KR101030429B1 - Composition for induction of bone formation - Google Patents

Abstract

PURPOSE: A composition containing a mixture of sodium carboxy methyl cellulose and collagen for quickly inducing osteogenesis is provided. CONSTITUTION: A composition for inducing osteogenesis contains sodium carboxy methyl cellulose and collagen. The mixture of sodium carboxy methyl cellulose and collagen is in gel form. An osteogenic protein is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, or BMP-13. The composition contains 5-100 ug/ml of osteogenic protein.

Description

Composition for induction of bone formation

Demineralized bone matrix; A mixture of sodium carboxymethyl cellulose and collagen; And a bone formation inducing composition comprising a bone morphogenic protein.

Many experimental and clinical trials for the rapid and complete recovery of the missing bone have been underway for a long time, and various bone grafts, substitutes and bone growth factors have been studied and developed. However, the ideal and perfect bone substitutes have not been developed yet, and various studies have been conducted to find them.

Bone grafts are often used to augment the natural regeneration process when there are defects or wounds in the bone. Such bone grafts must be biocompatible. In addition, an ideal bone graft should be osteogenic, ie bone conductive and bone inductive, easily manipulated by a surgeon prior to transplantation, and be able to maintain strength and properties in vivo after transplantation. do. In terms of certain regenerative capacity, natural bone is a potential implant. However, autogenic, allogenic and xenogeneic bone use is complicated by concomitant disease metastasis, immune graft rejection, patient morbidity and complex surgical procedures. Thus, attention has been focused on synthetic bone graft materials.

In general, widely used bone inducing substances include demineralized bone matrix and recombinant human bone morphogenetic proteins. These organic bone inducers are typically delivered to the implant site with a liquid or gelatin carrier. An ideal bone graft contains abundant amounts of bone inducing substances to maximize regeneration.

Atelo-collagen sponge, known under the trade name TERUPLUG, is a US FDA approved material for bone supplement expansion. It is mainly used as a supplement for bone supplementation of collagen ingredient used to insert for conservative bone healing. The raw material is collagen extracted from bovine skin, and it is absorbent atelo-collagen that is heat treated to improve biocompatibility and minimize antigenicity. It is made by processing. The main component consists of type 1 collagen (85-95%) and type 3 collagen (5-15%) and is designed to be easily applied to bone defects in the form of a sponge.

The related art discloses the bone formation inducing effect of atelo-collagen sponges mixed with BMP-2. However, in the process of mixing BMP-2 with collagen sponge, when the physical strength is applied by the elastic property of the sponge, the amount of BMP-2 injected is frequently lost to the outside, so that the correct amount of BMP-2 There is a disadvantage of not transplanting. These drawbacks can pose a risk of causing significant side effects when BMP-2 lost from collagen sponges enters the body undesirably. In addition, there is a hassle that the surgeon has to operate a lot, there is a possibility that it can be contaminated by the operation, the collagen sponge is limited in size and shape compared to the conditions of various bone defects of patients. In addition, as the amount of bone-inducing substance bound increases, the mechanical strength of the bone graft becomes weaker, and even transplantation containing a desirable amount of bone-inducing substance is difficult to operate and loses adhesion and shape in vivo. easy to do.

Thus, even with the prior art, there is still a need to develop a bone formation inducing composition that can effectively induce bone formation in a short time without an immune response.

Demineralized bone matrix; A mixture of sodium carboxymethyl cellulose and collagen; And it provides a bone formation inducing composition comprising a bone morphogenic protein.

One aspect is demineralized matrix; A mixture of sodium carboxymethyl cellulose and collagen; And it provides a bone formation inducing composition comprising a bone morphogenic protein.

The bone formation inducing composition according to one embodiment may comprise a demineralized bone matrix.

The term “demineralized bone matrix (DBM)” refers to a substance that contains factors associated with bone formation by removing cells and minerals from the bone. Demineralized bone matrix is used as a filler in areas where bone regeneration is slow or impossible in orthopedic or dental fields due to its excellent bone regeneration ability. However, the DBM itself is incomplete to replace the bone, and because it is difficult to maintain the shape of the bone, a combination with other materials is required for use as a bone-forming filler.

Various demineralized bone matrices can be used in one embodiment. Demineralized bone matrices can be used, such as, for example, mixtures of whole or partially desalted formulations, including particulate or fiber-based formulations, mixtures of fiber and particulate formulations, totally or partially desalted formulations, surface desalted formulations. .

The bone used to make the demineralized bone matrix can be obtained from any source of living or dead tissue. For example, human cortical bone can be used. Suitably, the source of bone is suitable for the final recipient of the bone formation inducing composition according to one embodiment. Heterologous sources are possible, but to minimize immune rejection, it is desirable that the donor and recipient of bone be the same species.

The demineralized bone matrix can be made into fine particles by milling or grinding after obtaining a bone sample. In one embodiment, the particles of the demineralized bone matrix can have a minimum dimension of 100 to 2000 microns. After atomization, the demineralized bone matrix can be treated with a desalting agent known in the art such as hydrochloric acid to remove inorganics from the bone particles. Alternatively, desalting may be first carried out in the form of blocks or whole bone and then atomized. On the other hand, the demineralized bone substrate may be sterilized by lyophilization before preparation of the composition according to one embodiment.

In addition, the composition may comprise a mixture of sodium carboxymethyl cellulose and collagen. Specifically, the mixture may be of gel type.

Sodium carboxymethyl cellulose is a representative hydrophilic and biocompatible polymer, and may serve as a binder to increase the adhesion of the components included in the bone formation inducing composition according to one embodiment. In addition, collagen is one of proteins that is a major component of connective tissue, has inherently excellent immunological suitability, and is easy to process so that it can function as a carrier in a bone formation inducing composition according to one embodiment. For example, collagen of type I, IV, V and V can be used.

In particular, since collagen is degraded only by collagenase, which is present in a relatively low distribution in the tissue to which the composition can be implanted, it is highly stable and contains the following bone morphogenic proteins contained in the bone formation inducing composition according to one embodiment. By delaying the proliferation of the protein may serve to protect until the function of the protein in the transplanted tissue is activated.

The mixture may be suitably mixed with sodium carboxymethyl cellulose and collagen to form a gel type carrier. In the bone formation inducing composition according to one embodiment, the mixture may be mixed such that the weight ratio of sodium carboxymethyl cellulose and collagen is 20:80 to 40:60, most preferably to 30:70.

In addition, the bone formation inducing composition according to one embodiment may include a bone morphogenic protein.

Bone morphogenic protein (BMP) is a homodimeric protein derived from the bone matrix and is known to play a role in regenerating bone through inducing bone differentiation of stem cells or progenitor cells and promoting osteoblast migration. Examples thereof include BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, and BMP-13. In addition, the BMP herein includes natural and recombinant BMP. The bone morphogenic protein included in the bone formation inducing composition according to one embodiment may be prepared by genetic recombination techniques according to methods known in the art using prokaryotic and eukaryotic cell expression systems.

Of these BMPs, in particular, BMP-2 is currently used in clinical practice with US and European FDA approval. However, when BMP-2 protein is used in the clinic, the half-life in the body is short and requires a relatively large amount. The bone formation inducing composition according to one embodiment is to minimize the amount of the BMP-2 (eg, BMP-2 at a concentration of 5 μg / ml to 100 μg / ml), the maximum amount of bone formation induction effect Can be represented. However, the amount of BMP-2 may vary somewhat depending on the individual patient or the disease requiring treatment.

The bone formation inducing composition according to one embodiment may be a mixture of the above-mentioned material, that is, demineralized bone matrix, sodium carboxymethyl cellulose and collagen, and bone morphogenic proteins in an appropriate proportion.

The composition may have a weight ratio of the demineralized bone substrate and the mixture of sodium carboxymethyl cellulose and collagen in a range of 20:80 to 40:60, or 20:80 to 25:75, wherein 5 μg / ml to 100 μg / ml Concentrations of BMP-2 protein can be included. The mixed composition as described above may be prepared in the form of a product by dividing in an appropriate amount as necessary, for example, the composition can be prepared in a sterile syringe filled with 0.3 to 30 cc.

Demineralized bone matrix according to one or more embodiments; A mixture of sodium carboxymethyl cellulose and collagen; And a bone formation inducing composition comprising a bone morphogenic protein can effectively induce bone formation in a short time without an immune response as compared to conventional products.

Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are provided to illustrate one or more embodiments illustratively and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Bone Formation Inducing Compositions

First, the human cortical bone (processed in Korabobonbank) was treated with 83% ethanol and 6% hydrogen peroxide solution to remove bone marrow and lipids. The bone was then cut into blocks using a band saw machine. The cut block is put into bone mill_1 (M20, Fritsch, Germany), and firstly pulverized, and then the first pulverized product is put into bone mill_2 (M35, IKA, Germany) and then crushed secondly to make the first crushing. The size of the water was crushed smaller. To remove debris from the crushed bone powder, 15 ml of purified water per 1 g of bone powder was added to the container and washed for 10 minutes, and the purified water was exchanged for three times. The washed bone powder was placed in a stirrer and 15 ml of 0.6 N HCl was added per 1 g of bone powder, followed by stirring at 24 ° C. at a speed of 200 rpm for 6 hours. After the stirring, the washing operation was repeated until the pH of the solution containing the bone powder was 6.0 or more. The bone powder was then sterilized by treatment with 83% ethanol and 6% hydrogen peroxide, the supernatant was removed, and stored frozen at -70 ° C for 12 hours. The resultant was freeze-dried to prepare a demineralized bone matrix.

The demineralized bone matrix was appropriately applied to a sterile plate, then 50 μg / ml of BMP-2 was mixed and subjected to lyophilization. At the same time, 5.7% sodium carboxymethyl cellulose (Spectrum Chemical MFG) and 3% forcin collagen (Bioland, Korea) were mixed at a ratio of 30:70 based on the weight ratio to prepare a collagen gel carrier. . The mixture of the prepared demineralized bone substrate and BMP-2 and the collagen gel carrier were mixed in a ratio of 25:75 to 20:80 based on the weight ratio, and the mixture was filled in a sterile syringe by an appropriate amount. The doses filled in sterile syringes were 0.3, 0.5, 1, 3, 5, 10, 15 and 30 cc, respectively.

Example  2: Experiment of Transplantation of Bone Formation Inducing Composition in Animal

Animal transplantation experiments using rats were performed to confirm the degree and efficiency of bone fusion according to the composition ratio of each component of the bone formation inducing composition prepared in Example 1 above.

Sprague-Dawley rats (male, average body weight 350 g) were anesthetized by intraperitoneal injection of 0.05 ml of medemidine and 0.2 ml of zoletil. The rats were then dorsal midline incision and two-part median fascial incision to expose the transverse processes of L4 and L5 of the spine. The cortex was then removed from the exposed travertine, the composition was implanted between the cortex with the cortex removed in the paraspinal bed, and the incision was closed. Six rats were used for each experimental group, and there was no death of the rat. The animal experiment was performed in a sterile state according to conventional methods known in the art. Rats implanted with the composition were then bred, and then 4 and 6 weeks after transplantation via radiographic images (Listem, Model REX-525RF, Korea), manual palpation and micro CT (Skyscan 1072, Aartselaar, Belgium). The degree of bone fusion was checked at the time point.

On the other hand, as a control of the experiment was used a rat transplanted with a collagen sponge (Bioland, South Korea) mixed with 1 μg, 3 μg, 10 μg or 50 μg of BMP-2 in the same manner as above, respectively. Rats implanted with the composition prepared by the method of Example 1, wherein 1 μg, 3 μg, 10 μg or 50 μg of BMP-2 were mixed, were used.

As shown in the radiographs of FIGS. 1 and 2, the spine of the control rats were transplanted with the collagen sponge mixed with 50 μg of BMP-2 even 6 weeks after the implantation of the collagen sponge mixed with BMP-2. Only about 20% of bone fusion was observed in the spinal column of the present invention. In the case of transplanting the composition of the present invention, approximately 50% of the bone fusion was achieved in the rat transplanted with the composition containing 10 μg of BMP-2 within 4 weeks after transplantation. It was confirmed that the rat implanted with a composition containing 50 μg of BMP-2 showed a degree of bone fusion of 100%.

In addition, as shown in the micro-CT imaging results of FIG. 3, the spine of the rats to which the collagen sponge mixed with 10 μg of BMP-2 was mixed did not show bone fusion even after 6 weeks after transplantation. In the rat transplanted with the composition of the present invention, it was confirmed that the bone was completely fused.

Table 1 below shows the results of confirming the degree of bone fusion through palpation of the collagen sponge mixed with 1 μg, 3 μg, 10 μg or 50 μg of BMP-2 into the spine of rats, and 4 and 6 weeks after the injection.

Content of BMP-2 4 Weeks 6 weeks 1 μg Not fused Not fused 3 μg Not fused Not fused 10 μg Not fused Not fused 50 μg Not fused 20% fusion

In addition, Table 2 below shows that the bone formation inducing composition of the present invention mixed with 1 μg, 3 μg, 10 μg or 50 μg of BMP-2 was implanted into the spine of rats, respectively, and after 4 and 6 weeks, respectively, This is the result of checking the degree of fusion.

Content of BMP-2 4 Weeks 6 weeks 1 μg Not fused Not fused 3 μg Not fused 20% fusion 10 μg 50% fusion 80% fusion 50 μg 100% fusion 100% fusion

In summary, it was confirmed that the bone fusion efficiency of the composition of the present invention is much better than the collagen sponge-type composition in which BMP-2, which is a conventional product (INFUSE, Medtronic), is mixed.

1 is a radiographic image showing the degree of bone fusion in the spine (control) of rats 6 weeks after implantation of collagen sponges mixed with BMP-2.

Figure 2 is a radiographic picture showing the degree of bone fusion in the spine (experimental group) of rats 6 weeks after implantation of the composition of the present invention.

Figure 3 is a micro-CT image showing the degree of bone fusion in the spine of rats implanted with collagen sponge mixed with BMP-2 and the spine of rats implanted with the composition of the present invention.

Claims (6)

The weight ratio of the demineralized bone substrate and the mixture of sodium carboxymethyl cellulose and collagen is 20:80 to 40:60, wherein the mixture of sodium carboxymethyl cellulose and collagen has a weight ratio of 20:80 to 40 to sodium carboxymethyl cellulose and collagen. : A mixture which is 60; And bone morphogenetic protein. delete The bone formation inducing composition of claim 1, wherein the mixture of sodium carboxymethyl cellulose and collagen is of gel type. According to claim 1, wherein the bone morphogenic protein BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12 and BMP-13 bone selected from the group consisting of Induction composition. The bone formation inducing composition of claim 1, wherein the composition comprises a bone morphogenic protein at a concentration of 5 μg / ml to 100 μg / ml. delete

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