WO2018225907A1

WO2018225907A1 - Method for preparing injectable adhesive composition for fixing bone graft material, and injectable adhesive composition for fixing bone graft material prepared therefrom

Info

Publication number: WO2018225907A1
Application number: PCT/KR2017/012248
Authority: WO
Inventors: 최다미; 심진형; 허중보; 김창환; 안근선; 윤원수
Original assignee: 주식회사 티앤알바이오팹; 부산대학교 산학협력단
Priority date: 2017-06-05
Filing date: 2017-11-01
Publication date: 2018-12-13
Also published as: KR20180133172A

Abstract

The present invention relates to a method for preparing an injectable adhesive composition for fixing a bone graft material, which fixes a bone graft material or a shielding membrane that are filled in the body in order to restore defective parts in bone tissues, and an injectable adhesive composition for fixing a bone graft material prepared by the same method. As the injectable adhesive composition comprises a decellularized extracellular matrix (dECM) having excellent biocompatibility and a calcium phosphate compound having similar inorganic ingredients as those of bone tissues, the injectable adhesive composition has advantages in that the decellularized extracellular matrix undergoes a gelation reaction due to a protein component such as collagen at a temperature similar to body temperature, thereby being capable of fixing a bone graft material and a shielding membrane through physical fibrosis when inserted into the body, and also the decellularized extracellular matrix is completely dissolved and absorbed in the body without any foreign body reaction after a certain period of time, thereby requiring no secondary removal procedure.

Description

[Specifications

[Name of invention]

Method for preparing an injection-type adhesive composition for fixing bone grafts and an injection-type adhesive composition for fixing bone grafts

Technical Field

The present invention relates to a method for preparing an implantable adhesive composition for fixation of bone graft material for fixing a bone graft material or a shielding membrane filled in the body for restoration of a defect of bone tissue, and to an implantable adhesive composition for fixation of bone graft material prepared through the same. will be.

Background Art

Bone tissue is a hard tissue that maintains the skeleton of the human body, and bone grafting substems and soft tissue blocking membranes (hereinafter referred to as bone tissue) when bone tissue is damaged by trauma, tumors, malformations, or physiological phenomena. New bone can be formed by inserting a barrier, guided tissue regenerat ion or guided bone regener at ion.

Detailed description of the invention

[Technical problem]

In general, bone grafts are intended to replace missing bones, and not just to compensate for the missing bones, but also to help new bones form in the areas where bones are lost. For orthopedic surgery to promote bone formation and to replace bone tissue defects in bone defects left behind or bone fractures remaining in the dentist's bone or spine after removal of fracture or fracture dislocation, nonunion or delayed union, tumor or osteomyelitis, etc. Can be used.

Such bone graft can be implanted into the bone defects in the body for the recovery of damaged bone defects, the bone graft transplant method is a method of autologous bone graft to extract and transplant a portion of their own bone in different areas according to the type of bone graft material, Allogeneic bone graft method for chemically transplanting bone of another person, there is a xenograft graft method for chemically transplanting bone of animal.

The best method of transplantation is autologous bone graft, which is used for transplanting its own bones, and the use of autologous bone is likely to cause bone formation or bone induction, healing of the bone to be transplanted, rapid conversion to living bone, and immune response caused by autologous bone There is a merit that there is no reaction, but it requires secondary surgery, it is difficult to obtain the required amount, and it is difficult to carry out in general private clinics.

Allograft and xenograft methods, which are other transplantation methods, do not require a secondary surgical part, and have advantages such as fast operation time, recovery period, and low cost compared to plywood implants.

Alternative Site (Article 26) However, compared to autologous bone, the bone induction period is about twice that of bone, the bone quality of the bone to be formed is likely to be lowered, immune reactions can occur, and the probability of introducing viruses such as AIDS or hepatitis is low. There is a risk that it can.

After implanting the bone graft into the damaged bone defect in the body, a barrier should be formed to prevent the surrounding soft tissue from penetrating into the space of the bone defect so that sufficient new bone can be formed in the bone defect space during the formation of new bone. The bone graft material and the barrier membrane implanted in the bone defect should be stably fixed in the body during the formation of new bone.

Generally, fibrin glue or sealant is used to fix the bone graft and the shield, or a metal bone screw is used to fix the shield directly. .

However, in the case of fibrin glue or sealant, it is possible to fix between the bone graft and the shielding membrane, but after about 4 weeks in the body is decomposed and can not help the formation of new bone, there is a problem that the bone defect is not completely formed into new bone. In addition, when fixing the shielding membrane using a bone screw made of a metal material, there is a problem that additional surgery is required to remove it after bone formation is completed.

Technical Solution

In the present invention, the bone graft material or the shielding layer layered in the body for the restoration of the defect of the bone tissue is stably fixed without being shaken by the external stratification, the bone graft fixation injectable adhesive composition for the new bone can be formed smoothly It is to provide a method of manufacturing and an injection-type adhesive composition for fixing bone graft material produced through the same.

One embodiment of the present invention for achieving the above object, relates to a method for producing an injection-type adhesive composition for fixing bone graft material, decellularized extracellular matrix (dece l lul ar i zed ext racel lul ar mat r ix, dECM); Grinding the calcium phosphate compound; A first mixing step of mixing the pulverized calcium phosphate compound with water to prepare a first mixture; And a second mixing step of preparing the second mixture by mixing the first mixture with the decellularized extracellular matrix, and preparing an injection adhesive composition for fixing bone graft materials.

After the secondary mixing step, it is preferable to further include; a step of cooling the secondary mixture to a temperature of 2 ~ 8 ° C.

Specifically, in the grinding step, the calcium phosphate compound may be ground to a particle size of 10 ~ 2,000 μια.

In addition, the first mixing step, the mixed calcium phosphate compound may be mixed at a ratio of 80 to 90 wt and water 10 to 20 wt%, the second mixing step, the first mixture 50 to 80 wt and decellularization It is desirable to mix at 20-50 wt% ratio of extracellular matrix.

The second mixing step, the decellularized extracellular matrix and a further natural polymer material is further mixed in the first mixture, the natural polymer material, carboxyl methyl cel l ul ose Heparan sul fate, One or more selected from the group consisting of hyaluronic acid, collagen, col lagen, dextran, and alginate may be used. In addition, the second mixing step, the decellularized extracellular matrix and additional functional substances in the first mixture is further mixed, and the functional substance is a bone formation protein (BMP), ^' epithelial cell growth factor ( EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-β), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), thioredoxin (TRX), stem cell factor (SCF) ), Hepatocyte proliferation factor (HGF), human growth hormone (hGH) and angiogenin can be used at least one selected from the group consisting of.

On the other hand, another embodiment of the present invention, an injection-type adhesive composition for fixing bone graft material produced by the above-mentioned manufacturing method.

Specifically, the injectable adhesive composition for fixation of bone graft material includes a decellularized extracellular matrix (dECM), a calcium phosphate compound, a natural polymer material, a functional material, and water, preferably the decellularized extracellular matrix. 20 to 50 wt of the substrate, 40 to 72% of the calcium phosphate compound, wherein the sum of the decellularized extracellular matrix, the phosphate compound, the natural polymer, the functional material, and the water does not exceed 100 _w t%.

Specifically, the calcium phosphate compound has a particle size of 10 to 2,000, and the natural polymer material is carboxymethyl cellulose, carboxyl methyl cellulose, heparan sulfate, hyaluronic acid, and collagen. (col lagen dextran and alginate) is one or more selected from the group consisting of, the functional substance, bone morphogenic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), Transforming growth factor (TGF-β), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), thioredoxin (TRX), stem cell factor (SCF), hepatocyte growth factor (HGF), human growth One or more selected from the group consisting of hormones (hGH) and angiogenin may be used.

【Effects of the Invention】

Injectable adhesive composition for fixation of bone graft material according to the present invention includes a decellularized extracellular matrix (dECM) having excellent biocompatibility and a calcium phosphate compound similar to bone tissue and an inorganic component. Substrate causes gelation reaction at temperature similar to body temperature due to protein components such as collagen, and it is possible to fix bone graft material and shielding membrane through physical fibrosis when inserted into body, It is completely decomposed and absorbed without the need for a secondary removal procedure.

In addition, the use of the decellularized extracellular matrix as an adhesive material completely removes the heterologous cells, thereby significantly lowering the immune rejection response to the subjects, as well as collagen, glycoprotein, and BMP-2 in the decellularized extracellular matrix. Including bone-derived proteins that can help bone formation, such as ALP, has an effect of having excellent bone conduction and osteoinduction. On the other hand, in the mixing process of the calcium phosphate compound and decellularized extracellular matrix, respectively, dissolved in a solvent and then mixed so that the calcium phosphate compound can be uniformly dispersed without aggregation and new bone can be formed uniformly in the bone defect. .

[Brief Description of Drawings]

1 is a photograph showing the decellularized extracellular matrix prepared through the preparation of the present invention.

Figure 2 is a photograph showing the injection adhesive composition for fixing the bone graft prepared through the preparation of the present invention.

Figure 3 is a photograph showing the result of measuring the hydration state holding force of the injection-type adhesive composition for fixing bone graft material of the present invention.

Figure 4 is a graph showing the adhesion measurement test results of the bone graft fixing injectable adhesive composition of the present invention.

Figure 5 is a schematic diagram schematically showing an experimental method for measuring the fixing performance of the injectable adhesive composition for fixing bone graft material of the present invention.

Figure 6 is a graph showing the results of the fixation measurement test of the injection adhesive composition for fixing bone graft material of the present invention.

Figure 7 is a graph showing the results of measuring the compressive strength of the adhesive composition according to the content of the decellularized extracellular matrix.

Figure 8 is a photograph sequentially showing the test method for confirming the bone conduction and osteoinduction ability of the bone adhesive fixable injection composition for bone graft of the present invention.

Figure 9 is a photograph showing the results of measuring the effectiveness of bone conduction and osteoinduction when using the injection adhesive composition for fixing the bone graft material of the present invention.

[Form for implementation of invention]

Before describing in detail through the preferred embodiments of the present invention, the terms or words used in the present specification and claims should not be construed as being limited to the conventional or dictionary meanings, meanings corresponding to the technical spirit of the present invention To be interpreted as

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding other components unless specifically stated otherwise.

Terms such as "first" and "second" are intended to distinguish one component from another component, and the scope of rights should not be limited by these terms. For example, the first component may be referred to as a second component, and similarly, the second component may also be referred to as a U component.

In each step, the identification code is used for convenience of explanation, and the identification code does not describe the order of each step, and each step may be performed differently from the stated order unless the context clearly indicates a specific order. have. That is, each step may be performed in the order specified or substantially simultaneously It may be practiced or in the reverse order.

As used herein, an "extracellular matrix" (ECM) is a complex aggregate of biopolymers filling a space in or outside a tissue, such as fibrous proteins, complex proteins such as proteoglycans, fibronectin, laminin, etc. It is composed of various kinds of molecules synthesized by cells such as proteins and secreted and accumulated besides cells. "Decellularization" also means the removal of cellular components other than extracellular matrix, such as nuclei, membranes, nucleic acids, etc. from cells or tissues, and "decellularized extracellular matrix. , dECM) '' means the extracellular matrix remaining after removal of cellular components such as nuclei, membranes and nucleic acids from tissues or cells.

Hereinafter will be described in more detail with respect to the method for producing a bone graft fixable injectable adhesive composition and the bone graft fixed injectable adhesive composition prepared by the present invention.

First, a method for preparing an implantable adhesive composition for fixing bone grafts includes preparing a decellularized extracellular matrix (dECM); Grinding the calcium phosphate compound; A first mixing step of mixing the pulverized calcium phosphate compound with water to prepare a first mixture; And a second mixing step of preparing the second mixture by mixing the first mixture with the decellularized extracellular matrix.

Preparing the decellularized extracellular matrix may include tissues obtained from mammals such as humans, pigs, cattle, rabbits, dogs, goats, sheep, chickens, and horses (eg, heart tissues, cartilage tissues, bone tissues, Adipose tissue, muscle tissue, skin tissue, mucosal epithelial tissue, amniotic tissue, corneal tissue, etc.) may be decellularized by methods known to those skilled in the art or by appropriate modifications thereof to prepare decellularized extracellular matrix.

However, since the components of the extracellular matrix are different depending on the type of cells or the degree of differentiation of the cells as raw materials, the decellularized extracellular matrix used in the injectable adhesive composition for fixation of bone grafts of the present invention has improved bone conduction. And decellularized extracellular matrix derived from bone tissue obtained through decellularization method in bone tissue, preferably swine, pretty, xenograft obtained from horse bone or allogenous bone obtained from human litter, to have osteoinductive ability. have.

The decellularization method is a method of removing the remaining cellular components other than the extracellular matrix in the tissue, may be a physical method, a chemical method or a combination thereof, preferably a chemical method may be used. .

For example, when preparing the decellularized extracellular matrix by using a chemical method, sterilizing the xenograft or allogeneic bone, and then immersing the cells cultured in the decellularization solution for a predetermined time, then leaving it to decellularize, Degreasing extracellular matrix can be prepared by sequentially degreasing.

In this case, the decellularization solution used may be an acid solution, a basic solution, a non-ionic detergent or an ionic detergent, but a nonionic detergent may be preferably used. For example, when using Triton X-100, a nonionic detergent, the cells to be decellularized are immersed in Triton X-100 to remove the cell membrane. It can be removed to remove intracellular components, or to remove the nucleus components using DNase and RNase, but preferably using trypsin and using EDTA at 35 ~ 39 ⁰ C, pH 7.5 ~ 8.5 conditions of the bone tissue By breaking the cell and removing the contents of the cell, it can be decellularized. .

Preparing the decellularized extracellular matrix of the present invention, preferably by removing only the nucleus, cell membrane, etc. of the cells from bone tissue such as xenograft or allogeneic bone to prepare a decellularized extracellular matrix ᅳ the decellularized extracellular compared to normal bone In addition, the DNA content contained in the vial is remarkably low (<50 ng / mg), which can significantly reduce immunity rejection when introduced into the body, as well as collagen and glycoproteins, bone-derived proteins that are extracellular matrix components of bone tissue cells. Glycosaminoglycans, GAGs), BMP-2 (Bone Morphogenetic Protein-2), ALP (Alkaline Phosphatase), etc. can be utilized, and can have excellent bone conduction and osteoinduction ability when filling the defective bone tissue.

In addition, when the decellularized extracellular matrix is introduced into the body, gelation (gelation) at a temperature similar to the body temperature due to collagen and protein components contained in the decellularized extracellular matrix, binding between the bone graft and bone graft material, bone graft material Binding between the membrane and the barrier membrane, the barrier membrane and the surrounding cell tissues can be fixed, and the phosphate compound and other substances included in the injectable adhesive composition for fixing bone graft material of the present invention can be aggregated.

The pulverizing step is a step of pulverizing the calcium phosphate compound, the calcium phosphate compound with a predetermined particle size to include the calcium phosphate compound having bone conduction ability, bone induction ability in the bone graft material fixing injectable adhesive composition of the present invention May be ground. The finer the particle size of the calcium phosphate compound may have excellent bone conduction and osteoinductive ability. However, if the particle size of the chamomile compound is too fine, the chamomile compound is scattered in a fine form, and thus it is difficult to use.

The calcium phosphate compound is a compound in which phosphoric acid and calcium are formed through chemical bonding, and natural bone and inorganic components are similar, and as long as the calcium phosphate compound can help the bone grow by conduction, it can be used without particular limitation, for example, hydroxy Apatite (Hydroxyapatite, HAp), Carbonated apatite, Tricalcium Phosphate (TCP), Ca lcium Hydrogen Phosphate, Monocalcium Phosphate (FCnocalcium phosphate) At least one selected from the group consisting of Dicalckmi phosphate, Calcium di hydrogen phosphate, Tricalcium phosphate, Oct 10 calcium phosphate, and Calcium pyrophosphate The above can be used. Preferably, β-tri calcium phosphate (β-TCP) or hydroxyapatite (HAp) may be used.

Specifically, in the grinding step, the calcium phosphate compound may be pulverized to a particle size of 10-2,000 μπι, but if the particle size of the compound of the calcium phosphate is less than 10 μια, the cost and time for finely pulverizing the particles may be advantageous. Economic efficiency can be reduced, and when the particle size exceeds 2,000, Too large, it is difficult to homogeneously disperse in the injection adhesive composition for fixing the bone graft material, it is difficult to agglomerate the composition when it is introduced into the body can lower the fixing force. Since the lower the particle size of the calcium phosphate compound is improved bone conduction and bone conduction properties, preferably, the particle size of the calcium phosphate compound in the grinding step may be ground to 20 ~ 200 urn particle size.

The method for pulverizing the calcium phosphate compound in the pulverizing step is not particularly limited as long as it is a method for pulverizing the compound to a predetermined particle size, such as wet pulverization, dry pulverization, preferably triggered pulverization or manure sieve using induction It can be ground to a calcium phosphate compound having the desired particle size using.

The pulverized chamophosphate compound is mixed with water to prepare a first mixture, followed by a second mixing step of preparing a second mixture by mixing the first mixture with the decellularized extracellular matrix. Can be rough. This is to ensure that the calcium phosphate compound and the decellularized extracellular matrix contained in the injectable adhesive composition for fixing bone grafts of the present invention can be uniformly dispersed and mixed.

In detail, the first mixing step may be prepared by mixing 80 to 90 wt% of the pulverized calcium phosphate compound and 10 to 20 wt% of water to prepare a primary mixture, wherein the mixing ratio of the calcium phosphate compound and water is Outside of the stated range. In the case of the second mixing step, the decellularized extracellular matrix and the calcium phosphate compound are not uniformly mixed, or the moisture content is too high, so that the injection adhesive composition for fixing the bone graft material of the present invention is laminated in a syringe and injected into the body completely. It may take a long time to bond, and thus the convenience of the procedure may be reduced, or the adhesion may be degraded, and thus the binding force between the bone graft and the bone graft, between the bone graft and the shield, and the shielding membrane and the surrounding tissue may be reduced. Agitation may occur.

To prepare an injection-type adhesive composition for fixation of bone graft material according to the present invention through a second mixing step of preparing a second mixture by mixing the decellularized extracellular matrix with the first mixture prepared through the first mixing step Can be.

Specifically, the secondary mixing step is to prepare a secondary mixture by mixing the first complex and the decellularized extracellular matrix, the first mixture 50 ~ 80 wt% and the decellularized extracellular matrix 20 It is preferable to control the mixing temperature so that the protein contained in the decellularized extracellular matrix is mixed, but more preferably collagen and other proteins contained in the decellularized extracellular matrix, Because gels are geled at temperatures similar to body temperature, they should be mixed with proper temperature control to ensure that the mixing temperature does not exceed 37 ° C.

In addition, it is preferable to be carried out in a temperature range exceeding at least 1 ° C, because when less than 1 ⁰ C water freezing occurs in the formulation it is impossible to formulate.

This, in the second mixing step, is stirred using a stirrer to prepare a homogeneous mixture when mixing the primary mixture and the decellularized extracellular matrix, wherein the stirring speed so that the mixed materials can be well dispersed Increased agitation, increased agitation The temperature of the mixture rises in proportion to the speed.

The decellularized extracellular matrix included in the injectable adhesive composition for fixation of bone graft material of the present invention contains a protein component, which is susceptible to heat. In particular, a protein component such as collagen may be at a temperature of about 35 to 39 ⁰ C. Due to the problem of deformation, coarse or fibrosis reaction, the formulation is denatured, the second mixing step is preferably mixed while controlling the temperature appropriately to maintain a temperature range of more than 1 ⁰ C but not more than 37 ° C. It is preferable to store the injectable adhesive composition for fixation of bone graft material through an angle step in which the silver mixture of the secondary mixture prepared after the step is angled to a temperature of 2 to 8 ° C.

In the second mixing step, the ratio of 50 to 80% of the first mixture and 20 to 50 wt% of the decellularized extracellular matrix is preferable. As the content of the decellularized extracellular matrix contained in the prepared secondary mixture increases, the fibrosis and physical crosslinking progress when the injectable adhesive composition for fixation of bone graft material of the present invention is introduced into the body, thereby increasing the intermolecular binding force. Although the viscosity improves, the fixation force of the bone graft material and the shielding membrane is improved, but when the content of the decellularized extracellular matrix is excessive, the content of calcium phosphate compound contained in the primary mixture is relatively low, leading to the bone defect. This is because it is difficult to expect a good bone formation effect after gelation, and the tensile strength of the gelled bone graft implantable injection adhesive composition is significantly lowered, which may be damaged by external physical lamination.

In order to improve the adhesion of the bone graft material and the shielding membrane by improving the adhesive force in the injection adhesive composition for fixing the bone graft material of the present invention in the second mixing step, and to improve the bone conduction ability, bone induction ability, bone formation ability of the bone defects It may further comprise a natural polymer material or a functional material.

The natural polymer material or functional material is also susceptible to heat, and thus, it is preferable to add additionally in the second mixing step, rather than the first mixing step.

The natural polymer material is carboxylmethyl cel lulose, heparan sul fate, hyaluronic acid (hyaluroni c acid), collagen (col lgen), dextran and alginate ( alginate). One or more selected from the group consisting of, and the functional material is bone morphogenetic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), conversion growth factor (TGF-β) , Platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), thioredoxin (TRX), stem cell factor (SCF), hepatocyte growth factor (HGF), human growth hormone (hGH) and engiogenin ( Angio enin) may be used one or more selected from the group consisting of.

The natural polymer material and the functional material are preferably used in the range of 0.02 to 2 parts by weight based on 100 parts by weight of the secondary mixture in which the primary mixture and the decellularized extracellular matrix are mixed, each of which is 0.02 parts by weight. If less than, the effect of growth factors that are natural polymers or functional materials can not be exerted, and if it exceeds 2 parts by weight, rather than inhibiting the formation of bone-derived decellularized extracellular matrix, Because it becomes impossible.

On the other hand, in another embodiment of the present invention, there may be mentioned an implantable adhesive composition for fixation of bone graft material prepared by the above-mentioned manufacturing method, the implantable adhesive composition for fixation of bone graft material according to the present invention, decellularized extracellular matrix Due to the collagen and other protein components contained in the gel is ionized at a temperature of about the body temperature (gel at ion), it is physical fibrosis by body temperature when injecting the implantable adhesive composition for fixing the bone graft into the body through a syringe (fibrillogenesis) occurs, which improves the bond between the bone graft and the bone graft, the bond between the bone graft and the barrier, and the fixation force between the barrier and the surrounding tissues, so that the bone graft and the barrier are not shaken in the body during the formation of new bone. New bone can be formed stably fixed.

In addition, there is no need for a separate fixation device used to fix a bone graft, a barrier, etc., for example, a bone screw, and a second operation for removing a bone screw after a bone graft operation. Surgical prognosis may therefore be improved.

In addition, decellularized extracellular matrix extracted from bone tissue contained in the implantable adhesive composition for fixation of bone graft material is included, which contains various components that can help to form new bone such as bone-derived proteins. It may have induction ability. In addition, by including a phosphate compound similar to natural bone and inorganic components, it is possible to enjoy the formation of new bone. Hereinafter, an embodiment of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art may implement various modified forms of the contents described herein within the scope of the present invention.

Preparation Example Preparation of Injectable Adhesive Composition for Fixing Bone Graft Material

First, a decellularized extracellular matrix (dECM) was prepared from tibia in 12-24 month-old calves.

Specifically, the tibia is divided into pieces, separated into a cancellous part and a cortical part, and then to remove foreign substances and fine tissues of the separated spongy part.

Washing was performed with Phosphate-buffered ssaline (PBS) containing gentamicin (centamicin, Invitrogen, Carlsbad, Calif., USA). The spongy part was then frozen in liquid nitrogen and cut into sections of 4x4x4 mm or less, and then the cut sections were washed once more with distilled water. The washed sections were immersed in liquid nitrogen and then ground into a powder using a coffee mill (Kordia Co., Daegu, Gyeongbuk, Korea). 0.5 N HCK25 powder of ground sponge powder was used. After dissolving, the phase was stirred for 24 hours (300 rpm) to demineralize the spongy in powder form, after demineralization, the filter was filtered under vacuum and rinsed with distilled water, followed by chloroform (Chloroform, Fisher). Scientific, Loughborough, UK) and methane (Methanol, Fisher Scientific) using a mixed solvent mixed in a 1: 1 volume ratio to extract the lipid from the demineralized powder for 1 hour, and then sequentially using methane, distilled water After washing, snap frozen and then for a long time Lyophilized under reduced pressure and then stored at -20 ° C. The stored lyophilized powder was washed with distilled water, and then dissolved in a mixed solvent containing 0.05% trypsin (Trypsin, Sigma-Aldr ich) and 0.02% EDTACethylenedi aminetetraacet ic acid, Sigma-Aldr i ch) at 37 ° C, The cells were decellularized by stirring for 24 hours in a 5% C02 atmosphere. After decellularization, the cells were washed with PBS (Phosphate_buf fered ssal ine) containing 1% (w / v) penicillin / streptomycin for 24 hours at 4 ° C. to remove residual cellular material. Instant freeze after washing, and freeze-dried under a reduced pressure atmosphere for a sufficient time-stored at 20 ⁰ C to prepare a decellularized extracellular matrix, prepared decellularized extracellular matrix is shown in FIG.

-TCP (-tri calcium) was used to induce pulverization and powdering, followed by sieving to prepare a pulverized calcium phosphate compound having an average particle size of 75. 85 g of the pulverized calcium phosphate compound and 15 g of purified water were mixed and mixed for 10 minutes using a paste mixer to obtain a primary mixture.

After mixing the obtained primary mixture, the prepared decellularized extracellular matrix and the functional substance in the ratio as shown in Table 1 below, the secondary mixture was secondly mixed for 15 minutes using a paste mixer. Prepared. Care was taken not to exceed 37 ⁰ C in the second mixing. After mixing, the secondary mixture was angled at 5 ° C., and then the angled secondary mixture was layered in a syringe to prepare an injectable adhesive composition for fixing bone grafts.

Table 11

protein)

Total 100 100 100 100 100 100 100.2 102 100 103

(Unit g)

Experimental Example 1 Measurement of Hydration Retention of Adhesive

In order to measure the holding force of the adhesive prepared according to the preparation example, the Comparative Examples 1 and 2 were laminated in a syringe, and then injected into conical tubes containing water, respectively, at 37 ⁰ C, 60 rpm. Was observed for 12 weeks, and the results are shown in FIG. 3. Specifically, referring to Figure 3, in the case of Comparative Example 1 was confirmed that the form collapsed and sinked at the same time as the adhesive is injected into the conical tube containing water, in the case of Example 2 still injected through a syringe after 12 weeks It could be confirmed that it remains as it is. This is expected in the case of an injection-type adhesive composition for fixation of bone graft material including decellularized extracellular matrix and calcium phosphate, which can help to maintain the fixation and shape by adhering the bone graft material and the shielding membrane to each other at the bone defect.

Experimental Example 2 Adhesive Force Measurement Experiment

Comparative example using a Brookfield viscometer (Brookf i eld RVDV-3, Brookf ie ld, US) for 40 minutes at 37 ^° C. for 5 minutes to check the adhesive strength of the adhesive prepared in the preparation example as time passes The viscosity of 1 and Examples 1 to 4 was measured, and the results are shown in FIG. 4.

Referring to the results of FIG. 4, in Comparative Example 1 containing carboxymethyl cellulose instead of decellularized extracellular matrix, the viscosity was approximately 1, 200 cps without change in time, but the decellularized cells Examples 1 to 4 containing the external substrate was confirmed that the viscosity is gradually increased over time.

In particular, in the case of Examples 1 to 4, the viscosity increase of about 12 times compared to the initial time (0 min) was confirmed at the time when 15 minutes elapsed, and in Example 4, which showed the highest viscosity increase rate, An increase in viscosity could be confirmed.

This suggests that the decellularized extracellular matrix has increased viscosity due to increased binding force between molecules due to fibrosis, ie, physical crosslinking reaction, at a temperature similar to body temperature, that is, 37 ° C. In addition, as the content of the decellularized extracellular matrix gradually increased, it was confirmed that the viscosity increase rate was improved.

Therefore, it can be seen that Examples 1 to 4, which are the injection-type adhesive composition for fixing bone grafts, prepared in the above-described examples, have a sufficient viscosity for adhesion or fixation between the bone graft material and the shielding membrane.

[Experimental Example 3] Fixed performance measurement experiment of the adhesive

In order to confirm the fixation ability of the bone graft or the shielding membrane of Comparative Example 1 and Examples 1 to 4 prepared through the previous production example as shown in FIG. 5 (10) (In '0ss, Biomat lante Biologies Solutions (FRN) and the shielding membrane 30 (Col lagen Membrane, Genoss, KOR) were placed in Comparative Examples 1 and Examples 1 to 4, respectively, and the suture ((ETHILON Nylon Suture 4) -0, Ethi con, US) was connected to Instron (3343 / B10948, UK) and subjected to suture pulse out test, and the results are shown in FIG.

Looking at the results of Figure 6, when using the adhesive of Examples 1 to 4 compared to Comparative Example 1 was able to confirm the fixing force of about 10 times or more. In particular, in the case of Example 4 having a high content of the decellularized extracellular matrix, a high fixing force was confirmed.

Thus, the embodiment is an injection-type adhesive composition for fixing bone graft material prepared in Example

1 to 4 was confirmed that the bone graft and the barrier membrane can be sufficiently fixed so as not to be shaken in the body.

Experimental Example 4 Experiment for Measuring Compressive Strength of Adhesive

In order to confirm the compressive strength according to the content change of the decellularized extracellular matrix contained in the adhesive prepared in the preparation example, the compressive strength was measured using an compressive strength device (Instron) based on ASTM D 790, the international standard. . The sample was placed on the measuring jig and the strength was measured by applying a constant force at 10 kW / inin downward, and the result is shown in FIG. 7.

Referring to the results of FIG. 7, the comparative strength of the decellularized extracellular matrix content of Comparative Example 2 or the decellularized extracellular matrix content of Comparative Example 3, which is lower than that of the calcium phosphate compound, was significantly higher than that of Examples 2 or 4. It was confirmed that the low.

Specifically, in the case of Comparative Example 2 having a low content of the decellularized extracellular matrix, due to the low protein content, the gelation effect was insignificant, resulting in insufficient adhesion when introduced into the body, and included in the adhesive composition. There was a problem in that the formulation is not due to the aggregation of the calcium phosphate compound.

On the other hand, in Comparative Example 3, which contains an excessive amount of decellularized extracellular matrix, the calcium phosphate compound is relatively low, and thus, it is difficult to expect a fine bone formation, and the strength decreases, which may be damaged by external physical stratification. There is.

On the other hand, in Examples 5 and 6 in which the bone-forming protein, which is a functional material in the adhesive, is further included, even if the functional material is further included, the strength of the adhesive is insignificant. It was confirmed that the formulation is not inhibited by inhibiting the aggregation of the extracellular matrix.

Experimental Example 5 Validation Experiment of Adhesive

The bone graft formation was confirmed through animal experiments to confirm the excellent bone conduction and osteoinduction ability when the bone graft fixation injection adhesive composition prepared above was applied to the bone defect.

First, as shown in Figure 8 using a trephine bur in the skull of the rat to form a skull defect of 8ι 誦 diameter (Fig. 8a), bone graft material (In 'Oss Os Biomat lante Biologies Solutions, FRN) 0.2ce was applied (FIG. 8B). After applying the Comparative Example 1 and Example 2 prepared in the above manufacturing example where the bone graft material is laminated (Fig. 8c), the shielding film (Col lagen Membrane, Genoss, KOR) was cut to 10 × 10 × and adhered to the injectable adhesive (FIG. 8D). Thereafter, the periosteum and soft tissue were sutured and the individual was regenerated at 6 weeks to observe the degree of bone formation.

In order to observe the degree of bone formation, the specimens to which Comparative Example 1 and Example 2 were applied were tissue stained to confirm the degree of bone formation. The results are shown in FIG. 9.

Looking at the results of Figure 9, compared to the skull to which Comparative Example 1 was applied, it was confirmed that the skull bonded using Example 2 had a high density of bone formation, especially when the bone graft material was not bonded when using Example 2 It was confirmed that it is fixed as it is in the implanted position.

In particular, the bone graft material and the shielding membrane are stably fixed and stable, and have excellent bone conduction and osteoinduction ability of the decellularized extracellular matrix included in Example 2, thereby confirming that new bone is well formed from the periphery of normal bone. Could.

Industrial Applicability

The present invention relates to a method for preparing an implantable adhesive composition for fixation of bone graft material for fixing a bone graft material or a shielding membrane filled in the body for restoration of a defect of bone tissue, and to an implantable adhesive composition for fixation of bone graft material prepared through the same. As it is, it is not shaken by the external stratification and is stably fixed, there is an effect that the new bone can be formed smoothly, there is industrial applicability.

Claims

[Range of request]

[Claim 1]

Preparing a decellularized extracellular matrix (decel lularized extracel lular matr ix, dECM);

Grinding the phosphate compound;

A first mixing step of preparing a primary mixture by mixing the pulverized calcium phosphate compound and water; And a second mixing step of preparing the second mixture by mixing the first mixture with the decellularized extracellular matrix. 2.

[Claim 2]

According to claim 1, After the second mixing step,

Further comprising a cooling step of the second mixture to the temperature of 2 ~ 8 ⁰ C; Method for producing a bone graft fixable injectable adhesive composition.

[Claim 3]

The method of claim 1, wherein the grinding step,

A method for producing a bone graft fixable injectable adhesive composition, characterized in that the calcium phosphate compound is ground to a particle size of 10 to 2,000.

[Claim 4]

The method of claim 1, wherein the first mixing step,

A method for producing a bone graft fixable injectable adhesive composition, characterized in that the mixed calcium phosphate compound 80 ~ 90 wt% and water 10 ~ 20 wt% ratio.

[Claim 5]

The method according to claim U, wherein the second mixing step,

A method for producing a bone graft fixable injectable adhesive composition, characterized in that it is mixed at a ratio of 50 to 80 wt% of primary mixture and 20 to 50 wt% of decellularized extracellular matrix.

[Claim 6]

The method of claim 1, wherein the second mixing step is

The first mixture is further mixed with the decellularized extracellular matrix in addition to the natural polymer material,

The natural polymer material is carboxyl methyl cel lulose, heparan sul fate, hyaluronic acid, hyaluroni c acid, col lagen, dextran. And alginate (alginate), characterized in that at least one selected from the group consisting of, graft implant fixing method for preparing an adhesive composition.

[Claim 7]

The method of claim 1, wherein the second mixing step,

The first mixture is mixed with the decellularized extracellular matrix and further includes a functional material,

The functional substance may include bone morphogenetic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), conversion growth factor (TGF-β), platelet derived growth factor (PDGF), insulin-like growth factor ( IGF-1), thioredoxin (TRX), stem cell factor (SCF), hepatocyte proliferation factor (HGF), human growth hormone (hGH), and angiogenin (Angiogenin) The method of producing an injection-type adhesive composition for fixing bone graft materials, characterized in that above.

[Claim 8]

An implantable adhesive composition for fixing bone graft materials prepared by the method of any one of claims 1 to 7.

[Claim 9]

Decel hilarized extracellular matrix (dECM), calcium phosphate compounds, natural polymers, functional materials and water;

20-50 wt% of the decellularized extracellular matrix, 40-72% of the calcium phosphate compound, wherein the sum of the decellularized extracellular matrix, the phosphate decalcium compound, a natural polymer, a functional substance, and water does not exceed 100%,

The calcium phosphate compound has a particle size of 10 to 2,000 βΆ,

The natural polymer material is composed of carboxyl methyl cellulose, heparansulfate, hyaluronic acid, collagen, dextran and alginate. At least one selected from the group,

The functional substance, bone morphogenetic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), conversion growth factor (TGF-β), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), thioredoxin (TRX), stem cell factor (SCF), hepatocyte proliferation factor (HGF) 간 human growth hormone (hGH) and angiogenin (Angiogenin) characterized in that at least one selected from the group consisting of ， Injectable adhesive composition for fixing bone graft materials.