KR100676945B1 - Bone graft and scaffolding materials immobilized with osteogenesis enhancing peptides on the surface - Google Patents

Abstract

The present invention relates to a bone graft material and tissue engineering scaffold fixed to the bone tissue formation enhancing peptide on the surface, and more particularly to a bone graft material and tissue engineering scaffold fixed on the surface derived peptides and / or tissue growth factor-derived peptide will be.

The bone graft material and support for tissue engineering of the present invention can maximize the tissue regeneration ability by promoting the transfer, proliferation and differentiation of cells involved in regeneration by the bone tissue formation promoting peptide attached to the surface. In addition, peptides immobilized on the surface have a low molecular weight, which reduces the risk of immune response when applied in the body, and can exist in a stable form in the body, which enables the continuation of the drug, and facilitates surgery during periodontal tissue, alveolar bone regeneration, and other bone tissue regeneration. Excellent convenience and high therapeutic effect can be expected.

Tissue growth factor, bioactive peptide, immobilization, bone graft material, support, bone regeneration

Description

Bone Graft And Scaffolding Materials Immobilized With Osteogenesis Enhancing Peptides On The Surface}

Figure 1 shows the results of the electronic surface analysis of the peptide immobilized on the bone graft material according to the invention, Figure 1a shows the surface analysis results of the bone graft material is not fixed peptide, Figure 1b is a peptide containing sulfur is fixed It shows the surface analysis results of the bone graft material.

Figure 2 is a confocal scanning micrograph showing the cell adhesion pattern of bone graft material of the present invention, Figure 2a shows the cell adhesion pattern on the bone graft material is not fixed peptide, Figure 2b and Figure 2c is BMP, respectively And cell adhesion to the bone graft surface to which the peptide derived from bone sialoprotein is fixed.

Figure 3 is a graph quantitatively analyzing the cell adhesion of the surface of the bone graft immobilized peptide of the present invention.

4 is a photograph showing the result of measuring the amount of smad 1,5,8 protein, which is a bone tissue differentiation marker, in the cells by culturing cells for a certain period of time by dispensing the cells in the bone graft material of the present invention. (Con: bone graft material with no peptide immobilized; BMP: bone graft material with BMP-derived peptide fixed; and BSP: bone graft material with peptide derived from bone sialoprotein).

Figure 5 is a photograph showing the bone regeneration in the skull defect of the rabbit using the bone graft material according to the invention, Figure 5a shows the bone regeneration in the skull defect of the bone graft material (HA) is not fixed peptide , Figure 5b shows the bone regeneration in the skull defects of the bone graft material (HA) fixed peptide of the present invention New Bone shows the new bone generated by the peptide fixed on the bone graft surface.

The present invention relates to a bone graft material and a tissue engineering support for fixing bone tissue formation peptides on the surface, and more specifically, a bone graft material and a tissue engineering support for fixing the cell adhesion-derived peptide and / or tissue growth factor-derived peptide to the surface (hereinafter , Support).

The periodontal tissue supporting the tooth is composed of the alveolar bone, the periodontal ligament tissue, epithelial tissue and connective tissue that constitute the periodontal membrane between the alveolar bone and the tooth. The loss of alveolar bone due to the progression of periodontitis is accompanied by the loss of periodontal ligament tissues, and normal recovery of the alveolar bone and periodontal ligament tissues is impossible due to overgrowth of connective tissues in the tissue region lost after periodontitis treatment. In addition, even if new bone is produced, periodontal ligament tissues are not normally differentiated, which can lead to tooth loss.

Therefore, in order to solve this problem, attempts to induce complete tissue regeneration or neoplasia using an artificial shielding membrane along with autografting with alveolar bone regeneration have been actively made. In addition, tissue engineering culture scaffolds are also utilized as bone graft material for bone tissue regeneration. Since the introduction of bone graft materials and scaffolds in recent decades, the effective induction of periodontal and bone tissue regeneration (Camelo, M. et.al, International J. Periodont. Restorative Dent . 21: 109, 2001) Various materials have been used as tissue scaffolds for bone grafts and tissue regeneration purposes, including bone powder particles composed of bovine bone.

On the other hand, in order to improve the regeneration efficiency of the bone graft material and the support, research is being carried out to attach substances capable of improving tissue regeneration to the bone graft material and the support. Among these, extracellular matrix or specific tissue growth factors have been reported to be excellent in repairing and regenerating tissue damage in vivo, and their excellent tissue regeneration ability has been confirmed in many clinical trials. have.

However, it has been pointed out that most of these extracellular matrix and growth factors are relatively expensive and have high molecular weight of several tens of kDa. In particular, since it is lost within a few minutes within the body, it is pointed out that a large amount should be administered in order to obtain a desired therapeutic effect, and that side effects caused by this are caused.

Recently, attempts have been made to alleviate the shortcomings of simple application by incorporating the tissue growth factors into the scaffolds used in bone graft materials and tissue engineering techniques for bone regeneration. The effect was proved to the extent. However, these bone grafts or scaffolds themselves are in a state in which tissue growth factors are physically mixed, resulting in a burst release upon initial application, which makes it difficult to maintain an effective concentration during treatment.

Thus, the present inventors have made efforts to solve the problems of the prior art, as a result, the bone growth material and the support on which the peptide of the active site of the tissue growth factor and extracellular matrix protein that can obtain the tissue regeneration effect attached to the surface is low concentration It was confirmed that only the peptide of the stable and sustained pharmacological effect was completed the present invention.

It is an object of the present invention to provide a bone graft material and a support for tissue engineering that can obtain a desired tissue regeneration effect even at a low concentration of the cell adhesion-derived peptide and / or tissue growth factor-derived peptide dose.

In order to achieve the above object, the present invention provides a support for bone graft material and tissue engineering in which the cell adhesion inducing peptide and / or tissue growth factor-derived peptide is fixed to the surface.

That is, the present invention by fixing the cell adhesion inducing peptide and / or tissue growth factor-derived peptide having a pharmacological activity on the surface of the bone graft material for bone tissue regeneration and support for tissue engineering, to have pharmacological activity to increase bone tissue and other tissue regeneration efficiency Provided are bone graft materials and supports.

The cell adhesion-derived peptide or tissue growth factor-derived peptide is an amino acid sequence of an active site separated from a physiologically active cytokine (cytokine) to maintain an active structure through chemical modification after extraction.

Specifically, the cell adhesion inducing peptide is a peptide having an amino acid sequence of the commonly used RGD, more preferably CGGRGDS (SEQ ID NO: 1) or CGGVACDCRGDCFC (designed to maintain structurally stable amino acid sequence of the RGD) The peptide of SEQ ID NO: 2) is used.

In addition, the tissue growth factor-derived peptide is used by chemically synthesized by identifying that derived from the active region of the tissue growth factor, specifically (a) bone morphogenetic protein (BMP) -2, 4 and 6 Amino acid sequences at positions 2-18 of each of the amino acid sequences of [BMP-2 (SEQ ID NO: 3), BMP-4 (SEQ ID NO: 4), and BMP-6 (SEQ ID NO: 5)]. Amino acid sequence 16-34 (SEQ ID NO: 6), amino acid sequence 47-71 (SEQ ID NO: 7), amino acid sequence 73-92 (SEQ ID NO: 8), amino acid sequence 88-105 (SEQ ID NO: 9 ), An amino acid sequence of positions 283-302 (SEQ ID NO: 10), an amino acid sequence of positions 335-353 (SEQ ID NO: 11), and an amino acid sequence of positions 370-390 (SEQ ID NO: 12); Amino acid sequence at positions 74-93 (SEQ ID NO: 13), amino acid sequence at positions 293-313 (SEQ ID NO: 14), amino acid sequence at positions 360-379 (SEQ ID NO: 15), and amino acid sequence at positions 382-402; (SEQ ID NO: 16) Amino acid sequence at positions 91-110 of the BMP-6 (SEQ ID NO: 17), amino acid sequence at positions 397-418 (SEQ ID NO: 18), amino acid sequence at positions 472-490 (SEQ ID NO: 19), and 487- Amino acid sequence at position 510 (SEQ ID NO: 20), amino acid sequence at position 98-117 (SEQ ID NO: 21) at BMP-7, amino acid sequence at position 320-340 (SEQ ID NO: 22), amino acid sequence at position 390-409 (SEQ ID NO: 23) No. 23) and amino acid sequences at positions 405-423 (SEQ ID NO: 24);

(b) amino acid sequence at position 62-69 (SEQ ID NO: 25), amino acid sequence at position 139-148 (SEQ ID NO: 26), amino acid sequence at position 259-277 (SEQ ID NO: 27), position 199-204 Amino acid sequence (SEQ ID NO: 28), amino acid sequence at positions 151-158 (SEQ ID NO: 29), amino acid sequence at positions 275-291 (SEQ ID NO: 30), amino acid sequence at positions 20-28 (SEQ ID NO: 31), 65-90 Amino acid sequence of position (SEQ ID NO: 32), amino acid sequence of position 150-170 (SEQ ID NO: 33) and amino acid sequence of position 280-290 (SEQ ID NO: 34);

(c) the amino acid sequence at positions 24-2-250 (SEQ ID NO: 35), the amino acid sequence at positions 279-299 (SEQ ID NO: 36) and the amino acid sequence at positions 343-361 (SEQ ID NO: 37) of the transforming growth factor; ;

(d) amino acid sequences at positions 100-120 (SEQ ID NO: 38) and amino acid sequences 121-140 (SEQ ID NO: 39) of platelet derived growth factors;

(e) amino acid sequences 23-31 of the acidic fibroblast growth factor (SEQ ID NO: 40) and amino acid sequences 97-105 (SEQ ID NO: 41);

(f) the amino acid sequence at positions 16-27 (SEQ ID NO: 42), the amino acid sequence at positions 37-42 (SEQ ID NO: 43), and the amino acid sequence at positions 78-84 of the basic fibroblast growth factor; No. 44) and amino acid sequences 107-112 (SEQ ID NO: 45);

(g) the amino acid sequence of positions 255-275 (SEQ ID NO: 46), amino acid sequence of positions 475-494 (SEQ ID NO: 47) and amino acid sequence of positions 551-573 (SEQ ID NO: 48) of the dentin sialoprotein;

(h) amino acid sequence at position 63-83 (SEQ ID NO: 49), amino acid sequence position 84-103 (SEQ ID NO: 50), 104-116 position of heparin binding EGF-lke growth factor; Amino acid sequence of SEQ ID NO: 51 and amino acid sequence of position 121-140 (SEQ ID NO: 52);

(i) the amino acid sequence of positions 326-350 (SEQ ID NO: 53), the amino acid sequence of positions 351-371 (SEQ ID NO: 54), and the amino acid sequence of positions 372-400 of the cadherin EGF LAG seven-pass G-type receptor 3 55), amino acid sequence of positions 401-423 (SEQ ID NO: 56), amino acid sequence of positions 434-545 (SEQ ID NO: 57), amino acid sequence of positions 546-651 (SEQ ID NO: 58), amino acid sequence of positions 1375-1433 (SEQ ID NO: 59), amino acid sequence at position 1435-1471 (SEQ ID NO: 60), amino acid sequence at position 1475-1514 (SEQ ID NO: 61), amino acid sequence at position 1515-1719 (SEQ ID NO: 62), position 1764-1944 Amino acid sequence (SEQ ID NO: 63) and amino acid sequence at positions 2096-2529 (SEQ ID NO: 64); And

(j) amino acid sequence at position 54-159 (SEQ ID NO: 65), amino acid sequence at position 160-268 (SEQ ID NO: 66), amino acid sequence at position 269-383 (SEQ ID NO: 67) of osteoblast specific cadherin (OB-cadherin); It is preferable to use any one or more selected from the group consisting of peptides having amino acid sequences at positions 384-486 (SEQ ID NO: 68) and amino acid sequences at positions 487-612 (SEQ ID NO: 69). More preferably, the peptide has a spacer (CGG-) consisting of cysteine and two molecules of glycine at the N-terminus to facilitate fixation to the bone graft material and the support chemically.

The active peptide according to the present invention synthesizes the amino acid sequence of 10-20 units of the amino acid sequence of the entire tissue growth factor, and performs the cell adhesion experiment using these to select the highest activity amino acid sequence, these amino acid sequences The chemical formula is added back to the end of the bone graft material and is designed to facilitate fixing. As a result, the bone graft material can maintain the activity with only the minimum amino acid sequence on the surface and at the same time can reduce the loss and side effects of the drug due to the physical incorporation and application of tissue growth factors can provide additional benefits to the therapeutic effect.

The bone graft material and the support for use in the present invention may be any kind and type of bone graft material and polymer support used in the art, preferably bio-derived bone mineral powder and porous block derived from autologous bone, bovine bone and pork bone , Synthetic Apatite Powder and Porous Block, Tricalcium Phosphate Powder and Porous Block, Monocalcium Phosphate Powder and Porous Block, Bone Graft Material with Silicon Dioxide (Silica), Bone Filling Implant with a Mixture of Silica and Polymer, Chitosan, Polylactic Acid Particulate and porous supports made of biocompatible polymers, including titanium and three-dimensional porous supports. At this time, the surface of the bone graft material and the support is preferably modified surface to facilitate the attachment of the active peptide.

According to the present invention, since the peptide is less sensitive to enzyme reactions in the body and lowers immunogenicity in the body than the tissue growth factor itself, when the peptide is fixed to the surface of bone graft material, support, shielding membrane or implant for tissue regeneration, The active peptide is present locally and can be active to increase the therapeutic effect, thus having characteristics suitable for treating bone tissue and periodontal tissue regeneration.

The peptide having a free amino group or cysteine at the N-terminal of the present invention is easily fixed to the surface by a bone graft material and a support by a crosslinking agent. Crosslinkers suitable for the present invention include 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM [PEO] 4), 1-ethyl-3- [3-dimethyl aminopropyl] carbodiimide hydrochloride (EDC), succinimidyl-4- [N-maleimidomethyl cyclohexane-1-carboxy- [6-amidocaproate]] (SMCC) and its sulfonates (sulfo-SMCC), succimidyl 6- [3- (2-pyridyldithio) -ropionamido] hexanoate] ( SPDP) and its sulfonate (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and its sulfonate (sulfo-MBS), succimidyl [4- (p-maleimidophenyl) butyrate] (SMPB) and its sulfone Flames (sulfo-SMPB), but are not limited thereto.

In addition, the peptide is preferably chemically bonded to the surface of the bone graft material and the support so that 0.1 ~ 10mg per cm 2 fixed on the surface, more preferably bone is to fix 1 ~ 5mg per cm 2 surface of the plant and support. .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Fixation of Cell-Adhesive RGD Peptides to Bovine-Derived Bone Mineral Particles

The bovine bone-derived bone mineral particles were washed under reduced pressure with ethanol, and left to stand at 100 ° C. in a vacuum oven for 20 hours to remove impurities from the surface. The bone mineral particle surface was treated with 3-aminopropyl triethoxysilane (APTES) dissolved in hexane, and then washed. As a result, an amine moiety was formed on the surface, and the crosslinking agent BMB was added thereto to bond. The bone mineral particles bound to the crosslinking agent were reacted with the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 having RGD for 12 hours, and then washed to obtain bone mineral particles fixed with the peptide.

Example 2: Fixation of Cell-Adhesive RGD Peptides to Synthetic Hydroxyapatite and Tricalcium Phosphate

Synthetic hydroxide apatite and tricalcium phosphate bone graft powder was washed with ethanol under reduced pressure, and left to stand in a vacuum oven at 100 ° C. for 20 hours to remove impurities from the surface. The particle surface was treated with 3-aminopropyl triethoxy silane (APTES) dissolved in hexane, and then washed. As a result, an amine moiety was formed on the surface, and the crosslinking agent BMB was added thereto to bond. The bone graft particles to which the crosslinker was bound were reacted with the peptides of SEQ ID NO: 1 and SEQ ID NO: 12 for 12 hours, and then washed to obtain the bone graft particles having the peptide fixed thereto.

Example 3: Fixation of Cell-Adhesive RGD Peptides to Chitosan Bone Graft

Chitosan bone graft prepared in the form of powder or porous support was added to 2 ml of phosphate buffer (pH 7.4) to hydrate the surface, and sulfo-SMCC was added thereto as a crosslinking agent at a concentration of 5 mg / ml and stirred for 2 hours. A functional group was introduced to the chitosan bone graft surface. After 2 hours of room temperature reaction, the chitosan bone graft was washed, and 10 mg of the peptide of SEQ ID NO: 1 was dissolved in 100 μl of phosphate buffer, and then reacted for 24 hours. Then, the peptide was fixed to prepare chitosan bone graft.

Example 4 Fixation of Cell-Adhesive RGD Peptides to Polylactic Acid Bone Grafts

Polylactic acid graft powder or porous support was added to phosphate buffer (pH 4.7) to hydrate the surface, and then reacted with 20 mg / ml hydrochloric acid cystamine solution. EDC was added dropwise thereto as a crosslinking agent to activate carboxylic acid on the bone graft surface. After reacting for 24 hours, washing, 1 ml of 30 mg / ml dithiothreniol (DTT) solution was added thereto, and reacted for another 24 hours to allow sulfhydryl groups to be introduced on the surface of the polylactic acid. When the modified polylactic acid bone graft material was mixed with the cell-adhesive RGD peptide (SEQ ID NO: 1), the peptide was fixed by inducing S-S binding between the sulfhydryl group and the peptide of the bone graft material.

Example 5: Fixation of Tissue-derived Peptides to Bone Mineral Particles

The tissue growth factor-derived peptide used in the present example has a cysteine at the amino acid sequence N-terminus of SEQ ID NO: 3 and SEQ ID NOs: 6-9, which are peptides containing the cell adhesion and active domains of bone forming protein (BMP-2). A chemically synthesized peptide was used with the addition of a CGG-spacer.

The bovine bone-derived bone mineral particles were washed with ethanol under reduced pressure, and left to stand at 100 ° C. in a vacuum oven for 20 hours to remove impurities from the surface. The surface of the bone mineral particles was treated with 3-aminopropyl triethoxysilane (APTES) dissolved in hexane and then washed. An amine residue was formed on the surface, and sulfo-SMCC as a crosslinking agent was 5 mg / g. The solution was added at a concentration of ml, stirred for 2 hours, and a reactor was introduced to the surface of the bone graft material. After the reaction at room temperature for 2 hours, the bone graft material was washed, and the solution of 10 mg of the peptide dissolved in 100 μl of phosphate buffer was added thereto, followed by reaction for 24 hours, followed by washing to obtain bone mineral particles fixed with peptide.

Example 6 Fixation of Tissue Growth Factor-Derived Peptides to Synthetic Bone Graft Particles

In the present example, the same peptide as the peptide used in Example 5 was used as the peptide derived from the tissue growth factor.

Synthetic bone dichroic materials were used as synthetic apatite hydroxide and tricalcium phosphate minerals, and the bone graft particles were washed under reduced pressure with ethanol and stored in a vacuum oven for 20 hours to remove impurities on the surface. The particle surface was washed with 3-aminopropyl triethoxysilane (APTES) dissolved in hexane. As a result, an amine residue was formed on the surface, and 5 mg / ml sulfo-SMCC was added thereto as a crosslinking agent, followed by stirring for 2 hours to introduce a functional group to the surface of the bone graft material. After the reaction at room temperature for 2 hours, the bone graft material was washed, and the solution of 10 mg of the peptide dissolved in 100 μl of phosphate buffer was added thereto for reaction for 24 hours, followed by washing to obtain bone graft particles having the tissue growth factor peptide fixed thereto.

Example 7: Fixation of Tissue-Derived Peptides on Chitosan Bone Graft Materials and Supports

Chitosan bone graft and tissue regeneration support were added to 2 ml of phosphate buffer (pH 7.4) to hydrate the surface, and sulfo-SMCC was added to the concentration of 5 mg / ml as a crosslinking agent and stirred for 2 hours. A functional group was introduced to the surface of the reactor. After 2 hours of room temperature reaction, the chitosan bone graft was washed, and 10 mg of the same peptide as the tissue growth factor-derived peptide used in Example 5 was dissolved in 100 μl of phosphate buffer solution, followed by reaction for 24 hours, followed by washing to fix the peptide. Chitosan bone graft material and support were prepared.

Example 8: Fixation of tissue growth factor-derived peptides to polylactic acid bone graft and support

Polylactic acid graft powder or porous support was added to phosphate buffer (pH 4.7) to hydrate the surface and reacted with 20 mg / ml hydrochloric acid cystamine solution. EDC was added dropwise thereto as a crosslinking agent to activate the carboxylic acid on the surface of the polylactic acid bone graft material. After washing for 24 hours, the mixture was washed, and then 1 ml of dithiothreniol (DTT) solution (30 mg / ml) was added thereto, and reacted for another 24 hours to allow sulfhydryl groups to be introduced on the surface of the polylactic acid. The bone graft material was immobilized by spontaneously inducing the binding of S-S between the bone graft material and the peptide by mixing with a peptide having a CGG spacer coupled to the amino acid sequence of SEQ ID NO: 8, the tissue growth factor-derived peptide.

Example 9 Fixation of Bone Sialoprotein-Derived Peptides to Bone Mineral Particles

The bone sialoprotein-derived peptide used in this example was a chemical synthesis of the peptide of SEQ ID NO: 15, which is a peptide containing a calcification-inducing active domain structure, and the peptide of SEQ ID NO: 27, which is a peptide containing a cell adhesion function site.

The bovine bone-derived bone mineral particles were washed with ethanol under reduced pressure, and left to stand at 100 ° C. in a vacuum oven for 20 hours to remove impurities from the surface. The bone mineral particle surface was washed with 3-aminopropyl ethoxysilane (APTES) dissolved in hexane. As a result, an amine residue was formed on the surface, and 5 mg / ml of Sulfo-SMCC was added thereto as a crosslinking agent, followed by stirring for 2 hours to introduce a functional group to the surface of the bone graft material. After washing, 10 mg of the bone sialoprotein-derived peptide was dissolved in 100 μl of phosphate buffer solution, and then reacted for 24 hours, followed by washing to obtain bone mineral particles fixed with peptide.

Example 10 Fixation of Bone Sialoprotein-Derived Peptides to Synthetic Bone Graft Particles

In this example, the same peptide as that used in Example 9 was used.

The apatite hydroxide and tricalcium phosphate mineral particles were washed with ethanol under reduced pressure, and left to stand at 100 ° C. in a vacuum oven for 20 hours to remove impurities from the surface. The particle surface was washed with 3-aminopropyl triethoxysilane (APTES) dissolved in hexane. As a result, an amine residue was formed on the surface, and sulfo-SMCC at a concentration of 5 mg / ml was added thereto as a crosslinking agent and stirred for 2 hours to introduce a functional group to the surface of the bone graft material. After completion of the reaction, the bone graft material was washed, and 10 mg of the peptide used in Example 9 was dissolved in 100 µl of phosphate buffer solution, followed by reaction for 24 hours, followed by washing to obtain bone graft particles having the peptide fixed thereto.

Example 11: Adhesion of Bone Sialoprotein to Chitosan Bone Graft and Fixation of Peptide Containing Active Sites

In this example, the same peptide as that used in Example 9 was used.

Chitosan bone graft and support were added to 2 ml of phosphate buffer (pH 7.4) to hydrate the surface, and then 5 mg / ml sulfo-SMCC was added thereto as a crosslinking agent, followed by stirring for 2 hours. ) Was introduced. After completion of the reaction, the chitosan bone graft was washed, and the solution of 10 mg of the peptide dissolved in 100 μl of phosphate buffer was added thereto, followed by reaction for 24 hours, followed by washing to prepare chitosan bone graft material and the support to which the peptide was fixed.

Example 12 Attachment of Bone Sialoprotein to Polylactic Acid Bone Graft Materials and Supports and Fixation of Peptides Containing Active Sites

In this example, the same peptide as that used in Example 9 was used.

The polylactic acid bone graft material and the support were added to a phosphate buffer (pH 4.7) to hydrate the surface and then reacted with a solution of 20 mg / ml of cystamine hydrochloride. EDAC as a crosslinking agent was added dropwise to activate the carboxylic acid on the surface of the polylactic acid. After reaction for 24 hours, the solution was washed, and then, 1 ml of 30 mg / ml DTT solution was added and reacted again for 24 hours. The sulfhydryl group was introduced to the surface of the. The bone graft material and the support were mixed with the peptide to spontaneously induce S-S binding between the bone graft material and the peptide to fix the peptide.

 Experimental Example 1: Surface analysis of bone graft material according to the present invention

In order to analyze the surface of the bone graft material fixed with each peptide prepared in Examples 1 to 12, the bone graft material was fixed with 2% glutaraldehyde solution. The fixed bone graft material was treated with 1% osmium tetroxide solution, washed, dehydrated and dried.

The surface of the prepared bone graft material was analyzed by XPS method. The method identifies the elements fixed on the surface of the material to determine the presence or absence of the bond, since the presence of sulfur bond (disulfide bond) between the peptide and the bone graft material immobilized by the present invention confirmed the presence of sulfur It was.

Figure 1 shows the analysis results of the peptides immobilized on chitosan bone graft material according to the present invention, Figure 1a shows the surface of the bone graft material made of chitosan unmodified peptide, Figure 1b is a peptide containing sulfur It shows a fixed bone graft material. As shown, the presence of sulfur on the surface of the bone graft material is fixed peptide was confirmed that the peptide was fixed. In addition, the fixation rate of the peptide per total surface area of the implant was confirmed by measuring the sulfur content of the bone-grafted bone graft material. As a result, as shown in Table 1, sulfur was not detected in chitosan that was not modified with peptides, whereas 8.66% of sulfur was detected in chitosans in which peptides were fixed.

Element O (%) N (%) C (%) S (%) O / C N / C Chitosan with no peptide 31.83 6.18 61.99 0 0.513 0.0997 Peptide immobilized chitosan 32.33 2.96 60.05 8.66 0.605 0.033

Experimental Example 2: Cell adhesion of bone graft material according to the present invention

After inoculating osteoblasts (MC3T3 cell line) to the peptide-attached bone graft prepared in Examples 3, 7 and 11 and cultured for 4 hours and 1 day, respectively. Osteoblasts cultured with osteoblasts were fixed with 2% glutaraldehyde solution. The immobilized bone graft material was stained with cytoplasm by adding a hydrofluorescent paloidine solution treated with 1% triton X-100. After staining, the specimens were fixed by washing, and the cells attached to the bone graft material were observed with a confocal laser scanning microscope (FIG. 2).

Figure 2a shows the cell attachment to the unmodified bone graft material, Figures 2b and 2c shows the cell attachment to the bone graft material fixed peptides derived from BMP and bone sialoprotein, respectively. As a result of cell adhesion, the cell adhesion pattern of the bone graft material in which the peptide was not immobilized was observed to be unstable in spherical form. On the surface of the bone graft material in which the peptide derived from BMP and bone sialoprotein were fixed, most of the cells were already 4 hours after cell culture. Stable cell adhesion was observed, such as cytoplasmic elongation observed in the cells.

In addition, Figure 3 shows the results of quantitative analysis of the cell adhesion, the degree of adhesion of the cells in the peptide modified chitosan bone graft significantly more than the chitosan bone graft not modified with the peptide, this increase is modified The concentration was proportional to the amount of peptide.

Experimental Example 3 Expression of Differentiation Marker Proteins in Osteoblasts Cultured on the Surface of Peptide Fixed Bone Graft Material According to the Present Invention

In order to confirm the expression of the differentiation marker protein of osteoblasts cultured on the bone graft material on which the peptide was immobilized according to the present invention, the expression levels of the differentiation marker proteins smad 1, 5 and 8 were confirmed by Western blot.

Osteoblasts were inoculated on the surface of the bone graft material and the bone graft material to which the peptides were fixed and cultured for 2 weeks. After incubation, the total protein in the cells was extracted and its amount was quantified by measuring the absorbance at 280 nm. 2 μl of a 1 mg / ml protein solution was taken and electrophoresed on an acrylamide gel, followed by reaction with antibodies of smads 1, 5 and 8, which are differentiation marker proteins. Thereafter, the secondary antibody binding to the antibody was labeled and reacted with a marker, and the gel band was developed to observe the protein bands and the density thereof was measured (FIG. 4).

As shown in FIG. 4, the expression of smad was significantly increased in osteoblasts cultured on the surface of the bone graft material in which the peptide was immobilized, compared with the case of cells cultured in the bone graft material in which the peptide was not immobilized. Cells grown on the surface of the bone graft on which the factor-derived peptides were immobilized were found to promote differentiation into bone tissue.

Experimental Example 4: Rabbit skull regeneration effect by peptide fixation bone graft

The bone graft material in which the peptides prepared in Examples 1 to 5 were immobilized was implanted in a skull bone defect of a rabbit to confirm bone regeneration. A circular bone defect with an diameter of 8 mm was formed on the skull portion of the anesthetized rabbit, and a bone graft material modified with a bone graft material and a peptide modified in the bone defect part was implanted at 50 mg per defect part, and the periosteum and the skin were double-sealed. Two weeks after the transplantation, the animals were sacrificed, and the collected samples were fixed in formalin solution and embedded in tissue to prepare specimens having a thickness of 20 μm. The prepared specimens were stained with basic fuchsin and toluidine blue to prepare non-limeous specimens. The produced specimen was observed by optical microscope and photographed.

Figure 5 shows the bone regeneration effect by the bone graft material and the bone graft material to which the peptide is fixed, compared to the bone graft material without the peptide (FIG. 5a), bone graft material with bone tissue formation promoting peptides attached to the surface according to the present invention When applied to the cranial defect of the rabbit, it was confirmed that a significant bone regeneration ability within 2 weeks (Fig. 5b).

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has an effect of providing a bone graft material and a support for tissue engineering in which a cell adhesion inducing peptide and / or a tissue growth factor-derived peptide are immobilized on the surface, even at low doses, to obtain a desired tissue regeneration effect.

The bone graft material and the scaffold for tissue regeneration fixed with the bone tissue formation promoting peptides according to the present invention promote cell adhesion and differentiation into bone tissue, and rapidly decompose and leak in the body by simple incorporation of tissue growth factors by conventional methods. Side effects can be prevented, and there is an advantage that can reduce the enormous cost of applying a large amount to increase the local concentration.

Attach sequence list electronic file  

Claims (17)

delete delete In bone graft material in which a tissue growth factor-derived peptide is immobilized on the surface, The tissue growth factor-derived peptide is (a) bone morphogenetic protein (BMP) -2, amino acid sequences of positions 2-18 of the amino acids of 4 and 6 [BMP-2 (SEQ ID NO: 3), For BMP-4 (SEQ ID NO: 4) and for BMP-6 (SEQ ID NO: 5)] Amino acid sequence at positions 16-34 of BMP-2 (SEQ ID NO: 6), amino acid sequence at positions 47-71 (SEQ ID NO: 7) ), Amino acid sequence at positions 73-92 (SEQ ID NO: 8), amino acid sequence at positions 88-105 (SEQ ID NO: 9), amino acid sequence at positions 283-302 (SEQ ID NO: 10), amino acid sequence at positions 335-353 (SEQ ID NO: 8) Number 11) and the amino acid sequence at positions 370-390 (SEQ ID NO: 12); Amino acid sequence at positions 74-93 (SEQ ID NO: 13), amino acid sequence at positions 293-313 (SEQ ID NO: 14), amino acid sequence at positions 360-379 (SEQ ID NO: 15), and amino acid sequence at positions 382-402; (SEQ ID NO: 16) Amino acid sequence at positions 91-110 of the BMP-6 (SEQ ID NO: 17), amino acid sequence at positions 397-418 (SEQ ID NO: 18), amino acid sequence at positions 472-490 (SEQ ID NO: 19), and 487- Amino acid sequence at position 510 (SEQ ID NO: 20), amino acid sequence at position 98-117 (SEQ ID NO: 21) at BMP-7, amino acid sequence at position 320-340 (SEQ ID NO: 22), amino acid sequence at position 390-409 (SEQ ID NO: 23) No. 23) and amino acid sequences at positions 405-423 (SEQ ID NO: 24); (b) amino acid sequence at position 62-69 (SEQ ID NO: 25), amino acid sequence at position 139-148 (SEQ ID NO: 26), amino acid sequence at position 259-277 (SEQ ID NO: 27), position 199-204 Amino acid sequence (SEQ ID NO: 28), amino acid sequence at positions 151-158 (SEQ ID NO: 29), amino acid sequence at positions 275-291 (SEQ ID NO: 30), amino acid sequence at positions 20-28 (SEQ ID NO: 31), 65-90 Amino acid sequence of position (SEQ ID NO: 32), amino acid sequence of position 150-170 (SEQ ID NO: 33) and amino acid sequence of position 280-290 (SEQ ID NO: 34); (c) the amino acid sequence at positions 24-2-250 (SEQ ID NO: 35), the amino acid sequence at positions 279-299 (SEQ ID NO: 36) and the amino acid sequence at positions 343-361 (SEQ ID NO: 37) of the transforming growth factor; ; (d) amino acid sequences at positions 100-120 (SEQ ID NO: 38) and amino acid sequences 121-140 (SEQ ID NO: 39) of platelet derived growth factors; (e) the amino acid sequence at positions 23-31 (SEQ ID NO: 40) and the amino acid sequence at positions 97-105 of the acidic fibroblast growth factor (SEQ ID NO: 41); (f) the amino acid sequence at positions 16-27 (SEQ ID NO: 42), the amino acid sequence at positions 37-42 (SEQ ID NO: 43), and the amino acid sequence at positions 78-84 of the basic fibroblast growth factor; No. 44) and amino acid sequences 107-112 (SEQ ID NO: 45); (g) the amino acid sequence of positions 255-275 (SEQ ID NO: 46), amino acid sequence of positions 475-494 (SEQ ID NO: 47) and amino acid sequence of positions 551-573 (SEQ ID NO: 48) of the dentin sialoprotein; (h) amino acid sequence at position 63-83 (SEQ ID NO: 49), amino acid sequence position 84-103 (SEQ ID NO: 50), 104-116 position of heparin binding EGF-lke growth factor; Amino acid sequence of SEQ ID NO: 51 and amino acid sequence of position 121-140 (SEQ ID NO: 52); (i) the amino acid sequence of positions 326-350 (SEQ ID NO: 53), the amino acid sequence of positions 351-371 (SEQ ID NO: 54), and the amino acid sequence of positions 372-400 of the cadherin EGF LAG seven-pass G-type receptor 3 55), amino acid sequence of positions 401-423 (SEQ ID NO: 56), amino acid sequence of positions 434-545 (SEQ ID NO: 57), amino acid sequence of positions 546-651 (SEQ ID NO: 58), amino acid sequence of positions 1375-1433 (SEQ ID NO: 59), amino acid sequence at position 1435-1471 (SEQ ID NO: 60), amino acid sequence at position 1475-1514 (SEQ ID NO: 61), amino acid sequence at position 1515-1719 (SEQ ID NO: 62), position 1764-1944 Amino acid sequence (SEQ ID NO: 63) and amino acid sequence at positions 2096-2529 (SEQ ID NO: 64); And (j) amino acid sequence at position 54-159 (SEQ ID NO: 65), amino acid sequence at position 160-268 (SEQ ID NO: 66), amino acid sequence at position 269-383 (SEQ ID NO: 67) of osteoblast specific cadherin (OB-cadherin); , Bone graft material, characterized in that any one or more peptides selected from the group consisting of amino acid sequence of position 384-486 (SEQ ID NO: 68) and amino acid sequence of position 487-612 (SEQ ID NO: 69). The bone graft material according to claim 3, wherein the cell adhesion inducing peptide having the amino acid sequence of SEQ ID NO: 1 (CGGRGDS) or SEQ ID NO: 2 (CGGVACDCRGDCFC) is further fixed. 5. The bone graft material of claim 4, wherein the tissue growth factor-derived peptide has cysteine added to the N-terminus. The bone graft material according to claim 5, wherein the cysteine is added in the form of CGG. The bone graft material according to any one of claims 4 to 6, wherein the bone graft material is a powder and porous block of bio-derived bone mineral, powder and porous block of synthetic apatite, powder and porous block of tricalcium phosphate, powder of monocalcium phosphate And microparticles and porous supports, titanium and three-dimensional porous supports, consisting of a porous block, a bone graft material made of silicon dioxide (silica), a bone filler implant made of a mixture of silica and a polymer, a biocompatible polymer including chitosan, polylactic acid, Graft material, characterized in that any one selected from the group consisting of. The bone graft material according to claim 4, wherein the surface of the bone graft material has a crosslinking agent bonded thereto. The method of claim 8, wherein the crosslinking agent is 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM [PEO] 4), 1-ethyl-3- [3-dimethyl aminopropyl] carbodiimide hydrochloride ( EDC), succinimidyl-4- [N-maleimidomethylcyclohexane-1-carboxy- [6-amidocaproate]] (SMCC) and its sulfonates (sulfo-SMCC), succimidyl-6- [3- (2-pyridyldithio) -propionamido] hexanoate] (SPDP) and its sulfonate (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and its sulfonate (sulfo-MBS), succimidyl [4- (p-maleimidophenyl) butyrate] (SMPB) And its sulfonate (sulfo-SMPB) bone graft material, characterized in that any one or more selected from the group consisting of. delete delete In the support for tissue engineering in which a tissue growth factor-derived peptide is immobilized on the surface, The tissue growth factor-derived peptide is (a) bone morphogenetic protein (BMP) -2, amino acid sequences of positions 2-18 of the amino acids of 4 and 6 [BMP-2 (SEQ ID NO: 3), For BMP-4 (SEQ ID NO: 4) and for BMP-6 (SEQ ID NO: 5)] Amino acid sequence at positions 16-34 of BMP-2 (SEQ ID NO: 6), amino acid sequence at positions 47-71 (SEQ ID NO: 7) ), Amino acid sequence at positions 73-92 (SEQ ID NO: 8), amino acid sequence at positions 88-105 (SEQ ID NO: 9), amino acid sequence at positions 283-302 (SEQ ID NO: 10), amino acid sequence at positions 335-353 (SEQ ID NO: 8) Number 11) and the amino acid sequence at positions 370-390 (SEQ ID NO: 12); Amino acid sequence at positions 74-93 (SEQ ID NO: 13), amino acid sequence at positions 293-313 (SEQ ID NO: 14), amino acid sequence at positions 360-379 (SEQ ID NO: 15), and amino acid sequence at positions 382-402; (SEQ ID NO: 16) Amino acid sequence at positions 91-110 of the BMP-6 (SEQ ID NO: 17), amino acid sequence at positions 397-418 (SEQ ID NO: 18), amino acid sequence at positions 472-490 (SEQ ID NO: 19), and 487- Amino acid sequence at position 510 (SEQ ID NO: 20), amino acid sequence at position 98-117 (SEQ ID NO: 21) at BMP-7, amino acid sequence at position 320-340 (SEQ ID NO: 22), amino acid sequence at position 390-409 (SEQ ID NO: 23) No. 23) and amino acid sequences at positions 405-423 (SEQ ID NO: 24); (b) amino acid sequence at position 62-69 (SEQ ID NO: 25), amino acid sequence at position 139-148 (SEQ ID NO: 26), amino acid sequence at position 259-277 (SEQ ID NO: 27), position 199-204 Amino acid sequence (SEQ ID NO: 28), amino acid sequence at positions 151-158 (SEQ ID NO: 29), amino acid sequence at positions 275-291 (SEQ ID NO: 30), amino acid sequence at positions 20-28 (SEQ ID NO: 31), 65-90 Amino acid sequence of position (SEQ ID NO: 32), amino acid sequence of position 150-170 (SEQ ID NO: 33) and amino acid sequence of position 280-290 (SEQ ID NO: 34); (c) the amino acid sequence at positions 24-2-250 (SEQ ID NO: 35), the amino acid sequence at positions 279-299 (SEQ ID NO: 36) and the amino acid sequence at positions 343-361 (SEQ ID NO: 37) of the transforming growth factor; ; (d) amino acid sequences at positions 100-120 (SEQ ID NO: 38) and amino acid sequences 121-140 (SEQ ID NO: 39) of platelet derived growth factors; (e) the amino acid sequence at positions 23-31 (SEQ ID NO: 40) and the amino acid sequence at positions 97-105 of the acidic fibroblast growth factor (SEQ ID NO: 41); (f) the amino acid sequence at positions 16-27 (SEQ ID NO: 42), the amino acid sequence at positions 37-42 (SEQ ID NO: 43), and the amino acid sequence at positions 78-84 of the basic fibroblast growth factor; No. 44) and amino acid sequences 107-112 (SEQ ID NO: 45); (g) the amino acid sequence of positions 255-275 (SEQ ID NO: 46), amino acid sequence of positions 475-494 (SEQ ID NO: 47) and amino acid sequence of positions 551-573 (SEQ ID NO: 48) of the dentin sialoprotein; (h) amino acid sequence at position 63-83 (SEQ ID NO: 49), amino acid sequence position 84-103 (SEQ ID NO: 50), 104-116 position of heparin binding EGF-lke growth factor; Amino acid sequence of SEQ ID NO: 51 and amino acid sequence of position 121-140 (SEQ ID NO: 52); (i) the amino acid sequence of positions 326-350 (SEQ ID NO: 53), the amino acid sequence of positions 351-371 (SEQ ID NO: 54), and the amino acid sequence of positions 372-400 of the cadherin EGF LAG seven-pass G-type receptor 3 55), amino acid sequence of positions 401-423 (SEQ ID NO: 56), amino acid sequence of positions 434-545 (SEQ ID NO: 57), amino acid sequence of positions 546-651 (SEQ ID NO: 58), amino acid sequence of positions 1375-1433 (SEQ ID NO: 59), amino acid sequence at position 1435-1471 (SEQ ID NO: 60), amino acid sequence at position 1475-1514 (SEQ ID NO: 61), amino acid sequence at position 1515-1719 (SEQ ID NO: 62), position 1764-1944 Amino acid sequence (SEQ ID NO: 63) and amino acid sequence at positions 2096-2529 (SEQ ID NO: 64); And (j) amino acid sequence at position 54-159 (SEQ ID NO: 65), amino acid sequence at position 160-268 (SEQ ID NO: 66), amino acid sequence at position 269-383 (SEQ ID NO: 67) of osteoblast specific cadherin (OB-cadherin); , Amino acid sequence of position 384-486 (SEQ ID NO: 68) and amino acid sequence of position 487-612 (SEQ ID NO: 69) any one or more peptides selected from the group consisting of. The support for tissue engineering according to claim 12, wherein the cell adhesion inducing peptide having the amino acid sequence of SEQ ID NO: 1 (CGGRGDS) or SEQ ID NO: 2 (CGGVACDCRGDCFC) is further fixed. The support for tissue engineering according to claim 13, wherein the tissue growth factor-derived peptide has cysteine added to the N-terminus. 15. The tissue engineering scaffold according to claim 14, wherein the cysteine is added in the form of CGG. The support for tissue engineering of claim 12, wherein the surface of the support for tissue engineering has a crosslinking agent bound thereto. The method of claim 16, wherein the crosslinking agent is 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM [PEO] 4), 1-ethyl-3- [3-dimethyl aminopropyl] carbodiimide hydrochloride ( EDC), succinimidyl-4- [N-maleimidomethylcyclohexane-1-carboxy- [6-amidocaproate]] (SMCC) and its sulfonates (sulfo-SMCC), succimidyl-6- [3- (2-pyridyldithio) -propionamido] hexanoate] (SPDP) and its sulfonate (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and its sulfonate (sulfo-MBS), succimidyl [4- (p-maleimidophenyl) butyrate] (SMPB) And a sulfonate salt thereof (sulfo-SMPB).

Images