KR102054978B1 - Method of producing injectable adhesive composition for fixing bone grafting materials and a injectable adhesive composition for fixing bone grafting materials produced thereby - Google Patents

Abstract

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. The decellularized extracellular matrix may be prepared by preparing an injectable adhesive composition for fixing bone grafts including a decellularized extracellular matrix (dECM) having excellent biocompatibility and a calcium phosphate compound similar to bone tissue and an inorganic component. Protein components, such as collagen, cause gelation reaction at a temperature similar to body temperature, and can be used to fix bone grafts and shielding membranes through physical fibrosis when inserted into the body. Absorbed, no secondary removal procedure is required. In addition, by using the decellularized extracellular matrix as an adhesive material, the heterologous cells are completely removed and the immune rejection reaction is significantly lowered to the operator, as well as collagen, glycoprotein, and BMP-2 in the decellularized extracellular matrix. Contains bone-derived proteins that can help bone formation, such as ALP, has excellent bone conduction and osteoinduction.

Description

Method of producing injectable adhesive composition for fixing bone grafting materials and a injectable adhesive composition for fixing bone grafting materials produced thereby

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.

Bone tissue is a hard tissue that maintains the skeleton of the human body, and bone grafting substitue and soft tissue blocking membranes (hereinafter, blocking membranes) in the damaged bone tissue when the bone tissue is damaged by trauma, tumor, malformation or physiological phenomenon. New bone can be formed by inserting guided tissue regeneration or guided bone regeneration.

In general, bone grafts are intended to replace missing bones, and not just to compensate for lost bones, but to help new bones form in areas of bone loss. Orthopedic surgery used to promote bone formation and replacement of bone tissue defects in bone defects remaining in the limbs and vertebrae, fracture dislocations, disunion or delayed unions, tumors or osteomyelitis, or in the dental alveolar bone defects. Can be.

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 transplantation method to transplant the bone of another person by chemical treatment, xenograft transplantation method to transplant the bone of the animal by chemical treatment.

The best method of transplantation is autologous bone graft, which is used for transplanting its own bones. The use of autologous bone is likely to cause bone formation or bone induction, the healing of bones to be transplanted, the rapid conversion to living bones, and the immune response due to autologous bones. There is a disadvantage that there is no response, but secondary surgery is required, it is difficult to obtain the required amount, and it is difficult to perform in a general medical clinic.

Allografts and allografts, which are other transplantation methods, do not require a secondary surgical unit, which has advantages such as fast operation time, recovery period, and low cost compared to synthetic bone grafts, but have about twice as much bone induction compared to autologous bone. There is a risk that the bone quality of the bone to be formed may be reduced, an immune response may occur, and although the probability is low, a virus such as AIDS or hepatitis may be introduced into the patient.

After implanting a bone graft into a 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 while new bone is formed. In other words, bone grafts and barriers implanted in bone defects should be fixed stably without shaking during the formation of new bone.

In general, a fibrin glue or sealant is used to fix the bone graft and the shielding membrane, or a metal bone screw is used to directly fix the shielding membrane.

However, in the case of fibrin glue or sealant, although it can be fixed between the bone graft material and the shielding membrane, it is decomposed after about 4 weeks in the body, and there is a problem that the bone defect cannot be completely formed into new bone because it cannot help formation of 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.

Publication No. 2016-0093520 (2016.08.08.)

In the present invention, the bone graft material or the shielding membrane filled in the body for the restoration of the defect of the bone tissue is stably fixed without being shaken by an external impact, the bone graft fixation injectable adhesive composition can be formed smoothly new bone It is to provide a method for preparing and injectable adhesive composition for fixing bone graft material prepared through the same.

In order to achieve the object as described above, a method for preparing an implantable adhesive composition for fixation of bone graft material according to an embodiment of the present invention is decellularized extracellular matrix from a xenograft obtained from porcine bone, bovine bone or horse bone. matrix, dECM); Grinding the calcium phosphate compound to a particle size of 20 ~ 200㎛; A first mixing step of preparing a primary mixture by mixing the pulverized calcium phosphate compound with 80 to 90 wt% and 10 to 20 wt% of water; A secondary mixing step of preparing a secondary mixture by mixing 50 to 80 wt% of the primary mixture and 20 to 50 wt% of the decellularized extracellular matrix; And maintaining the secondary mixture at a temperature of 2 to 8 ° C.

The second mixing step, 1 ℃ than preparing a to substrates other preferred, the evasion saturated cells is carried out at a temperature range of not more than 37 ℃ is, the heterogeneous bone using trypsin and EDTA other than the substrate extracellular Decellularization is performed to remove cellular components.

In addition to the secondary mixture may be further mixed with a natural polymer material or a functional material, it is preferably used in the range of 0.02 to 2 parts by weight based on 100 parts by weight of the secondary mixture.

At this time, the natural polymer used is composed of carboxyl methyl cellulose, heparan sulfate, hyaluronic acid, collagen, dextran and alginate. It may be one or more selected from the group, the functional material, bone morphogenetic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), conversion growth factor (TGF), platelet-derived growth factor (PDGF), It may be at least one selected from the group consisting of insulin-like growth factor (IGF), thioredoxin (TRX), stem cell factor (SCF), hepatocyte proliferation factor (HGF), human growth hormone (hGH) and angiogenin. .

In another embodiment of the present invention, an injectable adhesive composition for fixing bone graft materials produced by such a method is mentioned.

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 not only to fix bone graft material and barrier membrane through physical fibrosis when inserted into the body, but also to foreign body reaction after a certain period of time. It is completely decomposed and absorbed without the need for a secondary removal procedure.

In addition, the use of 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 process of mixing the calcium phosphate compound and the 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. .

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 bone graft material of the present 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 otherwise stated.

The terms "first", "second", and the like 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 named a second component, and similarly, the second component may also be named a first 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 same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order.

As used herein, an "extracellular matrix" (ECM) is a complex aggregate of biopolymers filling a space within or outside a tissue, such as fibrous proteins, complex proteins such as proteoglycans, and cell adhesion of fibronectin, laminin and the like. 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, 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 graft materials includes preparing a decellularized extracellular matrix (dECM); Grinding the calcium 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 a second mixture by mixing the first mixture and 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 preferably different depending on the type of cells or the degree of differentiation of the cells, the decellularized extracellular matrix used in the injectable adhesive composition for fixation of bone graft material according to the present invention has improved bone conduction. And decellularized extracellular matrix derived from bone tissue obtained through decellularization method in bone tissue, preferably xenograft obtained from pork bone, bovine bone, horse bone or allogene bone obtained from human bone, in order to have osteoinductive ability. have.

The decellularization method is a method of removing the remaining cellular components except for the extracellular matrix in the tissue of the tissue, it may be used a physical method, a chemical method or a mixture thereof, preferably a chemical method may be used. .

For example, when preparing a decellularized extracellular matrix using a chemical method, sterilizing a xenograft or allogeneic bone, and then immersing cells cultured in a decellularization solution for a predetermined time, and 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 break the cell membranes of the cells to remove intracellular components, or DNase and RNase to remove the nucleus components. However, preferably, trypsin may be used together with EDTA at 35 to 39 ° C. and pH 7.5 to 8.5 to break the cells of bone tissue and remove the contents in the cells, thereby decellularizing.

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, the decellularized extracellular compared to normal bone The DNA content in the substrate is significantly lower (<50 ng / mg), which can significantly reduce the immune rejection reaction when introduced into the body, as well as the 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 the collagen and protein components contained in the decellularized extracellular matrix, binding between the bone graft and bone graft material, bone Binding between the implant and the barrier, fixation between the barrier and the surrounding tissue, and also aggregate the calcium phosphate compounds and other substances included in the injectable adhesive composition for fixation of bone grafts of the present invention.

The pulverizing step is a step of pulverizing the calcium phosphate compound, the calcium phosphate compound with a predetermined particle size in order to include the calcium phosphate compound having bone conduction ability, bone induction ability in the bone graft fixation injection 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, when the particle size of the calcium phosphate compound becomes too fine, the calcium phosphate compound is scattered in the form of fine powder, so that it is difficult to use, and therefore, it is preferable to use it by pulverizing it to the desired particle size.

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), Calcium Hydrogen Phosphate, Monocalcium Phosphate, Dicalcium Phosphate Dibasic At least one selected from the group consisting of calcium dihydrogen phosphate, tricalcium phosphate, tricalcium phosphate, calcium phosphate 10, and calcium pyrophosphate may be used. . Preferably, β-tricalcium phosphate (β-TCP) or hydroxyapatite (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 ㎛, if the particle size of the calcium phosphate compound is less than 10 ㎛ benefit compared to the cost and time required to finely crush the particles If the particle size exceeds 2,000 μm, the particle size is so large that it is difficult to disperse homogeneously in the injection-type adhesive composition for fixation of bone graft material, and when it is incorporated into the body, the composition is aggregated. This difficulty can reduce the holding force. As the particle size of the calcium phosphate compound is lower, the bone conduction ability and the bone induction property are improved. Preferably, the particle size of the calcium phosphate compound may be pulverized to a particle size of 20 to 200 μm.

The method of pulverizing the calcium phosphate compound in the pulverizing step is not particularly limited as long as it is a method of pulverizing the compound to a predetermined particle size to be obtained, such as wet grinding or dry grinding. It can be ground to a calcium phosphate compound having the desired particle size using.

The pulverized calcium phosphate compound may be subjected to a second mixing step of preparing a second mixture by mixing the first mixture and the decellularized extracellular matrix after the first mixing step of mixing the water with the first mixture. . This is to allow the calcium phosphate compound and the decellularized extracellular matrix contained in the injectable adhesive composition for fixing bone grafts of the present invention to be uniformly dispersed and mixed.

In detail, the primary mixing step may be prepared by mixing the pulverized calcium phosphate compound in a ratio of 80 to 90 wt% and 10 to 20 wt% of water, wherein the mixing ratio of the calcium phosphate compound and water is If outside the stated range, the decellularized extracellular matrix and calcium phosphate compound may not be uniformly mixed in the second mixing step, or the moisture content is too high to fill the syringe with the injectable adhesive composition for fixing the bone graft material of the present invention. It takes a lot of time to inject into the body and completely adheres to it, so the convenience of the procedure is lowered, or the adhesion is lowered, so that the binding force between the bone graft and the bone graft, between the bone graft and the shield, between the shielding membrane and the surrounding tissue is lowered. Agitation of the bone graft and the shield may occur.

Injectable adhesive composition for fixation of bone graft material according to the present invention may be prepared through a secondary mixing step of preparing a secondary mixture by mixing the decellularized extracellular matrix with the primary mixture prepared through the primary mixing step. .

Specifically, the secondary mixing step is to prepare a secondary mixture by mixing the primary mixture and the decellularized extracellular matrix, the primary mixture 50 ~ 80 wt% and the decellularized extracellular matrix 20 ~ 50 wt The mixing temperature is preferably mixed in a ratio of%, so that the mixing temperature is controlled so that the protein contained in the decellularized extracellular matrix is not denatured, and more preferably collagen and other proteins contained in the decellularized extracellular matrix are similar to body temperature. Since gelation is performed at the temperature, the mixing should be performed while controlling the temperature appropriately so as not to exceed 37 ° C.

It is also preferred to be carried out in a temperature range exceeding at least 1 ° C., because below 1 ° C. condensation of moisture present in the formulation occurs, making formulation impossible.

In the second mixing step, the mixture is stirred using a stirrer to prepare a homogeneous mixture when mixing the primary mixture and the decellularized extracellular matrix. Increasing increases the temperature of the mixture in proportion to the increased stirring 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 is modified at a temperature of about 35 to 39 ° C. Due to the problem that the formulation is denatured due to the coagulation or fibrosis reaction, the second mixing step is preferably mixed while controlling the temperature appropriately so as to maintain a temperature range of more than 1 ° C and less than 37 ° C, and is prepared after the second mixing step. It is preferable to store the injectable adhesive composition for fixation of bone graft through the step of maintaining the temperature of the prepared secondary mixture at a temperature of 2 to 8 ° C.

In the second mixing step, the ratio of 50 to 80 wt% of the primary mixture and 20 to 50 wt% of the decellularized extracellular matrix is preferred. As the content of the decellularized extracellular matrix contained in the prepared secondary mixture increases, the fibrous and physical crosslinking progresses when the injectable adhesive composition for fixing the bone graft material of the present invention is introduced into the body, thereby increasing the intermolecular adhesion. The viscosity improves the fixation force of the bone graft material and the shielding membrane, 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. Not only is it difficult to expect a sufficient bone formation effect afterwards, but the tensile strength of the gelled bone graft fixable injection-type adhesive composition is significantly lowered, which may be damaged by external physical shocks.

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, 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 therefore, it is preferable to add additionally in the second mixing step, rather than the first mixing step.

The natural polymer is selected from the group consisting of carboxyl methyl cellulose, heparan sulfate, hyaluronic acid, collagen, dextran and alginate. One or more selected may be used, and the functional substance may include bone morphogenetic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), conversion growth factor (TGF), platelet-derived growth factor (PDGF), One or more selected from the group consisting of insulin-like growth factor (IGF), thioredoxin (TRX), stem cell factor (SCF), hepatocyte growth factor (HGF), human growth hormone (hGH) and angiogenin Can be.

The natural polymer 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 less than 0.02 parts by weight. In this case, the effect of growth factors that are natural polymeric materials or functional materials cannot be exerted, and when it exceeds 2 parts by weight, it is rather impossible to formulate by inhibiting the aggregation of bone-derived decellularized extracellular matrix.

On the other hand, in another embodiment of the present invention, there may be mentioned an injection-type adhesive composition for fixing bone graft material prepared by the above-mentioned manufacturing method, the bone graft material-injectable injection adhesive composition according to the present invention, decellularized extracellular matrix Because of the collagen and other protein components contained in the gelation (gelation) in a temperature atmosphere of about the body temperature, the physical fiberization (fibrillogenesis) by the body temperature when the injection implant composition for fixing the bone graft implant into the body through a syringe This improves the bond between bone graft and bone graft, the bond between bone graft and barrier, and the fixation force between barrier and surrounding tissues, so that bone graft and barrier are not fixed in the body during the formation of new bone. New bone can be formed stably.

In addition, there is no need for a separate fixing device, for example, a bone screw, which is generally used to fix a bone graft material, a barrier, and the like, after a bone graft operation, a second operation for removing a bone screw is not necessary. Prognosis can be improved.

In addition, the decellularized extracellular matrix extracted from the bone tissue contained in the implantable adhesive composition for fixation of bone grafts is included, which contains various components that can help to form new bone such as bone-derived proteins. It can have osteoinductive ability. In addition, by including a calcium phosphate compound similar to natural bone and inorganic components, it can help in 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 can implement various modified forms of the contents described herein within the scope of the present invention.

[Production example]

Preparation of Injectable Adhesive Composition for Fixing Bone Graft

First, 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 0.1% (w / v) centamicin (w / v) centamicin, in order to remove foreign matter and fine tissue of the separated spongy part. Washing was performed using Phosphate-buffered ssaline (PBS) containing Invitrogen, Carlsbad, CA, USA. The spongy part was then frozen in liquid nitrogen and cut into sections of 4 × 4 × 4 mm or less, and then the cut sections were washed once more with distilled water. The washed sections are immersed in liquid nitrogen and ground using a coffee mill (Kordia Co., Daegu, Gyeongbuk, Korea). The powdered spongy was dissolved using 0.5 N HCl (25 mL / g powder), and then stirred at room temperature for 24 hours (300 rpm) to demineralize the spongy in powder state. After demineralization, it was filtered through a vacuum and rinsed with distilled water. Thereafter, lipids were extracted from the demineralized powder for 1 hour using a mixed solvent of chloroform (Chloroform, Fisher Scientific, Loughborough, UK) and methanol (Methanol, Fisher Scientific) in a 1: 1 volume ratio, and then sequentially Methanol, washed with distilled water. After washing, snap frozen and then lyophilized under a reduced pressure atmosphere for a sufficient time and stored at -20 ° C. After the stored lyophilized powder was washed with distilled water, it was dissolved in a mixed solution containing 0.05% Trypsin (Sigma-Aldrich) and 0.02% EDTA (ethylenediaminetetraacetic acid, Sigma-Aldrich) to 37 ° C. and 5% CO ₂ atmosphere. It was decellularized by stirring for 24 hours under. After decellularization, the cells were washed with PBS (Phosphate-buffered ssaline) 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 and then stored at-20 ℃ to prepare a decellularized extracellular matrix, the decellularized extracellular matrix prepared as shown in FIG.

After pulverizing β-TCP (β-tricalcium phosphate) by induction and powdering, a pulverized calcium phosphate compound having an average particle size of 75 μm was prepared using a sieve. 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.

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

Comparative Example 1 Comparative Example 2 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 3 Comparative Example 4 Adhesive

matter Decellularized Extracellular Matrix - 10 20 30 40 50 30 30 70 30 Carboxymethyl Cellulose 30 - - - - - - - - - Primary mixture 70 90 80 70 60 50 70 70 30 70 Functional
matter
(Bone morphogenetic protein) - - - - - - 0.02 2 - 3 Sum 100 100 100 100 100 100 100.2 102 100 103

(Unit: g)

Experimental Example 1

Hydration retention test of adhesive

In order to measure the holding force of the adhesive prepared according to the above preparation example, each of Comparative Examples 1 and 2 was filled into a syringe, and then 5 g of conical tube containing water was injected, and then 37 ° C. and 60 rpm. Observations were made for 12 weeks under conditions, 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. In the case of the injection-type adhesive composition for fixation of bone graft material including decellularized extracellular matrix and calcium phosphate, it is expected that the bone graft and the shielding membrane may be adhered to each other in bone defects to help maintain fixation and shape.

Experimental Example 2

Adhesive force measurement experiment

Comparative Example 1 and Example 1 using a Brookfield Viscometer (Brookfield RVDV-3, Brookfield, US) for 40 minutes at 37 ℃ 5 minutes intervals in order to check the adhesive strength of the adhesive prepared in the above Preparation Example as time passes The viscosity of 4 to 4 was measured, and the results are shown in FIG. 4.

Referring to the results of FIG. 4, the comparative example 1 including carboxymethyl cellulose instead of the decellularized extracellular matrix exhibited a viscosity of approximately 1,200 cps without change in viscosity over time, but the decellularized extracellular matrix was included. In Examples 1 to 4, it was confirmed that the viscosity gradually increased as time passed.

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 the case of Example 4, which showed the highest viscosity increase rate, about 16 times compared to the initial time. An increase in viscosity could be confirmed.

This suggests that the decellularized extracellular matrix increases the binding strength between molecules due to fibrosis, i.e., 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.

Accordingly, 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 preparations, have sufficient viscosity for adhesion or fixation between the bone grafts and the shielding membrane.

Experimental Example 3

Test of Fixation Performance of Adhesives

Bone graft 10 (In'Oss, Biomatlante Biologics Solutions, FRN), shielding membrane as shown in Figure 5 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 manufacturing example (30) (Collagen Membrane, Genoss, KOR) was placed in Comparative Examples 1 and Examples 1 to 4, respectively, the adhesive 100, and the suture ((ETHILON Nylon Suture 4-0, Ethicon, US) to Instron (3343) / B10948, UK) and then suture pull out test was performed, 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 it was confirmed that the fixing force of about 10 times or more. In particular, in the case of Example 4 having a high content of decellularized extracellular matrix, a high fixation force was confirmed.

Therefore, Examples 1 to 4 of the injection adhesive composition for fixing the bone graft material prepared in the preparation example was able to confirm that the bone graft material and the shielding 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 change in the content of the decellularized extracellular matrix contained in the adhesive prepared in the preparation example, the compressive strength was measured by using a compressive strength device (Instron) based on the international standard ASTM D 790. . The sample was placed on the measuring jig and the strength was measured by applying a constant force downwardly at 10 mm / min. The result is shown in FIG. 7.

Referring to the results of FIG. 7, the comparative strength of the decellularized extracellular matrix in comparison example 2 or the comparative example 3 in which the content of the decellularized extracellular matrix was excessive compared to 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 decellularized extracellular matrix, due to the low protein content, the gelation effect was insignificant, resulting in insufficient adhesion to the body, and calcium phosphate contained in the adhesive composition. There was a problem that the formulation is not agglomerated due to the aggregation of the compound.

On the other hand, Comparative Example 3, which contains an excessive amount of decellularized extracellular matrix, has a relatively low content of calcium phosphate compound, making it difficult to expect sufficient bone formation, and a decrease in strength, which may be damaged by external physical shocks. have.

On the other hand, in Examples 5 and 6 in which the bone-forming protein as a functional material in the adhesive is further included, even if the functional material is further included, the strength of the adhesive is minimal, but in the case of Comparative Example 4 in which the functional material is added in excess, decellularization It was confirmed that the formulation is not inhibited by inhibiting the aggregation of the extracellular matrix.

Experimental Example 5

Experiment of Validation of Adhesive

In order to confirm the excellent bone conduction and osteoinduction ability applied to the bone graft fixing injectable adhesive composition prepared in the above manufacturing example to the bone defect was confirmed the degree of bone formation through animal experiments.

First, as shown in Figure 8 using a trephine bur in the skull of the rat to form a skull defect of 8mm diameter (Fig. 8a), bone graft material (In'Oss, Biomatlante Biologics Solutions, FRN) 0.2cc Applied (FIG. 8B). After applying Comparative Example 1 and Example 2 prepared in the above manufacturing example in the bone graft material filled (Fig. 8c), cut the shielding film (Collagen Membrane, Genoss, KOR) to 10mm × 10mm to adhere to the injection adhesive (FIG. 8D). Thereafter, the periosteum and soft tissue were sutured, and at 6 weeks, the sacrifice was performed 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 dispersed 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 were stably fixed and stable, and had excellent bone conduction and osteoinduction ability of the decellularized extracellular matrix included in Example 2, thereby confirming that new bone was well formed from the periphery of normal bone. Could.

Claims (6)

Preparing a decellularized extracellular matrix (dECM) from xenografts obtained from porcine bone, bovine bone or horse bone;
Grinding the calcium phosphate compound to a particle size of 20 ~ 200㎛;
A first mixing step of preparing a primary mixture by mixing 80 to 90 wt% of the pulverized calcium phosphate compound and 10 to 20 wt% of water;
A second mixing step of preparing a second mixture by mixing 50 to 80 wt% of the first mixture and 20 to 50 wt% of the decellularized extracellular matrix; And
Maintaining the secondary mixture at a temperature of 2-8 ° C .;
Preparing the decellularized extracellular matrix,
Dividing the xenograft into pieces, separating it into a cancellous part and a cortical part, followed by washing using a Phosphate-buffered ssaline (PBS) to remove foreign matter and fine tissue from the separated spongy part;
Cutting the spongy portion to cut into sections, powdering and demineralizing the spongy portion; And
And a decellularization step of mixing with tryptic and EDTA, followed by stirring for 24 hours at 37 ° C. and 5% CO ₂ atmosphere to remove other cellular components except the extracellular matrix.
In addition to the secondary mixture is further mixed with a natural polymer material or a functional material, based on 100 parts by weight of the secondary mixture is used in the range of 0.02 to 2 parts by weight,
The second mixing step, characterized in that carried out in a temperature range of more than 1 ℃ 37 ℃, method of producing a bone graft fixable injection adhesive composition. delete delete The method of claim 1,
The natural polymer is selected from the group consisting of carboxyl methyl cellulose, heparan sulfate, hyaluronic acid, collagen, dextran, and alginate. A method for producing a bone graft fixable injectable adhesive composition, characterized in that at least one selected. The method of claim 1,
The functional substance is a bone morphogenetic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), conversion growth factor (TGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) , Thioredoxin (TRX), stem cell factor (SCF), hepatocyte proliferation factor (HGF), human growth hormone (hGH) and angiogenin (Angiogenin), characterized in that one or more selected from the group consisting of Process for the preparation of injectable adhesive compositions.
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