COMPOSITION FOR BONE GRAFTING, COMPRISING NUCLEIC ACIDS, BONE GRAFT MATERIAL AND CATIONIC POLYMER, AND BONE GRAFT KIT FOR MANUFACTURING SAME

Abstract

The present invention relates to a composition for bone grafting, comprising nucleic acids, a bone graft material, and a cationic polymer, and a bone graft kit for manufacturing the same. The composition for bone grafting, of the present invention, has been confirmed to promote the formation of a cushioning force that can respond to physiological stress and the formation of new bones at grafted sites, and has been confirmed to improve bone grafting convenience, and thus is expected to be effectively usable in the treatment of bone diseases.

Claims

1. A bone grafting composition, comprising: a) a nucleic acid; b) a bone graft; and c) a cationic polymer, wherein a) the nucleic acid and c) the cationic polymer form a bond which holds b) the bone graft to form an aggregate, said bone grafting composition being prepared by: a process comprising a first step of mixing a) the nucleic acid with b) the bone graft to give a nucleic acid-bone graft mixture and a second step of adding c) the cationic polymer to the nucleic acid-bone graft mixture to hold the bone graft to form an aggregate; or a process comprising a first step of mixing c) the cationic polymer with b) the bone graft to give a cationic polymer-bone graft mixture and a second step of adding a) the nucleic acid to the cationic polymer-bone graft mixture to hold the bone graft to form an aggregate.

2. The bone grafting composition of claim 1, wherein the bond is an ionic bond.

3. The bone grafting composition of claim 1, wherein the nucleic acid is mixed at a weight ratio of 1-20:1 with the cationic polymer.

4. The bone grafting composition of claim 1, wherein the nucleic acid is contained in an amount of 0.001-2% by weight, based on the total weight of the bone grafting composition.

5. The bone grafting composition of claim 1, wherein the nucleic acid is a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a mixture thereof.

6. The bone grafting composition of claim 1, wherein the nucleic acid has a molecular weight of 1-100,000 kDa.

7. The bone grafting composition of claim 1, wherein the cationic polymer is contained in an amount of 0.001-2% by weight, based on the total weight of the bone grafting composition.

8. The bone grafting composition of claim 1, wherein the cationic polymer is at least one selected from the group consisting of chitosan, polyethylene amine, poly-L-lysine, and polyallylamine.

9. The bone grafting composition of claim 1, wherein the cationic polymer is chitosan.

10. The bone grafting composition of claim 1, wherein the bone graft is an autogeneic demineralized bone matrix, an allogeneic demineralized bone matrix, a xenogeneic demineralized bone matrix, a synthetic bone graft, an autologous bone homogenate, or a mixture thereof.

11. The bone grafting composition of claim 1, further comprising a bone morphogenesis activator.

12. The bone grafting composition of claim 11, wherein the bone morphogenesis activator is a growth factor, a hemostatic, or a mixture thereof.

13. The bone grafting composition of claim 12, wherein the growth factor is at least one selected from the group consisting of bone morphogenetic protein (BMP), bone sialoprotein, transforming growth factor (TGF), platelet-derived growth factor (PDGF), platelet rich plasma (PRP), fibroblast growth factor (FGF), dentin sialoprotein, polydeoxyribonucleotide, heparin-binding EGF-like growth factor (HB-EGF), cadherin EGF LAG seven-pass G-type receptor 3, and osteoblast specific cadherin (OB-cadherin).

14. The bone grafting composition of claim 12, wherein the hemostatic is at least one selected from the group consisting of thrombin, thromboplastin, fibrinogen, casein-kinase II, tissue factor, epinephrine, gelatin, vitamin K, calcium chloride, aluminum chloride-hexahydrate, and aluminum sulfate.

15. A bone grafting kit for preparing the bone grafting composition of claim 1, the bone grafting kit comprising: a-1) a nucleic acid solution; b) a bone graft; and c-1) a cationic polymer solution.

16. The bone grafting kit of claim 15, further comprising a bone morphogenesis activator.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1 shows whether an aggregate is formed from (A) a bone graft alone, (B) composition 1 comprising a mixture of a nucleic acid and a chitosan, (C) composition 2 prepared by mixing a nucleic acid with a chitosan and then with a bone graft, and (D) composition 3 prepared by mixing a nucleic acid with a bone graft and then with a chitosan.

[0076] FIG. 2 shows whether a bone graft is aggregated according to mixing ratios of a nucleic acid and a cationic polymer.

[0077] FIG. 3 shows whether a bone graft is aggregated according to concentrations and mixing ratios of a nucleic acid and a cationic polymer.

[0078] FIG. 4 shows cohesiveness and cushioning force of bone grafting compositions prepared from (A) a bone graft alone, without a nucleic acid and a cationic polymer, (B) composition 1 comprising a mixture of a nucleic acid and a chitosan, and (C) composition 3 prepared by mixing a nucleic acid with a bone graft and then with a chitosan.

MODE FOR CARRYING OUT THE INVENTION

[0079] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Experimental Example 1: Aggregation of Bone Graft by Nucleic Acid and Cationic Polymer

[0080] A polynucleotide (hereinafter, referred to as nucleic acid), commercially available from PharmaResearch Products, Co. Ltd., was used as a nucleic acid. In 9.8 ml of a buffer (0.8% sodium chloride, 0.25% sodium hydrogen phosphate, 0.028% sodium dihydrogen phosphate dihydrate), 0.2 g of the nucleic acid was dissolved at 40-70 C. for 30-60 min using a heating stirrer to give a 2% by weight nucleic acid solution.

[0081] Chitosan was used as a cationic polymer. In 9.8 ml of 0.12 M lactic acid, 0.2 g of chitosan was dissolved using a stirrer to give a 2% by weight chitosan solution.

[0082] A demineralized xenogeneic bone derived from cattle was used as a bone graft.

[0083] The nucleic acid solution, the chitosan solution, and the bone graft prepared above were mixed as shown in Table 1, below. The resulting compositions were analyzed for physical properties and for aggregation of the bone graft, and the results are depicted in FIG. 1.

TABLE-US-00001 TABLE 1 Composition Preparation Composition Mix 1.25 ml of 2% by weight nucleic acid 1 solution with 0.25 ml of 2% by weight of chitosan solution Composition Mix 1.25 ml of 2% by weight nucleic acid 2 solution with 0.25 ml of 2% by weight of chitosan solution and then with 0.5 g of bone graft Composition Mix 0.5 g of bone graft with 1.25 ml of 2% 3 by weight nucleic acid solution and then with 0.25 ml of 2% by weight chitosan solution

[0084] As shown in FIG. 1, composition 1, which was prepared by mixing the nucleic acid solution and the chitosan solution (FIG. 1B), might appear to be a gel, but was not a gel and showed a physical property such that it could be easily handled with tweezers. In composition 2 (FIG. 1C), which was prepared by mixing the nucleic acid solution with the chitosan solution and then with the bone graft, the bone graft was not aggregated by the nucleic acid solution and chitosan solution mixture and was observed to be dispersed, like the bone graft not mixed with any material. In contrast, composition 3 (FIG. 1D), which was prepared by mixing the bone graft with the nucleic acid solution and then with the chitosan solution was observed to aggregate the bone graft.

[0085] Although not shown in FIG. 1, a composition prepared by mixing the bone graft with the chitosan solution and then with the nucleic acid solution was also observed to aggregate the bone graft, as in composition 3.

[0086] Even when chitin, polyethylene amine, poly-L-lysine, and polyallylamine were used as cationic polymers, the same results were obtained although not stated in detail.

[0087] Taken together, the data obtained in this Example indicated that the bond between the nucleic acid and the cationic polymer induces the aggregation of the bone graft material and the order of mixing the nucleic acid, the cationic polymer, and the bone graft material is also important for the aggregation of the bone graft aggregation.

Experimental Example 2: Aggregation of Bone Graft According to Mixing Ratio of Nucleic Acid and Cationic Polymer

[0088] An examination was made to see whether or not the nucleic acid and chitosan mixture-induced bone graft aggregation confirmed in Experimental Example 1 was influenced by mixing ratios of nucleic acid and chitosan. To this end, the bone graft was analyzed for aggregation in the presence of nucleic acid and chitosan mixed at various ratios. In this regard, the concentration of chitosan was changed while the concentration of nucleic acid was fixed.

[0089] A 2% by weight nucleic acid solution and 2% by weight, 1% by weight, 0.5% by weight, and 0.1% by weight chitosan solutions were prepared in the same manner as in Experimental Example 1. Compositions containing nucleic acid and chitosan at the concentrations indicated in Table 2, below, were made by mixing 0.5 g of bone graft with 1.25 ml of a 2% by weight nucleic acid solution, followed by adding 0.25 ml of each of the chitosan solutions having the concentrations while aggregation of the bone graft was monitored. The results are depicted in FIG. 2.

TABLE-US-00002 TABLE 2 Final Conc. in Mixing ratio Composition (wt %) (weight ratio) Composition Nucleic acid Chitosan Nucleic acid Chitosan Composition 3 1.667 0.333 5 1 Composition 3-1 1.667 0.167 10 1 Composition 3-2 1.667 0.083 20 1 Composition 3-3 1.667 0.017 100 1

[0090] As shown in FIG. 2, aggregation of the bone graft was found to be dependent on mixing ratios between nucleic acid and chitosan. That is, when nucleic acid and chitosan in bone grafting compositions were mixed at weight ratios of 5:1 (composition 3), 10:1 (composition 3-1), and 20:1 (composition 3-2), the bone graft aggregated. In contrast, when nucleic acid and chitosan was mixed at 100:1 (composition 3-3), the bone graft did not aggregate, but was dispersed.

[0091] The data obtained in this experiment indicate that mixing ratios between nucleic acids and cationic polymers have important influences on the aggregation of the bone graft.

Experimental Example 3: Aggregation of Bone Graft According to Concentration of Nucleic Acid and Cationic Polymer

[0092] Through Experimental Example 2, it was observed that mixing ratios between nucleic acid and chitosan are important for the aggregation of bone grafts. In addition, an experiment was made to confirm the aggregation of bone grafts according to concentrations of nucleic acids and chitosan.

[0093] Chitosan solutions having 2% by weight, 1% by weight, and 0.5% by weight concentrations and nucleic acid solutions having 2% by weight, 1.5% by weight, 1% by weight, and 0.5% by weight concentrations were prepared in the same manner as in Experimental Example 1 for the nucleic acid and chitosan solutions. Compositions containing nucleic acid and chitosan at the concentrations indicated in Table 3, below, were made by mixing 0.5 g of bone graft with 1.25 ml of each of the nucleic acid solutions having the concentrations, followed by adding 0.25 ml of each of the chitosan solutions having the concentrations while aggregation of the bone graft was monitored. The results are depicted in FIG. 3.

TABLE-US-00003 TABLE 3 Final Conc. in Mix Ratio Composition (wt %) weight ratio) Composition nucleic acid chitosan nucleic acid chitosan Composition 3 1.667 0.333 5 1 Composition 3-1 1.667 0.167 10 1 Composition 3-2 1.667 0.083 20 1 Composition 4-1 1.250 0.333 3.6 1 Composition 4-2 1.250 0.167 7.2 1 Composition 4-3 1.250 0.083 15 1 Composition 4-4 0.830 0.333 2.4 1 Composition 4-5 0.830 0.167 4.7 1 Composition 4-6 0.830 0.083 10 1 Composition 4-7 0.420 0.333 1.2 1 Composition 4-8 0.420 0.167 2.3 1 Composition 4-9 0.420 0.083 5 1

[0094] Compositions 3, 4-5, and 4-9 were different in concentrations of nucleic acid and chitosan, but similar in mixing ratios therebetween. As shown in FIG. 3, the bone graft was not aggregated, but dispersed in composition 4-9 whereas the bone graft was aggregated in compositions 4-5 and 3. These results were true of compositions 4-4 and 4-8 and compositions 3-1 and 4-6.

[0095] In this experiment, it was found that the aggregation of the bone graft was dependent on the final concentrations of nucleic acid and cationic polymer as well as the mixing ratios of nucleic acid and cationic polymer.

[0096] The data obtained in Experimental Examples 1 to 3 show that the bone grafting composition of the present invention preferably contains a nucleic acid in an amount of 0.001-2% by weight and a cationic polymer in an amount of 0.001-2% by weight, based on the total weight thereof, with the nucleic acid and the cationic polymer mixed at a ratio of 1-20:1 (weight ratio).

Experimental Example 4: Effect of Nucleic Acid and Cationic Polymer on Cohesiveness of Bone Graft and Buffering Powder of Bone Grafting Composition

[0097] An examination was made of effects of the nucleic acid and cationic polymer on the cohesiveness of the bone graft and the cushioning force of the bone grafting composition. In this regard, compositions 1 and 3 of Experimental Example 1 were employed and monitored for morphological change upon application of a force thereto. The results are depicted in FIG. 4.

[0098] As can be seen in FIG. 4, the bone graft, when existing alone (FIG. 4A), did not aggregate and was easily dispersed with the application of even a small force thereto. Composition 1 which contained a mixture of nucleic acid and chitosan, but not bone graft (FIG. 4B), was not dispersed even in the presence of a force applied thereto and was observed to have a restoring force to return back to the original shape and a cushioning force upon removal of the applied force. For composition 3 containing a mixture of nucleic acid, chitosan, and bone graft (FIG. 4C), the bone graft in the mixture was aggregated in contrast to the bone graft alone and retained the morphology thereof without being dispersed even when a force was applied thereto. Thus, the composition was observed to have a cohesive force and a cushioning force.

[0099] In this experiment, it was thus found that the bone grafting composition comprising a nucleic acid, a cationic polymer, and a bone graft according to the present invention has a cohesive force to aggregate the bone graft which can be thus prevented from being dispersed by an external force, and a cushioning force to counteract a physical impact in the course of bone grafting and bone regeneration, whereby the bone graft can retain a necessary shape and improve the convenience of bone grafting.

Experimental Example 5: Maintenance of Cohesiveness of Bone Grafting Composition Over Time

[0100] The bone grafting composition of the present invention was monitored for a decrease in cohesiveness with time. Changes in the total weight of the composition were used as criteria for the maintenance of cohesiveness because the bone graft is dispersed as the cohesiveness of the bone grafting composition is decreased.

[0101] In this experiment, composition 3 of Experimental Example 1 was employed. Just after being prepared, composition 3 was measured for total weight every 30 minutes. The results are given in Table 4, below.

TABLE-US-00004 TABLE 4 Time (min) 0 30 60 90 120 150 Weight (g) 0.7 0.69 0.7 0.69 0.68 0.69

[0102] As can be understood from the data of Table 4, the total weight of the bone grafting composition according to the present invention did not undergo a large change with time. Thus, the cohesiveness of the bone grafting composition according to the present invention lasted for a long period of time.

[0103] Accordingly, the bone grafting composition of the present invention guarantees high stability without a morphological change during a grafting procedure for a long time, and allows the bone graft to last in an aggregate form without being broken for a long period of time, thereby making the bone grafting procedure convenient.

Experimental Example 6: Induction of Bone Morphogenesis

[0104] In order to examine the effect of the bone grafting composition of the present invention on bone morphogenesis, the bone grafting composition was implanted to a site free of bone growth and observed for the formation of new bone.

[0105] In this regard, compositions 2 and 3 of Example 1 were implanted to bone growth-free abdominal muscles in rats, followed by suturing the muscles and skins. As a control, the bone graft was used alone. After the rats were bred, the entire implanted abdominal muscle area was excised, demineralized, and embedded into paraffin to construct tissue specimens. The tissue specimens were subjected to Giemsa staining and H&E staining and quantitatively analyzed for new bone tissue to determine the formation of new bone.

[0106] In the control treated with the bone graft alone, no new bones were found. For composition 2, new bones were almost not found, like the control. In contrast, composition 3 was found to significantly increase the amount of new bones, unlike the control and composition 2.

[0107] Taken together, the data obtained in this experiment indicate that the bone grafting composition of the present invention has the effect of inducing bone morphogenesis.

Example 1: Construction of Bone Grafting Kit

Example 1-1: Preparation of Nucleic Acid Solution

[0108] In 9.8 ml of a buffer (0.8% sodium chloride, 0.25% sodium hydrogen phosphate, 0.028% sodium dihydrogen phosphate dihydrate), 0.2 g of a nucleic acid was dissolved at 40-70 C. for 30 to 60 minutes using a heating stirrer to prepare a 2% by weight nucleic acid solution.

Example 1-2: Preparation of Cationic Polymer

[0109] As a cationic polymer, chitosan was used.

[0110] In 9.8 ml of a 0.12 M lactic acid solution, 0.2 g of chitosan was dissolved using a stirrer to prepare a 2% by weight chitosan solution.

Example 1-3: Construction of Bone Grafting Kit

[0111] A bone grafting kit according to the present invention was constructed by combining a vial storing the nucleic acid solution prepared in Example 1-1 and a vial storing the chitosan (cationic polymer) solution prepared in Example 1-2. In this regard, a vial storing a bone graft may be included in the kit.