KR20200136563A - Peptides from marine resources capable of silica formation and use thereof - Google Patents

Abstract

The present invention relates to a marine resource-derived silica synthetic peptide and a use thereof. The novel marine resource-derived peptide according to the present invention has excellent silica synthesis activity from silicic acid and can be used in the manufacture of a composite bone graft material in which silica and collagen are laminated. The composite bone graft material has an improved carrying capacity of biomolecules compared to an existing graft material and continuously releases supported molecules, thereby promoting bone regeneration when transplanted to a damaged bone area. Therefore, the peptide according to the present invention can be usefully utilized in the fields related to silica synthesis and bone grafting.

Description

[Peptides from marine resources capable of silica formation and use thereof]

The present invention relates to marine-derived silica synthetic peptides and uses thereof.

Silica is important for synthesizing a new hybrid material (organic-inorganic complex) because it can covalently bond with specific chemical species, and as a biocompatible material, it has excellent properties that can compensate for the disadvantages of individual materials. Currently, the industrial production process of silica requires high temperature, strong acid or strong base conditions, and there is a problem of producing environmentally harmful by-products. However, because of the discovery of enzymes and peptides that biosynthesize silica based on the extracts or genetic information of sponges and diatoms, which are marine organisms, environmentally friendly bio-silica produced by them and its applicability to various uses, it is derived from oceans worldwide. The development of bio-silica composite materials is becoming an issue. Therefore, it is very important to possess the original technology for securing bio-silica materials through biological synthesis methods.

In order to repair damaged bone, a replacement agent or bone graft material is needed. This supports the bone defect area through graft and filling in the damaged or defective bone area. As the bone tissue expands from the current bone to the graft material, the damaged area becomes a new bone. It serves to help you recover. This role of most bone graft materials is called bone conduction. However, for sufficient bone regeneration, a function of inducing or regenerating a bone graft material to form new autologous bone tissue from surrounding autologous bone tissue to the bone defect is required. To this end, active factors such as BMP, PDGF, TGF, FGF, TGF, GDF, which induce bone growth, are introduced into the conductive bone graft material, and these active factors convert stem cells into osteogenic cells to induce bone formation. In transplantation to replace damaged bone, the best method for bone repair is to use bone conduction and osteoinductive properties, and autogenous bone containing stem cells. However, in the case of secondary surgery and large damaged areas, the amount of autogenous bone is also problematic. The use of synthetic bone grafts incorporating island growth factors is recognized as an alternative.

In the United States, which successfully commercialized rhBMP-2 for the first time in the world, the size of the bone graft material market has grown to more than double the existing size due to bone-inducing substances. However, the bone grafting material loaded with the growth factor physically binds rhBMP2 to the bone grafting material and adsorbs it. Due to the absence of a chemical bond between the protein and the bone grafting material, side effects such as soft tissue expansion and abnormal bone formation due to immediate release at the initial stage of transplantation have been reported. Due to the manufacturing cost, it does not have a large market share, especially in Korea.

As described above, products containing bone morphogenetic proteins are on the market, but because the half-life of BMP2 is short, the released BMP2 may quickly lose its biological activity, and it is a form in which growth factors are absorbed into the carrier by simple immersion. BMP2, which was released immediately at the beginning of transplantation due to the absence of a chemical bond, is rapidly lost through diffusion, and a large amount of BMP-2 is used for therapeutic effect during clinical treatment. This not only incurs high treatment costs, but also causes bone regeneration such as soft tissue expansion and ectopic bone formation. It can cause undesirable side effects, including an immune response. Therefore, it is required to develop a carrier capable of not only carrying growth factors well, but also releasing growth factors in a sustained-release form after transplantation. In addition, patents exist for formulations in which a bone graft material and a growth factor are integrally supported for sustained release of growth factors, but in the case of an integrated medical device in which a growth factor and a bone graft material are combined in the actual commercialization, growth factor when sterilization is performed at the same time. As there is a risk of destruction of the bone graft material and distribution and storage of the bone graft material is limited, the development of a carrier that allows the growth factor to be effectively loaded on the graft material even if mixed immediately before transplantation and release the supported growth factor in a sustained-release form after transplantation will be more advantageous for commercialization.

Domestic patent application number: 10-2012-0109601

Shimizu, Cha et al. 1998; Silicatein alpha: cathepsin L-like protein in sponge biosilica. Proc Natl Acad Sci USA 95:6234-6238 Kroger, Deutzmann et al. 1999; Polycationic peptides from diatom biosilica that direct silica nanosphere formation. Science 286:1129-1132 Cha, Stucky et al. 2000; Biomimetic synthesis of ordered silica structures mediated by block copolypeptides. Nature 403:289-292.

Accordingly, as a result of continuing efforts to develop a new bone grafting material, the present inventors discovered a silica synthetic peptide derived from the ocean and coated it on the surface of the bone grafting material to form a structure in which silica and collagen were stacked. The present invention was completed by confirming that the carrying capacity is improved and can be used as a carrier for a solvent to continuously release the supported molecules.

Accordingly, an object of the present invention is to provide a novel peptide having silica synthesis activity.

Another object of the present invention is to provide a composition for synthesizing silica, including the peptide, for bone grafting or for bone filling.

Another object of the present invention is to provide a method for producing a bone graft material using the peptide.

In order to achieve the above object, the present invention provides a peptide having silica synthesis activity consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a composition for synthesis of silica, including the peptide.

In addition, the present invention provides a composition for bone grafting or bone filling, comprising the peptide.

In addition, the present invention provides a method for producing a bone graft material comprising the step of coating the peptide.

The novel marine-derived peptide according to the present invention has excellent silica synthesis activity from silicic acid, and can be used in the manufacture of a composite bone graft material in which silica and collagen are laminated. The composite bone graft material has an improved carrying capacity of biomolecules compared to the existing bone graft material, and has an effect of continuously releasing the supported molecule, and thus has an excellent effect of promoting bone regeneration when transplanted to a damaged bone site. Therefore, the peptide according to the present invention can be usefully utilized in the fields related to silica synthesis and bone graft.

1 is a diagram showing a schematic diagram of the production of a bone graft material in which peptides and silica/collagen are stacked according to the present invention.
2 is a diagram illustrating the surface of a bone graft material according to silica/collagen coating.
Figure 3 is a diagram (A) confirming the silica-forming ability of the peptide used in the present invention and a diagram (B) confirming the modified GFP binding ability of the silica-laminated bone graft material.
Figure 4 is a view confirming the growth factor binding method according to the surface coating of the bone graft material.
5 is a diagram illustrating the loading effect of modified GFP and BMP2 on the silica/collagen composite bone graft material.
6 is a diagram confirming the bone regeneration rate of a bone graft material using a damaged bone model.
7 is a diagram showing bone formation of regenerated bone tissue through MT-Goldner collagen/bone staining.
Figure 8 is a diagram confirming the ratio of bone graft material, hard tissue, and soft tissue in the regenerated tissue.
9 is a diagram comparing an enlarged image of regenerated bone tissue through MT-Goldner and H&E staining.
10 is a diagram confirming the speed of bone regeneration by a bone graft material in an animal model.

Hereinafter, the present invention will be described in more detail.

The present invention provides a peptide having silica synthesis activity consisting of the amino acid sequence of SEQ ID NO: 1.

In the present invention, the term "peptide having silica synthesis activity" or "silica forming peptide (SFP)" refers to a peptide capable of synthesizing (forming) silica by reacting with a silica precursor.

In the present invention, the term "silica formation reaction" or "silica mineralization reaction" refers to a reaction of forming silica minerals by organic materials or organic molecules.

The peptide having silica synthesis activity according to the present invention is also referred to as E6Ectp1, and 8 amino acids are added to the N-terminus of Ect1, a peptide derived from marine organisms, with a glutamic acid residue capable of calcium binding and two glycines, a linking sequence. It was prepared by combining with.

The specific amino acid sequence is as follows.

E6Ectp1 : EEEEEEGGSSRSSSHRRHDHHDHRRGS (SEQ ID NO: 1)

The peptide can be prepared by a method known in the art for synthesizing the peptide. For example, it can be produced by in vitro synthesis through gene recombination and protein expression systems or peptide synthesizers.

The peptide can synthesize silica by reacting with a silica precursor under conditions of room temperature and pressure and in an aqueous solution of a weak acid or a weak base near neutral, and in particular, through the modification of the end of the peptide, the reactive groups react to form silica rather than bind to the bone graft material. It may be more involved in, has a more excellent activity compared to the known silica synthetic peptide.

In addition, the present invention provides a composition for synthesis of silica, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a composition for bone grafting or bone filling, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The composition may further include a silica precursor.

The silica precursors are tetramethoxy silane, tetramethyl orthosilicate, tetraethyl orthosilicate, methyltriethoxysilane, phenyl triethoxysilane, dimethyl dimethoxy silane, ethyl triethoxysilane, titania tetraisopropoxide and tetra It may be one or more selected from the group consisting of ethyl germanium, but is not limited thereto.

The composition may further include collagen.

The composition may further include hydroxyapatite or tricalcium phosphate. The hydroxyapatite or tricalcium phosphate may be a synthetic powder or a porous block, but is not limited thereto.

The composition may further contain a physiologically active substance related to bone formation.

In the present invention, the term "bioactive substance" refers to a biological substance that promotes healing ability during tissue regeneration.

The physiologically active substances related to bone formation include bone morphogenetic protein (BMP), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (Platelet). derived growth factor, PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) may be one or more selected from the group consisting of, but is not limited thereto.

The composition according to the present invention may be in a form in which a peptide consisting of an amino acid of SEQ ID NO: 1, a silica precursor, and collagen are coated on the surface of hydroxyapatite or tricalcium phosphate, and the collagen is hydroxyapatite or a hydroxy It may be coated with apatite or tricalcium phosphate, but is not limited thereto.

The composition may release a physiologically active substance in a sustained release form.

The composition may be for dental bone grafting or for bone filling, but is not limited thereto.

In addition, the present invention provides a method for producing a bone grafting material comprising the step of coating a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on the surface of the bone grafting material.

The method comprises the steps of: (1) coating a bone graft material with a peptide consisting of the amino acid sequence of SEQ ID NO: 1; (2) coating a silica precursor on the bone graft material of step 1; And (3) coating collagen on the bone grafting material of step 2, or (1) coating collagen on the bone grafting material; (2) coating the bone graft material of step 1 with a peptide consisting of the amino acid sequence of SEQ ID NO: 1; And (3) coating a silica precursor on the bone graft material of step 2; can be performed.

At this time, when the final step ends with the step of coating the silica precursor, the binding strength of the physiologically active material slightly increases compared to the case where the step of coating with collagen ends,

When the final step ends with the step of coating with collagen, it appears that the aggregation with the blood is better when the bone graft material is transplanted than when the step ends with the step of coating the silica precursor.The final step ends with the step of coating with collagen. The case is preferred.

The steps (1) to (3) may be repeated 1 to 20 times, preferably twice, but are not limited thereto.

The method may further include (4) coating a physiologically active material related to bone formation on the surface of the bone graft material, and the step (4) may be performed at the time of use.

Hereinafter, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following Examples and Experimental Examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the Examples and Experimental Examples.

Example 1. Novel with marine-derived silica synthesis activity Peptide Produce

A new peptide was prepared by additionally binding 8 amino acids (a glutamic acid residue capable of calcium binding and two glycines, a linking sequence) to the N terminal of Ect1, a peptide derived from marine organisms. Hereinafter, it is indicated as E6Ectp1 or silica synthetic peptide (SFP).

The specific amino acid sequence is as follows.

E6Ectp1 : EEEEEEGGSSRSSSHRRHDHHDHRRGS (SEQ ID NO: 1)

Example 2. New marine origin Peptide number Coated silica/collagen composite bone Of the graft material (Col/E6Ectp1Si@HA) Produce

1 mL of the marine-derived peptide (E6Ectp1) prepared in Example 1 at 0.1 to 0.5 mg/mL was added to 1 g of granular type hydroxyapatite (HA) having a size of 0.6 to 1 mm, and the entire reaction was carried out overnight at 4°C. . Thereafter, the unbound peptide was removed by lightly washing with 3 distilled water once. Next, for silica coating, 0.75 ml of TEOS was dissolved in 25 ml of ethanol, and 3% silicic acid containing 10 ml of 1M HCl was prepared there, and then 25 ml of 72 mM cholin chloride to stabilize the silicic acid. Was added to prepare 1.5% silicic acid with a final volume of 50 mL. 10 mL of the 1.5% silicic acid was added to the washed bone graft material and subjected to shaking reaction at room temperature for 6 to 24 hours. Then, it was washed with distilled water 10 times and then dried at room temperature, and in order to perform collagen coating, a granular type of hydroxyapatite (HA) was performed in 1×PBS containing 0.01 to 0.05% type 1 collagen. ) And shaken at room temperature for 6 to 24 hours. After the reaction was terminated, the unbound collagen was removed, dried, washed with distilled water 3 times, and the reaction was repeated to laminate the silica-synthetic peptide/silica/collagen again on the HA surface. This reaction can be repeated once again, and a total of two or three coatings were applied. After the final reaction was terminated, dried, washed with distilled water three times, and dried again in a biosafety cabinet. The above experimental process is shown in the right process of FIG. 1, and all processes were performed as aseptic processes.

Alternatively, a process for coating the HA surface with collagen first and then coating silica is shown in the left process of FIG. 1. To this end, the granular type of hydroxyapatite (HA) was added to 1×PBS containing 0.01 to 0.05% type 1 collagen, and shaken at room temperature for 6 to 24 hours to coat with collagen, and then marine-derived peptide It was reacted with (E6Ectp1). After that, after applying silica coating, washing and drying, in order to repeat the above process, collagen is coated again, reacted with silica synthetic peptide, and then dried through a silica coating process. It was dried after final coating once again.

The control group used hydroxyapatite without a coating process, and the comparative group applied only the step of laminating silica through a silica formation reaction after silica synthesis peptide coating in the left process of FIG. 1, and dried without collagen coating.

The surface of the prepared bone graft material Col-E6Ectp1Si-HA was observed by scanning electron microscope observation (SEM), and the results are shown in FIG. 2.

As shown in FIG. 2, the surface of the control group, hydroxyapatite (HA) granules without any coating, exhibited a smooth surface, and the comparative group, the HA coated with only silica after the silica synthesis peptide coating, exhibited a rough surface. In contrast, the surface roughness of the HA surface coated with the silica synthetic peptide, silica, and collagen according to the present invention twice compared to the control group was increased, and a fibrous structure due to collagen fibers was observed.

Example 3. New marine origin Peptide number Coated βTCP Preparation of silica-based/collagen composite bone graft material (Col/E6Ectp1Si@βTCP/Col)

Instead of 0.6mm to 1mm HA, 1g of beta-TCP granules was prepared by repeating the silica synthesis peptide/silica/collagen coating twice in the same manner as in Example 2 according to the process on the right side of FIG. 1. After that, after dissolving 0.07g to 0.15g of type 1 collagen in 3 to 5 mL of 0.2M acetic acid to form a gel, mix it with the βTCP-based bone graft material coated with the silica/collagen and knead it, then a 2 mL syringe, or 2 mm It was placed in a silicone mold with x 8 mm holes and lyophilized, placed in a dialysis membrane and dialyzed twice with PBS for 24 hours each. Finally, it was used after dialysis with distilled water for 24 hours once and lyophilized.

As a control group, 1 g of beta-TCP granules of 0.6 mm to 1 mm were used, and 0.07 g to 0.15 g of type 1 collagen was dissolved in 3 to 5 mL of 0.2 M acetic acid to form a gel, and then the silica/collagen coated βTCP-based The mixture was kneaded with a bone graft material, and freeze-dried by putting it in a 2 mL syringe or a silicone mold with a 2 mm x 8 mm hole. This was put into a dialysis membrane and dialyzed twice with PBS for 24 hours each. Finally, it was used after dialysis with distilled water for 24 hours once and lyophilized.

Experimental Example 1 . Marine origin new Peptide Silica synthesis effect verification

Prepared in Example 1 In order to confirm the silica-forming activity of E6Ectp1, the peptide of SEQ ID NO: 1, the following experiment was performed. First, hydroxyapatite granules were used as a control (HA), and 5 mg/mL of the peptide of SEQ ID NO: 1 (E6Ectp1) was added to 1 g of HA and reacted at 4° C. for 12 hours. The peptide-bound HA was immersed in a PBS solution containing 20mM silicic acid and shaken overnight (E6Ectp1Si@HA). As a comparative group, HA was immersed in the same conditions without a peptide and subjected to shaking reaction (Si-HA), and then the amount of silica in each bone graft was analyzed by XPS. The results are shown in Fig. 3A.

As shown in FIG. 3A, silica was not detected in the control group (HA), and in the case of the comparative group, silica occupied about 0.19% of the bone graft components, whereas in the bone graft material coated with the peptide of the present invention, about 3.7% of silica was detected. Through this, the ability of the marine-derived peptide according to the present invention to form silica was confirmed.

Next, in order to confirm the active molecule binding ability by silica coating, a modified GFP (BMP2-like protein) prepared to have a surface charge similar to BMP2 was used to bind to the bone graft material, and then the bound and unbound amount Was compared. The results are shown in Fig. 3B.

As shown in FIG. 3B, as a result of comparing the amount released from the bound protein after 30 minutes of reaction and the amount remaining in the bone graft material, the increase in binding of the modified GFP protein in the group coated with the peptide of the present invention compared to the control group. Confirmed.

Experimental example 2. New marine origin Peptide Used silica/collagen composite bone Of the graft material (Col/E6Ectp1Si@HA) Verification of growth factor loading and release effect

An experiment was conducted to verify the effect of supporting and releasing growth factors of the bone graft material coated with the marine-derived peptide prepared in Example 2. First, FITC-BMP2 was prepared by labeling FITC on rhBMP2 as a supporting material. FITC-BMP2 was loaded on each bone graft material, and 30 minutes later, FITC-BMP2 that was not bound to the bone graft material was measured with a fluorescence photometer (excitation 485 nm/emission 535 nm), and the amount of bound FITC-BMP2 was compared. The results are shown in Fig. 4A.

As shown in FIG. 4A, only 10% of the amount of HA was bound, but 35% of FITC-BMP2 was bound to Col/E6Ectp1Si@HA of the present invention. That is, it was confirmed that the supporting power of FITC-BMP2 was increased by 3 times or more compared to the control by the marine-derived peptide and silica/collagen composite coating of the present invention.

Next, the release pattern of the bound FITC-BMP2 from the bone graft material was observed. The results are shown in Fig. 4B.

As shown in Figure 4B, HA was released 20% after 1 day and 95% after 9 days, and the control group E6Ectp1Si@HA was released less than 5% after 1 day and 30% after 10 days, and then almost released. Didn't. On the contrary, Col/E6Ectp1Si@HA of the present invention was released less than 5% after 1 day, showed a similar release pattern to E6Ectp1Si@HA until 3 days, and then showed a rapid increase in release after 3 days. Through the above experimental results, it was confirmed that the problem of the silica alone coating, that is, the release rate is too slow, can be controlled through the collagen/silica coating.

In order to quantify the pattern, the release behavior and rate of FITC-BMP2 were measured using the Korsemeyer-Peppas Model equation (the equation below), and the results are shown in Table 1.

[n=release exponent, k=release rate constant, R2=correlation co-efficient, Mt=released amount at time t, and M∞loaded amount]

As mentioned above, the present inventors determined that the initial release rate ( K ) could be adjusted through the combination of silica and collagen, as well as the n value related to sustained release. For this, protein binding and release patterns were investigated using a modified GFP whose isoelectric point is similar to that of hBMP2. The results are shown in FIG. 5.

As shown in Figure 5, it was confirmed that the binding pattern of rhBMP2 and the modified GFP appeared similar according to the coating method of the bone graft material.

Therefore, using the modified GFP, the release pattern over time was investigated, and the bone graft material showing the optimal binding and release rate was investigated. The release rate and pattern of the modified GFP for each bone graft were calculated by the Korsemeyer-Peppas Model equation and shown in Table 2.

As shown in Table 2, when coated with silica and collagen once, the initial release rate of the model protein decreases by 50% or more, and after the initial release, almost no release occurs similar to that of HA, but when silica and collagen are coated twice, the initial release rate Was found to decrease while the sustained diffusion rate increased. In addition, when the silica and collagen were coated three times, the initial release rate increased slightly compared to the second coating, but the diffusion rate was low. Therefore, silica and collagen No. 2 coating was optimally determined.

As a result of measuring binding affinity through Microscale Thermophoresis using Monolith NT.115, a meaningful binding pattern was not found between the silica-synthetic peptide and BMP2, a bone growth factor, through which the peptide coating according to the present invention was It was judged that it promotes the formation of silica, but does not affect the binding of BMP2. In addition, analysis of the effect on bone differentiation and bone formation using MC3T3 E1 mouse osteoblasts showed that the peptide coating according to the present invention did not have a significant effect. However, in the case of the organic-inorganic composite silica formed by the mediation of the peptide compared to the silica composed of only inorganic substances, it was confirmed that the peptide affects the decomposition rate of the silica and thus the release rate of bound BMP2.

Experimental example 3. New marine origin Peptide Used silica/collagen composite bone Graft material (Col/Si) ₂ @HA)'s bone regeneration ability verification

Using the bone graft material coated with marine-derived peptide-mediated silica and collagen prepared in Example 2, the bone regeneration ability was confirmed. At this time, silica and collagen coating were performed twice based on the results of Experimental Example 2. Novosis HA, a commercially untreated synthetic bone graft material, was used as a control carrier (HA).

Specifically, a bone defect site (8 mm) was made on the skull of a SD (Sprague-Dawley) rat, and a bone graft material carrying rhBMP2 was transplanted to the HA prepared in the same manner as in Experimental Example 2. The concentration of rhBMP2 dissolved in PBS was adjusted so that the final 1, 2.5, and 5 μg of rhBMP2 per animal was carried on the defect site, respectively, and mixed with a carrier 30 minutes before transplantation. After implantation of the bone graft material, the recovery pattern of the damaged bone was confirmed through microCT over a period of 1 to 6 weeks, and tissues of the final sacrificed animal were observed at 8 weeks. Bone regeneration between the Novosis HA group without rhBMP2 treatment, the Novosis HA group adsorbing rhBMP2 (HA+BMP), and the group adsorbing rhBMP2 to the bone graft material according to the present invention ((Col/Si)2@HA+BMP2) The speed is shown in Figure 6.

As shown in FIG. 6, it was confirmed that the relationship between the amount of growth factor and bone regeneration ability is not a proportional relationship, and an optimum concentration for bone regeneration is required. This is also related to the characteristics of the carrier. Specifically, the HA treatment group, the control group, showed the highest bone regeneration when 2.5 μg was used, and the bone regeneration was high in the first week. On the other hand, when 5 μg was used, bone regeneration almost stopped at 4 weeks due to early bone regeneration, and sufficient bone regeneration was inhibited. When 1 μg was used, it was determined that growth factors were insufficient because bone regeneration was initially released and progressed for the remaining period. Became. On the other hand, the bone graft material ((Col/Si)2@HA) coated with the marine peptide of the present invention showed the highest bone regeneration when 2.5 μg was used, and there was no significant difference from the case of 1 μg. In addition, when 2.5 ㎍ was used, the initial bone formation rate was lower than that of HA, but after 2 weeks, the bone formation rate was faster, and the highest bone formation rate was shown at 6 weeks. In addition, when 1 μg was used, bone regeneration was increased by 22% or more compared to bone regeneration by HA. This phenomenon is due to the strong binding force between the carrier and the growth factor, and it is possible to continuously promote bone regeneration by supporting a small amount of growth factor for a long time and releasing it in a sustained release form.

Next, bone regeneration was observed histologically after 8 weeks of the animals transplanted with 1 µg of rhBMP2-supported bone graft material. The results are shown in FIGS. 7 to 9.

7 and 8, as a result of MT-Goldner collagen/bone staining, the bone tissue regenerated by the marine-derived peptide-coated bone graft material ((Col/Si)2@HA+BMP2) of the present invention is a graft material. It was confirmed that the binding between the and bone was excellent, and the graft material was buried in the mature bone. On the other hand, bone tissue regenerated by HA+BMP, a control group, had new bone formation, but the fibrous connective tissue proliferated clearly, and the ratio of hard tissue was lower than that of the composite bone graft material.

In addition, when comparing the enlarged images of each treatment group as shown in FIG. 9, new bones are hardly formed in the group (HA) not treated with BMP2, but the bone graft material coated with the marine-derived peptide of the present invention ((Col In the /Si)2@HA+BMP2) treatment group, it was confirmed that the bone was formed smoothly below the periosteum, which is believed to have been continuously released from the graft material to induce bone.

Experimental example 4. New marine origin Peptide number Coated βTCP Based silica/collagen composite bone Of the graft material ((Col/Si)@βTCP/Col) Validation of activity in animal models

A βTCP-based silica/collagen composite bone graft material coated with a novel marine peptide prepared in Example 3 was used, and at this time, silica and collagen coating were performed twice based on the results of Experimental Example 2 to produce. Here, the bone regeneration ability was confirmed by using a bone graft material (a disc-shaped 2mm x 8 mm size) in which 10% bovine type 1 collagen was mixed with respect to the weight of the bone graft material. ΒTCP/Col mixed with untreated βTCP and 10% collagen was used as a control carrier.

Specifically, a bone defect site (8 mm) was made on the skull of SD (Sprague-Dawley) rats, and a bone graft material carrying rhBMP2 on the bone graft material was transplanted to the defect site in a similar manner to that of Experimental Example 2. The concentration of rhBMP2 dissolved in PBS was adjusted so that the final 1 μg of rhBMP2 was carried on the defect site per animal, and mixed with a carrier 30 minutes before transplantation. After implantation of the bone graft material, bone regeneration was confirmed through microCT imaging from 1 week to 8 weeks for the recovery pattern of the damaged bone. βTCP/Col group without rhBMP2 treatment, βTCP/Col group adsorbing rhBMP2 (βTCP/Col+BMP), and group adsorbing rhBMP2 to the bone graft material according to the present invention ((Col/Si)@βTCP/Col+ BMP) liver bone regeneration rate is shown in Tables 3 to 5, and Fig. 10.

% new bone formation through μCT measurement of βTCP/collagen One 2 4 6 8 βTCP/Col +PBS-1 48.29412 52.56049 67.36846 60.74038 62.34507 βTCP/Col +PBS-2 47.67299 57.21153 62.26699 64.94307 67.1265 βTCP/Col +PBS-3 50.78956 57.30784 67.52425 69.71064 69.54137 βTCP/Col +PBS-4 48.09954 45.36222 53.75279 52.58632 60.26937 mean 48.71405 53.11052 62.72812 61.9951 64.82058 sd 1.407777 5.620631 6.462838 7.264515 4.260078

% new bone formation through μCT measurement of βTCP/collagen+rhBMP2 (1 μg) One 2 4 6 8 βTCP/Col +BMP-1 40.91572 48.91413 62.39724 67.024 70.45852 βTCP/Col +BMP-2 52.40068 59.54967 69.86605 71.33 72.70087 βTCP/Col +BMP-3 44.97122 57.84023 69.23038 72.48051 75.44627 βTCP/Col +BMP-4 54.96161 42.33732 41.9939 43.10462 42.89169 mean 48.31231 52.16034 60.87189 63.48478 65.37434 sd 6.501292 8.03934 13.03156 13.78825 15.12658

% New bone formation through μCT measurement of (Col/Si)@βTCP/collagen+rhBMP2(1 μg) One 2 4 6 8 (Col/Si)@βTCP/collagen+BMP-1 41.57968 49.83504 60.94675 68.67336 73.39701 (Col/Si)@βTCP/collagen+BMP-2 46.55464 50.7691 60.97675 66.01051 75.51372 (Col/Si)@βTCP/collagen+BMP-3 49.0798 41.97737 55.59293 63.37739 74.42508 (Col/Si)@βTCP/collagen+BMP-4 47.16493 54.63935 71.08763 78.98929 83.37954 mean 46.09476 49.30522 62.15102 69.26264 76.67884 sd 3.196517 5.309551 6.473039 6.835386 4.549972

As shown in Tables 3 to 5 and FIG. 10, as a result of comparing the bone regeneration rate by rhBMP2 support after 8 weeks of experiment, the (Col/Si)@βTCP/Col treatment group according to the present invention was in the βTCP/Col treatment group Compared to this, bone regeneration rate increased by about 17%. Specifically, the βTCP/Col+BMP group initially showed faster bone formation and then very slowly bone formation after 4 weeks, whereas the (Col/Si)@βTCP/Col+BMP group according to the present invention was initially Although the rate of formation was slow, new bones were formed more rapidly after 4 weeks. This is because the supported rhBMP2 was released early from the βTCP/Col+BMP group, whereas the (Col/Si)@βTCP/Col+ BMP group showed rhBMP2 from the early stage. It is a result of the increase in the formation of new bones as it is released gradually and continuously until the second half.

Through the above experimental results, the novel marine-derived peptide according to the present invention has excellent silica synthesis activity from silicic acid, and can be used in the manufacture of a composite bone graft material in which silica and collagen are laminated, and the composite bone graft material is Compared to this, it was confirmed that the biomolecule's carrying capacity was improved as well as the effect of continuously releasing the carried molecule, and thus the effect of promoting bone regeneration when transplanted to a damaged bone site was excellent.

<110> Korea University Research and Business Foundation, Sejong Campus <120> Peptides from marine resources capable of silica formation and use thereof <130> 1-1 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> E6Ectp1 <400> 1 Glu Glu Glu Glu Glu Glu Gly Gly Ser Ser Arg Ser Ser Ser His Arg 1 5 10 15 Arg His Asp His His Asp His Arg Arg Gly Ser 20 25

Claims (22)

Peptide having silica synthesis activity consisting of the amino acid sequence of SEQ ID NO: 1. A composition for synthesis of silica, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1. The method according to claim 2,
The composition further comprises a silica precursor, a composition for silica synthesis. The method of claim 3,
The silica precursors are tetramethoxy silane, tetramethyl orthosilicate, tetraethyl orthosilicate, methyl triethoxysilane, phenyl triethoxysilane, dimethyl dimethoxy silane, ethyl triethoxysilane, titania tetraisopropoxide and tetra At least one selected from the group consisting of ethyl germanium, silica synthesis composition. The method according to claim 2,
The composition is a gun that further comprises collagen, a composition for the synthesis of silica. The method according to claim 2,
The composition further comprises hydroxyapatite or tricalcium phosphate, a composition for silica synthesis. The method according to claim 2,
The composition further comprises a physiologically active substance related to bone formation, a composition for silica synthesis. The method of claim 7,
The physiologically active substances related to bone formation include bone morphogenetic protein (BMP), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (Platelet). Derived growth factor, PDGF), epidermal growth factor (Epidermal growth factor, EGF), and fibroblast growth factor (fibroblast growth factor, FGF) that is one or more selected from the group consisting of, silica synthesis composition. A composition for bone grafting or bone filling, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 9,
The composition further comprises a silica precursor, bone grafting or bone filling composition. The method of claim 9,
The composition further comprises collagen, bone grafting or bone filling composition. The method of claim 9,
The composition further comprises hydroxyapatite or tricalcium phosphate, a composition for bone graft or bone filling. The method of claim 9,
The composition is that the hydroxyapatite or tricalcium phosphate surface comprising the peptide consisting of the amino acid of SEQ ID NO: 1, silica precursor and collagen is coated, bone grafting or bone filling composition. The method of claim 9,
The composition further comprises a physiologically active substance related to bone formation, a composition for bone graft or bone filling. The method of claim 14,
The physiologically active substances related to bone formation include bone morphogenetic protein (BMP), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (Platelet). Derived growth factor, PDGF), epidermal growth factor (Epidermal growth factor, EGF), and fibroblast growth factor (fibroblast growth factor, FGF) that is one or more selected from the group consisting of, bone grafting or bone filling composition. The method of claim 14,
The composition is characterized in that the release of the physiologically active substance related to bone formation in a sustained release, bone grafting or bone filling composition. The method of claim 9,
The composition is for dental bone grafting or for bone filling, bone grafting or bone filling composition. Comprising the step of coating a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on the surface of the bone graft material, bone graft manufacturing method. The method of claim 18,
The above method is
(1) coating a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on the bone graft material;
(2) coating a silica precursor on the bone graft material of step 1; And
(3) comprising the step of coating collagen on the bone graft material of step 2, and repeating the steps of (1) to (3) 1 to 20 times. The method of claim 18,
The above method is
(1) coating collagen on the bone graft material;
(2) coating a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on the bone graft material of step 1; And
(3) coating a silica precursor on the bone graft material of step 2; containing, and repeating the steps of (1) to (3) 1 to 20 times, bone graft manufacturing method. The method of claim 19 or 20,
(4) coating a physiologically active substance related to bone formation on the surface of the bone grafting material; which further comprises a method for producing a bone grafting material. The method of claim 21,
The step (4) is to be carried out at the time of melting, bone graft manufacturing method.

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