold according to one embodiment of the present invention.

Referring to Figure 1, first, a recombinant silk protein and a biocompatible polymer can be mixed. The types and characteristics of the recombinant silk protein and the biocompatible polymer are as described above.

Additionally, ammonium persulfate and N, N, N', N'-tetramethylethylenediamine may be added in the mixture manufacturing step, and the mixture may be stored at 0 to 6°C, and more specifically, at 4°C.

Next, the above mixture can be frozen.

According to one embodiment of the present invention, the freezing may be performed at a temperature of -50 to -10°C for 10 to 50 hours. Specifically, the freezing may be performed at a temperature of -25 to -15°C for 15 to 25 hours. If the temperature is outside the above range, cross-linking and polymerization of the recombinant silk protein and the biocompatible polymer may not occur properly.

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

Specifically, lyophilization using a freeze dryer can form pores within the scaffold as the biocompatible polymer and recombinant silk protein are cross-linked by thawing or removing ice crystals. The lyophilization can be performed for 15 to 30 hours. Additionally, after pore formation, the scaffold can be washed with sterile PBS to remove unreacted residues, and sterilized using UV.

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

Hereinafter, the present invention will be described in more detail through examples according to one 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 was prepared in which a peptide having an amino acid sequence represented by the following sequence number 1 was repeated 48 times.

Sequence number 1: NH ₂ -SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT-COOH

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

Experimental Example 2: Fabrication of a Porous Cryogel Scaffold

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

Example 1.

A 20% (w/v) solution of polyethylene glycol diacrylate (PEGDA) dissolved in phosphate-buffered saline (PBS) and a 2 μg/mL solution of recombinant silk protein were mixed in a volume ratio of 1:1 so that the final concentration of PEGDA was 10% (w/v) and the final concentration of the recombinant silk protein was 1 μg/mL. The mixed solution was stored at 4°C, and ammonium persulfate (10% (w/v), Ammonium persulfate, APS; Sigma-Aldrich, USA) and N,N,N',N'-tetramethylethylenediamine (TEMED; Sigma-Aldrich, USA) were added to the final concentrations of 0.4% (w/v) and 0.25% (v/v), respectively. The mixed solution containing ammonium persulfate and TEMED was pipetted into a mold and frozen at -20°C for 20 hours to slowly promote crosslinking. Cryogel scaffolds with cross-linked pore structures were fabricated by removing ice crystals from the scaffold using a freeze dryer. The resulting cryogel scaffolds were washed several times with sterile PBS to remove unreacted residues and then sterilized under UV for 1 hour.

Example 2.

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

Example 3.

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

Example 4.

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

Example 5.

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

Comparative Example 1: Manufacturing of a Porous Cryogel Scaffold

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 manufactured in the same manner as in Example 1, except that the recombinant silk protein was mixed.

Experimental Example 3: Measurement of the Physical Properties of Porous Cryogel Scaffolds

1) Pore measurement

The morphology and pore size of the cryogel scaffolds were analyzed using SEM (Scanning Electron Microscopy). Figure 2 shows SEM images (X120) of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. The average pore size of the cryogel scaffold manufactured in Comparative Example 1, which has been widely used, was 60 to 150 μm.

The average pore size of the cryogel scaffolds manufactured in Examples 3 and 5 containing recombinant silk protein was also measured to be 60 to 150 μm, similar to that of Comparative Example 2. In addition, it was confirmed through SEM images that numerous pores with a size of 60 to 150 μm were formed.

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

2) Measurement of swelling rate

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

FIG. 3 is a graph showing the swelling ratio of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. Referring to FIG. 3, the highest swelling ratio was observed when the recombinant silk protein concentration was 1000 μg/mL. As a result of calculating the swelling ratio obtained by comparing the weight before and after moisture retention, it was confirmed that the PEGDA-cryogel of Comparative Example 1, which is widely used in the past, exhibits a high moisture retention capacity like a hydrogel of about 15, and when PEGDA and recombinant spider silk protein were mixed, it was confirmed that the moisture retention capacity increased, and in particular, when the silk protein concentration was 1000 μg/mL, it was confirmed that these properties were further improved.

3) Measurement of compressive strength and elasticity

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

Figure 4 shows the compressive strength of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. All of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5 showed a similar trend in compressive strength for loads up to 18 N.

Therefore, it was found that there was no difference in compressive strength between the scaffold using PEGDA and the scaffold containing the recombinant silk protein.

In addition, the slope value at the inflection point was obtained using the compressive strength to calculate the Young's modulus value. Figure 5 shows the Young's modulus of the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. Young's modulus is a mechanical property that measures the stiffness of a solid material, and is an elastic coefficient that defines the relationship between the stress (force per unit area) and strain of a linear elastic material in the uniaxial deformation region.

As a result, as confirmed in Fig. 5, it was confirmed that the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5 all had similar Young's moduli of approximately 64 to 68 kPa. Therefore, it was confirmed that the scaffolds of Examples 3 and 5 also had suitable physical properties for inducing osteogenic differentiation, similar to Comparative Example 1.

Next, since it has been reported that bone differentiation can be promoted when the young’s modulus is 60 to 70 kPa, we attempted to confirm the effect of enhancing bone differentiation of adipose-derived stem cells using the scaffold and to confirm its usability as a bone graft material.

Experimental Example 4: Measuring the Effectiveness of a Porous Cryogel Scaffold as a Bone Graft Material

1) Cytotoxicity testing

A cytotoxicity test was conducted according to the concentration of recombinant spider silk protein using a Live/Dead viability kit. Approximately 500,000 human adipose-derived stem cells (hADSCs) were seeded onto the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. After 2 days, a Live/dead assay was performed, in which surviving cells fluoresce green and dead cells fluoresce red.

Figures 6 and 7 show the results of cytotoxicity experiments on the cryogel scaffolds manufactured in Comparative Example 1 and Examples 2 to 5. Referring to Figures 6 and 7, similar to the scaffold of Comparative Example 1 that does not include spider silk protein, it was confirmed that there was no cytotoxicity with a survival rate of 98% or more when the concentration of the recombinant spider silk protein was 1 ㎍/ml (not shown), 10 ㎍/ml to 100 ㎍/ml. In contrast, when the concentration of the recombinant spider silk protein exceeded 500 ㎍/ml, the proportion of dead cells (red) was high. This was confirmed to be due to the urea solution used to dissolve the high-molecular-weight protein at a high concentration, rather than the cytotoxicity of the spider silk protein itself.

2) Cell adhesion assay

GFP-tagged 293T cells were seeded at a constant concentration on the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. Then, as the scaffold expanded, whether the cells attached well or penetrated and were located evenly was evaluated using confocal microscopy.

Figure 8 shows the results of cell attachment experiments on cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. As a result, it was confirmed through confocal imaging that cells attached well and spread evenly on all scaffolds manufactured in Comparative Example 1, Example 3, and Example 5.

Therefore, it was found that the scaffolds of Examples 3 and 5 containing recombinant silk proteins were also effective in cell attachment, similar to Comparative Example 1.

3) Wound healing assay

To determine the interaction with growth factors and the effect on cell growth, wound healing assay and RT-PCR were performed.

Figure 9 shows the results of a wound healing assay performed using confocal microscopy to confirm the interaction between recombinant silk protein and growth factors and their effects on cell growth.

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

As shown in Fig. 9, when scratches were made with a certain width on confluently grown cells and cell regeneration was observed for 2 days, it was confirmed that cells in the Spider silk+GF group, which was treated with recombinant spider silk protein and growth factors together, regenerated quickly to a similar degree as the positive control group, which was cultured in a general growth medium. Therefore, it was found that recombinant silk protein promotes wound healing or regeneration.

Figure 10 shows the results of RT-PCR performed to confirm the interaction between recombinant silk protein and growth factors and their effects 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, the Silk group (silk+DMEM) was cultured in DMEM medium with spider silk protein, and the silk_GF group (DMEM+silk protein+GF) was cultured in DMEM medium containing spider silk protein and growth factors. The growth factors used in the experiment were TGF-beta, SCF, FGF-alpha, FGF-beta, IGF, VEGF, and KGF.

As confirmed in Fig. 10, the results of RT-PCR confirmed that the expression of vinculin, NF-kB, etc. 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 connection with the actin cytoskeleton, and NF-kB is also known to be involved in cell survival.

The results in Figures 9 and 10 confirmed that the recombinant spider silk protein increases cell differentiation and proliferation and increases the expression of proteins involved in focal adhesion.

4) Evaluation of osteogenic differentiation potential (Alizarin red S staining)

Finally, to confirm the improvement in osteogenic differentiation in vitro, cells were seeded on the scaffolds manufactured in Comparative Example 1, Examples 3 and 5, and treated with Serum Free Medium (SF) and osteogenic factor BMP2 (osteogenic factor), and after 2 weeks, Alizarin reds S staining was performed to confirm calcium deposition.

Figure 11 shows the results of an Alizarin red S staining experiment on the cryogel scaffolds manufactured in Comparative Example 1, Example 3, and Example 5. As a result, it was confirmed that the scaffold of Example 3, which included 100 μg/ml of spider silk protein, had more red, clustered calcium deposition spots than the other groups. Therefore, it was confirmed that the concentration of recombinant spider silk protein at 100 μg/ml was the most effective in inducing osteogenic differentiation due to the high calcium deposition level.

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

Although the present invention has been described above with reference to embodiments thereof, it will be readily understood by those skilled in the art that various modifications and changes to the present invention can be made without departing from the spirit and scope of the present invention as set forth in the claims below.

<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 porous cryogel scaffold for bone tissue and wound regeneration comprising recombinant silk protein and polyethylene glycol diacrylate (PEGDA).
The recombinant silk protein is contained at a concentration of 100 μg/mL to 1000 μg/mL,
A porous cryogel scaffold for bone tissue and wound regeneration, wherein the porous cryogel scaffold has a Young's modulus of 60 to 80 kPa and an average pore size of 60 to 150 μm. In the first paragraph,
A porous cryogel scaffold for bone tissue and wound regeneration, wherein the recombinant silk protein comprises a peptide having an amino acid sequence of sequence number 1 repeated 8 to 128 times. delete delete In the first paragraph,
A porous cryogel scaffold for bone tissue and wound regeneration, wherein the polyethylene glycol diacrylate (PEGDA) is contained at a concentration of 5% (w/v) to 20% (w/v). delete delete A method for manufacturing a porous cryogel scaffold of the first clause,
A step of mixing recombinant silk protein and polyethylene glycol diacrylate (PEGDA);
a step of freezing the mixture; and
A step of forming pores by freeze-drying the above frozen mixture;
A method for manufacturing a porous cryogel scaffold for bone tissue and wound regeneration, comprising: In paragraph 8,
A method for manufacturing a porous cryogel scaffold for bone tissue and wound regeneration, wherein the freezing step is performed at -50 to -10°C for 10 to 50 hours. delete delete