KR102019939B1 - Sustained release bioactive substance-carrying bone graft and manufacturing method thereof - Google Patents

Abstract

The present invention is a technique for binding a bioactive material to a bone graft using a bio-mineralization reaction formed by using a marine organism-derived gene source that can be used for bone regeneration as a catalytic template. It provides a bone graft material (bone regeneration material) by combining the bioactive material to various carriers through the mineralization by these. In particular, it is possible to produce a bioactive material-carrying bone graft material in which the release of the bioactive substance (growth factor) is controlled in a sustained release form for 4 to 6 weeks after transplantation.

Description

TECHNICAL FIELD [Sustained release bioactive substance-carrying bone graft and manufacturing method thereof]

The present invention relates to a sustained-release physiologically active material-supported bone graft material and a method for producing the same.

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 shortcomings of each individual material. 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, the discovery of enzymes and peptides that biosynthesize silica based on the extracts or genetic information of sponges and diatoms, which are marine organisms, have been discovered. The development of bio-silica composite materials is becoming an issue (non-patent documents 1 to 4). Therefore, it is very important to possess the original technology for securing biosilica materials through biological synthesis methods.

Currently, products containing bone-forming protein are on the market, but swelling due to protein immediate release at the initial stage of transplantation is reported as a form in which a physiologically active substance (growth factor) is absorbed into the bone graft material by simple soaking. And the release rate could not be controlled, so a large amount was lost at the beginning of the transplant and side effects occurred. There is an urgent need to develop a graft material that can minimize side effects caused by mass release of physiologically active substances (growth factors) by reducing the release rate of a large amount of physiologically active substances (growth factors) due to initial immediate release.

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. Poulsen and Kroger 2004; Silica morphogenesis by alternative processing of silaffins in the diatom Thalassiosira pseudonana. J Biol Chem 279: 42993-42999.

Accordingly, the present inventors have studied to solve the conventional problems as described above, as a result of the sustained-release physiology in which a physiologically active substance is bound to the surface of a bone graft material through a silica-forming reaction of a silica-forming organic molecule (peptide or polymer) derived from marine organisms. The present invention was completed by preparing a bone graft material carrying an active material.

Accordingly, an object of the present invention is to provide a sustained-release physiologically active material-supported bone graft material and a method for producing the same, in which a physiologically active material is bound to the surface of a bone graft material through a silica-forming reaction of a silica-forming peptide derived from marine organisms or a polymer.

As a means for solving the above problems, the present invention provides a composition for synthesis of silica, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

As another means for solving the above problems, the present invention is a sustained-release physiologically active material-supported bone graft material and a method for producing the same, in which a physiologically active material is bound to the surface of a bone graft material through a silica-forming reaction of a silica-forming peptide derived from marine organisms or a polymer Provides.

The present invention utilizes genetic resources derived from marine organisms that can be used for bone regeneration, and binds physiologically active molecules to the bone graft material through a biomineral reaction formed by using them as a catalytic template. Bone graft material (bone scaffold) supported by sustained-release physiologically active substances that is easy to immobilize to inorganic substances in biologically mild conditions, that is, in aqueous solutions at room temperature, normal pressure, and near neutral (pH 6 to 8), and release of physiologically active substances (growth factors) is controlled. ).

That is, the bone graft material according to the present invention can minimize side effects due to mass release of physiologically active substances (growth factors) even if it is only able to reduce the release rate of a large amount of physiologically active substances (growth factors) due to initial immediate release, and has an expensive physiological activity. The burden of economic treatment can be reduced by reducing the amount of substance (growth factor) used.

Figure 1 shows the mineralization of HAp (A) and the formation of silica of CBP (B) by a silica-mediated silica-forming reaction to CBP in which six glutamic acids are bound to increase the binding power of SFPs derived from marine organisms to HAp. will be.
Figure 2 shows the mineralization of HAp and immobilization of BMP2 peptide to HAp by the silica formation reaction of SFP derived from marine organisms.
3 is an analysis of the immobilization effect of BMP2 to HAp by the silica formation reaction of SFP derived from marine organisms through gene expression analysis.
4 is an analysis of the bone mineralization analysis using Alizarin red S staining and the effect of immobilizing BMP2 to HAp by the silica formation reaction of SFP derived from marine organisms through the analysis.
Figure 5 shows the immobilization of GFP to HAp bone graft material by the silica-forming reaction of SFP derived from marine organisms.
6 shows the behavior of GFP release after HAp binding of GFP by the silica formation reaction of SFP derived from marine organisms.
7 shows a wettability test of a dental bone graft material.
8 shows the surface change of the silica mineralized bone graft material of a commercial bone graft material D product.
9 shows a schematic diagram of silica mineralization and binding of physiologically active substances to the bone graft material by silica-forming organic molecules derived from marine organisms [(A) Sustained-release physiologically active substances by immobilizing physiologically active substances to the bone graft material by silica formation reaction The process of manufacturing a supported bone graft material, (B) A process of manufacturing a sustained-release physiologically active material-supported bone graft material by loading a physiologically active material on a bone graft material that has been mineralized by silica minerals by organic molecules.
Figure 10 shows the rhBMP2 release behavior until the initial 4 hours (A) and 312 hours (B) after the support of rhBMP2 in the silica mineralized bone graft material by the silica-forming organic molecules derived from marine organisms.
11 is a comparison of the loading amount of rhBMP2 in the bone graft material coated with silica mineralized and polymer.
12 shows the bone regeneration ability of a silica mineralized bone graft material and rhBMP2 supported bone graft material by silica-forming organic molecules derived from marine organisms using a damaged bone model.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

The term "Silica forming peptide (SFP)" or "a peptide having silica synthesis activity" refers to a peptide capable of synthesizing (forming) silica by reacting with a silica precursor.

The term "silica-forming polymer" generically refers to a polymer capable of synthesizing (forming) silica by reacting with a silica precursor.

The term "bioactive substance" refers to a biological substance that promotes healing capacity in the process of tissue regeneration.

The term “silica mineralization reaction” or “silica formation reaction” refers to a reaction of forming silica minerals by organic materials or organic molecules.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for synthesis of silica, comprising a silica synthesis peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The silica synthesis peptide is a peptide CBP represented by SEQ ID NO: 1 (SSRSSSHRRHDHHDHRRGSEEEEEE) in which a glutamic acid residue capable of calcium binding is added to a peptide derived from marine organisms (Ect_p1 in SEQ ID NO: 3). Have.

The composition may further include a silica precursor.

The silica precursors are tetramethoxy silane, tetramethyl orthosilicate, tetraethyl orthosilicate, methyl triethoxysilane, 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.

Meanwhile, the composition for synthesizing silica according to the present invention may further include hydroxyapatite.

Silica may be synthesized by reacting with the silica precursor in an aqueous solution of neutral, that is, pH 6 to 8 under conditions of room temperature and pressure. To explain the silica synthesis method of the present invention in more detail, a silica precursor and a silica-forming peptide dissolved in a Tris-HCl solution or a phosphate buffer solution (pH 6 to 8) are mixed, reacted at room temperature and pressure, and then centrifuged. It can be prepared by separating and precipitating silica.

In addition, the present invention relates to a sustained-release physiologically active material-bearing bone graft material, in which a physiologically active material is bound to the surface of the bone graft material through a silica-forming reaction of a silica-forming peptide derived from marine organisms or a polymer.

The marine organism-derived silica-forming peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, a peptide consisting of the amino acid sequence of SEQ ID NO: 3 derived from Ectocarpus siliculosus, a brown algae (Ect_p1) (Patent No. 10- 1473059, Silica synthetic peptide and its use), Peptide consisting of the amino acid sequence of SEQ ID NO: 4 derived from Ectocarpus siliculosus, a brown algae (Ect_p2) (Patent No. 10-1473059, Silica Synthetic Peptide and Use thereof ), a peptide consisting of the amino acid sequence of SEQ ID NO: 5 derived from Salpingoeca sp. (Sal_p1) (Patent No. 10-1473059, a silica synthetic peptide and its use), SEQ ID NOs: 6 and 7 derived from Volvox , And peptides consisting of the amino acid sequence of 8 (Vol_p1, Vol_p2, and Vol_p3) (Patent No. 10-1473059, silica synthetic peptides and uses thereof) may be one or more selected from the group consisting of, but is not limited thereto.

The silica-forming polymer derived from marine organisms is chitosan, chitosan oligosaccharide, collagen, hyaluronic acid, alginic acid, alginic acid alkali metal salt, fucoidan, pullulan, cellulose, hydroxymethylpropylcellulose, dextrin, dextran, polydextrose, xanthan gum, It may be one or more selected from the group consisting of gellan gum and carrageenan, but is not limited thereto.

The silica formation reaction may be performed by adding a silica precursor. The silica precursors are tetramethoxy silane, tetramethyl orthosilicate, tetraethyl orthosilicate, methyl triethoxysilane, 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 physiologically active substances are type 2 bone morphogenic protein (Bone Morphogenic Protein-2, BMP2), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and fibroblast growth. Factors (fibroblast growth factor, FGF), transforming growth factor (Transforming growth factor beta 1, TGF-beta1), and epidermal growth factor (Epidermal growth factor, EGF) may be one or more selected from the group consisting of, but is not limited thereto.

The bone graft material is a bone graft material made of bio-derived bone mineral powder and porous block, synthetic hydroxyapatite powder and porous block, tricalcium phosphate powder and porous block, monocalcium phosphate powder and porous block, and silicon dioxide (silica) , It may be one or more selected from the group consisting of bone-filled implants consisting of a mixture of silica and polymer, but is not limited thereto.

In addition, the present invention relates to a method for producing a bone graft material carrying a sustained-release bioactive material, comprising the step of reacting a silica-forming peptide or a polymer, a silica precursor, and a physiologically active material to the bone graft material for silica formation.

The silica formation reaction may be performed by adding a silica precursor. Specifically, the silica formation reaction is 0.1 to 40 parts by weight (preferably 1 part by weight) of a silica precursor dissolved in a Tris-HCl solution or a phosphate buffer solution (pH 6 to 8) based on 100 parts by weight of the bone graft material. To 30 parts by weight), 0.001 to 0.1 parts by weight of a physiologically active substance, and 0.01 to 5 parts by weight of a silica-forming peptide or polymer (preferably 0.01 to 2 parts by weight), and at room temperature and atmospheric pressure. Shake reaction.

More specifically, 100 parts by weight of the bone graft material was added to ethanol, stirred, and then centrifuged to remove ethanol and washed with distilled water. Then, phosphate buffered saline (PBS) is added, and 0.01 parts by weight to 5 parts by weight (preferably 0.01 parts by weight to 2 parts by weight) of a silica-forming peptide or polymer derived from marine organisms is added thereto, followed by stirring to form a silica-forming peptide or After allowing the polymer to bind to the bone graft material, 0.001 to 0.1 parts by weight of a physiologically active substance is added, and 0.1 to 40 parts by weight of a silica precursor (preferably 1 to 30 parts by weight) is added and stirred. Then, unbound molecules and unreacted substances are removed through centrifugation, and the recovered bone graft material is washed again with PBS. Again, PBS was added and the preparation of the silica-peptide or polymer-bioactive material-bone graft material complex was repeated once to three times. Finally, the prepared complex is lyophilized and stored. All processes are carried out at room temperature, pressure, neutral and sterile conditions.

Figure 9A shows an example of the manufacturing process of a sustained-release physiologically active material-supported bone graft material using a silica-forming peptide or a polymer derived from marine organisms.

In addition, the present invention comprises the steps of preparing a silica-silica-forming peptide or a polymer-bone graft material complex by reacting silica-forming peptides or polymers and silica precursors derived from marine organisms to a bone graft material; And

Supporting a physiologically active substance on the complex

It relates to a method for producing a sustained-release physiologically active material-supported bone graft material comprising a.

More specifically, 100 parts by weight of the bone graft material is added to ethanol and stirred, followed by adding 0.01 parts by weight to 5 parts by weight of a silica-forming peptide or a polymer (preferably 0.01 parts by weight to 2 parts by weight) derived from marine organisms, followed by stirring. 0.1 to 40 parts by weight of a silica precursor (preferably 1 to 30 parts by weight) was added thereto, and after stirring at room temperature, 0.01 to 1 part by weight of ammonia water was added, followed by stirring for 12 hours or more. After stirring, unbound molecules and unreacted substances are removed through centrifugation, and the recovered bone graft material is washed again with distilled water. Put it in ethanol again and repeat the preparation of the silica-peptide or polymer-bone graft material complex once to three times. Finally, the prepared composite is freeze-dried and stored. All processes are carried out at room temperature, pressure, neutral and sterile conditions. 0.001 parts by weight to 0.1 parts by weight of a physiologically active substance is supported on the dried composite material thus prepared. The physiologically active substance may be carried by a commonly used support method such as a physical adsorption method (ex, immersion method) of the physiologically active substance on the bone graft material immobilized by a silica formation reaction.

FIG. 9B shows an example of a manufacturing process of a sustained-release physiologically active material-carrying bone graft material in which a physiologically active material is supported on a complex prepared by reacting a silica-forming peptide or a polymer derived from a marine organism to form a silica.

All the contents described above with respect to the sustained-release bioactive material-supported bone graft material may be applied or applied mutatis mutandis to the method of manufacturing the sustained-release bioactive material-supported bone graft material.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

[ Example ]

Manufacturing example One: CBP Produce

Six glutamic acids were added to the silica-forming peptide to enhance the binding ability to HAp. Peptides were synthesized and manufactured with a purity of 90% or more by requesting Genscript (GenScript USA Inc. NJ, USA) or Peptron (Peptron, Dajeon, Korea).

Manufacturing example 2: CBP Confirmation of silica formation

In order to confirm the silica-forming ability of CBP (SSRSSSHRRHDHHDHRRGSEEEEEE, SEQ ID NO: 1) to which a calcium-binding glutamic acid residue was added to Ect_p1, 1 ml of a phosphate buffer solution containing 0.1 mg/ml of CBP was reacted with 0.1 g of HAp overnight. Then, after adding 20 µl of a 1M concentration of hydrolyzed silica precursor (tetramethoxysilane, TMOS), the mixture was stirred for 2 hours. After stirring, unbound molecules and unreacted substances were removed through centrifugation, and the recovered HAp was washed again with distilled water 3 times. Again, 1 ml of PBS was added and the preparation of the peptide-silica-HAp complex was repeated. At this time, 50 µl of 1 mg/ml SFP was added, stirred for 30 minutes, and 10 µl of 1 M hydrolyzed silica precursor was added, followed by stirring for 2 hours. After stirring, unbound molecules and unreacted substances were removed through centrifugation, and the recovered HAp was washed again with distilled water 3 times. Finally, the prepared complex was lyophilized and stored. All procedures were performed in a sterile condition. As a control, a silica reaction was induced in the presence of a silica precursor under the same conditions without CBP during the silica reaction. The state in which silica was formed on the surface of HAp was confirmed by SEM image (FIG. 1A) and the amount of silica precipitated on the surface was quantified using the molybdate-blue method (FIG. 1B). To briefly describe the quantification method, a silica mineralized material was first dissolved in 1M NaOH at 95° C. for 30 minutes, and then 1M HCl was added to neutralize. To this mixture, a 2 mg/ml solution of ammonium heptamolybdate dissolved in 0.1N sulfuric acid was added in the same volume, and allowed to stand for 5 minutes, and then the same volume of 50 mg/ml oxalic acid solution and the same amount of a reduction buffer (100 mM ascorbic acid solution) was sequentially added. The absorbance of this mixture was measured at 700 nm using a micro plate reader (Infinite M200 PRO, TECAN, Austria). As the standard silica solution, sodium metasilicate was used. As shown in FIG. 1, it was confirmed that silica precipitated on the surface of the HAp bone graft material was clearly observed in the presence of CBP, and a 10-fold or more silica precipitation reaction was induced through quantification.

Example One: HAp BMP2 Peptide Immobilized and Molecular Mediated Silica Forming Silica from Marine Organisms In mineralization The effect of promoting bone regeneration

In 100 μl of 100 mM hydroxyapatite (HAp) powder (~200 nm size), 6 glutamic acid amino acid residues synthesized from Genscript (GenScript USA Inc. NJ, USA) to increase the binding power to HAp in 100 μl are BMP2 Prepare a BMP2 peptide (KIPKASSVPTELSAISTLYLGGKGSSEEEEEE, SEQ ID NO: 9) attached to the end of the peptide carboxyl group, add 5 µl of BMP2 peptide at a concentration of 2 mg/ml, conduct a pre-reaction for 10 minutes to bind to HAp, and then add 1 mg/mL of sea 50 µl of biologically-derived silica-forming peptide (SFP) and 20 µl of hydrolyzed 1 M tetramethoxysilane (TMOS) were added, and a final volume of 500 µl was shaken overnight in a 1×PBS (phosphate buffered saline) reaction system. At this time, three types of SFP were used as the SFP. The three types of SFP are CBP (SSRSSSHRRHDHHDHRRGSEEEEEE, SEQ ID NO: 1), R5 (SSKKSGSYSGSKGSKRRIL, SEQ ID NO: 2), Ect_p1 (SSRSSSHRRHDHHDHRRGS, SEQ ID NO: 3).

As a control, only 100 µl of 100 mM HAp, 100 µl of 100 mM HAp and 5 µl of 2 mg/ml BMP2 peptide were added, and 100 µl of 100 mM HAp, 5 µl of 2 mg/ml BMP2 peptide, and When 20 μl of the decomposed 1M TMOS was added, the final volume was shaken overnight in a 1×PBS reaction system having a final volume of 500 μl. After completion of the reaction, the reaction solution was centrifuged at 13000 rpm for 2 minutes, the reaction solution was removed, the precipitate was recovered, suspended with distilled water, and centrifuged again to remove the supernatant. This method was repeated three times to remove components not bound to HAp, and the precipitate was suspended in 70% ethanol and left to stand overnight to sterilize. Then, the ethanol was removed through centrifugation again, and the washing process using centrifugation was performed with sterilized distilled water. Repeated 3 times. The amount of precipitation of mineralized HAp by SFP increased by 1.5 times compared to the control group, and the sedimentation rate was in a suspended state compared to that of the reaction control group without SFP and then slowly settled (FIG. 2).

To see the effect on osteoblast differentiation, MC3T3 E1 cells, a preosteoblast, ^{were inoculated with 5 × 10 5} cells per well in a 12 well plate, and cultured by adding 100 µl of the prepared HAp in 500 µl suspension for one day after cultivation. As a culture medium, 10mM beta-glycerophosphate and 50 μM ascorbic acid were added to the same medium after 3 days incubation in alpha-MEM medium containing 10% Fetal bovine serum (FBS) and 1% antibiotic. It was cultured by changing. After culturing for 7 days and 14 days, gene expression was analyzed and shown in FIG. 2. As a result, it was observed that the expression of bone differentiation marker ALP (alkaline phosphatase) and osteocalcin involved in bone mineralization were significantly increased in the group treated with BMP2 immobilized HAp due to silica mineralization. Above all, when BMP2 was simply added to HAp, BMP2 was easily removed during the washing process, and thus there was no difference from the group treated with only HAp (FIG. 3). Analysis of the bone mineralization reaction with Alizarin Red S after 3 weeks showed that the amount of calcium precipitates stained red with Alizarin Red S significantly increased in the group treated with silica and BMP2 rather than HAp alone. It could be observed (Fig. 4). Bone mineralization was further increased when HAp was treated through silica mineralization using CBP and Ect_p1 than the silica formation reaction by the R5 peptide used as a control, which made the assumption that the functionality of the silica-forming molecule derived from marine organisms used in the present invention is superior. . In the case of CBP and Ect_p1, unlike R5, it was estimated that the presence of acidic amino acids capable of calcium binding promoted bone mineralization.

Example 2: Silica of silica-forming molecules derived from marine organisms Mineralization Bone of green fluorescent protein used Graft material Attachment and release

In order to confirm the binding of functional molecules to the bone graft material by silica mineralization reaction of silica-forming molecules, green fluorescent protein (GFP) is immobilized on the surface of the bone graft material by silica mineralization. It was compared with the method of bonding to the bone graft material by the immersion method (FIG. 5). The reaction was carried out with 50 μg of GFP, 10 μg of CBP, and 10 mg of bone graft material (HAp) in 100 μl of 50 mM phosphate buffer (pH 7.6) with a final hydrolyzed TMOS concentration of 10 mM and shaking for 30 minutes. Reacted. As shown in FIG. 4, the control is a HAp bone graft material without any treatment, and the second line of bone graft material is immersed in a GFP solution for adsorption, and the third line is the HAp bone graft material, silica-forming molecules, and GFP. This is a method in which GFP is bound to the bone graft material through silica mineralization. As a result of confirming the GFP bound to the bone graft material with a fluorescence microscope, the simple immersion method did not show much difference from the bone graft material without GFP, but the binding method by silica mineralization clearly observed that GFP was immobilized on the bone graft material. Could.

To check the release rate of GFP bound to HAp, 1 μmoles of HAp, 115 μg of GFP were mixed 100 μl of 50 mM phosphate buffer (pH 7.6), 1 μmoles of HAp, 115 μg of GFP, and acid hydrolyzed. 100 μl of 50 mM phosphate buffer (pH 7.6) mixed with 5 μmoles of TMOS, and 100 μl of 50 mM of 1 μmoles of HAp, 115 μg of GFP, 10 μg of CBP and 5 μmoles of acid hydrolyzed TMOS Phosphoric acid buffer solutions (pH 7.6) were prepared, respectively, to bind GFP and HAp through a silica formation reaction, and then remove unbound GFP through centrifugation. After centrifugation, the unbound GFP concentration was calculated and the amount of bound protein was calculated by subtracting it from the amount of protein used.When HAp and GFP were simply mixed, only 3% of GFP was bound to HAp by Sol-Gel reaction without a catalyst. About 65% of GFP was bound to HAp in the case where it was bound to HAp through the silica formation reaction that was formed and the case where it was bound to HAp through the silica formation reaction that was CBP-mediated (FIG. 6A). As a result of measuring the amount of GFP extracted each time by separating GFP from HAp through centrifugation, in the case of simple adsorption, GFP was completely released in 3 times, but when bound through silica, GFP was rapidly released up to 4 times, and then slowly released. It became (FIG. 6B). When the silica-forming CBP was used, the initial release rate was reduced by about 40% compared to the silica bonding method by a simple Sol-Gel reaction.

Example 3: Bone by silica-forming organic polymer derived from marine organisms Graft material Silica Mineralization And increase the wettability of bone graft material through this

The silica mineralization reaction of the bone graft material by organic polymers and biopolymers derived from marine organisms was performed using 1 g of synthetic bone and heterogeneous bone graft material (A: Daewoong Pharmaceutical Novosis, B: Oscotec InduCera, C: Naivec OCS-B, D: Genos OSTEONII. ) Was immersed in 10 ml of 100% ethanol, shaken at room temperature for 30 minutes, 1 ml of 1% chitosan solution (chitosan 10 mg) was added, and shaken at room temperature for 30 minutes. Thereafter, 200 µl (200 mg) of TEOS was added and the mixture was shaken at room temperature for 2 hours, and then 500 µl of 25% ammonia water was added thereto, followed by shaking at room temperature overnight. After removing the reaction solution through centrifugation, washing the bone graft material 3 times with distilled water, 10 ml of 100% ethanol was added again, and the reaction of the previous day was repeated. At this time, 1 ml of 1% chitosan solution (10 mg of chitosan) was added, stirred for 30 minutes, TEOS 100 µl (100 mg) was added, stirred for 2 hours at room temperature, and then 200 µl of 25% ammonia water was added, followed by shaking reaction overnight. Unbound molecules and unreacted substances were removed through centrifugation, and the recovered HAp was washed again with distilled water 3 times. Finally, the prepared composite was freeze-dried. When only silica was treated, silica was formed only by ammonia without chitosan, which is a polymer in the above method. The degree of increase in wettability of the bone graft material through silica mineralization of the commercially available bone graft material was confirmed through mixing with blood.

In FIG. 7, as a result of comparing the wettability of the blood after dropping a drop of mouse blood for 4 types of products, products A and B were well absorbed by the bone graft material as soon as a drop of blood was added, and product C was It was not absorbed immediately, but slowly absorbed, and product D did not mix over time. As a result of comparing the wettability of products A and D after silica mineral treatment and silica mineral treatment with chitosan, an organic polymer derived from marine organisms, there was no difference in blood absorption when A was not treated or treated, whereas D did not show any difference in blood absorption. When the silica mineral treatment and the silica mineral treatment through chitosan were treated, blood was immediately absorbed. In addition, in the case of silica mineral treatment through chitosan, it could be seen that agglomeration of the bone graft material by blood in both A and D occurs better (FIG. 7). The dental bone graft material is soaked in saline or blood before transplantation, and then transplanted. If it is well fused with blood, the treatment for transplantation can be easy, and the better it is, the better it is. It was confirmed that wettability was increased when silica was introduced to the surface through a silica mineralization reaction to the bone graft material, and as shown in FIG. 6, the surface of the bone graft material through silica mineralization by chitosan, a polymer derived from marine organisms, was observed through a scanning electron microscope. It was thought that the surface area increased due to the increase in the roughness of the cells, which resulted in not only mixing with blood, but also improving the agglomeration phenomenon (FIG. 8).

Example 4: Silica by organic molecules that form silica from the ocean Mineralized Support and release of active molecules of bone graft material

According to the method shown in FIG. 9A, 1 g of hydroxyapatite (HAp), which is an artificial bone porous granule, was added to 10 ml of 100% ethanol, stirred with a magnetic rod for 30 minutes, and then centrifuged to remove the ethanol and centrifuged 3 times with distilled water. HAp was washed using the separation method. Then, 9 ml of PBS was added, and 1 ml (CBP 1 mg) of silica-forming peptide (CBP) added with a calcium-binding moiety at a concentration of 1 mg/ml was added thereto and stirred for 30 minutes to allow the peptide to bind to HAp, and produced by Genscript. 0.5 ml (0.25 mg) of rhBMP2 was added, and 0.2 ml (30 mg) of 1M hydrolyzed TMOS was added, followed by stirring for 2 hours. After stirring, unbound molecules and unreacted substances were removed through centrifugation, and the recovered HAp was washed 3 times with PBS again. Again, PBS was added and the preparation of the CBP-silica-rhBMP2-HAp complex was repeated. At this time, 0.5 ml (SFP 0.5 mg) of 1 mg/ml SFP was added, stirred for 30 minutes, 0.5 ml (0.25 mg) of rhBMP2 at a concentration of 0.5 mg/ml was added, and 0.1 ml of the hydrolyzed silica precursor TMOS at a concentration of 1 M ( 15 mg) was added and stirred for 2 hours. After stirring, unbound molecules and unreacted substances were removed through centrifugation, and the recovered HAp was washed 3 times with PBS again. Finally, a freeze-dried bone graft material (CBP-rhBMP2-SiO2) was prepared from the prepared composite. All processes were performed at room temperature, normal pressure, neutral and sterile conditions.

When the bone graft material is produced by the method A of FIG. 9, the bone graft material can be produced only by the silica formation reaction by CBP without rhBMP2 (CBP-SiO2), and rhBMP2 at a concentration of 0.5 mg/ml per 1 g of the bone graft material thus made is 1 A bone graft material (CBP-SiO2+rhBMP2) was also prepared after physical adsorption by adding ml (0.5 mg) and removing unbound rhBMP2. A bone graft material (HA+rhBMP2) in which rhBMP2 was physically adsorbed to untreated HAp was prepared as a control.

According to the method shown in FIG. 9B, 1 g of artificial bone hydroxyapatite (HAp) as collagen was added to 10 ml of 100% ethanol, stirred with a magnetic bar for 30 minutes, and then 1 ml of collagen (collagen 10 mg) at a weight ratio of 1%. After adding, it was stirred for 30 minutes. After adding 0.2 ml (0.2 g) of TEOS (99% to 100%), stirring at room temperature for 2 more hours, 0.5 ml of 25% weight ratio of ammonia water was added thereto, followed by stirring for 12 hours or more. After stirring, unbound molecules and unreacted substances were removed through centrifugation, and the recovered HAp was washed again with distilled water 3 times. Again, 10 ml of 100% ethanol was added and the silica-collagen-HAp complex was prepared. At this time, 1 ml of collagen (10 mg of collagen) in a 1% weight ratio was added and stirred for 30 minutes. 0.1 ml (0.1 g) of TEOS (99% to 100%) was added thereto, followed by stirring at room temperature for 2 more hours, and then 0.2 ml of 25% weight ratio of ammonia water was added, followed by stirring for 12 hours or more. After stirring, unbound molecules and unreacted substances were removed through centrifugation, and the recovered HAp was washed again with distilled water 3 times. All processes were performed at room temperature, normal pressure, neutral and sterile conditions.

Finally, the prepared collagen=-silica-bone graft material complex was freeze-dried to prepare a collagen-silica-coated bone graft material, and 1 ml (0.5 mg) of rhBMP2 at a concentration of 0.5 mg/ml per 1 g graft material was added and physically adsorbed. After the unbound rhBMP2 was removed, a bone graft material (Collagen-SiO2+rhBMP2) was prepared and the release patterns of rhBMP2 were compared (FIG. 10).

When bound to the untreated control hydroxyapatite bone graft (HA+rhBMP2), more than 70% of the supported rhBMP2 was released within 30 minutes, and more than 80% was released in 3 hours, and the CBP-mediated silica formation reaction was performed. The silica-coated CBP-SiO2 graft material (CBP-SiO2+rhBMP2) formed through releases 50% rhBMP2 in 30 minutes and 65% rhBMP2 in 3 hours, reducing the initial release amount by 18%. When rhBMP2 was encapsulated with silica and immobilized on a bone graft material (CBP-rhBMP2-SiO2), the initial release amount was further reduced up to 3 hours, reducing it to about 75%. When collagen and silica were used together and coated (Collagen-SiO2+rhBMP2), the amount of rhBMP2 released during the initial 3 hours decreased by 50% compared to the control.

For each 50 mg of bone graft material, 0.5mg/ml of rhBMP2 was added to 100 µl and bound to the bone graft material for 30 minutes. The concentration of unbound rhBMP2 was measured by the Bradford method by centrifugation to calculate rhBMP2 bound to the bone graft material (BG). I did. The amount of rhBMP2 bound to the uncoated control HAp was 0.35 μg per mg bone graft material, 0.73 μg for silica-coated bone graft material (CBP-SiO2@HAp) by silica formation reaction by CBP, and 0.66 μg for collagen only (Collagen@HAp). , Collagen-silica-treated bone graft material (Collagen-SiO2@HAp) showed 0.75 μg (FIG. 11). The physical adsorption power of rhBMP2 was increased through silica coating and polymer bonding to the bone graft material. It was thought that the increase in adsorption power decreased the initial immediate release rate of rhBMP2 compared to the HA control.

Example 5: Silica from marine organisms-derived silica-forming organic molecules Mineralized Bone graft Bone regeneration ability

The skull of Sprague-Dawley rats purchased from Orient Bio (Orient Bio Co., Ltd., Seongnam City, Korea) with the approval of the Korea University Animal Experimental Ethics Committee was cut using a machine into a circular shape of 0.8 cm diameter, and then a graft material was inserted to repair the damaged bone. The recovery pattern was confirmed through microCT scans over 3-12 weeks. An untreated bone graft material was used as a control, and a graft material coated with silica (CBP-SiO2) on the surface of the HAp through a CBP-mediated silica-forming reaction, and rhBMP2 was added to form silica during the CBP-mediated silica-forming reaction. Fig. 12 shows microCT photographs of the bone formation process at the damaged site after 3, 6, 9, and 12 weeks by comparing the bone formation ability of the bone graft material (CBP-BMP2-SiO2) immobilized on the surface. In the control group, new bone formation grew slightly at the edge of the damaged bone and did not fill the inside, but it was confirmed that new bone was formed in the CBP-SiO2 treated group and CBP-BMP2-SiO2 and entered the damaged area. When BMP2 was immobilized, the rate of new bone formation appeared faster.As a result of calculating the area of the new bone using the Image J program, CBP-SiO2 was 29% at week 3, 47% at week 6, 50% at week 9, In addition, 82% of new bones were formed at week 12, and 49% of BMP2 immobilized bones were formed at week 3, 63% at week 6, 89% at week 9, and 92% at week 12.

<110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Sustained release bioactive substance-carrying bone graft and manufacturing method thereof <130> P1713C0254 <150> KR 10-2016-0042763 <151> 2016-04-07 <160> 9 <170> KopatentIn 2.0 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> CBP <400> 1 Ser Ser Arg Ser Ser Ser His Arg Arg His Asp His His Asp His Arg 1 5 10 15 Arg Gly Ser Glu Glu Glu Glu Glu Glu 20 25 <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> R5 <400> 2 Ser Ser Lys Lys Ser Gly Ser Tyr Ser Gly Ser Lys Gly Ser Lys Arg 1 5 10 15 Arg Ile Leu <210> 3 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Ect_p1 <400> 3 Ser Ser Arg Ser Ser Ser His Arg Arg His Asp His His Asp His Arg 1 5 10 15 Arg Gly Ser <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Ect_p2 <400> 4 Ser Ser Lys Lys Ser Gly Glu Arg His His Arg Ser Ala 1 5 10 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Sal_p1 <400> 5 Cys Gly Arg Arg Arg Gly Gly Arg Gly Gly Arg Gly Arg Gly Gly Cys 1 5 10 15 Gly Arg Arg Arg 20 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Vol_p1 <400> 6 Ser Gly Arg Arg Arg Gly Ser Arg Arg Arg Gly Ser Arg Arg Arg 1 5 10 15 <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Vol_p2 <400> 7 Ser Arg Leu Arg Gly Arg Arg Arg Arg Leu Ser Pro Gly Arg 1 5 10 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Vol_p3 <400> 8 Ser Ser His Arg His His His Asp His His Asp His His His 1 5 10 <210> 9 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> BMP2 <400> 9 Lys Ile Pro Lys Ala Ser Ser Val Pro Thr Glu Leu Ser Ala Ile Ser 1 5 10 15 Thr Leu Tyr Leu Gly Gly Lys Gly Ser Ser Glu Glu Glu Glu Glu Glu 20 25 30

Claims (12)

A sustained release bioactive substance-carrying bone graft material, in which a bioactive substance is bonded to a surface of a bone graft material through a silica formation reaction of a marine organism-derived silica-forming peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a silica precursor.
delete delete The method of claim 1,
The silica precursors are tetramethoxy silane, tetramethyl orthosilicate, tetraethyl orthosilicate, methyl triethoxysilane, phenyl triethoxysilane, dimethyl dimethoxy silane, ethyl triethoxysilane, titania tetraisopropoxide and tetra A sustained release bioactive substance-carrying bone graft material selected from the group consisting of ethyl germanium.
The method of claim 1,
The physiologically active substance is Bone Morphogenic Protein-2 (BMP2), Platelet derived growth factor (PDGF BB), Vascular endothelial growth factor (VEGF), Fibroblasts Support for sustained release bioactive substances, one or more selected from the group consisting of fibroblast growth factor (FGF), transforming growth factor beta 1 (TGF-beta1), and epidermal growth factor (EGF) Bone graft.
The method of claim 1,
The bone graft material is a bone graft material consisting of powder and porous block of bio-derived bone mineral, powder and porous block of synthetic apatite hydroxide, powder and porous block of tricalcium phosphate, powder and porous block of monocalcium phosphate, silicon dioxide (silica) And, one or more selected from the group consisting of a bone filling implant consisting of a mixture of silica and a polymer, sustained release bioactive material carrying bone graft.
A method for producing a sustained release bioactive material-carrying bone graft material, comprising the step of forming a silica-containing reaction of a marine organism-derived silica-forming peptide, a silica precursor, and a bioactive material comprising the amino acid sequence of SEQ ID NO: 1 on a bone graft.
Preparing a silica-silica-forming peptide complex by subjecting the bone graft to a silica-forming silica-forming peptide comprising an amino acid sequence of SEQ ID NO: 1, and a silica precursor; And
Supporting a bioactive substance in the complex
Comprising a, Method for producing a sustained release bioactive material carrying bone graft material.
delete The method according to claim 7 or 8,
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, a method for producing a sustained release bioactive substance carrying bone graft material.
The method according to claim 7 or 8,
The physiologically active substance is Bone Morphogenic Protein-2 (BMP2), Platelet derived growth factor (PDGF), Vascular endothelial growth factor (VEGF), Fibroblast growth Sustained release bioactive substance-bearing bone, which is one or more selected from the group consisting of fibroblast growth factor (FGF), transforming growth factor beta 1 (TGF-beta1), and epidermal growth factor (EGF) Method of Making Implants.
The method according to claim 7 or 8,
The bone graft material is a bone graft material consisting of powder and porous block of bio-derived bone mineral, powder and porous block of synthetic apatite hydroxide, powder and porous block of tricalcium phosphate, powder and porous block of monocalcium phosphate, silicon dioxide (silica) , The method for producing a sustained release bioactive material-carrying bone graft material, which is at least one selected from the group consisting of a bone filler graft comprising a mixture of silica and a polymer.

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