Composition for hard tissue regeneration

Abstract

The present invention relates to a composition for hard tissue regeneration.

Description

[Technical Field]

[0001] The present invention relates to a composition for hard tissue regeneration. [Background technology]

[0002] All materials implanted into the human body, especially those used for regenerating hard tissues such as bone and cartilage, must have good processability and moldability, as well as good in-situ polymerization to conform well to the wound. They must also provide a favorable environment for cell adhesion, growth, and differentiation, and their decomposition products must also be biocompatible. In particular, if a graft material for bone regeneration has too low a compressive strength or yield value, it becomes difficult to maintain its positional fixation and shape retention during the suturing and implant placement stages after injection or dense filling of the graft material. Furthermore, if the adhesiveness of a graft material is too high, it will adhere to surgical tools during surgery, making it difficult to fill the bone defect and resulting in poor workability.

[0003] In the early stages of development, implantable biomaterials for hard tissue regeneration relied on their inertness in vivo, but their use was limited by postoperative infection and inflammatory responses in surrounding tissues. Subsequently, with the rapid development of biomaterial technology using metals, ceramics, and polymers, biocompatible materials, rather than bioinert ones, were designed and developed, leading to the development of various types of bioactive scaffolds for bone tissue regeneration tailored to the site and purpose of use. These bioactive scaffolds for bone tissue regeneration have different physical properties depending on the implantation location, must not cause toxic reactions to surrounding tissues, and must have relatively high mechanical properties compared to other artificial organs. Various bioactive scaffolds for bone tissue regeneration are commercially available and under development depending on the characteristics of their raw materials and intended use.

[0004] On the other hand, in the case of hard tissues, the morphology of the damaged area is often irregular due to the characteristics of the tissue, making it difficult to select a transplant material that is appropriate for the defect site. Therefore, there is a need to find a material that not only has appropriate strength, but also has excellent adhesion to surrounding tissue, is easy to inject so that it can fill irregular damaged areas, and above all, promotes tissue regeneration. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a composition for hard tissue regeneration, which comprises crosslinked hyaluronic acid and transforming growth factor-beta (TGF-β), wherein the crosslinked hyaluronic acid is in the form of particles having an average diameter of 3 to 250 μm.

[0006] Another object of the present invention is to provide a kit for hard tissue regeneration comprising the composition for hard tissue regeneration, a mixing means, and an injection means. [Effects of the Invention]

[0007] The composition of the present invention exhibits high adhesive strength and viscosity, as well as low ejection force, and has physical properties suitable as a transplant material for hard tissue regeneration. Therefore, it can be easily injected into the tissue defect site, and its shape is maintained throughout the period during which the tissue is regenerated, and excellent clinical effects on hard tissue regeneration are expected. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a graph showing the adhesive force of hyaluronic acid in the composition of the present invention at various particle sizes. [Figure 2] FIG. 1 is a diagram showing the viscosity of hyaluronic acid at various particle sizes in the composition of the present invention. [Figure 3] FIG. 1 is a graph showing adhesive strength at various mixing ratios of hyaluronic acid and buffer solution in the composition of the present invention. [Figure 4] FIG. 1 is a graph showing the viscosity of the composition of the present invention at various mixing ratios of hyaluronic acid and buffer solution. [Figure 5] FIG. 1 is a graph showing the extrusion force at various mixing ratios of hyaluronic acid and buffer solution in the composition of the present invention. [Figure 6] FIG. 1 shows the preparation of a microfracture animal model having a defect site on the trochlear groove using a rabbit, and a tissue regeneration method using the same. [Figure 7] FIG. 1 is a diagram showing the scoring criteria for the OARSI evaluation system. [Figure 8] FIG. 1 shows the progress of OARSI score after administration of a composition for hard tissue regeneration in a microfracture animal model according to one embodiment of the present invention. [Figure 9] FIG. 1 shows the scoring criteria for the subchondral bone grading system. [Figure 10] FIG. 1 shows the progress of subchondral bone grade following administration of a composition for hard tissue regeneration in a microfracture animal model according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0009] Each description and embodiment disclosed in the present invention applies to other descriptions and embodiments. That is, any combination of various elements disclosed in the present invention is included in the present invention. Furthermore, the present invention is not limited to the following specific description.

[0010] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0011] Furthermore, throughout the specification of the present invention, when a part is said to "comprise" a certain component, this does not mean that it excludes other components, but that it may further include other components, unless otherwise specified.

[0012] The present invention will now be described in more detail. To achieve the above object, a first aspect of the present invention provides a composition for hard tissue regeneration comprising crosslinked hyaluronic acid and transforming growth factor-beta (TGF-β).

[0013] Here, the crosslinked hyaluronic acid is characterized by being particles having an average diameter of 3 to 250 μm. For example, tissues that can be regenerated by applying the composition of the present invention include, but are not limited to, cartilage tissue and bone tissue.

[0014] In the present invention, "hyaluronic acid (HA)," also known as hyaluronan, refers to an anionic, nonsulfated glycosaminoglycan widely distributed in connective, epithelial, and neural tissues. Because it is nonsulfated, it is unique among glycosaminoglycans. It is formed in the plasma membrane rather than the Golgi apparatus, and some forms are very large. The average 70 kg (150 lb) human contains approximately 15 g of hyaluronan in the body, of which approximately one-third is degraded and synthesized daily. It is one of the major components of the extracellular matrix, significantly contributing to cell proliferation and migration and also involved in the progression of many malignant tumors.

[0015] The term "crosslinked hyaluronic acid" as used herein refers to a category distinct from the linear hyaluronic acid described above, distinguished by the type of processing and acid composition. The term "crosslinking" refers to the process of linking linear hyaluronic acid molecules together to obtain a new structured material composed of multiple hyaluronic acid filaments with higher molecular weights. The high-molecular-weight hyaluronic acid formed by crosslinking loses the properties of individual molecules and reaches a gel-like state. While the linear hyaluronic acid described above was the first material to be used and commercially available and is composed of unaltered straight chains of hyaluronic acid, crosslinked hyaluronic acid is a more recently developed material characterized by the inclusion of crosslinks formed between multiple linear hyaluronic acid molecules during the production process. These crosslinks are transversal bridges that connect multiple chains to form hyaluronic acid macromolecules with larger size, higher molecular weight, and consequently higher density and durability.

[0016] For example, the crosslinked hyaluronic acid may be crosslinked with at least one crosslinking agent selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), bis ethyl carbodiimide (BCDI), polyethylene glycol (PEG), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0017] For example, the composition may contain cross-linked hyaluronic acid in a content of 15 mg / mL to 30 mg / mL based on the volume of the final composition. In the present invention, "Transforming Growth Factor-beta (TGF-β)" refers to a latent form of TGF-β complexed with two distinct polypeptides, LTBP (latent TGF-beta binding protein) and LAP (latency-associated peptide), secreted by many cell types, including macrophages. One of its main functions is to regulate inflammatory processes, particularly in the intestine. TGF-β also plays an important role in the regulation and differentiation of T cells as well as stem cells. Due to its role in the regulation and differentiation of immunity and stem cells, TGF-β is a cytokine that has been extensively studied in the fields of cancer, autoimmune diseases, and infectious diseases. The TGF-β superfamily includes endogenous growth-inhibitory proteins. Increased expression of TGF-β is usually associated with the malignant progression of many cancers and defective cytostatic responses to TGF-β. When its immunosuppressive function becomes dominant, it contributes to oncogenesis. Dysregulation of such immunosuppressive function is also associated with the pathogenesis of autoimmune diseases, but its effects are mediated by the environment of other cytokines present.

[0018] For example, the TGF-β includes all proteins belonging to the TGF-β superfamily, a large group of structurally related cell regulatory proteins that interact with the TGF-β receptor. Specifically, the TGF-β is at least one selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-5, BMP-6, and BMP-7. More specifically, the TGF-β is TGF-β3, and the TGF-β3 is human recombinant TGF-β3 derived from Escherichia coli, but is not limited thereto.

[0019] "TGF-β3" in the present invention is a protein encoded in humans by the TGFB3 gene. It is a type of protein known as a cytokine, which is involved in cell differentiation, embryonic development, and growth. It belongs to a large cytokine system called the transforming growth factor β superfamily, which includes the TGF-β family, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), inhibitory hormones (inhibins), and activins.

[0020] For example, the composition may contain the TGF-β, such as TGF-β3, at a content of 0.003 mg / mL to 1.0 mg / mL based on the volume of the final composition. For example, the composition may contain TGF-β at a concentration of 0.005 mg / mL to 1.0 mg / mL, 0.006 mg / mL to 1.0 mg / mL, 0.005 mg / mL to 0.5 mg / mL, 0.005 mg / mL to 0.3 mg / mL, or 0.006 mg / mL to 0.2 mg / mL based on the volume of the final composition, but is not limited to these. When the composition contains TGF-β at a content less than the aforementioned levels, it is difficult to achieve the synergistic effect of adding the desired level of TGF-β. When the composition contains TGF-β at a content greater than the aforementioned levels, further effects due to the added content cannot be expected.

[0021] For example, the composition has an adhesive strength of 2 N to 15 N. For example, the composition has an adhesive strength of 3 N to 12 N, or 4 N to 10 N, but is not limited to these.

[0022] The composition has a viscosity of 8,000 cP to 80,000 cP. For example, the composition has a viscosity of 10,000 cP to 60,000 cP, 15,000 cP to 50,000 cP, or 20,000 cP to 40,000 cP, but is not limited to these.

[0023] Furthermore, the composition has a discharge force of 3 N to 50 N. For example, the composition has a discharge force of 3 N to 50 N, 10 N to 50 N, 10 N to 45 N, 15 N to 45 N, or 15 N to 35 N, but is not limited to these.

[0024] Due to these physical properties, the composition of the present invention can be easily injected into damaged hard tissue, and even when injected into an environment where it is present in body fluids such as bone marrow, it has excellent adhesion to the tissue and can maintain its shape at the injection site.

[0025] For example, the composition may reduce the Osteoarthritis Research Society International (OARSI) score to 2.0 or less 12 weeks after injection into the site of a hard tissue defect.

[0026] The composition may also reduce subchondral bone grading to 1.0 or less 8 weeks after injection into the site of hard tissue defect. The term "osteoarthritis research society international (OARSI) score" as used herein refers to a score formulated by OARIS in 1998 to standardize the evaluation of osteoarthritis (OA) histopathology according to the results of Safranin O / fast green staining. The OARSI score indicates a comprehensive evaluation ranging from 0 to 6 based on the severity and extent of OA in the articular cartilage. The criteria for the OARSI score are the same as those previously reported, with a higher OARSI score indicating more severe cartilage damage in the joint.

[0027] The "subchondral bone grading" used in this invention is a rating system that indicates the condition of subchondral bone on a scale of 0 to 3 based on previous studies showing changes in subchondral bone and the general observation of osteoarthritis sample material by two researchers, OMA and PL. Specifically, the subchondral bone grade indicates structural changes in the bone connected to the cartilage, with higher scores indicating more sclerosis or thickening of the bone. The subchondral bone is located between the cartilage and the bone marrow, and thickening of the subchondral bone blocks the supply of active factors from the bone marrow required to maintain the cartilage structure, accelerating cartilage damage.

[0028] As described above, the composition of the present invention reduces the OARIS score to 2.0 or less 12 weeks after injection into a hard tissue defect site and / or reduces the subchondral bone grade to a very low level of 1.0 or less 8 weeks after injection. These values ​​mean that the cartilage and / or subchondral bone at the defect site have been regenerated to the same level as before the defect.

[0029] For example, the composition may be prepared by mixing cross-linked hyaluronic acid with TGF-β immediately before injection into the site of a hard tissue defect. A second aspect of the present invention provides a kit for hard tissue regeneration, comprising the composition for hard tissue regeneration of the first aspect, mixing means, and injection means.

[0030] For example, the mixing means is a mixing syringe, a vial transfer device or a connector, and the injection means is a syringe or an injection needle, but is not limited thereto. For example, the kit of the present invention may be used for injection into the site of hard tissue defect using an injection needle, or may be used for direct injection using a syringe or the like into the affected area opened by auxiliary means after a surgical operation.

[0031] Specifically, a vial containing TGF-β and an empty syringe are connected to a vial transfer device to transfer the TGF-β to the empty syringe, and then the syringe is separated and then connected to a syringe filled with cross-linked hyaluronic acid using a connector, and the TGF-β and cross-linked hyaluronic acid are mixed. An injection needle is connected to the mixed syringe and the mixture can be injected directly into the hard tissue defect site, or it can be injected into the hard tissue defect site after surgery. When used in conjunction with surgery, the kit is provided in double sterilized packaging to minimize the possibility of infection during handling, but the method and principle of use are the same. [Example]

[0032] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Preparation Example 1: Preparation of cross-linked hyaluronic acid Crosslinked hyaluronic acid was prepared by reacting hyaluronic acid (molecular weight 500,000–1.5 million Da) with 1,4-butanediol diglycidyl ether (BDDE) as a crosslinker. Specifically, 1 g / g hyaluronic acid, 0.8 g / g NaOH, 3.2 g / g purified water, and 0.02 g / g BDDE were mixed and stirred to form a gel. After synthesis, the mixture was crosslinked in a stirring incubator (stirring speed: 80 rpm, temperature: 30°C, reaction time: 19 hours). The crosslinked gel was coarsely crushed to a size of 15 mm x 10 mm and then dialyzed. The dialyzed gel was passed through a standard sieve to obtain particles of a consistent size. [Example]

[0033] Evaluation of physical properties of cross-linked hyaluronic acid at various sizes The crosslinked hyaluronic acid prepared in Preparation Example 1 was divided into six groups based on particle size. Specifically, samples containing particles with average particle sizes of 5 μm, 80 μm, 200 μm, 300 μm, 800 μm, and 1000 μm in PBS buffer at a concentration of 20 mg / mL were prepared, and adhesive force and viscosity were measured. The results are shown in Figures 1 and 2, respectively. Specifically, adhesive force was measured using an Anton Paar rheometer by injecting the sample into a plate and then lowering the spindle to a position of 0.25 mm so that it was in contact with the sample, at a speed of 200 μm / sec. Viscosity was measured using a Brookfield viscometer, with 0.5 cc of sample placed in a sample cup at a speed of 0.5 rpm.

[0034] As shown in Figures 1 and 2, samples containing crosslinked hyaluronic acid particles with an average size of 5 to 200 µm have higher adhesive strength and viscosity than samples containing larger particles. [Example]

[0035] Evaluation of the physical properties of a composition for hard tissue regeneration containing cross-linked hyaluronic acid and TGF-β3 at various mixing ratios A series of compositions were prepared by preparing a 20 mg / mL solution of the cross-linked hyaluronic acid prepared in Preparation Example 1 in PBS buffer and mixing it with recombinant human transforming growth factor-beta3 (rhTGF-β3) at volume ratios ranging from 10:0 to 6:4. The adhesive strength, viscosity, and extrusion force of the compositions were measured. The results are shown in Figures 3 to 5. Specifically, adhesive strength and viscosity were measured as in Example 1. Extrusion force was measured using a universal testing machine by filling a 3 cc syringe with 2 cc of sample, connecting a 21 G syringe, and then fixing the filled syringe to a jig and pushing it at a speed of 30 mm / min.

[0036] As shown in FIGS. 3 to 5, as the ratio of crosslinked hyaluronic acid decreased, the adhesive strength, viscosity, and ejection force all tended to decrease. [Example]

[0037] Confirmation of cartilage regeneration ability using experimental animals As shown in Figure 6, animal models with microfractures were prepared, and the prepared compositions were injected into the defect site. The animals were then observed for 4, 8, and 12 weeks to confirm the degree of regeneration. Control groups included an untreated group (A) and a group treated with the carrier, i.e., cross-linked hyaluronic acid only (B). Experimental groups included groups (C–G) injected with compositions containing 20 mg / mL cross-linked hyaluronic acid and TGF-β3 at final concentrations of 6 ppm, 30 ppm, 60 ppm, 100 ppm, and 200 ppm, respectively. Hematoxylin and eosin staining (H&E staining) and safranin O-fast green staining (SO-FG staining) were used to calculate the OARSI score and subchondral bone grade according to the criteria shown in Figures 7 and 8. The results are shown in Figures 9 and 10. Furthermore, the figures are comprehensively summarized in Table 1.

[0038] [Table 1]

[0039] As shown in Figures 9 and 10 and Table 1, some experimental groups showed worse scores than the control group 4 weeks after composition injection, but after 8 and 12 weeks, each experimental group showed significantly improved scores compared to the control group. This indicates that the composition of the present invention exerts an excellent regenerative effect on damaged cartilage when sufficient time for regeneration has passed after composition injection.

[0040] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalents.

Claims

1. It contains crosslinked hyaluronic acid and transforming growth factor-beta (TGF-β), The cross-linked hyaluronic acid is characterized in that it is in the form of particles having an average diameter of 3 to 250 μm. Composition for hard tissue regeneration.

2. The tissue is cartilage tissue or bone tissue. The composition of claim 1.

3. The crosslinked hyaluronic acid is crosslinked with at least one crosslinking agent selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), bis ethyl carbodiimide (BCDI), polyethylene glycol (PEG), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The composition of claim 1.

4. The composition contains cross-linked hyaluronic acid in an amount of 15 mg / mL to 30 mg / mL based on the volume of the final composition. The composition of claim 1.

5. The TGF-β is at least one selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-5, BMP-6, and BMP-7. The composition of claim 1.

6. The composition contains TGF-β at a content of 0.003 mg / mL to 1.0 mg / mL based on the volume of the final composition. The composition of claim 1.

7. The composition has an adhesive strength of 2N to 15N. The composition of claim 1.

8. The composition has a viscosity of 8,000 cP to 80,000 cP. The composition of claim 1.

9. The composition has a discharge force of 3N to 50N. The composition of claim 1.

10. The composition reduces the OARSI score to 2.0 or less 12 weeks after injection into the hard tissue defect site. The composition of claim 1.

11. The composition reduces the subchondral bone grade to 1.0 or less 8 weeks after injection into the hard tissue defect site. The composition of claim 1.

12. The composition is prepared by mixing cross-linked hyaluronic acid and TGF-β immediately before injection into a hard tissue defect site. The composition of claim 1.

13. The composition for hard tissue regeneration according to claim 1, a mixing means, and an injection means, Hard tissue regeneration kit.

14. The mixing means is a mixing syringe, a vial transfer device, or a connector; The kit of claim 13.

15. The injection means is a syringe or an injection needle. The kit of claim 13.

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