KR20140030605A - Kit for regenerating alveolar bone - Google Patents

Abstract

The present invention relates to a kit for regenerating an alveolar bone and, more specifically, to a kit for regenerating an alveolar bone which improves a regeneration speed of the alveolar bone by configuring growth factors or growth factor derivatives with tooth. The kit for regenerating the alveolar bones of the present invention has the outstanding regeneration speed for the alveolar bone than a case in which only the tooth are administered, and completeness and integrity of regenerated bone tissues are also significantly improved. The rapid and complete regeneration of the alveolar bones significantly alleviates discomfort of a patient and, in particular, the kit is more preferable for a case when shape maintenance is important for a wide range of osseous tissue defects since side effects due to deformation or shrink of the osseous tissue are reduced.

Description

Kit for Regenerating Alveolar Bone}

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a kit for regenerating alveolar bone, and more particularly, to a kit for alveolar bone regeneration in which alveolar bone regeneration speed is improved by constructing a growth factor or a growth factor derivative together with teeth.

BACKGROUND OF THE INVENTION [0002] In the medical field in recent years, regeneration of bone tissue defects due to diseases such as physical, chemical, and burn-related bone injuries or inflammation or tumors is regarded as important. The area where the bone tissue is missing is easily separated by external force or by osteomyelitis. In addition, skin, muscle, bone, and nerve tissue are damaged by multiple damages, and it is difficult to regenerate into normal tissues or organs in each layer, and it is replaced with scar tissue, resulting in restriction of mobility and functionality.

Conventionally, in order to solve this problem, the bone tissue of another part of the patient has been implanted and filled, but side effects such as bone defect in the other normal parts, mobility and functional restriction have been caused.

In addition, autogenous bone and other bone graft materials are required to have dense bone graft materials because of their high ability to maintain their bulk due to their high bone resorption during healing period, but it is difficult to achieve penetrating pores in these materials.

In addition, a shielding membrane may be used to block the growth of other tissues before the defective bone tissue is healed or to prevent invasion of external bacteria into the tissue, or a porous scaffold may be used to provide a space in which defective bone tissue cells may grow. (scaffold). In addition, a growth factor that allows stem cells necessary for healing or regeneration to move toward the defect site through chemotaxis, or by transplanting the stem cells into a defective site by manipulating or culturing the stem cells as in Korean Patent No. 644296, There is an increasing trend to try to do this.

On the other hand, recombinant human bone morphogenetic protein (BMP) is a growth factor that differentiates mesenchymal stem cells required for healing into various tissues by chemoattracting them to the defect site. In patent WO 1993/000050, BMP-2 has suggested that mesenchymal stem cells grow into bone tissue.

However, the technique using these stem cells also takes a lot of time to regenerate the alveolar bone, and the inconvenience of the patient is very serious.

Korean Registered Patent No. 644296 (Jeongpil Hoon et al.) WO 1993/000050 (Genentics Institute, LL.C.)

SUMMARY OF THE INVENTION It is an object of the present invention to provide a kit for regenerating alveolar bone, which comprises a growth factor or a growth factor derivative together with a tooth.

In order to achieve the above-mentioned object, the alveolar bone bone regeneration kit of the present invention comprises:

Human or animal teeth, and

A growth factor or a growth factor derivative.

In addition, the alveal bone repair kit may further include a hydrophilic polymer.

In addition, the alveolar bone bone regeneration kit may further include bone, demineralized bone, calcium phosphate, and a mixture thereof.

Also, the growth factors may be selected from the group consisting of recombinant human bone morphogenetic protein (BMP), amino acid binding constructs of BMP, fibroblast growth factor, growth differentiation factor (GDF) (TGF), platelet-derived growth factor, insulin-like growth factor, epithelial growth factor, keratinocyte growth factor 2 (KGF2), morphogenic protein 52 (MP52) And mixtures thereof.

In addition, the growth factor derivative may be polydeoxyribonucleotide (PDRN).

The hydrophilic polymer may be selected from alginic acid, chitosan, collagen, hyaluronic acid, cellulose, poly (vinylidene fluoride), poly (tetrafluoroethylene) poly (vinyl alcohol)], poly (hydroxyalkanoate), poly (ethylene terephthalate), poly (butylene terephthalate) ), Poly (methyl methacrylate), poly (hydroxyethyl methacrylate), poly (N-isopropylacrylamide), poly ), Poly (dimethyl siloxane), polydioxanone, polypyrrole, poly (glycolic acid), poly (lactic) acids), poly (ethylene oxide) [poly (ethylene oxides)], , Poly (lactide-co-glycolides), poly (s-caprolactone), polyanhydrides, polyphosphazenes polyphosphazenes, poly (ortho-esters), polyimides, and mixtures thereof.

The teeth may be selected from the group consisting of powders having an average particle size of 50 to 800 탆, chips having an average major axis of 800 to 2000 탆, membranes having an average thickness of 0.2 to 5.0 mm, scaffolds in the form of a root- have.

Further, the teeth can be removed by removing the soft tissue in the dental cavity.

In addition, the tooth may be further demineralized.

In addition, the alveolar bone bone regeneration kit may be freeze-dried.

In addition, the alveolar bone regeneration kit may separately store the tooth, the growth factor or the growth factor derivative, or may be stored together.

In addition, the alveolar bone regeneration kit may contain teeth, growth factors, growth factor derivatives, and hydrophilic polymers separately or together.

In addition, the hydrophilic polymer may be cross-linked with the tooth.

In addition, the hydrophilic polymer may be in a gel state.

The alveolar bone regeneration kit of the present invention including a growth factor or a growth factor derivative is superior to the synthetic bone administration group in which alveolar bone can not be regenerated at all, as well as the bone induction ability is superior to that of the dental administration group and alveolar bone regeneration speed is superior. And integrity are also significantly improved. This rapid and complete alveolar bone regeneration greatly alleviates patient discomfort, and is even more desirable because it reduces side effects due to deformation or contraction of the bone tissue, particularly when shape retention is important in a wide range of bone defect sites. In addition, there is an effect of drastically improving the bone defect of other normal parts due to bone grafting, side effects of mobility and functional restriction, and an additional effect of preventing infection due to prolonged regeneration. It is very big.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a photograph of a powder-like tooth constituting the alveolar bone bone regeneration kit of the present invention. Fig.
Fig. 2 is a photograph of a tooth in the form of a chip constituting the alveolar bone bone regeneration kit of the present invention.
3 is a photograph of a tooth in a film form constituting the alveolar bone bone regeneration kit of the present invention.
Fig. 4 is a photograph of tooth-shaped teeth constituting the alveolar bone bone regeneration kit of the present invention.
FIG. 5 is a photograph of a bone alveolar bone reconstruction kit according to the present invention comprising a vial in which teeth are stored and a vial in which a growth factor is stored.
FIG. 6 is a photograph of a bone alveolar bone reconstruction kit according to the present invention comprising vials in which a tooth and a growth factor are stored together.
FIG. 7 is a photograph of a bone alveolar bone reconstruction kit according to the present invention, which comprises a freeze-dried vial coated with a growth factor on teeth.
FIG. 8 is a photograph of a bone alveolar bone reconstruction kit according to the present invention, which is composed of a vial in which a tooth is stored, a calcium phosphate and a growth factor together.
9 is a photograph of a bone alveolar bone reconstruction kit according to the present invention, which is composed of vials lyophilized by mixing teeth, collagen, and growth factors.
10 is a photograph of the alveolar bone bone regeneration kit of the present invention comprising vials in which teeth are stored, vials containing growth factors and collagen together.
FIG. 11 is a photograph of a bone alveolar bone reconstruction kit according to the present invention comprising a vial in which teeth are stored, a vial in which a growth factor is stored, and a vial in which collagen is stored.
Figures 12 and 13 show results of SDS-PAGE analysis after quantification and purification of rhBMP-2, wherein U is non-reducing rhBMP-2, R is reduced rhBMP-2, Dimer is dimer fraction, Monomer is monomer Lt; / RTI >
14 is an HPLC profile of rhBMP-2.
15 is a graph showing the secretion level of alkaline phosphatase when rhBMP-2 dimer is applied to C2C12 cells.
FIG. 16 is a photograph showing that rhBMP-2 is added to C2C12 cells to change into an osteoblast form.
FIG. 17 is a photograph of a biopsy tissue taken 2 weeks after the contents of the kit for regenerating the alveolar bone of the present invention including rhBMP-2 were inserted.
18 is a photograph of a biopsy tissue taken 2 weeks after the contents of the kit for regenerating the alveolar bone of the present invention including the PDRN are inserted.
19 is a photograph of a biopsy tissue taken 2 weeks after the insertion of the tooth powder.
20 is a photograph of a biopsy tissue taken two weeks after the insertion of the synthetic bone.
21 is an enlarged photograph of a biopsy tissue taken 2 weeks after the contents of the kit for regenerating the alveolar bone of the present invention including rhBMP-2 were inserted.
FIG. 22 is an enlarged photograph of a biopsy tissue taken 2 weeks after the contents of the kit for regenerating the alveolar bone of the present invention including the PDRN are inserted.
23 is an enlarged photograph of a biopsy tissue taken 2 weeks after the insertion of the tooth powder.
24 is an enlarged photograph of a biopsy tissue taken 2 weeks after the insertion of the synthetic bone.
25 is a photograph of a biopsy tissue taken 4 weeks after the contents of the kit for regenerating the alveolar bone of the present invention including rhBMP-2 were inserted.
26 is a photograph of a biopsy tissue taken 4 weeks after the contents of the kit for regenerating the alveolar bone of the present invention including the PDRN are inserted.
27 is a photograph of a biopsy tissue taken 5 weeks after the insertion of the tooth powder.
28 is a photograph of a biopsy tissue taken four weeks after the insertion of the synthetic bone.
29 is a photograph of a biopsy tissue taken 8 weeks after the contents of the kit for regenerating the alveolar bone of the present invention containing rhBMP-2 were inserted.
30 is a photograph of a biopsy tissue taken 8 weeks after the insertion of the tooth powder.

Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, numerous specific details, such as specific elements, are set forth in order to provide a thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without these specific details, It will be obvious to those who have knowledge of. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

The teeth contain collagen and bone formation inducing protein which are the same ingredients as the alveolar bone, and have almost the same components, absorption capacity to be included in bone remodeling, and fine pores having collagen fibers of 5 to 200 탆 in thickness It is optimal for existing bone induction regeneration. In view of this, the present invention is a technique for changing a tooth that has been discarded since the past extraction into a graft material having the same function and effect as that of autologous bone graft. Using the point that the growth factor is contained in the penetrating pore, The most important feature is to increase the rate of alveolar bone regeneration after implantation by including factor derivatives.

That is, the alveolar bone regeneration kit of the present invention is characterized by comprising a growth factor or a growth factor derivative together with a human or animal tooth. The tooth may be an autogenous tooth which is a tooth of a patient, a homogenous tooth which is a tooth of another person, and a heterogeneous tooth which is a tooth of an animal other than a human.

The tooth removes foreign substances on the surface through cleaning and completely removes the soft tissues such as the caries area and the dental nerve. It is preferable to carry out the degreasing process. Removing the fat makes it easy to disinfect, reduces the possibility of contamination, and retains only inorganic substances such as minerals and collagen which are favorable for bone formation and can prevent the immune rejection reaction. The tooth used in the present invention may further be subjected to a demineralization process.

As a specific example, degreasing is carried out with a 5- to 10-fold volume of ethyl ether or chloroform methanol solution of dehydrated teeth for 10 minutes to 24 hours. The demineralization is carried out in a 20-fold volume of 0.2 to 0.7 N aqueous hydrochloric acid solution for 12 to 72 hours. The aqueous hydrochloric acid solution is used in an amount of 5 ml per 1 g of the degreased tooth.

The teeth may be selected from the group consisting of powders having an average particle diameter of 50 to 800 占 퐉, chips having an average major diameter of 800 to 2000 占 퐉, membranes having an average thickness of 0.2 to 5.0 mm, roots-like scaffolds and mixtures thereof . The teeth in the form of a powder are shown in Fig. 1, the teeth in the form of a chip are shown in Fig. 2, the teeth in a film form are shown in Fig. 3, and the supports in the form of a root form are respectively shown in Fig.

The membrane or block may be cut to a predetermined thickness using a microtome knife (DS Global, Korea), and its shape may be changed depending on the shape and shape of the defect. It is also possible to form a plurality of holes or through holes in the film or block. This hole or through-hole is more preferable in that it can act as a space where the growth factor or the growth factor derivative remains.

Furthermore, it is possible to shorten the time taken to regenerate the alveolar bone by using it as a support in a root form and to provide a basic supporting force from the initial stage of implantation. Specifically, the damaged alveolar bone can be restored to its original shape by implanting the originally used alveolar bone in place of the root of the extracted tooth. A number of holes or through-holes having an average diameter of 0.2 to 1 mm may be formed in such a root-shaped support.

The alveolar bone regeneration kit of the present invention includes a growth factor or a growth factor derivative in addition to the tooth. The growth factor or the growth factor derivative has an effect that the alveolar bone regeneration rate is remarkably faster than the case where only the teeth are used.

Herein, the growth factors include recombinant human bone morphogenetic protein (BMP), amino acid binding constructs of BMP, fibroblast growth factor, growth differentiation factor (GDF), modified growth factor Transforming growth factor (TGF), platelet derived growth factor, insulin-like growth factor, epithelial growth factor, keratinocyte growth factor 2 (KGF2), morphogenic protein 52 (MP52) And mixtures thereof. Various types of BMP have been developed and utilized so far, and these BMPs have approximately equivalent bone formation function. As such BMPs, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12 and BMP- Can be used in the present invention. The amino acid binding complex of the amino acid constituting the BMP refers to a complex formed by twisting the amino acid of BMP-2 and the amino acid of BMP-4.

These growth factors act on the alveolar bone of the patient together with the tooth components contained in the alveolar bone regeneration kit of the present invention to promote regeneration of alveolar bone. For example, BMP moves the mesenchymal stem cells required for healing chemically to the defect site, transforms stem cells into osteocytes and regenerates alveolar bone tissue.

In addition, one of the growth factor derivatives, polydeoxyribonucleotide (PDRN), activates the salvage pathway and selectively activates the A2 purinergic receptor To regenerate cells and then to tissues. First, by activating the bypass pathway, DNA biosynthesis is promoted and matrix regeneration is promoted. It acts selectively on the A2 purinergic receptors to increase the number of fibroblasts and to activate secretion, thereby activating the production of collagen and non-collagen proteins and other necessary components of tissue injury sites. Specifically, it acts on A2A receptors with antiinflammatory effect and A2B receptors which have the effect of preventing blood vessel leakage and edema, thereby inducing endothelial cell migration, VEGF (vascular endothelial growth factor) vascular endothelial growth factor (VEGF) Cells and tissues.

The alveolar bone regeneration kit of the present invention can achieve remarkably improved bone alveolar bone regeneration speeds as compared with bone alveolar bone regeneration materials containing bone growth factors or growth factor derivatives.

In addition, the alveolar bone bone regeneration kit of the present invention may further include a hydrophilic polymer in addition to a tooth and a growth factor or a derivative thereof. These polymers form a skeleton to which inorganic substances such as calcium, which constitute the alveolar bone are bonded, impart mechanical strength to the alveolar bone and enhance the regeneration speed.

The hydrophilic polymer may be selected from the group consisting of alginic acid, chitosan, collagen, hyaluronic acid, cellulose, poly (vinylidene fluoride), poly (tetrafluoroethylene) poly (vinyl alcohol)], poly (hydroxyalkanoate), poly (ethylene terephthalate), poly (butylene terephthalate) ), Poly (methyl methacrylate), poly (hydroxyethyl methacrylate), poly (N-isopropylacrylamide), poly ), Poly (dimethyl siloxane), polydioxanone, polypyrrole, poly (glycolic acid), poly (lactic) acids), poly (ethylene oxide) poly (s-caprolactone)], polyanhydrides, poly (s-caprolactone) s, poly (lactide-co-glycolides) May be selected from the group consisting of polyphosphazenes, poly (ortho-esters), polyimides, and mixtures thereof.

The alveolar bone bone regeneration kit may further include an inorganic material selected from the group consisting of bone, demineralized bone, calcium phosphate and a mixture thereof. These substances bind to the growth factor or growth factor derivative) to impart mechanical strength to the alveolar bone and increase the rate of regeneration.

In addition, it is preferable that the kit is freeze-dried in order to maintain the alveolar bone regenerating ability for a predetermined period or more. More preferably, prior to the freeze-drying, it is recommended to conduct sterilization with a method known in the art, such as radiation or ethylene oxide gas.

In addition, the kit of the present invention may separately store teeth, growth factors, growth factor derivatives, and optional components of hydrophilic polymers and minerals, which are components of the kit, and may store them in a proper combination. Furthermore, it is also possible that the growth factor, the growth factor derivative and the hydrophilic polymer are liquefied and then coated on teeth or teeth and inorganic materials and stored by lyophilization, or the hydrophilic polymer is cross-linked with the tooth. In addition, it is also possible that the hydrophilic polymer in the gel state contains the tooth, the growth factor or the growth factor derivative, etc. Specifically, the gel or the growth factor or both may be contained in the gelatinous hydrophilic polymer to fill the syringe or vial ), Followed by lyophilization.

For example, FIG. 5 is a photograph of a bone alveolar bone reconstruction kit of the present invention comprising a vial 1 in which teeth are stored and a vial 2 in which a growth factor is stored, FIG. 6 is a photograph showing teeth 3 and growth factors 4 ) Were taken together with the vials of the present invention.

FIG. 7 is a photograph of a kit of the present invention composed of a lyophilized vial 5 coated with a growth factor on a tooth, and FIG. 8 is a photograph showing a vial 6 in which a tooth is stored, calcium phosphate and a growth factor And a vial 7 according to the present invention.

FIG. 9 is a photograph of a kit according to the present invention composed of vials 8 prepared by mixing teeth, collagen and growth factors, and FIG. 10 is a photograph showing the vials 9 in which teeth are stored, growth factors and collagen And a vial 10 according to the present invention.

11 is a photograph of the kit of the present invention comprising a vial 11 in which teeth are stored, a vial 12 in which a growth factor is stored, and a vial 13 in which collagen is stored.

Hereinafter, embodiments of the present invention will be described.

Example

Example 1: Preparation of tooth components

The teeth were prepared from the patient himself, and the defects such as cavities and inflammation were removed.

The powder-like teeth were frozen for 40 minutes with liquefied nitrogen at -196 ° C or lower, and then crushed with a pulverizer to obtain powders having an average particle diameter of 50 to 800 μm as shown in FIG. The powder was washed with distilled water for 40 minutes to remove contaminants and residual soft tissue, and the washed powder was degreased with chloroform methanol solution mixed with chloroform and methanol in a ratio of 1: 1 by volume for 3 to 12 hours . The degreased powder was centrifuged to remove suspended fat and then washed with distilled water for 2 hours. The degreased powder was dehydrated with neutral ethyl alcohol for 30 minutes to 2 hours. And then demineralized for 45 hours in a 20-fold volume of 0.2 to 0.7 N aqueous hydrochloric acid solution.

Chip-shaped teeth first removed both enamel and soft tissues of the tooth, leaving only the dentin, and the root of the tooth also removed all of the soft tissues in the nerve. The washed teeth were washed with distilled water for 40 minutes to remove contaminants and residual soft tissues, and the washed teeth were degreased with chloroform methanol solution mixed with chloroform and methanol at a ratio of 1: 1 by volume for 12 to 48 hours . The degreased powder was centrifuged to remove suspended fat and then washed with distilled water for 2 hours. The degreased powder was dehydrated with neutral ethyl alcohol for 30 minutes to 2 hours. And then demineralized in a 20-fold volume of 0.2 to 0.7 N aqueous hydrochloric acid solution for 24 hours. The demineralized teeth were prepared using a bone mill (Aceuropa, Spain) in the form of chips having an average diameter of 800 to 2000 μm as shown in FIG.

The membranous teeth also removed the enamel and soft tissues of the tooth, leaving only the dentin, and the root of the tooth also removed all of the soft tissues in the nerve. The washed teeth were washed with distilled water for 40 minutes to remove contaminants and remaining soft tissues. The washed teeth were degreased with chloroform methanol solution mixed with chloroform and methanol at a ratio of 1: 1 by volume for 3 to 12 hours . The degreased powder was centrifuged to remove suspended fat and then washed with distilled water for 2 hours. The degreased powder was dehydrated with neutral ethyl alcohol for 30 minutes to 2 hours. And then demineralized in a 20-fold volume of 0.2 to 0.7 N aqueous hydrochloric acid solution for 24 hours. The demineralized teeth were cut into shapes and thicknesses determined according to the shape and shape of the defects using a microtome knife (DS Global, Korea).

The teeth of the block type were cut at the cemento enamel junction (CEJ) at the interface between the crown and root, and the crown was removed by trimming both the enamel and nerve soft tissue to leave only the dentin, Were all removed. The root-shaped teeth are, for example, as shown in FIG. 4, and penetration holes having a diameter of 0.8 mm can be drilled on each side as shown in the figure. The block was washed with distilled water for 40 minutes to remove contaminants and residual soft tissues, and the washed block was degreased with chloroform methanol solution mixed with chloroform and methanol at a ratio of 1: 1 by volume for 3 to 12 hours . The defatted blocks were centrifuged to remove floating fat and then washed with distilled water for 2 hours. The degreased block was dehydrated with neutral ethyl alcohol for 30 minutes to 2 hours. And then demineralized for 45 hours in a 20-fold volume of 0.2 to 0.7 N aqueous hydrochloric acid solution.

The degreased, dehydrated and demineralized tooth components were washed and freeze-dried and sterilized with ethylene oxide gas.

Example 2: Preparation of rhBMP-2

1. Production of rhBMP-2

(1) Expression and purification of human BMP-2 gene using E. coli

To obtain the human BMP-2 gene, total cellular RNA was extracted with Trizol (Gibco BRL, USA) solution in U2OS cells (Life technologies TM, Korea) and subjected to reverse transcription reaction. The polymerase chain reaction (PCR) was performed using 5'-AGAAGAACATATGCAAGCCAAACACAAACAGCGG-3 as a sense primer as a template and 5'-AATTTTACAGCTTCTAGCGACACCCACAACCCT-3 'as an antisense primer. The PCR product was isolated and inserted into pGEM-T vector (Promega, USA) and cloned using E. coli (DH5α) (Life technologies ™, Korea).

(2) High-density cell culture

Heat-induced production of human growth hormone by recombinant Escherichia coli by recombinant Escherichia coli. Biotechnol Lett 2004; 26: 245-250 (1998), using a fermenter (KoBioTec. Korea) . High-density culture was performed with a simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnology 1995; 39: 59-65.). A sterile nutrient medium (glucose 33.3 g / L, peptone 10 g / L, yeast extract _{5 g / L, MgSO 4 1} g / L, CaCl 2 0.048 g / L, ZnSO 4 0.0176 g / L, CuSO 4 0.008 g / L) was added thereto and cultured for 24 hours with stirring at a stirring speed of 250 rpm.

(3) Protein purification

The suspension was stored at -80 ° C in a cryogenic freezer (Nihon Freezer, Japan). The frozen suspension was thawed at refrigeration temperature, and the cells were disrupted by pressurization and then centrifuged at 5,500 xg at 4 ° C for 45 minutes. When the mature rhBMP-2 that has undergone the re-denaturation process has the original tertiary structure, the N-terminus has a heparin-binding site.

2. Biochemical properties of purified rhBMP-2

(1) Purity and identification test of rhBMP-2 stock solution

As a result of the SDS-PAGE test, as shown in Fig. 13, the rhBMP-2 stock solution showed the same migration distance as the standard solution and showed a purity of 95% or more.

(2) HPLC (High performance Liquid Chromatography) analysis

The purified rhBMP-2 dimer was dissolved in 0.1% TFA (trifluoroacetic acid) at a concentration of 1 μg / μl and subjected to C4 reversed-phase HPLC column (4.6 mm × 50 mm, 300 Å, 5 μm particle size; Gracevydac. The proteins of each fraction were detected and monitored. As shown in Fig. 14, the test results of the rhBMP-2 stock solution showed a retention time equivalent to that of the standard solution, confirming that it was purified with pure rhBMP-2.

3. Production and purification of rhBMP-2 protein

And the affinity chromatography was performed with a heparin column (Sigma Dow. Italy). As a result, as shown in FIG. 13, most monomers and a small amount of dimer were eluted from the 0.3 M NaCl fraction and the dimers eluted from the 0.5 M NaCl fraction. Purified rhBMP-2 monomers and dimers were identified by SDS-PAGE analysis. The size of the monomer produced is calculated to be about 114 amino acid residues and the molecular weight of the monomer is about 14 kDa. The dimer has the same size band as the monomer in the nonreducing condition because the two monomers are connected by disulfide bonds. The size of the dimer was about 28 kDa.

4. Biological activity of purified rhBMP-2 (in vitro test)

The rhBMP-2 dimer was applied to C2C12 cells (Sigma Dow, Italy), which was a myoblast cell. After 3 days of culture, alkaline phosphatase, a representative protein of osteoblasts, was secreted (Fig. 15) (Fig. 16). Thus, it was confirmed that rhBMP-2 functions as an osteogenic protein.

Example 3: Preparation of kit containing growth factor

The alveolar bone regeneration kit (Fig. 5) of the present invention was prepared by combining the vial 1 in which the teeth of Example 1 were stored and the vial 2 in which rhBMP-2 of Example 2 was stored, (FIG. 6) in which rhBMP-3 (3) and rhBMP-2 (4) of Example 2 were stored together. The kit of the present invention (Fig. 7) composed of the vial 5, which was prepared by coating rhBMP-2 of Example 2 on the tooth of Example 1 and lyophilized, was prepared and the vial 6 ), Ca ₃ (PO ₄ ) _2, and rhBMP-2 of Example 2 were stored together.

A kit (FIG. 9) in the form of a block (8) prepared by lyophilizing the teeth of Example 1, a 0.5% aqueous collagen solution (Sigma Aldrich, USA) and rhBMP-2 of Example 2 was prepared, The kit of the present invention (Fig. 10) composed of the vial 9 in which the teeth were stored and the block 10 in which the rhBMP-2 of Example 2 was freeze-dried together with the collagen solution was prepared. The vial 11 in which the tooth of Example 1 was stored and the vial 12 in which the BMP-2 of Example 2 was freeze-dried and the aqueous solution of collagen 0.5% (Sigma Aldrich, USA) (Fig. 11) of the present invention.

The method of coating the tooth of Example 1 and lyophilization will be described with reference to the kit of FIG. 7 as an example.

1 g of tooth implant was dispensed into glass vials, capped with a silicone plug, and sterilized with gamma rays. 1 mg of recombinant rhBMP-2 was dissolved in a solution consisting of 5 mM of glutamic acid, 2.5% by weight of glycine, 5 mM of sodium chloride, 0.015% by weight of Tween-80 and 0.5% by weight of D-sorbitol and added to a glass vial containing tooth granules Thereby immersing the teeth. The glass vials were placed in a cryocooler, immediately frozen, placed in a vacuum freeze dryer, held at -40 ° C for 3 hours, and then gradually heated to 20 ° C. The lyophilized glass vials were capped in an aseptic environment.

Example 4: Preparation of a kit containing a growth factor derivative

The same procedure as in Example 3 was carried out, and PDRN (MASTELLI _SRL, Italy) was used instead of rhBMP-2 in Example 2.

Test Example

Thirty-six athymic mice (average weight: 30 g) were used. The animals were divided into three groups (12 weeks, 2 weeks, 4 weeks or 5 weeks, 8 weeks), and each group was divided into growth factor administration group, growth factor derivative administration group and control group. General anesthesia was performed by intraperitoneal injection of pentobartibal sodium (Nembutal, 43 mg / kg, Dainabot Co., Japan) under aseptic conditions. The backs of each sample were incised and the right and left subcutaneous tissues were incised to a depth of 10 mm to make a subcutaneous bag for insertion of the alveolar bone regeneration kit of the present invention.

The growth factor administration group thawed the kit in which the tooth and the rhBMP-2 were separately stored in the kit of the present invention of Example 3, and 0.2 g of the tooth was hydrated in 0.2 ml of the rhBMP-2 aqueous solution and inserted into each site. The growth factor derivative-administered group thawed the kit of the present invention of Example 4 and hydrated 0.2 g of the tooth in 0.2 ml of rhBMP-2 aqueous solution and inserted into each site. Only 0.2 g of the tooth powder of Example 1 was inserted into each site, and only 0.2 g of synthetic bone (Cowell Medi, Korea) was inserted into each site in the synthetic bone administration group. After each implant was inserted, the incision was closed with a nylon suture. To protect the wound from infection, antibiotic ointment (Daewoong Pharm. Korea) was used throughout the study.

Soft tissue biopsies containing alveolar bone regeneration kit contents in subcutaneous pouches were obtained from athymic mice at 2, 4 or 5 and 8 weeks after insertion. A total of 72 biopsies were fixed in 10% formalin for 24 hours. After 12 hours of demineralization in Calci-Clear Rapid (National Diagnostics, Atlanta, GA), the specimens were washed with distilled water. The specimens were then treated with a Hypercentre XP tissue processor (Shandon, UK), submerged in paraffin, and cut into 4-5 μm thickness. Segmented specimens were observed using an optical microscope after hematoxylin (Figures 17-25 and 27-30) and eosin staining (Figure 26). Images were captured using a MagnaFire digital camera system (Optronics, Goleta, Calif.).

FIG. 17 to FIG. 20 show that the soft tissue production is most active in the synthetic bone administration group (FIG. 20) as the specimen photograph after 2 weeks, the growth factor administration group (FIG. 17) and the growth factor derivative administration group (FIG. 18) The soft tissue production was slowest in the tooth-receiving group (Fig. 19).

However, Figs. 21 to 22 showing enlarged images of the specimen after 2 weeks show that the alveolar bone is produced in a completely different manner. That is, FIG. 21, which is a growth factor administration group and FIG. 22 which is a growth factor derivative administration group, reproduce more bone than the original tooth particle, whereas the dental administration group of FIG. 23 only reproduces some alveolar bone on the left side only. In Fig. 24, which is a synthetic bone administration group, it can be seen that alveolar bone regeneration does not occur at all around the synthetic bone.

FIG. 25 to FIG. 28 are photographs of a specimen after 4 weeks or 5 weeks. FIG. 25, which is a group in which a growth factor is administered (after 4 weeks) and FIG. 26 which is a group in which a growth factor derivative is administered And that the alveolar bone regeneration was performed. On the contrary, FIG. 27, which is a group administered with a tooth (after 5 weeks), shows that a tooth part of Example 1 is still observed, indicating that alveolar bone regeneration speed is lower than that of the kit of the present invention. FIG. 28 shows that alveolar bone regeneration is still not performed.

Finally, in FIGS. 29 and 30, which are photographs of the specimen after 8 weeks, cartilage formation, which is evidence of bone induction, could be confirmed. Specifically, the cartilage is confirmed in the left side of FIG. 29, which is the group of the growth factor administration, as in the left lower side of FIG. 30, which is the tooth administration group, whereas the tooth particles are not found at all and it is confirmed that the cartilage is completely restored following the alveolar bone regeneration .

From the above drawings, it can be seen that the alveolar bone regeneration kit of the present invention including the growth factor or the growth factor derivative has superior alveolar bone regeneration speed as compared with the synthetic bone administration group which can not regenerate the alveolar bone at all. This rapid alveolar bone regeneration greatly alleviates the inconvenience of the patient and has an additional effect of preventing infection due to prolonged regeneration, which is very useful in the field of medical treatment.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course it is possible. Accordingly, the scope of the present invention should not be construed as being limited to the above-described embodiments, but should be determined by equivalents to the appended claims, as well as the following claims.

Claims (10)

Teeth of a person or animal, and
Alveolar bone regeneration kit comprising a growth factor or growth factor derivative. The method according to claim 1,
The alveolar bone regeneration kit is an alveolar bone regeneration kit, characterized in that it further comprises a hydrophilic polymer. The method according to claim 1,
Wherein the alveolar bone bone regeneration kit further comprises bone, demineralized bone, calcium phosphate, and a mixture thereof. The method according to claim 1,
The growth factors may be selected from the group consisting of recombinant human bone morphogenetic protein (BMP), amino acid binding constructs of BMP, fibroblast growth factor, growth differentiation factor (GDF) (TGF), platelet derived growth factor, insulin like growth factor, epithelial growth factor, keratinocyte growth factor 2 (KGF2), morphogenic protein 52 (MP52), their recombinant proteins and their mixtures Wherein the bone is a bone. The method according to claim 1,
Wherein the growth factor derivative is polydeoxyribonucleotide (PDRN). The method according to claim 2,
The hydrophilic polymer is alginic acid, chitosan, collagen, hyaluronic acid, cellulose, poly (vinylidene fluoride) [poly (vinylidene fluoride)], poly (tetrafluoroethylene) [poly (tetrafluoroethylene), poly (vinyl alcohol) [ poly (vinyl alcohol)], poly (hydroxyalkanoate) [poly (hydroxyalkanoate)], poly (ethylene terephthalate) [poly (ethylene terephthalate)], poly (butylene terephthalate) [poly (butylene terephthalate)] , Poly (methyl methacrylate), poly (hydroxyethyl methacrylate), poly (N-isopropylacrylamide) poly (N-isopropylacrylamide) , Poly (dimethyl siloxane) [poly (dimethyl siloxane)], polydioxanone, polypyrrole, poly (glycolic acid), poly (lactic acids) ], Poly (ethylene oxides), poly (Lactide-co-glycolides), poly (s-caprolactone), poly (s-caprolactone), polyanhydrides, polyphosphazenes , Poly (ortho-esters) [poly (ortho-esters)], polyimide (polyimides), Alveolar bone regeneration kit, characterized in that selected from the group consisting of. The method according to claim 1,
Characterized in that the tooth is a form selected from the group consisting of a powder having an average particle size of 50 to 800 탆, a chip having an average major axis of 800 to 2000 탆, a film having an average thickness of 0.2 to 5.0 mm, a scaffold of a root- The alveolar bone regeneration kit. The method according to claim 1,
Wherein the tooth has been removed by removing the soft tissue in the teeth of the alveolar bone. The method according to claim 8,
Wherein the teeth are demineralized. The method according to claim 1,
Wherein the kit is freeze-dried.

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