WO2025063708A1 - Injectable composition comprising crosslinked hyaluronic acid hydrogel and extracellular matrix, preparation method therefor, and use thereof - Google Patents

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

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.

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.

Cross-linked hyaluronic acid hydrogels have been mainly used as fillers for cosmetic purposes and for the purpose of relieving joint pain. In particular, in the field of fillers for cosmetic purposes, cross-linked hyaluronic acid hydrogels account for more than 80% of the entire market, and are injected into areas of tissue defects such as wrinkles to draw moisture into the tissue, thereby providing volume.

The reason why cross-linked hyaluronic acid hydrogels have a high market share in the plastic surgery 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, and they have the characteristic of being broken down in the body and eliminated as a non-toxic substance after a certain period of time, and they are easy to remove after injection, so if a doctor mistakenly administers it into a blood vessel or the patient wishes to return it to its original state, the injected filler can be easily removed by hyaluronidase. Accordingly, although numerous cross-linked hyaluronic acid hydrogels have been commercialized as plastic surgery fillers to date, cross-linked hyaluronic acid hydrogels only physically improve wrinkles, and they biodegrade and disappear after a certain period of time, and they do not provide the function of biologically regenerating tissues while they remain in the tissues.

Recently, polynucleotide (PN) or polydeoxyribonucleotide (PDRN) extracted from salmon testis have 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 function in providing volume, and relatively frequent administration intervals (every 1 to 2 weeks), so the use of other tissue regeneration raw materials is necessary.

Meanwhile, cross-linked hyaluronic acid hydrogels have also been used for the purpose of relieving joint pain. Based on the existing non-cross-linked hyaluronic acid solution being used as a joint lubricant, there is a case where cross-linked hyaluronic acid hydrogels were commercialized as joint lubricants with a long-term retention period in the body. This only showed pain relief by physically acting as a lubricant, and the effect of tissue regeneration was not confirmed.

As a result of extensive research efforts to solve the problems of the conventional cross-linked hyaluronic acid hydrogel material as described above, the inventors of the present invention have confirmed that an injectable composition containing a cross-linked hyaluronic acid hydrogel and an extracellular matrix simultaneously provides physical supplementation of a defective area in a tissue, surrounding tissue regeneration ability, and increased tissue repair period, thereby completing the present invention.

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

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

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention cannot be considered limited by the specific description described below.

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

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.

The term “cross-linked hyaluronic acid hydrogel” of the present invention means a form in which chemically cross-linked hyaluronic acid is pulverized in a form containing water or a buffer solution. Specifically, the cross-linked hyaluronic acid hydrogel of the present invention means 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.

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 a cross-linking agent to each hyaluronic acid hydrogel.

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 low cross-linking rate (hereinafter referred to as a first cross-linked hyaluronic acid hydrogel or an elastic gel in the present invention) may be added with a cross-linking agent in 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.

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.

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 in 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).

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

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.

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

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.

The mixing ratio of the first cross-linked hyaluronic acid hydrogel and the second cross-linked hyaluronic acid hydrogel among the mixture of two or more cross-linked hyaluronic acid hydrogels having different cross-linking rates of the present invention may be 99:1 to 1:99. However, in cases where the particle size of the finally formed hydrogel is not completely uniform and the cross-linking rates of each hyaluronic acid molecule are different, it is not limited thereto 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, because it is the same in terms of the composition principle of the present invention.

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 may be, but is not limited to, 2.4~2.5, 2.6~3.5, 2.6~3.0, or 2.6~2.8, and may be appropriately selected depending on the intended use. For example, in the case of dermal fillers, hyaluronic acid hydrogels having an extreme viscosity value of 1.6~1.8 are mainly used, and in the case of intra-articular injection, hyaluronic acid hydrogels having 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.

The cross-linked hyaluronic acid hydrogel of the present invention may have 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 storage modulus and G'' represents loss modulus.

The injectable composition comprising the cross-linked hyaluronic acid hydrogel and the extracellular matrix of the present invention contains 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 The concentration may be, but is not limited to, 10 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 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.

The term “extracellular matrix” of the present invention refers to an extracellular matrix including 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.

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.

The injectable composition of the present invention is characterized by including a liquid extracellular matrix. In a specific embodiment of the present invention, when adding a powdered extracellular matrix to the injectable composition of the present invention, the injection force when injected using a syringe is high and irregular, compared to when adding a liquid extracellular matrix. In addition, in a specific embodiment of the present invention, when adding a powdered extracellular matrix to the injectable composition of the present invention, compared to when adding a liquid extracellular matrix, the extracellular matrix may not be completely dissolved in the injection, so insoluble substances may exist.

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” means culturing connective tissue cells, epithelial tissue cells, or a combination thereof among human-derived cells.

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

In addition, the above 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 extracellular matrix derived from human cell culture, it has technical significance in that it can be used industrially because it is produced by culturing human cells in vitro.

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 interstitial matrix mainly composed of collagen types 1/2/3, elastin, and fibronectin, the provisional matrix composed of fibrinogen, fibrin, and fibronectin, and the granulation matrix composed of collagen types 1 to 3 and fibronectin, unlike other extracellular matrices, and is composed of the main component of the structural support of blood vessels (Biomaterials Science (Fourth Edition), 2020, Pages 701-715). It 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.

The basement membrane extracellular matrix is the main component of a thin membrane located between human epithelial tissue and connective tissue, and its main components are laminin and type IV collagen. Among these, laminin promotes angiogenesis and thus promotes the biosynthesis of collagen within the tissue, thereby playing an important role in tissue repair and re-epithelialization. It also interacts with integrins located on the cell surface to contribute to tissue integration.

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 The present invention can include, but is not limited to, an amount of from 0.1 to 90 μg/mL, from 0.1 to 70 μg/mL, from 0.1 to 50 μg/mL, from 0.1 to 40 μg/mL, from 0.1 to 30 μg/mL, from 0.1 to 20 μg/mL, from 0.1 to 10 μg/mL, from 1 to 90 μg/mL, from 1 to 70 μg/mL, from 1 to 50 μg/mL, from 1 to 40 μg/mL, from 1 to 30 μg/mL, from 1 to 20 μg/mL, or from 1 to 10 μg/mL.

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.

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 μg/mL, 0.001 to 110 μg/mL, 0.001 to 100 μg/mL, 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 120 μg/mL, 0.01 to 110 μg/mL, 0.01 to 100 μg/mL, 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 The present invention may include, but is not limited to, ㎍/㎖, 1 to 100 ㎍/㎖, 1 to 90 ㎍/㎖, 1 to 70 ㎍/㎖, 1 to 50 ㎍/㎖, 1 to 40 ㎍/㎖, 1 to 30 ㎍/㎖, 1 to 20 ㎍/㎖, or 1 to 10 ㎍/㎖.

The present inventors, considering that the concentration of laminin in the extracellular matrix that can be produced through human cell culture is at most about 1 mg/㎖, confirmed that the concentration of the final composition after mixing with a hyaluronic acid hydrogel can reach at most 300 μg/㎖, and confirmed that the final injectable composition of the present invention, consisting of a mixture of a 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.

According to one embodiment of the present invention, the hydration contact angle measured after applying and drying the injectable composition containing 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.

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 that it facilitates cell fixation, that is, 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 tissue regeneration ability of the injectable composition of the present invention is excellent from the excellent cell attachment ability.

According to one embodiment of the present invention, the excellent cell adhesion is not exhibited when only a single component of the extracellular matrix, such as laminin, is included. In addition, it can be seen that the decomposition rate of the hyaluronic acid hydrogel is reduced from the increase in the hydration contact angle, thereby increasing the tissue repair maintenance period of the injectable composition of the present invention.

The injectable composition comprising the cross-linked hyaluronic acid hydrogel and the extracellular matrix of the present invention can fix the extracellular matrix at the injection site due to the viscosity of the cross-linked hyaluronic acid hydrogel, and thus contributes to exhibiting a local tissue regeneration effect at the injection site. This secondary effect does not appear when non-cross-linked hyaluronic acid and the extracellular matrix are mixed.

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

In addition, in another specific embodiment of the present invention, the injectable composition containing the extracellular matrix of the present invention has much better collagen formation ability and angiogenic ability upon tissue examination after intradermal/subcutaneous injection than a composition composed only of a cross-linked hyaluronic acid hydrogel that does not contain an extracellular matrix. From the above-mentioned excellent angiogenic ability, it can be seen that the tissue regeneration ability of the composition of the present invention is excellent.

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 comprise 0.1 to 1.0% by weight of the injectable composition comprising the entire cross-linked hyaluronic acid hydrogel and the liquid human cell culture-derived extracellular matrix. At a concentration of 0.1% or less, the effect of local anesthesia is minimal, and pain may occur upon administration, and at a concentration of 1.0% or more, the amount administered is large, and safety cannot be ensured. As mentioned above, the local anesthetic of the present invention may be at least one local anesthetic 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.

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

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

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

The above preservative may be, but is not limited to, p-oxybenzoic acid esters, such as methyl p-oxybenzoate, thimerosal, chlorobutanol, benzyl alcohol, and the like.

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

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 carboxymethyl cellulose and polysorbate 80.

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 of 6.5 to 8.0, but is not limited thereto. Any pH range in which the extracellular matrix is not degraded is satisfactory.

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

In specific embodiments of the present invention, the injectable composition may be used for cosmetic purposes such as improving skin wrinkles, facial shaping, or increasing lip volume, but is not limited thereto.

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.

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

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.

The injectable composition of the present invention and the method for producing the injectable composition according to the above-described embodiment are product and method inventions including components common to each other, and the contents regarding the common components between the respective inventions can be equally applied.

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

A method for preparing an injectable composition of the present invention may include the following steps.

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

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.

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:

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

(b) A process for purifying a cross-linked hyaluronic acid hydrogel.

The cross-linking agent used in the process of cross-linking the above hyaluronic acid may be at least one selected from 1,4-butanediol diglycidyl ether, divinyl sulfone, and bis ethyl carbodiimide, preferably butanediol diglycidyl ether, but is not limited thereto.

In manufacturing the first cross-linked hyaluronic acid hydrogel, the cross-linking agent may be added in 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.

In producing the second cross-linked hyaluronic acid hydrogel, the cross-linking agent may be added in 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.

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.

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 homogeneously distributing the cross-linking agent in the hyaluronic acid aqueous solution.

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 for 8 hours or more at a temperature condition of 30℃ or higher. However, if the cross-linking reaction is sufficiently carried out to form a gel, it is not limited thereto. In addition, it may include any one cross-linking reaction selected from a single cross-linking reaction and two or more cross-linking reactions, but is not limited thereto.

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

The external dialysis fluid used in dialysis may be one or more selected from purified water, water for injection, a buffer solution, and an aqueous solution to which inorganic salts are added to correct osmotic pressure, 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.

When using the dialysis method, the dialysis time can last from 1 to 5 days, preferably from 1 to 3 days, but is not limited thereto. In this case, if the dialysis time is short, unreacted cross-linking agent may remain, which may cause the final product to cause toxicity.

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).

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

In one embodiment of the present invention, the particle size may be, but is not limited to, 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.

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).

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.

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

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 a first cross-linked hyaluronic acid hydrogel having high elasticity from large particles and a second cross-linked hyaluronic acid hydrogel having high viscosity from small particles at the same time, thereby providing an advantage in that physical properties can be variously controlled depending on the mixing ratio. 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 decomposition period of the second cross-linked hyaluronic acid having a large surface area that can be hydrolyzed due to its small particle size can be extended, thereby providing an advantage in that a decomposition rate similar to that of the first cross-linked hyaluronic acid can be provided.

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.

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 sterilized separately and then mixed.

The process for sterilizing the above-mentioned 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.

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

The above-mentioned sterilization of the 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.

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

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 the extracellular matrix of the present invention are not mixed after sterilization, but rather are subjected to a sterilization process after mixing, the heat applied during the sterilization process may cause denaturation of the heat-stable extracellular matrix component.

In one embodiment of the present invention, the process of mixing the cross-linked hyaluronic acid hydrogel and the extracellular matrix means 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.

The process for manufacturing the cross-linked hyaluronic acid hydrogel of the present invention may additionally include a process for correcting 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 correction and the stabilizer, preservative, isotonic agent, suspending agent and water for injection for regulating the osmotic pressure and improving the stability of the injectable composition are as described above.

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 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 It can be, but is not limited to, about 800 μg/ml, 100 μg/ml to 700 μg/ml, 100 μg/ml to 600 μg/ml, 100 μg/ml to 500 μg/ml, 100 μg/ml to 400 μg/ml, 100 μg/ml to 300 μg/ml, 100 μg/ml to 200 μg/ml, about 1 μg/ml, about 10 μg/ml, about 30 μg/ml, about 50 μg/ml, about 100 μg/ml, about 150 μg/ml, about 200 μg/ml, about 300 μg/ml, about 400 μg/ml, about 500 μg/ml, about 600 μg/ml, about 700 μg/ml, about 800 μg/ml, about 900 μg/ml, or about 1000 μg/ml.

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.

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.

The above prefilled syringe is a disposable syringe prefilled with a single dose of the composition for injection. The syringe may be a plastic syringe or a glass syringe, and a sterile needle (injection needle) may be attached to the inlet of the prefilled syringe to be applied for injection.

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

Since the method for producing an injectable composition of the present invention and the prefilled syringe are inventions relating to a method for producing an injectable composition according to the above-described aspect of the present invention and inventions comprising the injectable composition as a component, the contents relating to components common to each invention can be equally applied.

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, thereby allowing an extended administration interval, and can be completely biodegraded after a certain period of time, while simultaneously providing the physical function as a 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 the creation of blood vessels and the biosynthesis of collagen in tissue, thereby contributing to tissue regeneration.

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.

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).

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).

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).

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).

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.

Figure 5b is a diagram showing the results of a tissue examination analysis of a test comparing the blood vessel formation ability 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.

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.

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.

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).

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

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 having different crosslinking rates containing human culture-derived liquid extracellular matrices.

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.

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

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain 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 according to the gist of the present invention.

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

Example

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) into a cross-linked hyaluronic acid hydrogel

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 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.

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 prepare it. Afterwards, it was granulated using a 150 μm mesh, and the granulated cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature, humidified manner 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 crosslinked by standing reaction at 35°C for 24 hours. Then, the hyaluronic acid 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 cross-linked hyaluronic acid to 22.22 mg/㎖. After homogenization using a homogenizer, the homogenized cross-linked hyaluronic acid hydrogel was sterilized under high temperature and humidity at 121°C for 16 minutes.

And hyaluronic acid hydrogels with 4 mol% cross-linking agent added and hyaluronic acid hydrogels 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, to the mixed hyaluronic acid hydrogels, liquid-state sterile-filtered human cell culture-derived extracellular matrix (laminin concentration standard: 100 ug/㎖) was added in an amount corresponding to 1/9 of the weight of the cross-linked hyaluronic acid hydrogel and mixed.

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

The method of Example 1-4 was the same as that of Example 1, 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.

Comparative example

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

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

Comparative Example 2: Preparation of an injectable composition consisting only of cross-linked hyaluronic acid and laminin

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 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.

The pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed to 22.22 mg/㎖ to manufacture it. After that, 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℃ 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℃ for 24 hours. And the purified hyaluronic acid hydrogel was dialyzed for 144 hours using a 0.9% NaCl aqueous solution, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2 while fixing the concentration of the cross-linked hyaluronic acid to 22.22 mg/㎖. After that, it was homogenized using a homogenizer, and 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, respectively. 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.

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

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 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.

The pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed to 22.22 mg/㎖ to manufacture it. After that, 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℃ 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℃ for 24 hours. And the purified hyaluronic acid hydrogel was dialyzed for 144 hours using a 0.9% NaCl aqueous solution, and the pH of the purified hyaluronic acid hydrogel was adjusted to 7.2 while fixing the concentration of the cross-linked hyaluronic acid to 22.22 mg/㎖. After that, it was homogenized using a homogenizer, and the homogenized cross-linked hyaluronic acid hydrogel was sterilized in a high-temperature and wet condition at 121°C 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, respectively. 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/㎖.

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

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 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.

The purified hyaluronic acid hydrogel is manufactured by fixing the concentration of cross-linked hyaluronic acid to 22.22 mg/㎖ while adjusting the pH to 7.2. Afterwards, it is granulated using a 150 μm mesh. 10 g of sodium hyaluronate is 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°C for 24 hours. Then, it is purified by dialysis using 0.9% NaCl aqueous solution for 144 hours, and the purified hyaluronic acid hydrogel is manufactured by fixing the concentration of cross-linked hyaluronic acid to 22.22 mg/㎖ while adjusting the pH to 7.2. Afterwards, it is 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 at a ratio of 50:50, respectively. And 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. Thereafter, 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.

Comparative Example 5: Preparation of an injectable composition consisting solely of cross-linked hyaluronic acid hydrogel

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 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.

The pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed to 22.22 mg/㎖ to manufacture it. After that, 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℃ 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℃ 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 to 22.22 mg/㎖ to manufacture it. After that, it was homogenized using a homogenizer, and the homogenized cross-linked hyaluronic acid hydrogel was sterilized in high temperature and moisture 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, respectively. 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.

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

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 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.

The pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed to 22.22 mg/㎖ to manufacture it. After that, 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℃ 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℃ 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 to 22.22 mg/㎖ to manufacture it. After that, it was homogenized using a homogenizer, and the homogenized cross-linked hyaluronic acid hydrogel was sterilized in high temperature and moisture 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, respectively. 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.

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

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 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.

The pH of the purified hyaluronic acid hydrogel was adjusted to 7.2, and the concentration of cross-linked hyaluronic acid was fixed to 22.22 mg/㎖ to manufacture it. After that, 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℃ 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℃ 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 to 22.22 mg/㎖ to manufacture it. After that, it was homogenized using a homogenizer, and the homogenized cross-linked hyaluronic acid hydrogel was sterilized in high temperature and moisture 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, respectively. 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.

Experimental example

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)

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

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 the plate, and then dried inside the BSC for 4 to 5 days. Next, a water droplet was dropped onto the applied materials, and the contact angle was measured.

The results are shown in Figure 1.

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 commercialized 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 including 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.

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).

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.

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

1 mL of Examples 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 dried for 4 to 5 days inside a biological safety cabinet (BSC). Next, human vascular endothelial cells were seeded onto each plate at a concentration of 2.5 x 10 ⁵ cells/well, and the cells were observed and photographed after 0, 1, and 3 hours to confirm cell adhesion.

The results are shown in Fig. 2.

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

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)

The present inventors performed the following experiment to confirm the change in injection force depending on whether the extracellular matrix incorporated into a cross-linked hyaluronic acid hydrogel is in liquid or powder form.

Using an injection force meter, 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)), and Comparative Example 3 (cross-linked hyaluronic acid hydrogel + powdery extracellular matrix), 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.

The results are shown in Fig. 3.

As shown in Fig. 3, in the case of Example 2 and Comparative Example 6, where 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, where the extracellular matrix was mixed in powder form, it was confirmed that the injection force was not constant or high, making it difficult to inject a constant amount, indicating low convenience of use.

Experimental Example 4: Comparative Test on 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)

Similar to Experimental Example 3, the present inventors performed 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.

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 prefilled syringes, were observed for the presence of insoluble foreign substances in accordance with the Insoluble Foreign Substance Test Method 1 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.

The results are shown in Figures 4a and 4b.

As shown in Fig. 4a, Example 2 using a liquid extracellular matrix was homogeneously dissolved and transparent, and no insoluble foreign 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 foreign substances) were observed. Therefore, from the above results, it was confirmed that it is suitable to include a liquid extracellular matrix as an injection for tissue repair.

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

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, respectively, and administered intradermally/subcutaneously to BALB/c nude mice.

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

The results are shown in Figures 5a and 5b.

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.

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

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.

Figure 5c shows the results of quantitative analysis of the results of tissue examination after administration of the test substance. In the group administered Comparative Example 5 for both subcutaneous and intradermal administrations, fewer new blood vessels were observed. However, 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.

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.

Fig. 5d shows the results of observing the tissue repair ability after administration of the test substance. As shown in Fig. 5d, Comparative Example 1 (non-crosslinked hyaluronic acid + extracellular matrix) and Comparative Example 6 (liquid state 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 be rapidly decomposed and have low tissue repair ability. However, in the groups administered Examples 1-4 and Example 2 including human cell-derived extracellular matrix, it was confirmed that excellent tissue repair ability was exhibited compared to Comparative Example 5 due to the synergistic effect of the human cell-derived extracellular matrix and the crosslinked hyaluronic acid hydrogel.

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 including cross-linked hyaluronic acid showed high tissue repair ability because the transplanted material remained at the injection site, whereas Comparative Example 1 composed of a hyaluronic acid solution and a liquid-state human cell culture-derived extracellular matrix and Comparative Example 6 including 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 including both a cross-linked hyaluronic acid hydrogel and a human cell culture-derived basement membrane extracellular matrix were confirmed to show the best tissue repair ability and high utility as an injectable medical product.

Experimental Example 6: Comparative test of change in properties depending on whether mixing process is performed after sterilization or before sterilization (Example 2 vs. Comparative Example 4)

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.

In the case where the sterilization process was performed before mixing cross-linked hyaluronic acid and liquid extracellular matrix and then mixing (Example 2), and in the case where the cross-linked hyaluronic acid and 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.

The results are shown in Fig. 6.

As shown in Fig. 6, in the case of Example 2 where the extracellular matrix was mixed after sterilization, the appearance was transparent, but in the case of Comparative Example 4 where the extracellular matrix was mixed and then sterilized, the appearance was opaque and a large number of insoluble substances were confirmed due to denaturation of the extracellular matrix, which is unstable to heat, 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 an injection.

Experimental Example 7: Comparative Experiment According to the Tissue Origin of the Extracellular Matrix (Example 2 vs. Comparative Example 6) in vitro

In order to confirm the cell adhesiveness (tissue regeneration ability) according to the origin of the extracellular matrix tissue, 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, after which the cell adhesiveness was observed.

The results are shown in Fig. 7.

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.

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

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)

The present inventors conducted the following experiments to confirm the change in Tanδ value of mixtures of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrix.

Using a rheometer, the storage modulus (G') and loss modulus (G") of the prefilled syringes 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) were measured at a frequency of 0.02 Hz.

The results are shown in Fig. 8 and Table 1.

Elastic gel: Viscous gel Tanδ(G"/G') 100:0 0.140658 80:20 0.147625 60:40 0.158406 50:50 0.163076 40:60 0.167919 20:80 0.174209 0:100 0.178182

As shown in Fig. 8 and Table 1, the mixtures of hyaluronic acid hydrogels with different crosslinking rates containing human culture-derived liquid extracellular matrices maintained a constant slope of Tanδ values, allowing for the adjustment of viscosity and elasticity to suit tissue properties at various sites of administration, and were found to have an advantage in future quality control.

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)

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

Using an injection force meter, 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 by connecting them to 25G and 27G injection needles, respectively.

The results are shown in Fig. 9.

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 (20)

An injectable composition comprising a cross-linked hyaluronic acid hydrogel and an extracellular matrix. In claim 1, 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 contained therein before mixing is any one selected from 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 a cross-linking reaction twice or more, 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. 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, a 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 a second cross-linked hyaluronic acid hydrogel 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). An injectable composition in claim 3, wherein 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. 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. An injectable composition, wherein the composition further comprises 0.1 to 1 wt% of a local anesthetic based on the total weight of the composition. An injectable composition containing cross-linked hyaluronic acid at a concentration of 5 to 30 mg/ml in claim 1. An injectable composition comprising, in claim 1, a concentration of laminin in the extracellular matrix of 0.001 to 90 ㎍/㎖ based on the volume of the entire composition. In claim 1, the injectable composition is an injectable composition for improving skin wrinkles, facial plastic surgery, or increasing lip volume; replacing or supplementing joint synovial fluid, or improving joint pain; or peeling sphincter muscles, urethra, penis, vocal cords, or penile tissue. A method for preparing 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. A method for producing an injectable composition in 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. In the 11th paragraph, a method for producing an injectable composition, wherein the first cross-linked hyaluronic acid is produced by a method comprising the following processes: (a1) a process for cross-linking hyaluronic acid by adding a cross-linking agent at a concentration of 1 to 6 mol% relative to the amount (mole) of hyaluronic acid monomer (disaccharide); and (a2) A process for purifying a cross-linked hyaluronic acid hydrogel. A method for producing an injectable composition in claim 12, wherein the first cross-linked hyaluronic acid hydrogel is produced by a method additionally including a process of (a3) granulating a purified cross-linked hyaluronic acid hydrogel. In the 11th paragraph, a method for producing an injectable composition, 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) A process for purifying a cross-linked hyaluronic acid hydrogel. A method for producing an injectable composition in claim 14, wherein the second cross-linked hyaluronic acid hydrogel additionally includes a process of homogenizing (b3) a purified cross-linked hyaluronic acid hydrogel. A method for producing an injectable composition in 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. A method for producing an injectable composition in claim 10, wherein the extracellular matrix is sterilized or sterilized-filtered. 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. 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. A method for producing an injectable composition in claim 10, wherein the injectable composition contains a concentration of laminin in the extracellular matrix of 0.001 to 90 ㎍/㎖ based on the volume of the entire composition.

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