Composition for regenerating hard tissue

Abstract

The present invention provides a composition for hard tissue regeneration.

Description

Technical Field

[0001] The present disclosure relates to a composition for hard tissue regeneration.

Background Art

[0002] All materials to be transplanted into the human body, especially materials for the regeneration of hard tissues (such as bone tissue or cartilage tissue), should have good processability and plasticity or good in-situ polymerization properties to be suitable for wounds. These materials need to provide a suitable environment for cell adhesion, growth, and differentiation, and other products generated by material degradation also need to be biocompatible. In particular, when the compressive strength and yield value of a transplantation material for bone regeneration are too low, it is difficult to maintain the ability of the transplantation material to stay in place after injection or dense filling of the transplantation material and to maintain its external shape during the closing or implant implantation stage. In addition, if the adhesiveness of the transplantation material is too high, it is easily adhered to surgical tools during the operation, so it is difficult to fill bone defects, resulting in reduced processability.

[0003] In the initial stage of development, such transplantation biomaterials for hard tissue regeneration relied on their inert properties in the body, but their use has been significantly limited due to the infection and inflammatory reactions that occur in the surrounding tissues after implantation. Since then, with the rapid development of biomaterial technologies based on metals, ceramics, and polymers, materials with biocompatibility rather than bioinertness have been designed and developed, leading to the development of bioactive scaffolds for bone tissue regeneration that vary depending on the use site and purpose. Such bioactive scaffolds for bone tissue regeneration are required to have different physical properties depending on the location where the graft is placed; be non-toxic to the surrounding tissues; and have relatively high mechanical properties compared to other artificial organs. Such bioactive scaffolds for bone tissue regeneration have been sold and developed as various biomaterials according to the properties of the raw materials and their intended uses.

[0004] On the other hand, in the case of hard tissues, due to the nature of the tissues, the shape of the damaged area is usually irregular, making it difficult to select a transplantation material suitable for the defect area. Therefore, it is necessary to find a material with sufficient strength and excellent adhesiveness to the surrounding tissues, which is easy to inject to fill irregular damaged areas, and most importantly, can promote tissue regeneration.

Summary of the Invention

[0005]

Technical Problem

[0006] An object of the present disclosure is to provide a composition for hard tissue regeneration, which contains crosslinked hyaluronic acid and transforming growth factor-β (TGF-β), wherein the crosslinked hyaluronic acid is particles having an average diameter of 3 μm to 250 μm.

[0007] Another object of the present disclosure is to provide a kit for hard tissue regeneration, which includes a composition for hard tissue regeneration, a mixing tool, and an injection tool.

[0008]

Beneficial effects

[0009] The composition of the present disclosure exhibits high adhesiveness, viscosity, and low extrusion force, and thus has physical properties suitable as a graft material for hard tissue regeneration. Therefore, the composition can be easily injected into the defect area of the tissue and can also maintain its shape during the regeneration of the corresponding tissue. Therefore, excellent clinical effects on hard tissue regeneration can be expected.

[0010]

Brief description of the drawings

[0011] Figure 1 Shows the adhesiveness according to the particle size of hyaluronic acid in the composition of the present disclosure.

[0012] Figure 2 Shows the viscosity according to the particle size of hyaluronic acid in the composition of the present disclosure.

[0013] Figure 3 Shows the adhesiveness according to the mixing ratio of hyaluronic acid and buffer in the composition of the present disclosure.

[0014] Figure 4 Shows the viscosity according to the mixing ratio of hyaluronic acid and buffer in the composition of the present disclosure.

[0015] Figure 5 Shows the extrusion force according to the mixing ratio of hyaluronic acid and buffer in the composition of the present disclosure.

[0016] Figure 6 Shows the preparation of a microfracture animal model with a defect site on the trochlear groove using rabbits and a tissue regeneration method using the model.

[0017] Figure 7 Shows the scoring criteria of the OARSI evaluation system.

[0018] Figure 8 Shows the OARSI scoring process resulting from the administration of the composition for hard tissue regeneration in a microfracture animal model according to an embodiment of the present disclosure.

[0019] Figure 9 Shows the scoring criteria of the subchondral bone grading system.

[0020] Figure 10 Shows the process of the subchondral bone grade resulting from the administration of the composition for hard tissue regeneration in a microfracture animal model according to an embodiment of the present disclosure.

[0021]

Detailed Description of Preferred Embodiments

[0022] Each description and embodiment disclosed in this disclosure can also be applied to other descriptions and embodiments. That is to say, all combinations of various elements disclosed in this disclosure fall within the scope of this disclosure. In addition, the scope of this disclosure is not limited by the following specific descriptions.

[0023] In addition, those skilled in the art will recognize or be able to use, without more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Moreover, these equivalents should be construed as falling within the scope of this disclosure.

[0024] In addition, throughout the specification of this disclosure, when a part is referred to as "comprising" an element, it will be understood that other elements may also be included, unless otherwise specifically described, rather than excluding other elements.

[0025] Hereinafter, this disclosure will be described in more detail.

[0026] To achieve the above object, a first aspect of this disclosure provides a composition for hard tissue regeneration, which comprises cross-linked hyaluronic acid and transforming growth factor-β (TGF-β).

[0027] In this regard, the cross-linked hyaluronic acid can be characterized as particles having an average diameter of 3 μm to 250 μm.

[0028] For example, the tissue that can be regenerated by applying the composition of this disclosure can be cartilage tissue or bone tissue, but is not limited thereto.

[0029] As used herein, the term "hyaluronic acid (HA)", also known as hyaluronan, is an anionic, non-sulfated glycosaminoglycan that is widely distributed in connective tissue, epithelial tissue, and neural tissue. Hyaluronic acid is unique among glycosaminoglycans in being in a non-sulfated form, forming in the plasma membrane rather than the Golgi apparatus, and can be very large. There is approximately 15 g of hyaluronic acid in the body of an average 70 kg (150 lb) human, and one-third of it is degraded and synthesized every day. As one of the main components of the extracellular matrix, hyaluronic acid makes a significant contribution to cell proliferation and migration and is involved in the progression of many malignant tumors.

[0030] As used herein, the term "cross-linked hyaluronic acid" is classified as a different category from the linear hyaluronic acid described above, and can be distinguished according to the type of processing and the composition of the acid. Cross-linking refers to the process of obtaining a new structural material composed of multiple hyaluronic acid filaments with higher molecular weights by linking linear hyaluronic acid molecules together. The high-molecular-weight hyaluronic acid formed by cross-linking may lose the characteristics of individual molecules, thus reaching a state that is actually like a gel. The above-mentioned linear hyaluronic acid is the first substance to be used and commercialized, consisting of invariant linear hyaluronic acid chains, while cross-linked hyaluronic acid is a recently developed substance, characterized by containing cross-links formed between several linear hyaluronic acid molecules during the production stage. These cross-links are lateral bridges that connect multiple chains together, aiming to form hyaluronic acid macromolecules with larger sizes, higher molecular weights, and thus higher density and durability.

[0031] For example, cross-linked hyaluronic acid can be cross-linked by one or more cross-linking agents selected from the following: 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), bis-ethyl carbodiimide (BCDI), polyethylene glycol (PEG), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).

[0032] For example, the composition may contain cross-linked hyaluronic acid in an amount of 15 mg / mL to 30 mg / mL based on the volume of the final composition.

[0033] As used herein, the term "transforming growth factor-β (TGF-β)" is secreted in a latent form complexed with two different polypeptides, the latent TGF-β binding protein (LTBP) and the latency-associated peptide (LAP), by many cell types including macrophages. One of its key functions is to regulate the inflammatory process, especially in the gut. TGF-β plays an important role not only in T cell regulation and differentiation but also in stem cell differentiation. Due to its role in immune and stem cell regulation and differentiation, TGF-β is a highly studied cytokine in the fields of cancer, autoimmune diseases, and infectious diseases. The TGF-β superfamily contains endogenous growth inhibitory proteins. An increase in TGF-β expression is usually associated with the malignancy of many cancers and the defective response of cell growth inhibition to TGF-β. When its immunosuppressive function begins to dominate, it contributes to tumorigenesis. The dysregulation of its immunosuppressive function is also related to the pathogenesis of autoimmune diseases, although their effects are mediated by the environment of other cytokines present.

[0034] For example, TGF-β includes, but is not limited to, proteins belonging to the TGF-β superfamily (a large group of cell regulatory proteins that share a structural relatedness in interacting with TGF-β receptors). Specifically, TGF-β can be one or more selected from the following: TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-5, BMP-6, and BMP-7. More specifically, TGF-β can be TGF-β3, and TGF-β3 can include, but is not limited to, recombinant human TGF-β3 derived from Escherichia coli.

[0035] As used herein, the term "TGF-β3" refers to a protein encoded by the TGFB3 gene in humans. It is a protein called a cytokine that is involved in cell differentiation, embryogenesis, and development. It belongs to a large family of cytokines called the transforming growth factor-β superfamily (including the TGF-β family, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), inhibins, and activins).

[0036] For example, the composition can contain TGF-β (such as TGF-β3) in an amount of 0.003 mg / mL to 1.0 mg / mL based on the volume of the final composition. For example, the composition can contain TGF-β in an amount 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 thereto. When the content of TGF-β is less than the above amount, it may be difficult to achieve the desired level of synergistic effect depending on the added amount of TGF-β. Even if the added amount of TGF-β exceeds the above amount, it may be difficult to expect additional effects according to the added amount.

[0037] For example, the composition can have an adhesive force of 2 N to 15 N. For example, the composition can have an adhesive force of 3 N to 12 N or 4 N to 10 N, but is not limited thereto.

[0038] In addition, the composition can have a viscosity of 8,000 cP to 80,000 cP. For example, the viscosity of the composition can be 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 thereto.

[0039] In addition, the composition can have an extrusion force of 3 N to 50 N. For example, the extrusion force of the composition can be 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 thereto.

[0040] Due to the above physical properties, the compositions of the present disclosure can be easily injected into damaged hard tissues, and even when injected in an environment present in body fluids such as bone marrow, etc., it can have excellent adhesion to the corresponding tissues, thereby maintaining its shape at the injection site.

[0041] For example, the composition can reduce the Osteoarthritis Research Society International (OARSI) score to 2.0 or lower 12 weeks after injection into the hard tissue defect site.

[0042] In addition, the composition can reduce the subchondral bone grade to 1.0 or lower 8 weeks after injection into the hard tissue defect site.

[0043] As used herein, the term "Osteoarthritis Research Society International (OARSI) score" is a score established by OARSI in 1998 for standardizing the evaluation of osteoarthritis (OA) histopathology, based on the results of safranin O / fast green staining. The OARSI score represents a comprehensive evaluation based on the severity and extent of OA in articular cartilage, and the total score ranges from 0 to 6. The specific criteria are consistent with previous reports, and the higher the OARSI score, the more severe the articular cartilage damage.

[0044] As used herein, the term "subchondral bone grade" is an evaluation system that grades the state of subchondral bone on a scale of 0 to 3 based on previous studies describing subchondral bone changes and the gross observations of osteoarthritis sample materials by two researchers, OMA and PL. Specifically, the subchondral bone grade explains the structural changes in the bone connected to the cartilage, and as the grade increases, it indicates that the bone becomes harder and thicker. Subchondral bone is located between cartilage and bone marrow, and when subchondral bone thickens, the supply of active factors in the bone marrow that maintain the cartilage structure is blocked, which may accelerate cartilage damage.

[0045] As described above, the composition of the present disclosure reduces the OARIS score to a very low level of 2.0 or lower 12 weeks after injection into the hard tissue defect site, and / or reduces the subchondral bone grade to a very low level of 1.0 or lower at 8 weeks, which means that the defect site regenerates to a level similar to that before the cartilage and / or subchondral bone defect.

[0046] For example, the composition can be used by mixing cross-linked hyaluronic acid and TGF-β and immediately injecting it into the hard tissue defect site.

[0047] The second aspect of the present disclosure provides a kit for hard tissue regeneration, which includes a composition for hard tissue regeneration according to the first aspect, a mixing tool, and an injection tool.

[0048] For example, the mixing tool can be a mixing syringe, a vial transport device, or a connector, and the injection tool can be a syringe or an injection needle, but is not limited thereto.

[0049] For example, the kit of the present disclosure can use an injection needle to inject the composition into the hard tissue defect site, or use a syringe or the like as an auxiliary tool to directly inject the composition into the open affected part after surgery.

[0050] Specifically, a vial containing TGF-β is connected to a vial transport device, and TGF-β is delivered to an empty syringe. Then the syringe is separated and connected to a syringe containing crosslinked hyaluronic acid using a connector to mix TGF-β with crosslinked hyaluronic acid. An injection needle is connected to the mixed syringe and can be directly injected into the hard tissue defect site or injected into the hard tissue defect site after surgery. When used together with surgery, the kit can be provided by applying a double-sterile package or the like to minimize the possibility of infection that may occur during handling, but there is no difference in the usage method or principle.

[0051]

Mode for Carrying Out the Invention

[0052] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not intended to be limited by these exemplary embodiments.

[0053]

Preparation Example 1: Preparation of Crosslinked Hyaluronic Acid

[0054] Crosslinked hyaluronic acid is prepared by reacting hyaluronic acid (molecular weight of 500,000 Da to 1,500,000 Da) and 1,4-butanediol diglycidyl ether (BDDE) as a crosslinking agent. Specifically, 1 g / g hyaluronic acid, 0.8 g / g NaOH, 3.2 g / g purified water, and 0.02 g / g BDDE are mixed and stirred to form a gel. After the synthesis is completed, the gel is crosslinked in an orbital shaker (shaking speed: 80 rpm, temperature: 30 °C, reaction time: 19 hours). The crosslinked gel is coarsely crushed into a size of 15 mm × 10 mm and then dialyzed. The dialyzed gel is sieved through a standard sieve to obtain particles of regular size.

[0055]

Example 1: Evaluation of the Physical Properties of Crosslinked Hyaluronic Acid According to Size

[0056] The crosslinked hyaluronic acid prepared according to Preparation Example 1 is divided into six groups according to the particle size to prepare samples. Specifically, samples of particles with an average particle size of 5 μm, 80 μm, 200 μm, 300 μm, 800 μm, or 1000 μm and a concentration of 20 mg / mL are prepared in PBS buffer, and the adhesive force and viscosity are measured. The results are respectively as Figure 1 and Figure 2As shown. Specifically, after injecting the sample into the plate, using an Antopa rheometer, the rotor was lowered to a position of 0.25 mm so that they contacted the sample, and the adhesive force was measured at a speed of 200 μm / s. After placing 0.5 cc of the sample in the sample cup, the viscosity was measured using a Brookfield viscometer at a speed of 0.5 rpm.

[0057] As Figure 1 and Figure 2 shown, the sample containing cross-linked hyaluronic acid particles with an average size of 5 μm to 200 μm showed higher adhesive force and viscosity than the sample containing particles with a larger particle size.

[0058]

Example 2: Evaluation of the physical properties of a composition for hard tissue regeneration containing cross-linked hyaluronic acid and TGF-β3 according to the mixing ratio

[0059] A 20 mg / mL solution of cross-linked hyaluronic acid prepared according to Preparation Example 1 was prepared using PBS buffer, and a series of compositions were prepared by mixing the solution with recombinant human transforming growth factor-β3 (rhTGF-β3) in a predetermined volume ratio with the ratio changed from 10:0 to 6:4. The adhesive force, viscosity, and extrusion force of this series of compositions were measured, and the results are shown in Figures 3 to 5 respectively. Specifically, the adhesive force and viscosity were measured in the same manner as in Example 1, and using a universal testing machine, the extrusion force was measured by filling a 2 cc sample into a 3 cc syringe, connecting a 21G syringe, fixing the filled syringe to a fixture, and pressing it at a speed of 30 mm / min.

[0060] As Figures 3 to 5 shown, as the proportion of cross-linked hyaluronic acid decreased, the adhesive force, viscosity, and extrusion force all showed a decreasing trend.

[0061]

Example 3: Verification of cartilage regeneration ability using experimental animals

[0062] As Figure 6 shown, a microfracture animal experimental model was created, the prepared composition was injected into the corresponding defect site, and the degree of regeneration was examined by observing at 4 weeks, 8 weeks, and 12 weeks. As a control group, an untreated group (A) and a group treated only with the carrier (i.e., cross-linked hyaluronic acid) (B) were used. As experimental groups, groups 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, or 200 ppm (C to G in sequence) were used. Using hematoxylin and eosin staining (H&E staining) and safranin O-fast green staining (SO-FG staining), according to Figure 7 and Figure 8The OARSI score and the subchondral bone grade were calculated based on the standard scores shown, and the results are shown in Figure 9 and 10 respectively. In addition, these values are comprehensively summarized in Table 1 below.

[0063]

Table 1

[0064]

[0065] As Figure 9 and Figure 10 and Table 1 show, at 4 weeks after injection of the composition, some experimental groups showed slightly worse values compared to the control group. However, as the composition hardened at 8 weeks and 12 weeks, each experimental group showed significantly improved values compared to the control group, indicating that the composition of the present disclosure exhibits excellent regenerative effects on damaged cartilage after sufficient regeneration time after injection of the composition.

[0066] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in various aspects. The scope of the disclosure is defined by the appended claims rather than by the description preceding them, and thus, all changes and modifications within the scope and equivalence of such scope, or such scope, are intended to be included in the claims.

Claims

1. A composition for hard tissue regeneration, comprising cross-linked hyaluronic acid and transforming growth factor-β (TGF-β), wherein, the cross-linked hyaluronic acid is a particle having an average diameter of 3 μm to 250 μm.

2. The composition according to claim 1, wherein, the tissue is cartilage tissue or bone tissue.

3. The composition according to claim 1, wherein, the cross-linked hyaluronic acid is cross-linked by one or more cross-linking agents selected from the following: 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), bis-ethyl carbodiimide (BCDI), polyethylene glycol (PEG), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).

4. The composition according to claim 1, wherein, 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.

5. The composition according to claim 1, wherein, the TGF-β is one or more selected from the following: TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-5, BMP-6, and BMP-7.

6. The composition according to claim 1, wherein, the composition contains TGF-β in an amount of 0.003 mg / mL to 1.0 mg / mL based on the volume of the final composition.

7. The composition according to claim 1, wherein, the composition has an adhesive force of 2 N to 15 N.

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

9. The composition according to claim 1, wherein, the composition has an extrusion force of 3 N to 50 N.

10. The composition according to claim 1, wherein, the composition reduces the OARSI score to 2.0 or lower 12 weeks after being injected into the hard tissue defect site.

11. The composition according to claim 1, wherein, the composition reduces the subchondral bone grade to 1.0 or lower 8 weeks after being injected into the hard tissue defect site.

12. The composition according to claim 1, wherein, the cross-linked hyaluronic acid and TGF-β are mixed and immediately injected into the hard tissue defect site.

13. A kit for hard tissue regeneration, comprising the composition for hard tissue regeneration according to claim 1, a mixing tool, and an injection tool.

14. The kit according to claim 13, wherein, the mixing tool is a mixing syringe, a vial transport device, or a connector.

15. The kit according to claim 13, wherein, the injection tool is a syringe or an injection needle.

Images