Injectable composition comprising crosslinked hyaluronic acid hydrogel and extracellular matrix, preparation method therefor, and use thereof

Abstract

The present invention relates to an injectable composition comprising a crosslinked hyaluronic acid hydrogel and an extracellular matrix, a preparation method therefor, and use thereof for reducing wrinkles or alleviating joint pain. The injectable composition comprising a crosslinked hyaluronic acid hydrogel and an extracellular matrix derived from liquid human cell culture, of the present invention, can increase an administration interval due to a long residual time thereof in the body, can be completely biodegraded after a certain period of time, and can simultaneously impart physical functions as a tissue repair agent or lubricant and a tissue regeneration effect, and thus can be used for reducing wrinkles or alleviating joint pain. In particular, the injectable composition comprising a crosslinked hyaluronic acid hydrogel and an extracellular matrix, of the present invention, can promote angiogenesis and collagen biosynthesis in tissue, thus contributing to tissue regeneration.

Description

Injectable composition comprising cross-linked hyaluronic acid hydrogel and extracellular matrix, method for producing the same, and use thereof

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0125936, filed with the Korean Intellectual Property Office on September 20, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix, a method for producing the same, and a use thereof.

[0003]

[0004] Cross-linked hyaluronic acid hydrogels have been primarily used as cosmetic fillers and to relieve joint pain. In particular, cross-linked hyaluronic acid hydrogels account for over 80% of the cosmetic filler market. They are injected into areas of tissue defects, such as wrinkles, to attract moisture within the tissue, thereby providing volume.

[0005] The reason why cross-linked hyaluronic acid hydrogels have a high market share in the cosmetic filler market is because they utilize hyaluronic acid that exists in the extracellular matrix of the body, so they have a high level of safety. In addition, they have the characteristic of being broken down in the body and eliminated as a non-toxic substance after a certain period of time. In addition, since they are easy to remove after injection, if a doctor mistakenly administers the filler into a blood vessel or the patient wishes to return it to its original state, the injected filler can be easily removed by hyaluronidase. Therefore, although numerous cross-linked hyaluronic acid hydrogels have been commercialized as cosmetic fillers to date, cross-linked hyaluronic acid hydrogels only physically improve wrinkles. They are biodegraded after a certain period of time and do not provide the function of biologically regenerating tissues while remaining in the tissue.

[0006] Accordingly, polynucleotide (PN) or polydeoxyribonucleotide (PDRN) extracted from salmon testis has been commercialized for the purpose of tissue regeneration by activating the proliferation of fibroblasts in the human body. However, there are side effects such as severe pain during injection, unevenness at the injection site after injection, little volume-giving function, and relatively frequent administration intervals (every 1 to 2 weeks), so the use of other tissue regeneration raw materials is necessary.

[0007] Meanwhile, cross-linked hyaluronic acid hydrogels have also been used to alleviate joint pain. Building on the existing use of uncross-linked hyaluronic acid solutions as joint lubricants, cross-linked hyaluronic acid hydrogels have been commercialized as long-lasting joint lubricants in the body. However, these products only demonstrated pain relief through their physical lubricant function, and tissue regeneration effects have not been confirmed.

[0008]

[0009] The present inventors have conducted extensive research efforts to solve the problems of the conventional cross-linked hyaluronic acid hydrogel materials as described above, and have confirmed that an injectable composition containing a cross-linked hyaluronic acid hydrogel and an extracellular matrix simultaneously provides physical supplementation of a defect site in a tissue, regenerative ability to surrounding tissue, and increased tissue repair period, thereby completing the present invention.

[0010] Accordingly, an object of the present invention is to provide an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix.

[0011] Another object of the present invention is to provide a method for preparing an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix.

[0012]

[0013] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0014] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0015]

[0016] According to one aspect of the present invention, the present invention provides an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix.

[0017] The term “cross-linked hyaluronic acid hydrogel” of the present invention refers to a form in which chemically cross-linked hyaluronic acid is pulverized and supported by water or a buffer solution. Specifically, the cross-linked hyaluronic acid hydrogel of the present invention refers to a mixture of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates, and each cross-linked hyaluronic acid hydrogel included therein before mixing may include, but is not limited to, a cross-linked hyaluronic acid hydrogel obtained by performing a single cross-linking reaction in the manufacturing process, as well as a cross-linked hyaluronic acid hydrogel obtained through two or more cross-linking reactions, and a mixture of a cross-linked hyaluronic acid hydrogel and a non-cross-linked hyaluronic acid.

[0018] A mixture of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates can be prepared by mixing and adding different amounts and particle sizes of cross-linking agents to each hyaluronic acid hydrogel.

[0019] Among the mixture of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates, a cross-linked hyaluronic acid hydrogel having a low cross-linking rate (hereinafter referred to as a first cross-linked hyaluronic acid hydrogel or elastic gel in the present invention) may be added with a cross-linking agent at a concentration of 1 to 6 mol%, 1 to 5 mol%, 1 to 4 mol%, 1 to 3 mol%, 2 to 6 mol%, 2 to 5 mol%, 2 to 4 mol%, 2 to 3 mol%, 3 to 6 mol%, 3 to 5 mol%, 3 to 4 mol%, 1 mol%, 2 mol%, 3 mol%, or 4 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide), but is not limited thereto.

[0020] In addition, the first cross-linked hyaluronic acid hydrogel may be granulated into particles having a size of 150-600 μm, 150-500 μm, 150-400 μm, 150-350 μm, 150-300 μm, 150-250 μm, 150-200 μm, 200-600 μm, 200-500 μm, 200-400 μm, 200-350 μm, 200-300 μm, or 200-250 μm, but is not limited thereto. The granulated first cross-linked hyaluronic acid hydrogel maintains elasticity and can provide volume to the injection site when injected into the body.

[0021] Among the mixture of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates, the cross-linked hyaluronic acid hydrogel having a high cross-linking rate (hereinafter referred to as a second cross-linked hyaluronic acid hydrogel or viscous gel in the present invention) may be added with a cross-linking agent at a concentration of 5 to 10 mol%, 5 to 9 mol%, 5 to 8 mol%, 5 to 7 mol%, 5 to 6 mol%, 6 to 10 mol%, 6 to 9 mol%, 6 to 8 mol%, 6 to 7 mol%, 7 to 10 mol%, 7 to 9 mol%, 7 to 8 mol%, 5 mol%, 6 mol%, 7 mol%, or 8 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide), but is not limited thereto.

[0022] The particle size of the second cross-linked hyaluronic acid hydrogel of the present invention must be smaller than the size of the first cross-linked hyaluronic acid particles.

[0023] For example, the second cross-linked hyaluronic acid hydrogel has a particle size of 5-300 μm, 5-280 μm, 5-250 μm, 5-220 μm, 5-200 μm, 5-180 μm, 5-150 μm, 5-140 μm, 5-130 μm, 5-120 μm, 5-110 μm, 5-100 μm, 5-80 μm, 5-50 μm, 5-30 μm, 5-10 μm, 10-300 μm, 10-280 μm, 10-250 μm, 10-220 μm, 10-200 μm, 10-180 μm, 10-150 μm, 10-140 μm, 10-130 μm, 10-120 μm, 10-110 μm, 10-100 μm, 10-80 μm, 10-50 μm, 10-30 μm, 10-20 μm, 30-300 μm, 30-280 μm, 30-250 μm, 30-220 μm, 30-200 μm, 30-180 μm, 30-150 μm, 30-140 μm, 30-130 μm, 30-120 μm, 30-110 μm, 30-100 μm, 30-80 μm, 30-50 μm, 30-40 μm, 50-300 μm, 50-280 μm, 50-250 μm, 50-220 μm, 50-200 μm, 50-180 μm, 50-150 μm, 50-140 μm, 50-130 μm, 50-120 μm, 50-110 μm, 50-100 μm, 50-80 μm, 50-60 μm, 100-300 μm, 100-280 μm, 100-250 μm, 100-220 μm, 100-200 μm, 100-180 μm, 100-150 μm, 100-140 μm, 100-130 μm, 100-120 μm, 100-110 μm, It may have a particle size of, but is not limited to, 150-300 μm, 150-280 μm, 150-250 μm, 150-220 μm, 150-200 μm, 150-180 μm, 150-170 μm, or 150-160 μm.

[0024] The second cross-linked hyaluronic acid hydrogel can provide a soft feeling (less foreign body sensation) at the injection site due to the advantage of a smaller particle size compared to the first cross-linked hyaluronic acid hydrogel when injected into the body, and can maintain viscosity to prevent the hydrogel from detaching from the injection site.

[0025] The cross-linked hyaluronic acid hydrogel of the present invention may be a hyaluronic acid hydrogel cross-linked with one or more selected from 1,4-butanediol diglycidyl ether, divinyl sulfone, and bis ethyl carbodiimide, preferably butanediol diglycidyl ether, but is not limited thereto.

[0026] Among the mixtures of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates of the present invention, the mixing ratio of the first cross-linked hyaluronic acid hydrogel and the second cross-linked hyaluronic acid hydrogel may be 99:1 to 1:99. However, in cases where the particle sizes of the finally formed hydrogel are not completely uniform and the cross-linking rates of each hyaluronic acid molecule are different, the present invention is not limited thereto because it is the same in terms of the composition principle of the present invention even if it is composed only of the first cross-linked hyaluronic acid hydrogel or only of the second cross-linked hyaluronic acid hydrogel, such as 100:0 or 0:100.

[0027] The cross-linked hyaluronic acid hydrogel of the present invention has an intrinsic viscosity (e.g. dL / g) of 1.2~3.5, 1.2~3.0, 1.2~2.8, 1.2~2.5, 1.2~2.3, 1.2~2.0, 1.2~1.8, 1.2~1.6, 1.4~3.5, 1.4~3.0, 1.4~2.8, 1.4~2.5, 1.4~2.3, 1.4~2.0, 1.4~1.8, 1.4~1.6, 1.5~3.5, 1.5~3.0, 1.5~2.8, 1.5~2.5, 1.5~2.3, 1.5~2.0, 1.5~1.8, 1.5~1.6, 1.6~3.5, 1.6~3.0, 1.6~2.8, 1.6~2.5, 1.6~2.3, 1.6~2.0, 1.6~1.8, 1.8~3.5, 1.8~3.0, 1.8~2.8, 1.8~2.5, 1.8~2.3, 1.8~2.0, 2.0~3.5, 2.0~3.0, 2.0~2.8, 2.0~2.5, 2.0~2.3, 2.2~3.5, 2.2~3.0, 2.2~2.8, 2.2~2.5, 2.2~2.3, 2.4~3.5, 2.4~3.0, 2.4~2.8, It can be, but is not limited to, 2.4~2.5, 2.6~3.5, 2.6~3.0, or 2.6~2.8, and can be appropriately selected depending on the intended use. For example, in the case of dermal fillers, hyaluronic acid hydrogels with an extreme viscosity value of 1.6~1.8 are mainly used, and in the case of intra-articular injection, hyaluronic acid hydrogels with an extreme viscosity value of 2.4~3.0 can be used. It is known that the extreme viscosity of the hyaluronic acid hydrogel is correlated with the molecular weight of the hyaluronic acid.

[0028] The cross-linked hyaluronic acid hydrogel of the present invention may have a Tan δ (G'' / G') of 0.12 to 0.20 at a frequency of 0.02 Hz, depending on the mixing ratio of the first hyaluronic acid hydrogel and the second hyaluronic acid hydrogel. Here, G' represents the storage modulus, and G'' represents the loss modulus.

[0029] The injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix of the present invention comprises cross-linked hyaluronic acid at a concentration of 5 to 30 mg / ml. More specifically, the injectable composition of the present invention contains cross-linked hyaluronic acid in an amount of 5 to 30 mg / mL, 5 to 28 mg / mL, 5 to 25 mg / mL, 5 to 24 mg / mL, 5 to 23 mg / mL, 5 to 22 mg / mL, 5 to 20 mg / mL, 5 to 18 mg / mL, 5 to 16 mg / mL, 5 to 15 mg / mL, 5 to 14 mg / mL, 5 to 13 mg / mL, 5 to 12 mg / mL, 5 to 11 mg / mL, 5 to 10 mg / mL, 7 to 30 mg / mL, 7 to 28 mg / mL, 7 to 25 mg / mL, 7 to 24 mg / mL, 7 to 23 mg / mL, 7 to 22 ㎎ / ㎖, 7 to 20 ㎎ / ㎖, 7 to 18 ㎎ / ㎖, 7 to 16 ㎎ / ㎖, 7 to 15 ㎎ / ㎖, 7 to 14 ㎎ / ㎖, 7 to 13 ㎎ / ㎖, 7 to 12 ㎎ / ㎖, 7 to 11 ㎎ / ㎖, 7 to 10 ㎎ / ㎖, 10 to 30 ㎎ / ㎖, 10 to 28 ㎎ / ㎖, 10 to 25 ㎎ / ㎖, 10 to 24 ㎎ / ㎖, 10 to 23 ㎎ / ㎖, 10 to 22 ㎎ / ㎖, 10 to 20 ㎎ / ㎖, 10 to 18 ㎎ / ㎖, 10 to 16 ㎎ / ㎖, 10 to 15 ㎎ / ㎖, 10 to 14 ㎎ / ㎖, 10 to 13 ㎎ / ㎖, 10 to 12 ㎎ / ㎖, 10 to 11 ㎎ / ㎖, 12 to 30 ㎎ / ㎖, 12 to 28 ㎎ / ㎖, 12 to 25 ㎎ / ㎖, 12 to 24 ㎎ / ㎖, 12 to 23 ㎎ / ㎖, 12 to 22 ㎎ / ㎖, 12 to 20 ㎎ / ㎖, 12 to 18 ㎎ / ㎖, 12 to 16 ㎎ / ㎖, 12 to 15 ㎎ / ㎖, 12 to 14 ㎎ / ㎖, 12 to 13 ㎎ / ㎖, 14 to 30 ㎎ / ㎖, 14 to 28 ㎎ / ㎖, 14 to 25 ㎎ / ㎖, 14 to 24 ㎎ / ㎖, 14 to 23 ㎎ / ㎖, 14 to 22 ㎎ / ㎖, 14 to 20 mg / ml, 14 to 18 mg / ml, 14 to 16 mg / ml,14 to 15 mg / ml, 15 to 30 mg / ml, 15 to 28 mg / ml, 15 to 25 mg / ml, 15 to 24 mg / ml, 15 to 23 mg / ml, 15 to 22 mg / ml, 15 to 20 mg / ml, 15 to 18 mg / ml, 15 to 16 mg / ml, 16 to 30 mg / ml, 16 to 28 mg / ml, 16 to 25 mg / ml, 16 to 24 mg / ml, 16 to 23 mg / ml, 16 to 22 mg / ml, 16 to 20 mg / ml, 16 to 18 mg / ml, 18 to 30 mg / ml, 18 to 28 mg / ml, 18 to 25 mg / ml, 18 to 24 mg / ml, 18 to 23 mg / ml, 18 to 22 mg / ml, 18 to 20 mg / ml, 20 to 30 mg / ml, 20 to 28 mg / ml, 20 to 25 mg / ml, 20 to 24 mg / ml, 20 to 23 mg / ml, 20 to 22 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, May include, but is not limited to, a concentration of 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, or 30 mg / ml.

[0030] The term “extracellular matrix” of the present invention refers to an extracellular matrix comprising two or more components selected from laminin, type IV collagen, elastin, proteoglycan, fibronectin, fibrinogen, fibrin, perlecan, nidogen, and fibulin, but the components constituting the above-described extracellular matrix are not limited thereto.

[0031] In one embodiment of the present invention, the extracellular matrix included in the composition essentially comprises laminin. In another embodiment of the present invention, the extracellular matrix included in the composition essentially comprises laminin and type IV collagen. In yet another embodiment of the present invention, the extracellular matrix included in the composition essentially comprises laminin and fibronectin. In yet another embodiment of the present invention, the extracellular matrix included in the composition essentially comprises laminin, type IV collagen, and fibronectin. In yet another embodiment of the present invention, the extracellular matrix included in the composition includes, but is not limited to, laminin; and at least one component selected from the group consisting of type IV collagen, elastin, proteoglycan, fibronectin, fibrinogen, fibrin, perlecan, nidogen, and fibulin.

[0032] The injectable composition of the present invention is characterized by comprising an extracellular matrix in a liquid state. In a specific embodiment of the present invention, when the extracellular matrix in a powder form is added to the injectable composition of the present invention, the injection force when injected using a syringe is high and irregular compared to when the extracellular matrix in a liquid state is added. In addition, in a specific embodiment of the present invention, when the extracellular matrix in a powder form is added to the injectable composition of the present invention, compared to when the extracellular matrix in a liquid state is added, the extracellular matrix may not be completely dissolved in the injection, and thus insoluble substances may exist.

[0033] The extracellular matrix of the injectable composition of the present invention may be an extracellular matrix derived from human cell culture. The term "human cell culture" refers to culturing connective tissue cells, epithelial tissue cells, or a combination thereof among human-derived cells.

[0034] Specifically, the above connective tissue cells refer to at least one selected from the group consisting of fibroblasts, adipocytes, osteocytes, chondrocytes, ligament cells, tendon cells, and mesenchymal stem cells, but are not limited thereto.

[0035] In addition, the above-mentioned epithelial tissue cells refer to at least one selected from the group consisting of keratinocytes, vascular endothelial cells, intestinal epithelial cells, corneal epithelial cells, and hepatocytes, but are not limited thereto. In the case of human cell culture-derived extracellular matrix, since it is produced by culturing human cells in vitro, it has technical significance in that it can be used industrially.

[0036] The extracellular matrix of the present invention may be a basement membrane extracellular matrix. The basement membrane extracellular matrix is ​​one type of extracellular matrix, has a plate-like shape, supports cells and tissues, and is an extracellular matrix mainly located between epithelial tissue and connective tissue. In particular, among the known extracellular matrices, the basement membrane extracellular matrix is ​​composed of the main components of the structural support of blood vessels, unlike other extracellular matrices such as the interstitial matrix mainly composed of collagen types I / II / III, elastin, and fibronectin, the provisional matrix composed of fibrinogen, fibrin, and fibronectin, and the granulation matrix composed of collagen types I to III and fibronectin (Biomaterials Science (Fourth Edition), 2020, Pages 701-715), and is involved in the regeneration of connective tissue in damaged tissues and the growth, migration, and differentiation of keratinocytes, corneal epithelial cells, mucosal epithelial cells, hepatocytes, and pancreatic islet cells, thereby promoting re-epithelialization.

[0037] The basement membrane (BMM) is a thin membrane located between human epithelial and connective tissues, and its primary components are laminin and type IV collagen. Among these, laminin promotes angiogenesis, thereby stimulating collagen biosynthesis within tissues, thereby playing a crucial role in tissue repair and re-epithelialization. It also interacts with integrins located on the cell surface, contributing to tissue integration.

[0038] In one embodiment of the present invention, the injectable composition of the present invention has a concentration of laminin in the extracellular matrix included in the injectable composition based on the volume of the entire composition of 0.001 to 90 μg / mL, 0.001 to 70 μg / mL, 0.001 to 50 μg / mL, 0.001 to 40 μg / mL, 0.001 to 30 μg / mL, 0.001 to 20 μg / mL, 0.001 to 10 μg / mL, 0.01 to 90 μg / mL, 0.01 to 70 μg / mL, 0.01 to 50 μg / mL, 0.01 to 40 μg / mL, 0.01 to 30 μg / mL, 0.01 to 20 μg / mL, 0.01 to 10 μg / mL, 0.1 It may include, but is not limited to, 0.1 to 90 μg / ml, 0.1 to 70 μg / ml, 0.1 to 50 μg / ml, 0.1 to 40 μg / ml, 0.1 to 30 μg / ml, 0.1 to 20 μg / ml, 0.1 to 10 μg / ml, 1 to 90 μg / ml, 1 to 70 μg / ml, 1 to 50 μg / ml, 1 to 40 μg / ml, 1 to 30 μg / ml, 1 to 20 μg / ml, or 1 to 10 μg / ml.

[0039] In one embodiment of the present invention, the concentration of fibronectin in the extracellular matrix included in the injectable composition of the present invention may have a concentration equal to or greater than the concentration of laminin described above based on the volume of the entire composition, but is not limited thereto.

[0040] In a specific embodiment of the present invention, the injectable composition of the present invention has a concentration of fibronectin in the extracellular matrix included in the injectable composition based on the volume of the entire composition of 0.001 to 120 ㎍ / ㎖, 0.001 to 110 ㎍ / ㎖, 0.001 to 100 ㎍ / ㎖, 0.001 to 90 ㎍ / ㎖, 0.001 to 70 ㎍ / ㎖, 0.001 to 50 ㎍ / ㎖, 0.001 to 40 ㎍ / ㎖, 0.001 to 30 ㎍ / ㎖, 0.001 to 20 ㎍ / ㎖, 0.001 to 10 ㎍ / ㎖, 0.01 to 120 ㎍ / ㎖, 0.01 to 110 ㎍ / ㎖, 0.01 to 100 ㎍ / ㎖, 0.01 to 90 ㎍ / ㎖, 0.01 to 70 ㎍ / ㎖, 0.01 to 50 ㎍ / ㎖, 0.01 to 40 ㎍ / ㎖, 0.01 to 30 ㎍ / ㎖, 0.01 to 20 ㎍ / ㎖, 0.01 to 10 ㎍ / ㎖, 0.1 to 120 ㎍ / ㎖, 0.1 to 110 ㎍ / ㎖, 0.1 to 100 ㎍ / ㎖, 0.1 to 90 ㎍ / ㎖, 0.1 to 70 ㎍ / ㎖, 0.1 to 50 ㎍ / ㎖, 0.1 to 40 ㎍ / ㎖, 0.1 to 30 ㎍ / ㎖, 0.1 to 20 ㎍ / ㎖, 0.1 to 10 ㎍ / ㎖, 1 to 120 ㎍ / ㎖, 1 to 110 It may include, but is not limited to, 1 to 100 μg / ml, 1 to 90 μg / ml, 1 to 70 μg / ml, 1 to 50 μg / ml, 1 to 40 μg / ml, 1 to 30 μg / ml, 1 to 20 μg / ml, or 1 to 10 μg / ml.

[0041] Considering that the concentration of laminin in the extracellular matrix that can be produced through human cell culture is at most about 1 mg / ml, the present inventors confirmed that the concentration of the final composition after mixing with hyaluronic acid hydrogel can reach at most 300 μg / ml, and confirmed that the final injectable composition of the present invention, consisting of a mixture of hyaluronic acid hydrogel and the extracellular matrix, has sufficient tissue regeneration efficacy in a tissue regeneration ability test using the basement membrane extracellular matrix derived from human cell culture within the laminin concentration range.

[0042] According to one embodiment of the present invention, the hydration contact angle measured after applying and drying the injectable composition comprising the cross-linked hyaluronic acid hydrogel of the present invention and the extracellular matrix on a plate is 40-70°, 45-70°, 50-70°, 51-70°, 52-70°, 53-70°, 54-70°, 55-70°, 56-70°, 57-70°, 40-65°, 45-65°, 50-65°, 51-65°, 52-65°, 53-65°, 54-65°, 55-65°, 56-65°, 57-65°, 40-60°, 45-60°, 50-60°, 51-60°, 52-60°, 53-60°, 54-60°, 55-60°, 56-60°, 57-60°, 40-58°, 45-58°, 50-58°, 51-58°, 52-60°, 53-58°, 54-58°, 55-58°, 56-58° or 57-58°, but is not limited thereto.

[0043] In general, when the hydration contact angle of the substrate surface is 40° to 70°, it provides a characteristic that facilitates cell attachment to the substrate surface, thereby providing an advantage in facilitating cell fixation, i.e., cell attachment to the hydrogel surface, which affects tissue regeneration at the injection site in terms of tissue regeneration. Therefore, it can be seen that the injectable composition of the present invention has excellent tissue regeneration ability from the excellent cell adhesion.

[0044] According to one embodiment of the present invention, the excellent cell adhesion is not exhibited when the extracellular matrix comprises only a single component, such as laminin. Furthermore, the increased hydration contact angle reduces the degradation rate of the hyaluronic acid hydrogel, thereby increasing the tissue repair maintenance period of the injectable composition of the present invention.

[0045] The injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix of the present invention contributes to exhibiting a local tissue regeneration effect at the injection site because the viscosity of the cross-linked hyaluronic acid hydrogel can act to fix the extracellular matrix at the injection site. This secondary effect does not occur when uncross-linked hyaluronic acid and an extracellular matrix are mixed.

[0046] In one embodiment of the present invention, the mixing weight ratio of the cross-linked hyaluronic acid hydrogel and the extracellular matrix may be 7:3 to 9.9:0.1. Preferably, it may be 8:2 to 9:1, but is not limited thereto.

[0047] Furthermore, in another specific embodiment of the present invention, the injectable composition containing the extracellular matrix of the present invention exhibits significantly superior collagen formation and angiogenic potential upon tissue examination following intradermal / subcutaneous injection compared to a composition composed solely of a cross-linked hyaluronic acid hydrogel without the extracellular matrix. The superior angiogenic potential indicates that the composition of the present invention exhibits superior tissue regeneration potential.

[0048] The injectable composition comprising the cross-linked hyaluronic acid hydrogel and the extracellular matrix of the present invention may further comprise a local anesthetic. The local anesthetic of the present invention may be present in an amount of 0.1 to 1.0% by weight relative to the total weight of the injectable composition comprising the cross-linked hyaluronic acid hydrogel and the liquid human cell culture-derived extracellular matrix. At a concentration of 0.1% or less, the local anesthetic effect is minimal, and pain may occur upon administration. At a concentration of 1.0% or more, the dosage is large, making safety difficult to ensure. As mentioned above, the local anesthetic of the present invention may be one or more local anesthetics selected from benzocaine, oxybuprocaine, furoparacaine, procaine, ropivacaine, lidocaine, mepivacaine, bupivacaine, levobupivacaine, atacaine, prilocaine, cocaine, chloroprocaine, tetracaine, editocaine, and dibucaine, preferably lidocaine, but not limited thereto.

[0049] In one embodiment of the present invention, the injectable composition of the present invention may include a buffer, a preservative, an isotonic agent, a suspending agent, a solvent, or a combination thereof.

[0050] The above buffer may be, but is not limited to, sodium dihydrogen phosphate, disodium hydrogen phosphate, and the like.

[0051] The above stabilizer may be, but is not limited to, sodium bisulfite.

[0052] The above preservative may be, but is not limited to, p-oxybenzoic acid esters such as methyl p-oxybenzoate, thimerosal, chlorobutanol, benzyl alcohol, etc.

[0053] The above-mentioned isotropic agent may be D-Mannitol, Maltitol, Sorbitol, Lactitol, Xylitol, Sodium chloride or a combination thereof, and may be, for example, D-Mannitol, but is not limited thereto.

[0054] The above suspending agent may be sodium carboxymethylcellulose, polysorbate 80, starch, starch derivatives, polyhydric alcohols, chitosan, chitosan derivatives, cellulose, cellulose derivatives, collagen, gelatin, hyaluronic acid (HA), alginic acid, algin, pectin, carrageenan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, polylysine, titin, fibrin, agarose, fluran, xanthan gum, or a combination thereof. Examples thereof include, but are not limited to, sodium carboxymethylcellulose and polysorbate 80.

[0055] The solvent may be water for injection, and examples thereof include purified water for injection, saline solution, and glucose solution. However, any solvent that can be used as water for injection may be used without limitation. In one embodiment of the present invention, the composition for injection preferably has a pH in the range of 6.5 to 8.0, but is not limited thereto, and any pH range in which denaturation of the extracellular matrix does not occur is satisfactory.

[0056] In one embodiment of the present invention, the injectable composition can be used for tissue regeneration and tissue repair.

[0057] In a specific embodiment of the present invention, the injectable composition may be used for cosmetic purposes such as improving skin wrinkles, facial plastic surgery, or increasing lip volume, but is not limited thereto.

[0058] In another specific embodiment of the present invention, the injectable composition can be used to replace or supplement joint synovial fluid, or to improve joint pain.

[0059] In another specific embodiment of the present invention, the injectable composition may be used for filling of a sphincter, urethra, penis, vocal cords, or penile tissue, but is not limited thereto, and may be used for filling, enlargement, or repair of other tissues.

[0060]

[0061] According to another aspect of the present invention, the present invention provides a method for preparing an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix.

[0062] The injectable composition of the present invention and the method for producing the injectable composition according to the above-described aspect are product and method inventions that include common components, and the contents regarding the common components between the respective inventions can be applied equally.

[0063]

[0064] Hereinafter, a method for manufacturing an injectable composition comprising the cross-linked hyaluronic acid hydrogel and extracellular matrix of the present invention will be described in detail.

[0065] The method for preparing the injectable composition of the present invention may include the following steps.

[0066] i) A step of mixing cross-linked hyaluronic acid hydrogel and extracellular matrix.

[0067] The above cross-linked hyaluronic acid hydrogel is manufactured by a mixing process of a first cross-linked hyaluronic acid hydrogel having a low cross-linking rate and a second cross-linked hyaluronic acid hydrogel having a high cross-linking rate.

[0068] In one embodiment of the present invention, the process for manufacturing the first cross-linked hyaluronic acid hydrogel and the second cross-linked hyaluronic acid hydrogel each comprises the following steps:

[0069] (a) a process for cross-linking hyaluronic acid; and

[0070] (b) Process for purifying cross-linked hyaluronic acid hydrogel.

[0071] The crosslinking agent used in the process of crosslinking the hyaluronic acid may be one or more selected from 1,4-butanediol diglycidyl ether, divinyl sulfone, and bis ethyl carbodiimide, and is preferably butanediol diglycidyl ether, but is not limited thereto.

[0072] In manufacturing the first cross-linked hyaluronic acid hydrogel, the cross-linking agent may be added at a concentration of 1 to 6 mol%, 1 to 5 mol%, 1 to 4 mol%, 1 to 3 mol%, 2 to 6 mol%, 2 to 5 mol%, 2 to 4 mol%, 2 to 3 mol%, 3 to 6 mol%, 3 to 5 mol%, 3 to 4 mol%, 1 mol%, 2 mol%, 3 mol%, or 4 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide), but is not limited thereto.

[0073] In manufacturing the second cross-linked hyaluronic acid hydrogel, the cross-linking agent may be added at a concentration of 5 to 10 mol%, 5 to 9 mol%, 5 to 8 mol%, 5 to 7 mol%, 5 to 6 mol%, 6 to 10 mol%, 6 to 9 mol%, 6 to 8 mol%, 6 to 7 mol%, 7 to 10 mol%, 7 to 9 mol%, 7 to 8 mol%, 5 mol%, 6 mol%, 7 mol%, or 8 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide), but is not limited thereto.

[0074] When producing the first cross-linked hyaluronic acid hydrogel and the second cross-linked hyaluronic acid hydrogel, if the amount of the cross-linking agent is too large, the elasticity of the final product increases, which causes an increase in injection force during injection and may cause a foreign body sensation in the body. If the amount of the cross-linking agent is too small, the elasticity of the final product decreases, which causes a decrease in the volume of the injection site after injection and a decrease in the residual period in the body.

[0075] The method for adding the cross-linking agent is as follows: (1) adding the cross-linking agent to an aqueous solution having a pH of 10 or higher and stirring, and then dissolving the hyaluronic acid; (2) adding the cross-linking agent dropwise while dissolving the hyaluronic acid in an aqueous solution having a pH of 10 or higher and stirring; or (3) adding the cross-linking agent after dissolving the hyaluronic acid in an aqueous solution having a pH of 10 or higher and stirring, thereby distributing the cross-linking agent in a homogeneous state in the hyaluronic acid aqueous solution.

[0076] If the cross-linking reaction of hyaluronic acid is insufficient, unreacted residues of the cross-linking agent may remain, which may cause toxicity in the final product. Therefore, the cross-linking of hyaluronic acid is carried out by a stationary reaction at a temperature of 30°C or higher for 8 hours or longer. However, this is not limited to the case, as long as the cross-linking reaction is sufficiently carried out to form a gel. In addition, any one cross-linking reaction selected from a single cross-linking reaction and two or more cross-linking reactions may be included, but is not limited thereto.

[0077]

[0078] The process for purifying the above-mentioned (b) cross-linked hyaluronic acid hydrogel may be one or more selected from a dialysis method and a precipitation method, and may preferably be a dialysis method, but is not limited thereto.

[0079] The external dialysis fluid used during dialysis may be one or more selected from purified water, water for injection, a buffer solution, and an aqueous solution containing inorganic salts for osmotic pressure correction, and may be used interchangeably, but is not limited thereto. In addition, the external solvent used in the precipitation method may be one or more selected from ethanol and isopropyl alcohol, but is not limited thereto.

[0080] When using dialysis, the dialysis period can last from 1 to 5 days, preferably from 1 to 3 days, but is not limited thereto. If the dialysis period is too short, unreacted cross-linking agent may remain, potentially causing toxicity in the final product.

[0081] The process for manufacturing the first cross-linked hyaluronic acid hydrogel of the present invention may additionally include a particle formation process after purifying the cross-linked hyaluronic acid hydrogel in step (b).

[0082] The above granulation process may be one or more selected from a plunger mill or a physical crushing process by passing through a mesh sieve, but is not limited thereto.

[0083] In one embodiment of the present invention, the particle size may be 150-600 μm, 150-500 μm, 150-400 μm, 150-350 μm, 150-300 μm, 150-250 μm, 150-200 μm, 200-600 μm, 200-500 μm, 200-400 μm, 200-350 μm, 200-300 μm, or 200-250 μm, but is not limited thereto.

[0084] The process for manufacturing the second cross-linked hyaluronic acid hydrogel of the present invention may additionally include a homogenization process after purifying the cross-linked hyaluronic acid hydrogel in step (b).

[0085] The above homogenization process may be, but is not limited to, a physical homogenization process of gel particles by a homogenizer, a physical homogenization process of gel particles by sonication, or a homogenization process by milling.

[0086] The particle size of the second cross-linked hyaluronic acid hydrogel of the present invention manufactured by the above homogenization process has a particle size smaller than that of the first cross-linked hyaluronic acid cross-linked hyaluronic acid particles.

[0087] The method for producing a first cross-linked hyaluronic acid hydrogel and the method for producing a second cross-linked hyaluronic acid hydrogel of the present invention provide an advantage in that physical properties can be varied and adjusted depending on the mixing ratio by simultaneously providing a first cross-linked hyaluronic acid hydrogel having high elasticity characteristics provided by large particles and a second cross-linked hyaluronic acid hydrogel having high viscosity characteristics provided by small particles. In particular, by increasing the cross-linking ratio of the second cross-linked hyaluronic acid hydrogel compared to the first cross-linked hyaluronic acid, the in vivo degradation period of the second cross-linked hyaluronic acid, which has a large surface area that can be hydrolyzed due to its small particle size, can be extended, thereby providing an advantage in that a degradation rate similar to that of the first cross-linked hyaluronic acid can be provided.

[0088] The mixing ratio of the first cross-linked hyaluronic acid hydrogel and the second cross-linked hyaluronic acid hydrogel may be 99:1 to 1:99. However, if the particle size of the finally formed hydrogel is not completely uniform and the cross-linking rate of each hyaluronic acid molecule is different, it is not limited thereto because it is the same in terms of the composition principle of the present invention even if it is composed only of the first cross-linked hyaluronic acid hydrogel or only of the second cross-linked hyaluronic acid hydrogel, such as 100:0 or 0:100.

[0089]

[0090] In one embodiment of the present invention, the first cross-linked hyaluronic acid hydrogel and the second cross-linked hyaluronic acid hydrogel may be sterilized after mixing, or may be mixed after each is sterilized.

[0091] The process for sterilizing the above cross-linked hyaluronic acid hydrogel may be one selected from wet sterilization (autoclave), gamma irradiation sterilization, EO gas sterilization, dry heat sterilization, and electron beam irradiation sterilization, and is preferably wet sterilization, but is not limited thereto.

[0092]

[0093] In one embodiment of the present invention, the extracellular matrix may be sterilized or sterilized filtered.

[0094] The sterilization of the above extracellular matrix can be accomplished by using a membrane filtration method using a membrane having a pore size of 0.22㎛ or less, which is generally used in the field, or a cartridge filtration method using a cartridge filter, but is not limited thereto.

[0095] In addition, the sterilization of the extracellular matrix may be one selected from among virus inactivation methods generally known in the art, wet sterilization (autoclave), gamma irradiation sterilization, EO gas sterilization, dry heat sterilization, and electron beam irradiation sterilization, but is not limited thereto.

[0096] However, in a specific embodiment of the present invention, in the process of mixing the sterilized or sterilized filtered extracellular matrix and the hyaluronic acid hydrogel, if the cross-linked hyaluronic acid and extracellular matrix of the present invention are not mixed after sterilization, but rather are subjected to a sterilization process after mixing, denaturation of the heat-stable extracellular matrix component may occur due to the heat applied during the sterilization process.

[0097] In one embodiment of the present invention, the process of mixing the cross-linked hyaluronic acid hydrogel and the extracellular matrix refers to a process of adding a certain amount of the extracellular matrix to the purified cross-linked hyaluronic acid hydrogel and mixing using a stirrer or the like. At this time, the mixing weight ratio of the cross-linked hyaluronic acid hydrogel and the extracellular matrix may be a ratio of 7:3 to 9.9:0.1, preferably 8:2 to 9:1, but is not limited thereto. At this time, when the mixing weight ratio corresponds to 1:9 to 6:4, the weight ratio of the cross-linked hyaluronic acid hydrogel may be too low, which may reduce the tissue repair ability after administration.

[0098] The process for manufacturing the cross-linked hyaluronic acid hydrogel of the present invention may additionally include a process for adjusting the pH to a range of 6.5 to 8.0 after the process of mixing the extracellular matrix and the hyaluronic acid hydrogel. The buffer used for pH adjustment, and the stabilizer, preservative, isotonic agent, suspending agent, and water for injection for controlling the osmotic pressure and improving the stability of the injectable composition are as described above.

[0099] In one embodiment of the present invention, the concentration of laminin in the extracellular matrix mixed with the cross-linked hyaluronic acid hydrogel is 1 μg / ml to 1000 μg / ml, more specifically, 1 μg / ml to 1000 μg / ml, 1 μg / ml to 900 μg / ml, 1 μg / ml to 800 μg / ml, 1 μg / ml to 700 μg / ml, 1 μg / ml to 600 μg / ml, 1 μg / ml to 500 μg / ml, 1 μg / ml to 400 μg / ml, 1 μg / ml to 300 μg / ml, 1 μg / ml to 200 μg / ml, 1 μg / ml to 100 μg / ml, 1 μg / ml to 50 μg / ml, 10 μg / ml to 1000 μg / ml, 10 μg / ml to 900 μg / ml, 10 μg / ml to 800 ㎍ / ㎖, 10 ㎍ / ㎖ to 700 ㎍ / ㎖, 10 ㎍ / ㎖ to 600 ㎍ / ㎖, 10 ㎍ / ㎖ to 500 ㎍ / ㎖, 10 ㎍ / ㎖ to 400 ㎍ / ㎖, 10 ㎍ / ㎖ to 300 ㎍ / ㎖, 10 ㎍ / ㎖ to 200 ㎍ / ㎖, 10 ㎍ / ㎖ to 100 ㎍ / ㎖, 10 ㎍ / ㎖ to 50 ㎍ / ㎖, 30 ㎍ / ㎖ to 1000 ㎍ / ㎖, 30 ㎍ / ㎖ to 900 ㎍ / ㎖, 30 ㎍ / ㎖ to 800 ㎍ / ㎖, 30 ㎍ / ㎖ to 700 ㎍ / ㎖, 30 ㎍ / ㎖ to 600 ㎍ / ㎖, 30 ㎍ / ㎖ to 500 ㎍ / ㎖, 30 ㎍ / ㎖ to 400 ㎍ / ㎖, 30 ㎍ / ㎖ to 300 ㎍ / ㎖, 30 ㎍ / ㎖ to 200 ㎍ / ㎖, 30 ㎍ / ㎖ to 100 ㎍ / ㎖, 30 ㎍ / ㎖ to 50 ㎍ / ㎖, 50 ㎍ / ㎖ to 1000 ㎍ / ㎖, 50 ㎍ / ㎖ to 900 ㎍ / ㎖, 50 ㎍ / ㎖ to 800 ㎍ / ㎖, 50 ㎍ / ㎖ to 700 ㎍ / ㎖, 50 ㎍ / ㎖ to 600 ㎍ / ㎖, 50 ㎍ / ㎖ to 500 ㎍ / ㎖, 50 ㎍ / ㎖ to 400 ㎍ / ㎖, 50 ㎍ / ㎖ to 300 ㎍ / ㎖, 50 ㎍ / ㎖ to 200 ㎍ / ㎖, 50 ㎍ / ㎖ to 100 ㎍ / ㎖, 100 ㎍ / ㎖ to 1000 ㎍ / ㎖, 100 ㎍ / ㎖ to 900 ㎍ / ㎖, 100 ㎍ / ㎖ to 800 ㎍ / ㎖, 100 ㎍ / ㎖ to 700 ㎍ / ㎖, 100 ㎍ / ㎖ to 600 ㎍ / ㎖, 100 ㎍ / ㎖ to 500 ㎍ / ㎖, 100 ㎍ / ㎖ to 400 ㎍ / ㎖, 100 ㎍ / ㎖ to 300 ㎍ / ㎖, 100 ㎍ / ㎖ to 200 ㎍ / ㎖, about 1 ㎍ / ㎖, about 10 ㎍ / ㎖, about 30 ㎍ / ㎖,It may be about 50 ㎍ / ㎖, about 100 ㎍ / ㎖, about 150 ㎍ / ㎖, about 200 ㎍ / ㎖, about 300 ㎍ / ㎖, about 400 ㎍ / ㎖, about 500 ㎍ / ㎖, about 600 ㎍ / ㎖, about 700 ㎍ / ㎖, about 800 ㎍ / ㎖, about 900 ㎍ / ㎖, or about 1000 ㎍ / ㎖, but is not limited thereto.

[0100] The method for producing an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix of the present invention may additionally include a step of ii) adding a local anesthetic after the step of mixing the cross-linked hyaluronic acid hydrogel and the extracellular matrix. The local anesthetic of the present invention is as described above.

[0101]

[0102] According to another aspect of the present invention, the present invention provides a prefilled syringe comprising an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix.

[0103] The above prefilled syringe is a disposable syringe prefilled with a single-dose injectable composition. The syringe may be a plastic syringe or a glass syringe, and a sterile needle may be attached to the inlet of the prefilled syringe to be used for injection.

[0104] The above prefilled syringe may be provided in the form of a kit including a syringe containing the above injectable composition, a container, and instructions for use to be provided to the user.

[0105] The method for manufacturing an injectable composition of the present invention and the prefilled syringe are inventions relating to a method for manufacturing an injectable composition according to the above-described aspect of the present invention and inventions comprising the injectable composition as a component, and therefore, the contents regarding common components between each invention can be equally applied.

[0106]

[0107] The injectable composition comprising the cross-linked hyaluronic acid hydrogel and the extracellular matrix of the present invention has a long residual time in the body, allowing for an extended administration interval, and is capable of complete biodegradation after a certain period of time, while simultaneously providing the physical function of tissue repair or lubricant and the effect of tissue regeneration, and thus can be used for the purpose of improving wrinkles or joint pain. In particular, the injectable composition comprising the cross-linked hyaluronic acid hydrogel and the extracellular matrix of the present invention can promote blood vessel formation and collagen biosynthesis in tissues, thereby contributing to tissue regeneration.

[0108] In addition, the method for producing an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix of the present invention can be used to provide a method for producing a medical product that is sterile while providing a homogeneous mixture.

[0109]

[0110] Figure 1 is a diagram showing the results of a hydration contact angle comparison test of Examples 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration extracellular matrix), Example 2 (cross-linked hyaluronic acid hydrogel + high-concentration extracellular matrix), and Comparative Example 5 (cross-linked hyaluronic acid hydrogel).

[0111] Figure 2 is a diagram showing the results of a comparative test of human vascular endothelial cell adhesion of Examples 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration extracellular matrix), Comparative Example 2 (cross-linked hyaluronic acid hydrogel + laminin), and Comparative Example 5 (cross-linked hyaluronic acid hydrogel).

[0112] Figure 3 is a diagram showing the injection force results of Example 2 (cross-linked hyaluronic acid hydrogel + liquid extracellular matrix), Comparative Example 6 (cross-linked hyaluronic acid hydrogel + liquid extracellular matrix extracted from other tissues (simple collagen)), Comparative Example 3 (cross-linked hyaluronic acid hydrogel + powdery extracellular matrix), and Comparative Example 7 (cross-linked hyaluronic acid hydrogel + powdery extracellular matrix extracted from other tissues).

[0113] Figures 4a and 4b are diagrams showing the changes in properties of Example 2 (cross-linked hyaluronic acid hydrogel + liquid extracellular matrix), Comparative Example 3 (cross-linked hyaluronic acid hydrogel + powdery extracellular matrix), and Comparative Example 7 (cross-linked hyaluronic acid hydrogel + powdery collagen).

[0114] Figure 5a is a diagram showing the results of a comparative test of collagen production ability at the injection site after intradermal / subcutaneous administration of Examples 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration extracellular matrix) compared to Comparative Example 5 (cross-linked hyaluronic acid hydrogel) to BALB / c nude mice.

[0115] Figure 5b is a diagram showing the results of a tissue examination analysis of a comparative blood vessel formation ability test at the injection site after intradermal administration of Examples 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration extracellular matrix) compared to Comparative Example 5 (cross-linked hyaluronic acid hydrogel) to BALB / c nude mice.

[0116] Figure 5c is a diagram showing the results of a quantitative analysis of a test comparing the blood vessel formation ability at the injection site after intradermal / subcutaneous administration of Examples 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration extracellular matrix) compared to Comparative Example 5 (cross-linked hyaluronic acid hydrogel) to BALB / c nude mice.

[0117] Figure 5d is a diagram showing the results of a comparative test on the tissue repair ability at the injection site after intradermal administration of Comparative Example 6 (liquid-phase human cell culture-derived basement membrane extracellular matrix), Comparative Example 1 (non-crosslinked hyaluronic acid hydrogel + extracellular matrix), Comparative Example 5 (crosslinked hyaluronic acid hydrogel), Example 2 (crosslinked hyaluronic acid hydrogel + high-concentration extracellular matrix), and Examples 1-4 (crosslinked hyaluronic acid hydrogel + low-concentration extracellular matrix) to BALB / c nude mice.

[0118] Figure 6 is a diagram showing the change in properties when the sterilization process is performed before mixing cross-linked hyaluronic acid and liquid extracellular matrix and then mixing (Example 2), and when sterilization is performed after mixing cross-linked hyaluronic acid and liquid extracellular matrix (Comparative Example 4).

[0119] Figure 7 is a diagram showing the results of a comparative test of fibroblast adhesion between Example 2 (cross-linked hyaluronic acid hydrogel + high-concentration extracellular matrix) and Comparative Example 6 (cross-linked hyaluronic acid hydrogel + collagen).

[0120] Figure 8 is a diagram showing the Tanδ values ​​of Example 1-1 (mixing ratio 100:0), Example 1-2 (mixing ratio 80:20), Example 1-3 (mixing ratio 60:40), Example 1-4 (mixing ratio 50:50), Example 1-5 (mixing ratio 40:60), Example 1-6 (mixing ratio 20:80), and Example 1-7 (mixing ratio 0:100), which are mixtures of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrices.

[0121] Figure 9 is a diagram showing the injection power of Example 1-1 (mixing ratio 100:0), Example 1-2 (mixing ratio 80:20), Example 1-3 (mixing ratio 60:40), Example 1-4 (mixing ratio 50:50), Example 1-5 (mixing ratio 40:60), Example 1-6 (mixing ratio 20:80), and Example 1-7 (mixing ratio 0:100), which are mixtures of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrices.

[0122] Figure 10 is a diagram showing a manufacturing process for a mixture of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrix.

[0123]

[0124] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate 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, in accordance with the gist of the present invention.

[0125]

[0126] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0127]

[0128] Example

[0129]

[0130] Example 1: Preparation of an injectable composition by mixing a first cross-linked hyaluronic acid hydrogel with 4 mol% cross-linking agent and a second hyaluronic acid hydrogel with 6 mol% cross-linking agent in a ratio of 100:0 to 0:100 with a low-concentration extracellular matrix (laminin concentration of 10 ug / ㎖ in the final composition).

[0131] 10g of sodium hyaluronate was completely dissolved in a 0.25N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 4 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours.

[0132] The purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid at 22.22 mg / ㎖. Afterwards, the cross-linked hyaluronic acid hydrogel was granulated using a 150 μm mesh, and the granulated cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature humidified atmosphere at 121°C for 16 minutes.

[0133] 10 g of sodium hyaluronate was completely dissolved in a 0.25 N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 6 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, the purified hyaluronate hydrogel was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, while fixing the concentration of crosslinked hyaluronic acid to 22.22 mg / ㎖. After homogenization using a homogenizer, the homogenized crosslinked hyaluronic acid hydrogel was sterilized under high temperature and humidity at 121°C for 16 minutes.

[0134] And hyaluronic acid hydrogel with 4 mol% cross-linking agent added and hyaluronic acid hydrogel with 6 mol% cross-linking agent added were mixed in the ratios of 100:0 (Example 1-1), 80:20 (Example 1-2), 60:40 (Example 1-3), 50:50 (Example 1-4), 40:60 (Example 1-5), 20:80 (Example 1-6), and 0:100 (Example 1-7), respectively. And then, liquid-state sterile-filtered human cell culture-derived extracellular matrix (laminin concentration standard 100 ug / ㎖) was added to the mixed hyaluronic acid hydrogel in an amount corresponding to 1 / 9 of the weight of the cross-linked hyaluronic acid hydrogel and mixed.

[0135]

[0136] Example 2: Preparation of cross-linked hyaluronic acid hydrogel containing high concentration of human cell-derived extracellular matrix (laminin concentration of 20 ug / ㎖ in the final composition)

[0137] It was prepared in the same manner as in Example 1-4, except that the concentration of the extracellular matrix derived from human cell culture mixed with the hyaluronic acid hydrogel was 200 ug / ㎖ based on the laminin concentration.

[0138]

[0139] Comparative example

[0140] Comparative Example 1: Preparation of an injectable composition comprising a non-crosslinked hyaluronic acid solution and an extracellular matrix.

[0141] 2g of sodium hyaluronate was dissolved in 90mL of pH 7.4 phosphate buffer, and then titrated to 100g using pH 7.4 phosphate buffer. 11.11g of liquid, sterile, filtered extracellular matrix (laminin concentration 200ug / mL), corresponding to 1 / 9 of the above 100g, was added and mixed.

[0142]

[0143] Comparative Example 2: Preparation of an injectable composition composed only of cross-linked hyaluronic acid and laminin.

[0144] 10g of sodium hyaluronate was completely dissolved in a 0.25N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 4 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours.

[0145] The purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖. Afterwards, it was granulated using a 150 ㎛ mesh, and the granulated cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature, wet condition at 121°C for 16 minutes. 10 g of sodium hyaluronate was completely dissolved in a 0.25 N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 6 mol% of the monomer (disaccharide) of sodium hyaluronate, and then cross-linked by standing reaction at 35°C for 24 hours. And it was purified by dialysis using 0.9% NaCl aqueous solution for 144 hours, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2 while fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖ to manufacture it. After homogenization using a homogenizer, the homogenized cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature and wet condition at 121℃ for 16 minutes. And the hyaluronic acid hydrogel with 4 mol% cross-linking agent added and the hyaluronic acid hydrogel with 6 mol% cross-linking agent added were mixed at a ratio of 50:50. And the liquid-state sterile-filtered laminin (laminin concentration 100 ug / ㎖) was added to the mixed hyaluronic acid hydrogel in an amount corresponding to 1 / 9 of the weight of the cross-linked hyaluronic acid hydrogel and mixed.

[0146]

[0147]

[0148] Comparative Example 3: Preparation of an injectable composition comprising cross-linked hyaluronic acid and powdered extracellular matrix.

[0149] 10g of sodium hyaluronate was completely dissolved in a 0.25N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 4 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours.

[0150] The purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖. Afterwards, it was granulated using a 150 ㎛ mesh, and the granulated cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature, wet condition at 121°C for 16 minutes. 10 g of sodium hyaluronate was completely dissolved in a 0.25 N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 6 mol% of the monomer (disaccharide) of sodium hyaluronate, and then cross-linked by standing reaction at 35°C for 24 hours. And it was purified by dialysis using 0.9% NaCl aqueous solution for 144 hours, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2 while fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖ to manufacture. After homogenization using a homogenizer, the homogenized cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature wet condition at 121℃ for 16 minutes. And the hyaluronic acid hydrogel with 4 mol% cross-linking agent added and the hyaluronic acid hydrogel with 6 mol% cross-linking agent added were mixed at a ratio of 50:50. And the mixed hyaluronic acid hydrogel was added to the cross-linked hyaluronic acid hydrogel in a freeze-dried powder state and the extracellular matrix was mixed so that the laminin concentration in the final composition was 20 ug / ㎖.

[0151]

[0152] Comparative Example 4: Preparation of an injectable composition manufactured by mixing cross-linked hyaluronic acid and a liquid extracellular matrix and then sterilizing them.

[0153] 10g of sodium hyaluronate was completely dissolved in a 0.25N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 4 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours.

[0154] The purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖. Afterwards, it was granulated using a 150 ㎛ mesh. 10 g of sodium hyaluronate was completely dissolved in a 0.25 N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 6 mol% of the monomer (disaccharide) of sodium hyaluronate, and cross-linked by standing reaction at 35℃ for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours, and the purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖. Afterwards, it was homogenized using a homogenizer. And hyaluronic acid hydrogel with 4 mol% cross-linking agent added and hyaluronic acid hydrogel with 6 mol% cross-linking agent added were mixed in a 50:50 ratio, respectively. Then, liquid-state sterile-filtered human cell culture-derived extracellular matrix (laminin concentration standard 200 ug / ㎖) was added to the mixed hyaluronic acid hydrogel in an amount corresponding to 1 / 9 of the weight of the cross-linked hyaluronic acid hydrogel and mixed. Afterwards, the mixture of cross-linked hyaluronic acid hydrogel and liquid-state human cell culture-derived extracellular matrix was sterilized under high temperature and humidity at 121℃ for 16 minutes.

[0155]

[0156] Comparative Example 5: Preparation of an injectable composition composed solely of cross-linked hyaluronic acid hydrogel

[0157] 10g of sodium hyaluronate was completely dissolved in a 0.25N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 4 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours.

[0158] The purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖. Afterwards, it was granulated using a 150 ㎛ mesh, and the granulated cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature, wet condition at 121°C for 16 minutes. 10 g of sodium hyaluronate was completely dissolved in a 0.25 N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 6 mol% of the monomer (disaccharide) of sodium hyaluronate, and then cross-linked by standing reaction at 35°C for 24 hours. And it was purified by dialysis using 0.9% NaCl aqueous solution for 144 hours, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed at 22.22 mg / ㎖ to manufacture it. After homogenization using a homogenizer, the homogenized cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature wet condition at 121℃ for 16 minutes. And the hyaluronic acid hydrogel with 4 mol% cross-linking agent added and the hyaluronic acid hydrogel with 6 mol% cross-linking agent added were mixed at a ratio of 50:50. And the phosphate buffer of pH 7.4 was added in an amount corresponding to 1 / 9 of the weight of the cross-linked hyaluronic acid hydrogel and mixed.

[0159]

[0160] Comparative Example 6: Preparation of an injectable composition containing cross-linked hyaluronic acid and extracellular matrix derived from liquid tissue (skin tissue) extract

[0161] 10g of sodium hyaluronate was completely dissolved in a 0.25N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 4 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours.

[0162] The purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖. Afterwards, it was granulated using a 150 ㎛ mesh, and the granulated cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature, wet condition at 121°C for 16 minutes. 10 g of sodium hyaluronate was completely dissolved in a 0.25 N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 6 mol% of the monomer (disaccharide) of sodium hyaluronate, and then cross-linked by standing reaction at 35°C for 24 hours. And it was purified by dialysis using 0.9% NaCl aqueous solution for 144 hours, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed at 22.22 mg / ㎖ to manufacture it. After homogenization using a homogenizer, the homogenized cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature and wet condition at 121℃ for 16 minutes. And the hyaluronic acid hydrogel with 4 mol% cross-linking agent added and the hyaluronic acid hydrogel with 6 mol% cross-linking agent added were mixed at a ratio of 50:50. And the extracellular matrix (simple collagen) derived from liquid tissue (skin tissue) was added in an amount corresponding to 1 / 9 of the weight of the cross-linked hyaluronic acid hydrogel and mixed.

[0163]

[0164] Comparative Example 7: Preparation of an injectable composition containing cross-linked hyaluronic acid and extracellular matrix derived from powdered tissue (skin tissue) extract

[0165] 10g of sodium hyaluronate was completely dissolved in a 0.25N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 4 mol% of the monomer (disaccharide) of sodium hyaluronate, and crosslinked by standing reaction at 35°C for 24 hours. Then, it was purified by dialysis using a 0.9% NaCl aqueous solution for 144 hours.

[0166] The purified hyaluronic acid hydrogel was prepared by adjusting the pH to 7.2 and fixing the concentration of cross-linked hyaluronic acid to 22.22 mg / ㎖. Afterwards, it was granulated using a 150 ㎛ mesh, and the granulated cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature, wet condition at 121°C for 16 minutes. 10 g of sodium hyaluronate was completely dissolved in a 0.25 N sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (95%) corresponding to 6 mol% of the monomer (disaccharide) of sodium hyaluronate, and then cross-linked by standing reaction at 35°C for 24 hours. And it was purified by dialysis using 0.9% NaCl aqueous solution for 144 hours, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed at 22.22 mg / ㎖ to manufacture it. After homogenization using a homogenizer, the homogenized cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature and wet condition at 121℃ for 16 minutes. And the hyaluronic acid hydrogel with 4 mol% cross-linking agent added and the hyaluronic acid hydrogel with 6 mol% cross-linking agent added were mixed at a ratio of 50:50. And the extracellular matrix (simple collagen) derived from powdered tissue (skin tissue) was added in an amount corresponding to 1 / 9 of the weight of the cross-linked hyaluronic acid hydrogel and mixed.

[0167]

[0168]

[0169] Experimental example

[0170]

[0171] Experimental Example 1: Comparison test of hydration contact angle according to presence or absence of extracellular matrix (Examples 1-4, Example 2 vs. Comparative Example 5)

[0172] The present inventors conducted the following experiments to confirm cell adhesion and tissue regeneration ability according to the presence or absence of extracellular matrix.

[0173] First, 1 mL of each of the materials of Example 2 (cross-linked hyaluronic acid hydrogel + high-concentration human cell-derived extracellular matrix), Examples 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration human cell-derived extracellular matrix), and Comparative Example 5 (cross-linked hyaluronic acid hydrogel) was dropped and evenly applied onto a plate, and then dried inside a BSC for 4 to 5 days. Next, a water droplet was dropped onto the applied materials, and the contact angle was measured.

[0174] The results are shown in Figure 1.

[0175] In general, it is known that the ideal hydration contact angle of the surface of a cell culture dish for cell culture is within the range of about 40 to 70 degrees, and commercially available cell culture dishes have a hydration contact angle of about 56 degrees. As shown in Fig. 1, the materials of Examples 1-4 and 2 of the present invention containing human cell-derived extracellular matrices were measured to have a contact angle of 57.7 degrees, which is close to the hydration contact angle of an actual cell culture dish.

[0176] On the other hand, the contact angle of Comparative Example 5 was confirmed to be 26.98 degrees, which is significantly outside the ideal hydration contact angle range (40 to 70 degrees).

[0177] From the above results, it was confirmed that Examples 1-4 and 2 including extracellular matrix components exhibited surface properties more suitable for cell attachment than Comparative Example 5.

[0178]

[0179] Experimental Example 2: Comparative test of cell adhesion depending on the presence or absence of extracellular matrix (Examples 1-4 vs. Comparative Examples 2 and 5)

[0180] 1 mL of Example 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration human cell-derived extracellular matrix), Comparative Example 2 (cross-linked hyaluronic acid hydrogel + laminin), and Comparative Example 5 (cross-linked hyaluronic acid hydrogel) were evenly applied to each 6-well plate, and then dried for 4 to 5 days inside a biological safety cabinet (BSC). Next, 2.5 x 10 human vascular endothelial cells were seeded on each plate. 5 After inoculating at a concentration of 10 cells / well, the cells were observed and photographed after 0, 1, and 3 hours to confirm cell adhesion.

[0181] The results are shown in Fig. 2.

[0182] As shown in Fig. 2, in the case of Examples 1-4, the cell adhesion was excellent because the extracellular matrix derived from human cells was included, and in Comparative Example 2, which had the same laminin concentration, it was confirmed that the cell adhesion was reduced compared to Example 2. In addition, in Comparative Example 5, which only included cross-linked hyaluronic acid hydrogel, it was observed that the cells did not attach at all and formed a spherical shape of clumps. This confirmed that Examples 1-4, which included all extracellular matrix components, exhibited better cell adhesion than Comparative Example 2, which simply added laminin.

[0183]

[0184] Experimental Example 3: Comparison test of injection force according to extracellular matrix form (liquid / powder) (Example 2, Comparative Example 6 vs. Comparative Examples 3, 7)

[0185] The present inventors conducted the following experiment to determine the change in injection force depending on whether the extracellular matrix incorporated into the cross-linked hyaluronic acid hydrogel is in liquid or powder form.

[0186] Using an injection force measuring device, Example 2 (cross-linked hyaluronic acid hydrogel + liquid extracellular matrix), Comparative Example 6 (cross-linked hyaluronic acid hydrogel + liquid extracellular matrix extracted from other tissues (simple collagen)), Comparative Example 3 (cross-linked hyaluronic acid hydrogel + powdery extracellular matrix), and Comparative Example 7 (cross-linked hyaluronic acid hydrogel + powdery extracellular matrix extracted from other tissues) filled in a pre-filled syringe were each connected to a 27G injection needle, and the injection force (N) when the syringe plunger was pushed out at a speed of 20 mm / min was measured.

[0187] The results are shown in Fig. 3.

[0188] As shown in Fig. 3, in the case of Example 2 and Comparative Example 6, in which the extracellular matrix was mixed in liquid form, it was confirmed that the injection force was constant but low, indicating high convenience of use. On the other hand, in the case of Comparative Example 3 and Comparative Example 7, in which the extracellular matrix was mixed in powder form, it was confirmed that the injection force was inconsistent or high, making it difficult to inject a constant amount, indicating low convenience of use.

[0189]

[0190] Experimental Example 4: Comparative test of changes in properties according to the form (liquid / powder) of extracellular matrix derived from cell culture (Example 2 vs. Comparative Example 3, Comparative Example 7)

[0191] Similar to Experimental Example 3, the present inventors conducted the following experiment to confirm the change in product properties depending on whether the extracellular matrix incorporated into the cross-linked hyaluronic acid hydrogel is in liquid or powder form.

[0192] First, Example 2 (cross-linked hyaluronic acid hydrogel + liquid extracellular matrix), Comparative Example 3 (cross-linked hyaluronic acid hydrogel + powdery extracellular matrix), and Comparative Example 7 (cross-linked hyaluronic acid hydrogel + powdery collagen) filled in pre-filled syringes were observed for the presence of insoluble foreign substances in accordance with the first method of the insoluble foreign substance test method specified in the General Test Methods of the Korean Pharmacopoeia at a location with a brightness of approximately 1000 lux directly under a white light source.

[0193] The results are shown in Figures 4a and 4b.

[0194] As shown in Fig. 4a, Example 2 using a liquid extracellular matrix was homogeneously dissolved and transparent, and no insoluble substances were observed. On the other hand, in Comparative Example 3 using a powdered extracellular matrix and Comparative Example 7 using a powdered collagen, it was confirmed that the powdered extracellular matrix / collagen was not completely dissolved, and many powder lumps (insoluble substances) were observed. Therefore, from the above results, it was confirmed that it is suitable to include a liquid extracellular matrix as an injection agent for tissue repair.

[0195]

[0196] Experimental Example 5: Synergistic effect by mixing cross-linked hyaluronic acid hydrogel and liquid human cell culture-derived basement membrane extracellular matrix (Examples 1-4 vs. Comparative Example 5) - in vivo

[0197] To confirm the synergistic effect by mixing, Examples 1-4 (cross-linked hyaluronic acid hydrogel + low-concentration extracellular matrix) and Comparative Example 5 (cross-linked hyaluronic acid hydrogel) were prepared, and administered intradermally / subcutaneously to BALB / c nude mice.

[0198] Collagen production and angiogenesis were evaluated through tissue examination after autopsy 28 and 84 days after administration.

[0199] The results are shown in Figures 5a and 5b.

[0200] As shown in the left diagram of Fig. 5a, the results of tissue examination 28 days after intradermal administration of the test substance showed that Examples 1-4 were excellent in collagen production efficacy as a group containing human cell-derived extracellular matrix, but Comparative Example 5 had almost no collagen production efficacy.

[0201] In addition, as shown in the right side of Fig. 5a, 84 days after subcutaneous administration of the test substance, tissue examination results confirmed that the collagen production efficacy in Examples 1-4 was slightly increased compared to Comparative Example 5.

[0202] In addition, as shown in Fig. 5b, 28 days after administration of the test substance, almost no new blood vessels were observed in the group administered Comparative Example 5, but the formation of numerous new blood vessels (black arrows) was observed in the group administered Example 1-4 including human cell-derived extracellular matrix.

[0203] Figure 5c shows the results of quantitative analysis of the tissue examination results after administration of the test substance. In both subcutaneous and intradermal administrations, fewer new blood vessels were observed in the group administered Comparative Example 5, but in Examples 1-4 including human cell-derived extracellular matrix, about 1.5 times more new blood vessels were observed compared to Comparative Example 5.

[0204] From the above Figures 5a to 5c, it was confirmed that the injectable composition of the present invention exhibits excellent tissue regeneration ability by participating in collagen production and angiogenesis.

[0205] Figure 5d shows the results of observing tissue repair ability after administration of the test substance. As shown in Figure 5d, Comparative Example 1 (non-crosslinked hyaluronic acid + extracellular matrix) and Comparative Example 6 (liquid human cell-derived extracellular matrix) were confirmed to have no tissue repair ability at all. In addition, Comparative Example 5 (crosslinked hyaluronic acid hydrogel) was also confirmed to decompose quickly and have low tissue repair ability. However, in the groups administered Examples 1-4 and Example 2 containing human cell-derived extracellular matrix, it was confirmed that they exhibited superior tissue repair ability compared to Comparative Example 5 due to the synergistic effect of the human cell-derived extracellular matrix and the crosslinked hyaluronic acid hydrogel.

[0206] That is, as a result of measuring the volume of the material at the injection site 12 weeks after transplantation, Examples 1-4 and Comparative Example 5 containing cross-linked hyaluronic acid showed high tissue repair ability because the transplanted material remained at the injection site, while Comparative Example 1 composed of a hyaluronic acid solution and a liquid-state human cell culture-derived extracellular matrix and Comparative Example 6 containing only a liquid-state human cell culture-derived basement membrane extracellular matrix were confirmed to have decomposed and low tissue repair ability. In addition, among them, Examples 1-4 and Example 2 containing both a cross-linked hyaluronic acid hydrogel and a human cell culture-derived basement membrane extracellular matrix were confirmed to have the best tissue repair ability and high utility as an injectable medical product.

[0207]

[0208] Experimental Example 6: Comparative test of changes in properties depending on whether mixing process was performed after sterilization or before sterilization (Example 2 vs. Comparative Example 4)

[0209] The inventors of the present invention conducted the following test to determine whether there was a difference in properties when changing the order of the sterilization process before / after mixing the liquid extracellular matrix when manufacturing a filler using the cross-linked hyaluronic acid of the present invention.

[0210] In the case where the sterilization process was performed before mixing the cross-linked hyaluronic acid and the liquid extracellular matrix and then mixing (Example 2), and in the case where the cross-linked hyaluronic acid and the liquid extracellular matrix were mixed and then sterilized (Comparative Example 4), a test was performed according to the insoluble foreign matter test method specified in the General Test Methods of the Korean Pharmacopoeia.

[0211] The results are shown in Fig. 6.

[0212] As shown in Fig. 6, in Example 2, where the extracellular matrix was mixed after sterilization, the appearance was transparent, but in Comparative Example 4, where the extracellular matrix was mixed and then sterilized, the appearance was opaque and numerous insoluble substances were observed due to denaturation of the heat-unstable extracellular matrix during the sterilization process. From the above results, it was confirmed that the manufacturing process of Comparative Example 4 is unsuitable as a manufacturing process for injections.

[0213]

[0214] Experimental Example 7: Comparative experiment according to tissue origin of extracellular matrix (Example 2 vs. Comparative Example 6) in vitro

[0215] In order to confirm the cell adhesion (tissue regeneration ability) according to the tissue origin of the extracellular matrix, the inventors of the present invention treated Example 2 (cross-linked hyaluronic acid hydrogel + high-concentration extracellular matrix) and Comparative Example 6 (cross-linked hyaluronic acid hydrogel + extracellular matrix derived from other tissues (simple collagen)) in a well plate using the same method as Experimental Example 1, dried them, and then inoculated them with human fibroblasts, and observed the cell adhesion.

[0216] The results are shown in Fig. 7.

[0217] As shown in Fig. 7, Example 2 showed excellent adhesion between cells and human cell-derived extracellular matrix, and it was confirmed that normal cell morphology was maintained even 24 hours after cell inoculation, but Comparative Example 6 showed low adhesion between cells and collagen, and it was confirmed that normal cell morphology was not maintained 24 hours after cell inoculation, and the cells clumped into a spherical shape.

[0218] From the above results, it was confirmed that the liquid-phase human cell culture-derived basement membrane extracellular matrix has superior cell adhesion ability than the extracellular matrix extracted from other tissues (simple collagen).

[0219]

[0220] Experimental Example 8: Comparison of Tanδ values ​​of mixtures of hyaluronic acid hydrogels with different crosslinking rates containing extracellular matrix (Examples 1-1 to 1-7)

[0221] The present inventors conducted the following experiment to confirm the change in Tanδ value of a mixture of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrix.

[0222] Using a rheometer, the storage modulus (G') and loss modulus (G") of the prefilled syringes of Example 1-1 (mixing ratio 100:0), Example 1-2 (mixing ratio 80:20), Example 1-3 (mixing ratio 60:40), Example 1-4 (mixing ratio 50:50), Example 1-5 (mixing ratio 40:60), Example 1-6 (mixing ratio 20:80), and Example 1-7 (mixing ratio 0:100) were measured at a frequency of 0.02 Hz.

[0223] The results are shown in Fig. 8 and Table 1.

[0224] Elastic gel: Viscous gel Tanδ(G" / G') 100:00.14065880:200.14762560:400.15840650:500.16307640:600.16791920:800.1742090:1000.178182

[0225] As shown in Fig. 8 and Table 1, it was confirmed that the mixture of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrix maintained a constant slope of Tanδ value, thereby allowing for the adjustment of viscosity and elasticity to suit tissue properties at various sites of administration, and also had the advantage of being advantageous for future quality control.

[0226]

[0227] Experimental Example 9: Comparison of injection force of mixtures of hyaluronic acid hydrogels with different crosslinking rates containing extracellular matrix (Examples 1-1 to 1-7)

[0228] The present inventors conducted the following experiment to confirm the change in injection force of a mixture of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrix, according to the ratio.

[0229] Using an injection force measuring device, the prefilled syringes were filled with Examples 1-1 (mixing ratio 100:0), 1-2 (mixing ratio 80:20), 1-3 (mixing ratio 60:40), 1-4 (mixing ratio 50:50), 1-5 (mixing ratio 40:60), 1-6 (mixing ratio 20:80), and 1-7 (mixing ratio 0:100), and the injection force (N) was measured when the syringe plunger was pushed out at a speed of 15 mm / min and 20 mm / min, respectively, by connecting them to 25G and 27G injection needles.

[0230] The results are shown in Fig. 9.

[0231] As shown in Fig. 9, it was confirmed that the mixtures of hyaluronic acid hydrogels with different crosslinking ratios containing Hutrigel exhibited similar levels of injection force at all mixing ratios, thereby improving user convenience.

Claims

1. An injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix.

2. In the first paragraph, the cross-linked hyaluronic acid hydrogel is a mixture of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates, and each cross-linked hyaluronic acid hydrogel included therein before mixing is any one selected from among a cross-linked hyaluronic acid hydrogel obtained by performing a single cross-linking reaction in the manufacturing process, a cross-linked hyaluronic acid hydrogel obtained by performing two or more cross-linking reactions, and a hyaluronic acid hydrogel in the form of a mixture of a cross-linked hyaluronic acid hydrogel and a non-cross-linked hyaluronic acid.

3. In the second paragraph, an injectable composition, wherein among a mixture of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates, the first cross-linked hyaluronic acid hydrogel having a low cross-linking rate has a cross-linking agent added at a concentration of 1 to 6 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide), and the second cross-linked hyaluronic acid having a high cross-linking rate has a cross-linking agent added at a concentration of 5 to 10 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide).

4. In the third paragraph, the first cross-linked hyaluronic acid hydrogel is granulated with an average particle size of 150-600 μm, and the average particle size of the second cross-linked hyaluronic acid hydrogel is smaller than the average particle size of the first cross-linked hyaluronic acid hydrogel.

5. An injectable composition according to claim 1, characterized in that the extracellular matrix is ​​a liquid basement membrane extracellular matrix obtained by culturing connective tissue cells, epithelial tissue cells, or a combination thereof among human-derived cells.

6. An injectable composition according to claim 1, further comprising 0.1 to 1 wt% of a local anesthetic based on the total weight of the composition.

7. An injectable composition containing cross-linked hyaluronic acid at a concentration of 5 to 30 mg / ml in the first paragraph.

8. An injectable composition comprising, in paragraph 1, a concentration of laminin in the extracellular matrix of 0.001 to 90 μg / ml based on the volume of the entire composition.

9. In the first paragraph, the injectable composition is for improving skin wrinkles, facial plastic surgery, or increasing lip volume; for replacing or supplementing joint synovial fluid, or for improving joint pain; or for filling sphincter muscles, urethra, penis, vocal cords, or penile tissue.

10. A method for producing an injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix, comprising the following steps: i) A step of mixing cross-linked hyaluronic acid hydrogel and extracellular matrix.

11. A method for producing an injectable composition according to claim 10, wherein the cross-linked hyaluronic acid hydrogel is produced by a mixing process of a first cross-linked hyaluronic acid hydrogel having a low cross-linking rate and a second cross-linked hyaluronic acid hydrogel having a high cross-linking rate.

12. A method for producing an injectable composition according to claim 11, wherein the first cross-linked hyaluronic acid is produced by a method comprising the following process: (a1) a process for crosslinking hyaluronic acid by adding a crosslinking agent at a concentration of 1 to 6 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide); and (a2) Process for purifying cross-linked hyaluronic acid hydrogel.

13. A method for producing an injectable composition according to claim 12, wherein the first cross-linked hyaluronic acid hydrogel is produced by a method that additionally includes a process of granulating (a3) ​​a purified cross-linked hyaluronic acid hydrogel.

14. A method for producing an injectable composition according to claim 11, wherein the second cross-linked hyaluronic acid is produced by a method comprising the following processes: (b1) a process for crosslinking hyaluronic acid by adding a crosslinking agent at a concentration of 5 to 10 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide); and (b2) Process for purifying cross-linked hyaluronic acid hydrogel.

15. A method for producing an injectable composition according to claim 14, wherein the second cross-linked hyaluronic acid hydrogel further comprises a process of homogenizing (b3) the purified cross-linked hyaluronic acid hydrogel.

16. A method for producing an injectable composition according to claim 11, wherein the first cross-linked hyaluronic acid hydrogel and the second cross-linked hyaluronic acid hydrogel are sterilized after mixing, or are each sterilized and then mixed.

17. A method for producing an injectable composition according to claim 10, wherein the extracellular matrix is ​​sterilized or sterile filtered.

18. A method for producing an injectable composition in claim 10, wherein the mixing weight ratio of the cross-linked hyaluronic acid hydrogel and the extracellular matrix is ​​7:3 to 9.9:0.

1.

19. A method for producing an injectable composition in claim 10, wherein the concentration of laminin in the extracellular matrix mixed with the cross-linked hyaluronic acid hydrogel is 1 μg / ml to 1000 μg / ml.

20. A method for producing an injectable composition according to claim 10, wherein the injectable composition contains a concentration of laminin in the extracellular matrix of 0.001 to 90 μg / ml based on the volume of the entire composition.

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