Pharmaceutical composition for preventing, alleviating, or treating ligament or tendon diseases

Abstract

Provided is a pharmaceutical composition for preventing, alleviating, or treating ligament or tendon diseases, the composition comprising: a tendon-derived extracellular matrix powder; at least one biocompatible polymer selected from the group consisting of hyaluronic acid, gelatin, collagen, alginate, chitosan, elastin, fibronectin, laminin, and fibrinogen; and a pharmaceutically acceptable carrier.

Description

Pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases

[0001] The present invention relates to a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases.

[0002] Tendon and ligament disorders account for 45% of musculoskeletal injuries in modern society and typically refer to conditions such as Achilles tendon disease, patellar tendon disease, rotator cuff tendon disease, tenosynovitis, tendinitis, and tendon rupture. Specifically, biceps tendinitis and rotator cuff tendinitis, which most commonly occur in the shoulder area, frequently appear in workers who use excessive force during work or those engaged in tasks requiring unnatural postures, such as overhead work, raising the arms, or bending and raising the arms. In the case of the elbow area, known as lateral epicondylitis or tennis elbow, this refers to tendinitis occurring in the elbow due to inflammation of the tendons attached to the muscles located on the outer side of the humerus just above the elbow; it is associated with the repetitive performance of tasks that apply force to the fingers, wrists, and arms. Other examples include flexor tendinitis, which is inflammation of the palmar tendons of the wrist and hand; extensor tendinitis, which is inflammation of the dorsal tendons of the wrist and hand; flexor tendinitis, which is inflammation of the palmar tendon sheath of the wrist and hand; extensor tendinitis, which is inflammation of the dorsal tendon sheath of the wrist and hand; De Quervain's disease, which is inflammation of the tendon sheath at the base of the thumb; and Tuphidren's contracture, a disease in which fibrosis progresses in the subcutaneous tissue of the palm and causes flexion contracture mainly in the proximal phalanges.

[0003] Tendons and ligaments are fibrous soft tissues composed primarily of collagen. They are similar in both mechanical properties and structural aspects, differing only in their attachment points—whether between bone and bone or between bone and muscle, respectively. Among human tissues, tendons and ligaments receive a relatively insufficient blood supply compared to other tissues; therefore, once damaged, regeneration takes a considerable amount of time, and even after regeneration and treatment, their function is not fully restored to that of normal tendons or ligaments. It has been reported that even after regeneration, their biomechanical strength is reduced compared to normal tendons or ligaments, and this biomechanical strength is influenced by the collagen that constitutes these tissues. Meanwhile, studies on various growth factors affecting the treatment of tendons and ligaments have also been reported (Non-patent Literature 1). Traumatic damage to tendons or ligaments commonly occurs during exercise, work, or daily life, and inflammation or partial rupture of tendons caused by degenerative changes due to aging can occur even from minor trauma. However, to date, no specific treatment has been developed for tendons or ligaments damaged by inflammation, partial rupture (sprain), or complete rupture caused by aging (degenerative changes) and trauma, other than surgical suturing of the torn tendons or ligaments, and steroid injections and physical therapy that merely alleviate symptoms. Therefore, there is a need for treatment of tendon and ligament diseases that can substantially improve or cure these conditions by regenerating and repairing damaged tendons.

[0004] Recently, regenerative therapy using proteins or stem cells is being actively researched in relation to the treatment of such tendon and ligament diseases. Patent Document 1 discloses a composition containing autologous and allogeneic adipose-derived mesenchymal stem cells for the healing of tendon and ligament injuries, and a method for manufacturing the same. The document states that when a composition containing autologous and allogeneic adipose-derived mesenchymal stem cells is administered, collagen, extracellular matrix (ECM) proteins, and various growth factors are secreted, thereby healing tendon and ligament injuries; however, there is a technical limitation in that the effect is not sustained when the composition is administered only once. Patent Document 2 describes that tendon and ligament diseases can be treated by administering a composition containing platelet-derived growth factor (PDGF); however, the economic feasibility is very low because the process of isolating the platelet-derived growth factor from biological fluids, including blood, is complex, and obtaining it using recombinant DNA techniques also requires going through multiple processes. In addition, when artificial substances or substances not of homologous origin are applied clinically, there is a problem in that it cannot be guaranteed that they will be perfectly integrated within the organism and perform the expected functions without side effects.

[0005] To overcome the aforementioned limitations, there have been attempts to utilize the extracellular matrix (ECM) itself or to utilize ECM-related factors. For example, as a method utilizing the extracellular matrix (ECM) itself, a study on the tendon regeneration effect using a tendon-derived extracellular matrix (tECM) solution has been reported (Non-patent document 2), and a medical composition that promotes the production of autologous fat using adipose tissue-derived extracellular matrix powder is also known (Patent document 3). Separately, as a method of directly administering ECM-related factors as drugs, a technique for treating damaged tendons or ligaments by mixing TIMP-1, an isolated and purified MMP inhibitor, with a pharmaceutical carrier such as a gel or cream has been disclosed (Patent document 4).

[0006] However, the aforementioned prior art does not specifically consider the key physical property factors for ensuring the biological safety of dry tissue-derived extracellular matrix powder. In particular, it fails to recognize that crude fat components remaining during the production of dry tissue-derived ECM are key impurities that cause cytotoxicity, and that controlling this crude fat content to below a certain level is a minimum prerequisite for avoiding cytotoxicity. Furthermore, according to the inventors' research, even a powder that satisfies the above minimum safety standards can exhibit significant biocompatibility with a cell viability of 78% or more depending on the content of the inherent active ingredient, TIMP-1.

[0007] Furthermore, to accurately deliver these extracellular matrix powders to damaged tendons or ligaments within the human body, and to ensure they act as a physical support structure and exert long-term effects, mixing them with a carrier (polymer) possessing appropriate viscoelasticity and biocompatibility is essential. However, existing technologies merely involve mixing biocompatible polymers and fail to provide specific solutions regarding the optimal carrier conditions that can maximize tissue regeneration efficacy without inducing cytotoxicity when mixed with high-purity tendon-derived extracellular matrix powder. In fact, even some known biocompatible polymers may exhibit unexpected cytotoxicity or degrade physical properties when mixed with tendon-derived extracellular matrix powder.

[0008] Therefore, there is a need for research on new pharmaceutical compositions that can effectively regenerate tendon and ligament tissues while ensuring minimal safety by controlling crude fat content that induces cytotoxicity, and furthermore, maximizing both safety and tissue regeneration efficiency by selecting and applying biocompatible polymers optimized for this purpose.

[0009] The present invention aims to solve the problems of the aforementioned prior art, and one of the various objectives of the present invention is to provide a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases that has excellent regenerative effects and effects of reducing inflammation and pain.

[0010] In addition, another objective of the present invention is to provide a pharmaceutical composition with significantly improved usability and biological safety by selecting and including an optimized biocompatible polymer that provides physical support and excellent biocompatibility without causing cytotoxicity when mixed with dry-derived extracellular matrix powder.

[0011] Furthermore, another objective of the present invention is to provide a pharmaceutical composition capable of maximizing tissue regeneration efficiency by including a specific biocompatible polymer that promotes cell migration and secretome secretion, going beyond simple physical support.

[0012] According to one aspect, a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon disease is provided, comprising: a tendon-derived extracellular matrix powder; one or more biocompatible polymers selected from the group consisting of hyaluronic acid, gelatin, collagen, alginate, chitosan, elastin, fibronectin, laminin, and fibrinogen; and a pharmaceutically acceptable carrier.

[0013] In one embodiment, the dry-derived extracellular matrix powder may have a crude fat content of less than 5% by weight.

[0014] In one embodiment, the dry-derived extracellular matrix powder may have a TIMP-1 protein content of 20,000 pg or more per 1 mg of powder.

[0015] In one embodiment, the dry-derived extracellular matrix powder may be included in an amount of 0.5 to 15 weight percent relative to 100 weight percent of the total composition.

[0016] In one embodiment, the biocompatible polymer may be included in an amount of 0.5 to 15 weight percent relative to 100 weight percent of the total composition.

[0017] In one embodiment, the biocompatible polymer may be hyaluronic acid, gelatin, or a mixture thereof.

[0018] In one embodiment, at least a portion of the biocompatible polymer may be crosslinked by a crosslinking agent.

[0019] In one embodiment, the crosslinking agent is one selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and 1,2-(bis(2,3-epoxypropoxy)ethylene). It could be more than that.

[0020] In one embodiment, with respect to 100% by weight of the biocompatible polymer, 1 to 99% by weight of the biocompatible polymer crosslinked by the crosslinking agent may be included.

[0021] In one embodiment, the crude fat content of the dry-derived extracellular matrix powder may be 3% by weight or less.

[0022] In one embodiment, the TIMP-1 protein content contained in 1 mg of the dry-derived extracellular matrix powder may be 38,000 pg or more.

[0023] In one embodiment, the dry-derived extracellular matrix powder may have a biocompatibility index of 10,000 or more, which is the value obtained by dividing the TIMP-1 protein content (pg / mg) by the crude fat content (weight%).

[0024] In one embodiment, the biocompatibility index may be 30,000 or higher.

[0025] In one embodiment, the average particle size of the dry-derived extracellular matrix powder may be 425 μm or less.

[0026] In one embodiment, the dry-derived extracellular matrix powder may be prepared by a method comprising the following steps (a) to (e):

[0027] (a) Step of collecting tendon tissue;

[0028] (b) a step of decellularizing the above dry tissue to obtain a dry tissue-derived extracellular matrix;

[0029] (c) a step of freeze-drying the above-mentioned dry extracellular matrix;

[0030] (d) a step of degreasing the freeze-dried dry-derived extracellular matrix; and

[0031] (e) A step of obtaining a dry-derived extracellular matrix powder by degassing the above-mentioned degreased dry-derived extracellular matrix and then freeze-grinding it.

[0032] In one embodiment, the pharmaceutical composition may have a pH of 5.0 to 8.0.

[0033] In one embodiment, the pharmaceutical composition is an injectable hydrogel formulation and may have a viscosity of 2,000 to 5,000 cP.

[0034] The pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases according to the present invention has excellent regenerative, anti-inflammatory, and pain-relieving effects, and has the advantage of high user satisfaction.

[0035] In particular, the composition according to the present invention includes, by selecting and including a biocompatible polymer such as hyaluronic acid or gelatin that has excellent compatibility with tendon-derived extracellular matrix powder, not only forms a safe physical support without cytotoxicity but also has a synergistic effect that accelerates the regeneration of damaged ligament and tendon tissues by promoting the secretion and migration of cell growth factors.

[0036] In addition, the composition according to the present invention has the advantage of ensuring appropriate injectable viscosity and discharge power, thereby providing excellent convenience during the procedure, and maintaining its shape at the affected area to maximize the improvement effect.

[0037] Furthermore, according to a preferred embodiment of the present invention, the pharmaceutical composition can fundamentally eliminate concerns regarding cytotoxicity and ensure excellent biosafety by controlling the residual crude fat content in the dry-derived extracellular matrix powder to a level below a certain threshold.

[0038] In addition, according to a preferred embodiment of the present invention, by using a dry-derived extracellular matrix powder with a high intrinsic TIMP-1 content, the expression of MMP-1 and MMP-3, which are involved in pain and tissue destruction, can be suppressed, thereby further enhancing the effect of relieving inflammation and pain.

[0039] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration described in the description of the invention or the claims.

[0040] Figures 1 (a), (b), and (c) are the results of the analysis of a total of 48 types of growth factors for the extracellular matrix powders of Preparation Example 2, Comparative Preparation Example 5, and Comparative Preparation Example 6, respectively.

[0041] Figure 2 is a reference map showing the location of each growth factor corresponding to the analysis results of Figure 1.

[0042] Figure 3 is a graph comparing the TIMP-1 content in human dry tissue-derived extracellular matrix powders according to Comparative Examples 1 to 4 and Examples 1 to 3.

[0043] Figure 4 is a graph comparing the residual crude fat content in human dry tissue-derived extracellular matrix powder according to Comparative Manufacturing Examples 1 to 4 and Manufacturing Examples 1 to 3.

[0044] FIG. 5 is a graph showing the results of biological safety tests for compositions containing human dry tissue-derived extracellular matrix powders of Comparative Examples 1 to 4 and Examples 1 to 3.

[0045] Figure 6 is a graph showing the change in cell viability according to the residual crude fat content in the dry-derived extracellular matrix powder.

[0046] Figures 7 (a) and (b) are the results of the analysis of a total of 36 inflammation-related factors in human-derived fibroblast culture medium before and after treatment with the composition containing the human dry tissue-derived extracellular matrix powder of Preparation Example 2, respectively.

[0047] Figure 8 is a graph showing the change in the content of pro-inflammatory factors and anti-inflammatory factors before and after treatment of a composition containing the human dry tissue-derived extracellular matrix powder of Preparation Example 2.

[0048] Figure 9 is a graph showing the degree of inhibition of MMP-1 of Preparation Example 2.

[0049] Figure 10 is a graph showing the degree of inhibition of MMP-3 of Preparation Example 2.

[0050] Figure 11 is a graph comparing the viscosity and extrusion power of a composition mixed with the dry-derived extracellular matrix powder of the present invention and various biocompatible polymers (HA, PN, CMC, Gelatin).

[0051] Figure 12 is a graph showing the excellent biocompatibility of a mixture of hyaluronic acid (HA) and gelatin, as a result of evaluating the cytotoxicity of compositions mixed with various biocompatible polymers.

[0052] Figure 13 is a micrograph showing the cell migration patterns of a composition mixed with various biocompatible polymers over time (4hr, 8hr, 24hr, 48hr).

[0053] Figure 14 is a graph showing the cell area %) over time by quantifying the results of Figure 13.

[0054] Figure 15 shows the results of analyzing cell secretion factors secreted from compositions mixed with various biocompatible polymers, where (a) is the HA mixture group, (b) is the gelatin mixture group, (c) is the PN mixture group, (d) is the CMC mixture group, (e) is the control group, and (f) is an image of the reference map.

[0055] Figures 16 and 17 are graphs showing changes in gene expression related to fibrosis and tissue remodeling following treatment with a hyaluronic acid (HA) mixed composition.

[0056] Figures 18 to 23 are graphs showing changes in gene expression related to fibrosis and tissue remodeling following treatment with a gelatin mixed composition.

[0057] Figures 24 to 26 are graphs showing changes in the expression of wound healing-related genes following treatment with a hyaluronic acid (HA) mixed composition.

[0058] Figures 27 to 29 are graphs showing changes in the expression of wound healing-related genes following treatment with a gelatin mixed composition.

[0059] The present invention will be described below based on specific examples. However, the details described in this specification may be implemented in various different forms and are therefore not limited to the embodiments described herein.

[0060] definition

[0061] Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0062] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as generally understood by those skilled in the art to which the present invention pertains.

[0063] Where a range of numerical values ​​is described throughout the specification, unless a specific range is otherwise described, the value has the precision of significant figures provided in accordance with the standard rules for significant figures in chemistry. For example, 10 includes a range of 5.0 to 14.9, and the number 10.0 includes a range of 9.50 to 10.49.

[0064] Additionally, where a numerical range is described in this specification, unless otherwise specified, said range includes a starting value and an ending value and is interpreted to include all values ​​in between. For example, "1 to 5" includes not only 1 and 5 but also all values ​​between 1 and 5.

[0065] Throughout the specification, the term “in vivo” refers to events occurring in the body of the subject.

[0066] Throughout the specification, the term “in vitro” refers to events occurring outside the body of the subject. In vitro analysis includes cell-based analysis in which living or dead cells are used, and may include cell-free analysis in which intact cells are not used.

[0067] Throughout the specification, the terms “prevention,” “improvement,” and “treatment” are used interchangeably and include effects such as preventing, preventing, delaying, improving, resolving, alleviating, reducing, improving, or treating ligament or tendon disease, but are not necessarily limited to the complete inhibition of ligament or tendon disease.

[0068] Throughout the specification, the terms "ligament or tendon diseases" or "tendinopathy" are used to encompass all conditions resulting from damage, inflammation, rupture, or degenerative changes to tendons and ligaments. Specific diseases to which the present invention can be applied include, but are not limited to, Achilles tendon disease (Achilles tendinitis, etc.), patellar tendon disease (patellar tendinitis or Jumper's knee, etc.), rotator cuff tendon disease, tenosynovitis (trigger finger, De Quervain's tenosynovitis, etc.), tendinitis (lateral epicondylitis or tennis elbow, medial epicondylitis or golfer's elbow, biceps tendinitis, etc.), plantar fasciitis, Dupuytren's contracture, or tendon rupture due to trauma.

[0069] Throughout the specification, the term “biocompatible polymer” refers to a polymeric material that imparts appropriate viscoelasticity to the composition of the present invention, enhances the dispersion stability of dry-derived extracellular matrix powder, and serves as a physical support in vivo. Such biocompatible polymers may be dissolved or swollen in a pharmaceutically acceptable carrier described below to form a hydrogel.

[0070] Throughout the specification, the term “pharmaceuticalally acceptable carrier” means a liquid medium for dissolving, dispersing, or suspending the above-mentioned dry extracellular matrix powder and biocompatible polymer to form a formulation that can be administered to an individual. Specifically, it may be one or more selected from the group consisting of saline solution, sterile water, Ringer’s solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, and ethanol, and may further include other conventional additives such as antioxidants, buffers, and bacteriostatic agents as needed.

[0071] Throughout the specification, "TIMP-1 content" means the mass (pg, picogram) of TIMP-1 protein contained per 1 mg of dry-derived extracellular matrix powder (preferably freeze-dried dry powder), which can be measured by standard protein quantification methods such as ELISA (Enzyme-Linked Immunosorbent Assay).

[0072] Throughout the specification, “crude fat content” means the weight percentage (wt%) of lipid components measured based on 100% by weight of dry-derived extracellular matrix powder, unless otherwise specified, which can be measured by standard lipid quantification methods such as the Soxhlet extraction method.

[0073] Throughout the specification, “dry tissue” refers to dry tissue of homologous or xenologous origin, wherein “homologous” refers to humans and “xenologous” refers to mammals other than humans, such as pigs, horses, cattle, sheep, dogs, and rodents. However, since xenologous dry tissue carries the risk of animal-derived viruses and zoonotic cross-infection, homologous dry tissue may be more preferable for application to the human body.

[0074] Throughout the specification, the term “decellularization” refers to the general (80% or more), nearly complete (95% or more), or essentially complete (99% or more) removal of the cellular components of the tissue.

[0075] Throughout the specification, the term “individual” may be a mammal, such as a rat, livestock, mouse, or human, and preferably a human.

[0076] Pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases

[0077] A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon disease, which is one aspect of the present invention, comprises (a) a tendon-derived extracellular matrix powder, (b) a biocompatible polymer, and (c) a pharmaceutically acceptable carrier. Each component is described in detail below.

[0078] (a) Dry-derived extracellular matrix powder

[0079] The pharmaceutical composition of the present invention comprises a dry-derived extracellular matrix powder as an active ingredient.

[0080] The extracellular matrix primarily serves for structural support in animals, and more than 90% of its components are structural proteins such as collagen, while the remaining 10% consists of glycoproteins such as fibronectin, laminin, and glycosaminoglycans (GAGs). In addition, growth factors and some cytokines are present and are known to promote cell growth and tissue-specific differentiation of undifferentiated cells. These growth factors and cytokines also contain anti-inflammatory factors and pain-regulating factors, which can play an important role in pain relief and treatment. Specifically, they include TIMP-1 (tissue inhibitors of metalloproteinases-1) and TIMP-4, which are representative cytokines secreted in relation to pain and inflammation relief. These cytokines are known to induce the inhibition of inflammatory responses and to induce the inhibition of inflammatory cytokines.

[0081] A conventional therapeutic agent that utilizes the advantages of the extracellular matrix is ​​the acellular dermal matrix (ADM). This is a human-derived material consisting primarily of the extracellular matrix, obtained by undergoing only a decellularization process from human skin tissue donated after death. ADM was first developed in the 1970s during the advancement of emergency medicine, initially applied to burn patients. Currently, it is used for various tissue regeneration and reconstruction purposes beyond burns, including trauma, ulcers, abdominal wall reconstruction, breast reconstruction, vocal cord reconstructive surgery, and interdental papilla grafts. In particular, it is widely used in wound dressings and artificial skin. ADM products are available in sheet form, as well as in granular or microparticle liquid forms.

[0082] Meanwhile, it is known that extracellular matrix extracted from different tissues possesses tissue specificity, which reflects the characteristics of each tissue. This allows for the attachment of cytokines or growth factors suitable for that tissue, thereby creating the most optimal environment for cells to survive and constitute the tissue. For this reason, replenishing damaged tissue with its original components can lead to faster tissue regeneration.

[0083] Accordingly, the inventors have conducted extensive research to maximize regenerative effects, inflammation and pain reduction effects, and biological safety by freeze-drying and finely grinding the extracellular matrix obtained from dry tissue after a decellularization process, and controlling its physical properties.

[0084] As a result, the inventors confirmed that in order to ensure the biological safety (elimination of cytotoxicity) of dry-derived extracellular matrix powder, it is essential to control the residual crude fat content, and furthermore, that maintaining a high TIMP-1 content in the powder significantly increases the effect of inhibiting pain and tissue destruction.

[0085] Specifically, Patent Document 3 discloses a medical composition comprising extracellular matrix powder derived from adipose tissue, with the primary purpose of promoting the generation of autologous fat. This differs fundamentally in tissue origin and purpose from the present invention, which utilizes dry tissue-derived powder for the alleviation of inflammation and pain. Of course, Patent Document 3 discloses a delipidation step for removing lipid components from adipose tissue. However, under the technical concept of Patent Document 3, which aims for fat generation, residual lipid components can instead be utilized as biochemical cues to induce fat differentiation. In contrast, the technical concept of the present invention is opposite in that it regards residual crude fat as an impurity that causes cytotoxicity and seeks to ensure biological safety by actively removing it. In fact, in fields where fat generation is the objective, it has been reported that excessive lipid removal can impair the signals inducing fat generation, thereby causing a sharp decrease in tissue retention rates and hindering the achievement of the objective of the invention (Jiang et al., Theranostics (2021)).

[0086] In addition, Non-patent Literature 2 discloses a technique for preparing a water-soluble tECM solution from dried tECM through a urea extraction method. However, this method can result in the loss of specific proteins such as TIMP-1, and in fact, TIMP-1 was not detected in the protein component analysis results of the document. Furthermore, Non-patent Literature 2 states that MMP-1 and MMP-3 are upregulated upon tECM treatment, which is different from the mechanism of action of the present invention, which aims to alleviate inflammation and pain through MMP inhibition.

[0087] In addition, Patent Document 4 discloses a drug for the treatment of damaged tendons or ligaments in which isolated / purified TIMP is added externally and mixed with a pharmaceutical carrier such as a gel or cream. However, TIMP-1 protein injected externally in this manner has a fundamental limitation in that its efficacy is temporary due to low in vivo stability and a very short half-life of only a few hours (Chaturvedi et al., International Journal of Nanomedicine (2014)).

[0088] On the other hand, in the present invention, TIMP-1 is provided in the form of a complex embedded in a scaffold called ECM powder, thereby allowing for sustained release over a long period using the ECM as a reservoir (Rieber et al., Materials (2025)). Furthermore, while externally added TIMP-1 may be limited to a simple MMP inhibitory function, the ECM-embedded TIMP-1 of the present invention can exhibit complex synergistic effects, such as (1) the physical support function of the ECM powder itself, (2) the MMP inhibitory function of the embedded TIMP-1, and (3) cytokine-like activities in which excess TIMP-1 binds to cell surface receptors to promote cell proliferation and anti-apoptosis, thereby directly inducing tissue regeneration (Knight et al., Front. Mol. Neurosci. (2019)).

[0089] Based on these findings, the present invention provides a dry-derived extracellular matrix powder with novel properties that has a safe range of crude fat content without cytotoxicity and, preferably, contains a high amount of TIMP-1 to exhibit excellent efficacy.

[0090] Specifically, the dry-derived extracellular matrix powder used in the present invention is characterized by having a crude fat content of less than 5% by weight relative to 100% by weight of the powder. The crude fat content may preferably be 4.5% by weight or less, more preferably 4% by weight or less, even more preferably 3.5% by weight or less, even more preferably 3% by weight or less, and most preferably 2% by weight or less. For example, 4.99 wt%, 4.98 wt%, 4.97 wt%, 4.96 wt%, 4.95 wt%, 4.94 wt%, 4.93 wt%, 4.92 wt%, 4.91 wt%, 4.9 wt%, 4.8 wt%, 4.7 wt%, 4.6 wt%, 4.5 wt%, 4.4 wt%, 4.3 wt%, 4.2 wt%, 4.1 wt%, 4.0 wt%, 3.9 wt%, 3.8 wt%, 3.7 wt%, 3.6 wt%, 3.5 wt%, 3.4 wt%, 3.3 wt%, 3.2 wt%, 3.1 wt%, 3.0 wt%, 2.9 wt%, 2.8 wt%, 2.7 wt%, 2.6 wt%, 2.5 wt%, 2.4 wt%, 2.3 wt%, 2.2 wt%, 2.1 wt%, 2.0 wt%, 1.9 wt%, 1.8 wt%, 1.7 wt%, 1.6 wt%, 1.5 wt%, 1.4 wt%, 1.3 wt%, 1.2 wt%, 1.1 wt%, 1.0 wt%, 0.9 wt%, 0.8 wt%, 0.7 wt%, 0.6 wt%, 0.5 wt%, 0.4 wt%, 0.3 wt%, 0.2 wt%, 0.1 wt%, 0.09 wt%, 0.08 wt%, 0.07 wt%, 0.06 wt%, 0.05 wt%, 0.04 wt%, 0.03 wt%, 0.02 wt%, 0.01% by weight, a value between these two values, or less, but not limited thereto. When the crude fat content in the dry-derived extracellular matrix powder is controlled within the above range, the residue of immune response-inducing factors is minimized, and biological safety can be maximized.

[0091] According to one example, the TIMP-1 protein content contained in 1 mg of the above-mentioned dry-derived extracellular matrix powder may be 20,000 pg or more, preferably 25,000 pg or more, more preferably 28,000 pg or more, even more preferably 35,000 pg or more, and most preferably 38,000 pg or more. For example, 20,000pg, 21,000pg, 22,000pg, 23,000pg, 24,000pg, 25,000pg, 26,000pg, 27,000pg, 28,000pg, 29,000pg, 30,000pg, 31,000pg, 32,000pg, 33,000pg, 34,000pg, 35,000pg, 36,000pg, 37,000pg, 38,000pg, 39,000pg, 40,000pg, 41,000pg, 42,000pg, 43,000pg, 44,000pg, 45,000pg, 46,000pg, 47,000pg, 48,000pg, 49,000pg, 50,000pg, 51,000pg, 52,000pg, 53,000pg, 54,000pg, 55,000pg, 56,000pg, 57,000pg, 58,000pg, 59,000pg, 60,000pg, 61,000pg, 62,000pg, 63,000pg, 64,000pg, 65,000pg, 66,000pg, 67,000pg, 68,000pg, 69,000pg, 70,000pg, 71,000pg, 72,000pg, 73,000pg, 74,000pg, 75,000pg, 76,000pg, 77,000pg, 78,000pg, 79,000pg, 80,000pg, values ​​between these two values, or greater than or equal to, but not limited to.In the process of pulverizing the dry tissue-derived extracellular matrix, most of the TIMP-1 protein present in the dry tissue-derived extracellular matrix is ​​lost. However, in the present invention, by maintaining the TIMP-1 protein contained in the dry tissue-derived extracellular matrix powder at such a high concentration through a specific manufacturing process, superior regenerative effects and effects of reducing inflammation and pain can be expected.

[0092] According to one example, the dry-derived extracellular matrix powder may have a Biocompatibility Index of 10,000 or more, preferably 20,000 or more, more preferably 30,000 or more, even more preferably 40,000 or more, and most preferably 50,000 or more. The Biocompatibility Index has technical significance as a threshold that guarantees superior biocompatibility (cell viability of 78% or more) exceeding the minimum safety standard of 70% cell viability. The Biocompatibility Index may be defined by the following Formula 1.

[0093] [Equation 1]

[0094] Biocompatibility Index = (TIMP-1 content (pg / mg)) / (Crude fat content (weight%))

[0095] According to one example, the above-mentioned dry extracellular matrix powder may be included in an amount of 0.5 to 15 weight percent relative to 100 weight percent of the total composition. The content of the above-mentioned dry-derived extracellular matrix powder is, for example, 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, 4.5 wt%, 5.0 wt%, 5.5 wt%, 6.0 wt%, 6.5 wt%, 7.0 wt%, 7.5 wt%, 8.0 wt%, 8.5 wt%, 9.0 wt%, 9.5 wt%, 10.0 wt%, 10.5 wt%, 11.0 wt%, 11.5 wt%, 12.0 wt%, 12.5 wt%, 13.0 wt%, 13.5 wt%, 14.0 wt%, 14.5 wt%, 15.0 wt%, or two of these. The value may be between the values, preferably 0.6 to 13 weight%, more preferably 0.8 to 12 weight%, and even more preferably 1 to 10 weight%.

[0096] The inventors have discovered that a higher content of the dry-derived extracellular matrix powder within the total composition is not simply better, but that there exists an optimal effective concentration range that effectively inhibits the expression of MMP-1 and MMP-3, which are involved in pain and tissue destruction. Within this range, the pharmaceutical composition of the present invention exhibits excellent biocompatibility, along with excellent anti-inflammatory and pain-relieving effects, specifically an excellent MMP expression inhibition effect.

[0097] According to one example, the average particle size of the dry-derived extracellular matrix powder may be 425 μm or less, preferably 300 μm or less, more preferably 150 μm or less, and most preferably 100 μm or less. When the average particle size of the dry-derived extracellular matrix powder is controlled within the above range, injection needle dispensing is possible, thereby increasing convenience of use. Meanwhile, since a smaller average particle size of the dry-derived extracellular matrix powder is advantageous in terms of convenience of use, the present invention does not specifically limit the lower limit thereof.

[0098] As such, dry-derived extracellular matrix powder containing a high content of TIMP-1 protein can be manufactured in various ways, and the manufacturing method is not particularly limited. However, as one non-limiting example for achieving the physical properties of the present invention, the dry-derived extracellular matrix powder may be manufactured by a method comprising the steps of: (a) collecting dry tissue; (b) decellularizing the dry tissue; (c) freeze-drying the dry-derived extracellular matrix; (d) degreasing the freeze-dried dry-derived extracellular matrix; and (e) degreasing the degreased dry-derived extracellular matrix and then freeze-grinding it to obtain dry-derived extracellular matrix powder.

[0099] When a degreasing process is performed after freeze-drying as in step (d) above, compared to a method of performing degreasing before freeze-drying, the loss of TIMP-1 is minimized and residual crude fat is effectively removed, which may be advantageous for achieving the physical properties intended by the present invention. In addition, the crude fat content intended by the present invention can be achieved by adjusting the intensity or frequency of the degreasing process or by performing an additional degreasing process after powder preparation.

[0100] The detailed reaction conditions for each of the above steps are known in the art and are therefore not described separately here; for detailed manufacturing methods, refer to the manufacturing examples described below.

[0101] (b) Biocompatible polymer

[0102] The pharmaceutical composition of the present invention comprises a biocompatible polymer that is mixed with the dry-derived extracellular matrix powder to act as a physical scaffold in vivo, imparts appropriate viscoelasticity to the composition, and further creates a biological environment for tissue regeneration.

[0103] The inventors conducted experiments on various polymers to select the optimal biocompatible polymer that exhibits excellent physical properties and tissue regeneration efficacy without inducing cytotoxicity when mixed with dry-derived extracellular matrix powder. As a result, in the embodiments of the present invention, excellent effects were confirmed using hyaluronic acid, a polysaccharide, and gelatin, a protein, as representative biocompatible polymers. On the other hand, it was confirmed that nucleic acid-based polynucleotides (PN) or cellulose-based carboxymethylcellulose (CMC) caused problems such as inducing unexpected cytotoxicity or degrading physical properties when mixed with the dry-derived extracellular matrix powder of the present invention.

[0104] Therefore, for the biocompatible polymer of the present invention, it is preferable to use a natural polysaccharide (alginate, chitosan, etc.) having physical properties and biocompatibility similar to hyaluronic acid, or a bio-derived protein (collagen, elastin, fibronectin, etc.) similar to gelatin.

[0105] Specifically, the biocompatible polymer is preferably one or more selected from the group consisting of hyaluronic acid, gelatin, collagen, alginate, chitosan, elastin, fibronectin, laminin, and fibrinogen. More preferably, the biocompatible polymer may be hyaluronic acid, gelatin, or a mixture thereof, and most preferably, it may be hyaluronic acid, which has been proven to have a synergistic effect in promoting the secretion and migration of cell growth factors.

[0106] Hyaluronic acid (sodium hyaluronate) is a viscous polymer used for cell protection, maintenance of tissue interstitial spaces, and tissue lubrication. It refers to a composition consisting of long-chain glycosaminoglycans composed of N-acetylglucosamine and glucuronic acid, possessing hydrophilic groups with very large molecular weights and exhibiting high viscosity. Hyaluronic acid possesses excellent biocompatibility and does not exhibit interspecies, intertissue, or long-term specificity; instead, it acts as a lubricant to facilitate the movement of collagen between cells at the site of tissue injury. Common methods for obtaining hyaluronic acid include extraction from umbilical cords and tissues, extraction and purification from cultured bacteria, or purchasing commercially available products used for medical purposes.

[0107] Gelatin is a protein obtained by hydrolyzing collagen, and as it degrades in vivo, it provides an environment favorable for cell adhesion and proliferation. In this invention, gelatin ensures excellent biocompatibility when mixed with dry-derived extracellular matrix powder and can function as an economical and efficient support.

[0108] The above biocompatible polymer may be included in an amount of 0.5 to 15 weight% relative to 100 weight% of the total pharmaceutical composition. The content may preferably be 0.6 to 12 weight%, more preferably 0.8 to 10 weight%, even more preferably 1 to 8 weight%, and most preferably 1 to 5 weight% to maintain an optimal injectable viscosity, for example, 2,000 cP to 5,000 cP. For example, the content of the biocompatible polymer is 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, The content may be 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.5 wt%, 5.0 wt%, 5.5 wt%, 6.0 wt%, 6.5 wt%, 7.0 wt%, 7.5 wt%, 8.0 wt%, 8.5 wt%, 9.0 wt%, 9.5 wt%, 10.0 wt%, 11.0 wt%, 12.0 wt%, 13.0 wt%, 14.0 wt%, 15.0 wt%, or a value between these two values, but is not limited thereto. If the content of the biocompatible polymer is less than 0.5 wt%, the viscoelasticity of the composition may be insufficient, making it difficult to perform the role of a physical support in vivo, and if it exceeds 15 wt%, the viscosity may become excessively high, making injection through a needle impossible or requiring excessive force during the procedure.

[0109] Meanwhile, the above-mentioned biocompatible polymer may be in a cross-linked form by a cross-linking agent to extend the retention period of the composition in the body and to control its physical properties. Specifically, at least a portion of the above-mentioned biocompatible polymer may be cross-linked by a cross-linking agent.

[0110] The above crosslinking agent may be one or more selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and 1,2-(bis(2,3-epoxypropoxy)ethylene).

[0111] The content of the biocompatible polymer crosslinked by the above-mentioned crosslinking agent may be 1 to 99 weight% with respect to 100 weight% of the total biocompatible polymer. The content may preferably be 5 to 95 weight%, more preferably 10 to 90 weight%, even more preferably 20 to 80 weight%, and most preferably 30 to 70 weight%. For example, the content of the cross-linked biocompatible polymer may be 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, 96 wt%, 97 wt%, 98 wt%, 99 wt%, or a value between these two values. there is.

[0112] The content of the biocompatible polymer crosslinked by the above-mentioned crosslinking agent affects the viscoelasticity, extrusion capacity, and structural retention capacity of the composition, and differences in structural retention capacity also affect the expression and secretion levels of growth factors that assist in regeneration, inflammation, and pain relief. When the content of the biocompatible polymer crosslinked by the crosslinking agent is appropriately controlled, high viscoelasticity is maintained at the disease site, exhibiting excellent effects in buffering and lubricating surrounding tissues, and the effect of application is maximized as it does not scatter in powder or particle form at the application site. In addition, side effects such as pain and edema are minimized when injected into the disease site.

[0113] In contrast, if the content of the biocompatible polymer crosslinked by the crosslinking agent is not properly controlled, it may hinder the homogeneous dispersion of the dry extracellular matrix powder, causing clumping, increase the injection force due to heterogeneous discharge, decrease the cohesive force, accelerate degradation, and make it difficult to achieve the desired therapeutic effect.

[0114] (c) Pharmaceutically acceptable carrier

[0115] The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier for dissolving, dispersing, or suspending the above-mentioned dry-derived extracellular matrix powder and biocompatible polymer to form a formulation suitable for administration to an individual.

[0116] The above-mentioned pharmaceutically acceptable carrier refers to a liquid medium that does not significantly irritate the organism and does not impair the biological activity and properties of the administered compound. Specifically, the carrier may be one or more selected from the group consisting of saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, and ethanol. Preferably, phosphate-buffered saline (PBS) or physiological saline may be used to provide a pH and osmotic pressure similar to the in vivo environment.

[0117] The pH of the pharmaceutical composition of the present invention may be 5.0 to 8.0. The pH of the composition may be adjusted using a separate pH adjuster such as citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, or sodium monohydrogen phosphate, or it may be adjusted by controlling the weight ratio of each component.

[0118] In addition, the pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives commonly used in the manufacture of medical compositions. A pharmaceutically acceptable additive refers to a carrier or diluent that does not significantly irritate a living organism and does not impair the biological activity and properties of the administered compound. In addition, the above additives can improve the preparation, compressibility, appearance, and taste of the formulation, and, for example, stabilizers, surfactants, lubricants, solubilizers, buffers, sweeteners, bases, adsorbents, binders, binders, suspending agents, curing agents, antioxidants, glossers, flavoring agents, taste agents, pigments, coating agents, wetting agents, wetting regulators, fillers, defoaming agents, cooling agents, chewing agents, antistatic agents, coloring agents, sugaring agents, isotonic agents, softeners, emulsifiers, adhesives, thickeners, foaming agents, pH adjusters, excipients, dispersants, disintegrants, waterproofing agents, preservatives, preservatives, solubilizing agents, solvents, fluidizing agents, etc. may be added as needed.

[0119] In particular, in one embodiment, the pharmaceutical composition may additionally include a therapeutically effective amount of a local anesthetic, such as lidocaine, to relieve pain when injected into the affected area.

[0120] The content of the above pharmaceutically acceptable carriers and additives is not particularly limited, but can be appropriately adjusted considering the viscosity of the overall composition and the dispersibility of the dry-derived extracellular matrix powder.

[0121] The pharmaceutical composition of the present invention can be formulated into various forms according to conventional methods and used, for example, in the form of an aqueous solution, suspension, emulsion, paste, gel, powder, or injection solution.

[0122] The pharmaceutical composition of the present invention can be formulated in various forms for administration to an individual, and it is most preferable to formulate it in the form of an injectable hydrogel or an injectable solution to serve as a physical support by direct injection into the affected area. The injectable formulation can be prepared according to techniques known in the art using a suitable dispersant or wetting agent and a suspending agent. For example, each component can be dissolved in saline solution or a buffer solution to be formulated for injection.

[0123] The embodiments of this specification will be described in more detail below. However, the following experimental results represent only representative results among the above embodiments, and the scope and content of this specification should not be interpreted as being narrowed or limited by the embodiments. The respective effects of various embodiments of this specification not explicitly presented below will be described in detail in the relevant sections.

[0124] Comparative Manufacturing Example 1

[0125] The soft tissue of a human tendon was removed and washed with purified water, then pretreated by a freeze-drying process. The freeze-dried tissue was freeze-ground and sieved through a sieve with a mesh size of 75 to 425 μm. The grinding and sieving process was repeated several times to obtain finely ground extracellular matrix powder derived from a human tendon.

[0126] Comparative Manufacturing Example 2

[0127] A finely ground extracellular matrix powder derived from a human dry tissue was obtained in the same manner as Comparative Example 1, except that the human dry tissue from which soft tissue had been removed was washed with purified water, a degreasing process was performed once before freeze-drying, and a degassing process was performed after freeze-drying and before freeze-grinding.

[0128] Comparative Manufacturing Example 3

[0129] A finely ground extracellular matrix powder derived from a human dry tissue was obtained in the same manner as Comparative Example 1, except that the human dry tissue from which soft tissue had been removed was washed with purified water, a degreasing process was performed twice before freeze-drying, and a degassing process was performed after freeze-drying and before freeze-grinding.

[0130] Comparative Manufacturing Example 4

[0131] A finely ground extracellular matrix powder derived from a human dry tissue was obtained in the same manner as Comparative Example 1, except that the human dry tissue from which soft tissue had been removed was washed with purified water, a degreasing process was performed three times before freeze-drying, and a degassing process was performed after freeze-drying and before freeze-grinding.

[0132] Preparation Example 1

[0133] Finely ground extracellular matrix powder derived from human dry tissue was obtained using the same method as Comparative Example 1, except that a degreasing process was performed once and a degassing process was performed before freeze-grinding the dry tissue after freeze-drying was completed.

[0134] Preparation Example 2

[0135] Finely ground extracellular matrix powder derived from human dry tissue was obtained using the same method as Comparative Example 1, except that a degreasing process was performed twice and a degassing process was performed before freeze-grinding the dry tissue after freeze-drying was completed.

[0136] Preparation Example 3

[0137] Finely ground extracellular matrix powder derived from human dry tissue was obtained using the same method as Comparative Example 1, except that a degreasing process was performed three times and a degassing process was performed before freeze-grinding the dry tissue after freeze-drying was completed.

[0138] Comparative Manufacturing Example 5

[0139] The epidermis of human skin was removed and washed with a washing solution, after which a decellularization process was performed. The decellularized skin tissue was pretreated by a freeze-drying process. The freeze-dried skin tissue was freeze-ground and sieved through a sieve with a mesh size of 75 to 425 μm. The grinding and sieving process was repeated several times to obtain finely ground extracellular matrix powder derived from human skin.

[0140] Comparative Manufacturing Example 6

[0141] The soft tissue of human costal cartilage was removed and washed with a washing solution, then pretreated by a freeze-drying process. The freeze-dried costal cartilage tissue was freeze-ground and sieved through a sieve with a mesh size of 75 to 425 μm. The grinding and sieving process was repeated several times to obtain finely ground extracellular matrix powder derived from human skin.

[0142] Experimental Example 1: Analysis of Extracellular Matrix Components via Protein Array Method

[0143] A growth factor array was performed to analyze the components within the extracellular matrix powder of Preparation Example 2, Comparative Preparation Example 5, and Comparative Preparation Example 6. As a specific method for screening the components within the extracellular matrix powder, the extracellular matrix powder was lysed with a protein extraction solution, and then analyzed using the Proteome Profiler Human Angiogenesis Array Kit, which can identify 48 growth factors, followed by visualization using a Fluorescence Laser Scanner.

[0144] Figures 1 (a), (b), and (c) show the results of analyzing a total of 48 types of growth factors for the extracellular matrix powders of Preparation Example 2, Comparative Preparation Example 5, and Comparative Preparation Example 6, respectively, and Figure 2 is a reference map showing the location of each growth factor corresponding to the analysis results of Figure 1. When referring to Figures 1 and 2, it was confirmed that the extracellular matrix powder derived from human tendons of Preparation Example 2 contained a large amount of TIMP-1 and TIMP-4 proteins, which are known as pain regulators, specifically.

[0145] Experimental Example 2: Analysis of TIMP-1 Content in Extracellular Matrix

[0146] As it was confirmed through Experimental Example 1 that human dry tissue-derived extracellular matrix powder can contain a large amount of TIMP-1 protein, the TIMP-1 content was quantified by the ELISA method for the human dry tissue-derived extracellular matrix powders of Comparative Examples 1 to 4 and Examples 1 to 3 in order to determine the effect of the manufacturing method of the human dry tissue-derived extracellular matrix powder on the TIMP-1 protein content. Specifically, after lysing each extracellular matrix powder with a protein extraction solution, the procedure was performed using a Human TIMP-1 ELISA Kit, and the quantified values ​​were confirmed using a Spectrophotometer.

[0147] Figure 3 is a graph showing the TIMP-1 content of human dry-derived extracellular matrix powders of Comparative Examples 1 to 4 and Examples 1 to 3. Referring to Figure 3, it can be confirmed that the presence or absence of a degreasing process affects the TIMP-1 content. In particular, it can be confirmed that when a degreasing process is performed on dry tissue that does not contain moisture by freeze-drying, the remaining TIMP-1 content shows a significantly high value of 20,000 pg / mg protein or higher.

[0148] Experimental Example 3: Analysis of crude fat residue

[0149] The residual crude fat content was measured for the extracellular matrix powders derived from human dry tissue of Comparative Examples 1 to 4 and Examples 1 to 3. Specifically, the extracellular matrix powder was placed in a Soxhlet extractor, and after extraction, ethyl ether was circulated to extract ether-soluble substances (mainly lipids) and collected in a receiver. Then, the ether was drained and the extract was dried, and the weight of the extract was weighed to quantify the crude fat content.

[0150] Figure 4 is a graph showing the residual crude fat content of human dry tissue-derived extracellular matrix powders of Comparative Examples 1 to 4 and Examples 1 to 3. Referring to Figure 4, it can be confirmed that the presence or absence of a degreasing process affects the residual crude fat content. In particular, it can be confirmed that when a degreasing process is performed on dry tissue that does not contain moisture by freeze-drying, the crude fat content in the extracellular matrix powder is significantly reduced to 1% by weight or less.

[0151] Experimental Example 4: Cell viability test (1)

[0152] Biological safety was tested on compositions containing human tendon-derived extracellular matrix powders of Comparative Preparation Examples 1 to 4 and Preparation Examples 1 to 3. Specifically, cells (NCTC clone 929) were placed in a 96-well plate at a ratio of 1 x 10⁶ 5 After preparation at a cell / ml density, the samples were incubated at 37°C in a 5% CO2 incubator for 24 hours. Each composition was eluted in a medium solution at 37°C for 72 hours at a ratio of 20 ml per 4 g. Subsequently, the composition eluent, blank solution, negative and positive controls were incubated at 37°C in a 5% CO2 incubator for 48 hours. Biological safety was evaluated using the MTT method, and quantitative values ​​were confirmed using a spectrophotometer. The results are shown in Table 1 and Figure 5.

[0153] [Table 1]

[0154]

[0155] Referring to Table 1 and Figure 5, Comparative Examples 1 to 4 satisfied the minimum stability standard (70% or more) at a level of approximately 75-76%. Meanwhile, Examples 1 to 3, which had a biocompatibility index of 30,000 or more, showed a significantly superior cell viability of 78% or more, which clearly confirms that the biocompatibility index proposed in the present invention is a key indicator that guarantees superior biocompatibility beyond the minimum safety standard.

[0156] Experimental Example 5: Cell viability test (2)

[0157] In order to evaluate the effect of residual crude fat content itself on cytotoxicity, raw materials were prepared by intentionally varying the degree of mechanical removal of attached fat tissue when collecting the raw material dry tissue, and then samples with different residual crude fat content (Comparative Examples 7 to 11) were obtained by applying the same process as Comparative Example 1.

[0158] Cell viability was evaluated for Comparative Manufacturing Examples 7 to 11 using the same method as in Experimental Example 4. The residual crude fat content of Comparative Manufacturing Examples 7 to 11 was 45%, 30%, 20%, 10%, and 5%, respectively.

[0159] Figure 6 is a graph showing the results of biological safety tests for compositions containing human dry tissue-derived extracellular matrix powders of Comparative Examples 1 and 7 to 11. Referring to Figure 6, as the crude fat content increased to 5% (survival rate 62.14%), 10% (survival rate 48.56%), 20% (survival rate 23.45%), etc., the cell viability decreased sharply to less than 70%, exhibiting strong cytotoxicity. On the other hand, Comparative Example 1, with a crude fat content of 2.75%, showed a cell viability of 70% or more, confirming that there was no cytotoxicity.

[0160] Therefore, it was confirmed that the minimum residual crude fat content standard to avoid cytotoxicity of ECM powder is less than 5.0%.

[0161] Experimental Example 6: Inflammation Relief Efficacy Test

[0162] Changes in the expression of inflammation-related factors before and after treatment with a composition containing the human dry tissue-derived extracellular matrix powder of Preparation Example 2 were evaluated in a human-derived fibroblast culture medium. Specifically, human-derived fibroblasts (CCD-986sk) were cultured in a proliferation culture medium for one day and then treated with the composition at a concentration of 10% by weight. After culturing the cells for three days, the culture supernatant was obtained, and cytokine and chemokine expression in the cells following treatment with the composition was analyzed using the Proteome Profiler Human Cytokine Array Kit, followed by image verification using a Fluorescence Laser Scanner.

[0163] Figures 7 (a) and (b) are the results of the analysis of a total of 36 inflammation-related factors in human-derived fibroblast culture medium before and after treatment with the composition containing the human tendon-derived extracellular matrix powder of Preparation Example 2, respectively, and Figure 8 is a graph showing the changes in the content of pro-inflammatory factors and anti-inflammatory factors before and after treatment with the composition containing the human tendon-derived extracellular matrix powder of Preparation Example 2.

[0164] IL-8 is an anti-inflammatory factor as an inflammatory cytokine, IL-18 is an anti-inflammatory factor that promotes the production and expression of INF-γ, an inflammatory cytokine, and CXCL, a type of chemokine, is a pro-inflammatory factor that plays a role in gathering immune cells to the inflamed tissue when inflammation occurs. Referring to Figures 7 and 8, it can be confirmed that after treatment with the composition containing the human tendon-derived extracellular matrix powder of Preparation Example 2, the pro-inflammatory factor IL-8 decreased by 84.88% and IL-18 decreased by 85.03%, while the anti-inflammatory factor CXCL11 increased by 65.35% and CXCL12 increased by 65.33%.

[0165] Experimental Example 7: Pain Relief Efficacy Test

[0166] After establishing an inflammation model by treating human-derived fibroblast culture medium with an inflammation-inducing factor (IL-1β), the composition containing the human tendon-derived extracellular matrix powder of Preparation Example 2 was applied to evaluate the degree of inhibition of pain-related proteins MMP-1 and MMP-9. As a cell model, a pain-induced model was prepared by adding IL-1β, known as a pro-inflammatory factor, to human-derived fibroblasts (CCD-986sk) at a concentration of 10 ng / mL in a 6-well plate. The composition was treated at four different concentrations (1 wt%, 5 wt%, 10 wt%, and 20 wt%) and cultured for 3 days. Subsequently, the expression levels of pain-related factors (MMP-1, MMP-3) were measured using the Human MMP-1 ELISA Kit and Human MMP-3 ELISA Kit, and the quantitative values ​​were confirmed using a Spectrophotometer.

[0167] Figure 9 is a graph showing the degree of inhibition for MMP-1, and Figure 10 is a graph showing the degree of inhibition for MMP-3. In Figures 8 and 9, Con(-) represents the content of MMP-1 and MMP-3 contained in the human-derived fibroblast culture medium before treatment with inflammation-inducing factors, and Con(+) represents the content of MMP-1 and MMP-3 contained in the human-derived fibroblast culture medium after treatment with inflammation-inducing factors.

[0168] From Figures 9 and 10, it can be seen that the dry-derived extracellular matrix powder treatment group showed a significant decrease depending on the concentration from low concentration to 10% treatment group. Specifically, the expression level of MMP-1 decreased significantly in a concentration-dependent manner compared to Con(+) (3,891 pg / mg) at 1% (3,812), 5% (3,452), and 10% (2,916). The expression level of MMP-3 also decreased significantly in a concentration-dependent manner compared to Con(+) (35,110 pg / mg) at 1% (33,560), 5% (31,170), and 10% (24,010). However, in the 20% treatment group, the inhibitory effect on MMP-1 was negligible compared to the 10% treatment group (3,649), and in particular, a U-shaped curve was observed in which the expression level of MMP-3 increased significantly more than Con(+) to 61,410. This means that the ECM powder of the present invention exhibits an excellent pain relief (MMP inhibition) effect in the concentration range of 0.5 to 15%, and shows an optimal effect particularly at 10%. Furthermore, since high concentrations of 20% or more may actually reduce the effect or cause side effects, it was confirmed that the composition of the present invention has an optimal effective concentration range of 0.5 to 15% by weight.

[0169] Experimental Example 8: Evaluation of Viscosity and Ejection Power of Compositions According to Type of Biocompatible Polymer

[0170] Viscosity and injection force were measured to determine whether the mixture of dry-derived extracellular matrix powder and biocompatible polymers possessed suitable physical properties as an injectable formulation. Specifically, compositions as shown in Table 2 below were prepared by mixing 9 wt% of dry-derived extracellular matrix powder according to Preparation Example 2 and 3 wt% of various biocompatible polymers in phosphate-buffered saline (PBS). As a control group, a group was used in which only 9 wt% of the dry-derived extracellular matrix powder of Preparation Example 2 was mixed in phosphate-buffered saline (PBS) without adding polymers. The viscosity of each prepared composition was measured using a Rotational Rheometer at 25°C, and the injection force was measured by determining the load (N) when ejected through a 22G injection needle using a Universal Testing Machine (UTM).

[0171] [Table 2]

[0172]

[0173] Figures 11 (a) and (b) are graphs comparing the viscosity and ejection power of compositions mixed with the dry-derived extracellular matrix powder of the present invention and various biocompatible polymers (HA, PN, CMC, Gelatin), respectively. Referring to Figure 11, the viscosity was highest in the PN mixture group (6,400 cP) and the CMC mixture group (4,800 cP), followed by the HA mixture group (4,200 cP), the gelatin mixture group (2,500 cP), and the control group (2,030 cP). Accordingly, the ejection power (main force) also showed a similar trend to viscosity. Specifically, the PN mixture group (13.34 N) and the CMC mixture group (10.11 N) required excessive force of over 10 N during ejection, leading to the conclusion that this would increase operator fatigue and make precise injection difficult during actual clinical application. In fact, visual observation also revealed that the PN and CMC mixture groups exhibited poor fluidity and a stiff, dry texture.

[0174] On the other hand, the gelatin mixture group (4.80 N) showed a very low extrusion rate similar to the control group (4.73 N), suggesting that while injection is easy, its low viscosity may be somewhat insufficient to function as a physical scaffold in vivo.

[0175] However, in the case of the HA mixture group, it was confirmed that it exhibited the most suitable physical properties for maintaining appropriate volume and shape in the affected area after injection, by showing an ejection force of 7.07 N, which allows for smooth injection with about 53% of the force compared to the PN mixture group, while securing a viscosity (4200 cP) that is about twice as high as the control group.

[0176] Therefore, it was confirmed that hyaluronic acid (HA) is the optimal biocompatible polymer that simultaneously satisfies ease of use and physical support as an injectable hydrogel formulation.

[0177] Experimental Example 9: Evaluation of Cytotoxicity of Compositions According to Type of Biocompatible Polymer

[0178] To confirm whether the composition mixed with dried extracellular matrix powder and a biocompatible polymer is safe when applied to a living organism, a cell viability test was performed using the same method as in Experimental Example 4. Cells (NCTC clone 929) were placed in a 96-well plate at a ratio of 1 x 10⁶ 5 After preparation at a cell / ml density, the samples were cultured for 24 hours in a 37°C, 5% CO2 incubator. Each composition prepared in Experimental Example 8 (Control, HA group, PN group, CMC group, Gelatin group) was eluted in a 37°C medium solution at a ratio of 20 ml per 4 g for 72 hours to obtain the eluent. Subsequently, the composition eluent, blank solution, negative control, and positive control were cultured for 48 hours in a 37°C, 5% CO2 incubator to evaluate biological safety using the MTT method, and then quantitative values ​​were confirmed using a Spectrophotometer.

[0179] The results of measuring the cell viability of each composition are shown in Fig. 12. Referring to Fig. 12, it can be seen that even polymers commonly known as biocompatible materials exhibit significantly different cytotoxicity depending on their type when mixed with the dry-derived extracellular matrix powder according to the present invention.

[0180] Specifically, while the negative control group showed a survival rate of 100%, the positive control group showed a survival rate of 5.02%. Under these criteria, the CMC mixture group showed a cell survival rate of only 7.40%, confirming that it caused severe cytotoxicity similar to that of the positive control group (toxicity-inducing group), and the PN mixture group also showed a low survival rate of 48.07%, indicating that it is unsuitable for in vivo application. This is attributed to the fact that the polymers caused chemical and physical interactions with dry-derived extracellular matrix powder, creating an environment unfavorable to cell growth.

[0181] On the other hand, the HA mixture group showed a very high cell viability of 92.03%, and the gelatin mixture group showed 97.57%, confirming that excellent biosafety similar to that of the negative control was secured.

[0182] Accordingly, the biocompatible polymers suitable for use in combination with the dry-derived extracellular matrix powder of the present invention are hyaluronic acid (HA) and gelatin, and in particular, it has been proven that these are optimal carriers that do not induce cytotoxicity even when mixed with high concentrations of ECM powder.

[0183] Experimental Example 10: Evaluation of Cell Migration Inducing Ability of Compositions According to Type of Biocompatible Polymer

[0184] To determine whether the composition of the present invention possesses the ability to attract surrounding cells to the affected area (cell migration) to aid in the regeneration of damaged tissue, a transwell migration assay was performed. Specifically, measurements were taken using a polycarbonate membrane insert (Corning, Manassas, VA, USA) with 8.0 μm pores, and to analyze cell migration, 1 x 10⁶ human-derived fibroblast cells were used. 5 Cells / wells were placed in the upper chamber, and the lower chamber was treated with each composition (HA group, PN group, CMC group, Gelatin group) prepared in Experimental Example 8 and a control group (Positive / Negative Control) as a chemoattractant. Cells that had not migrated were carefully removed from the upper chamber with a cotton swab after 4, 8, 24, and 48 hours had elapsed following treatment. The migrated cells were stained with a 20% methanol solution containing 0.2% crystal violet (Sigma-Aldrich), and the extent of cell migration was observed under a microscope (Fig. 13). This was then quantified as cell area %) using an image analysis program (Fig. 14).

[0185] Referring to the micrograph in Fig. 13 and the graph in Fig. 14, it can be seen that there are distinct differences in cell migration patterns depending on the type of polymer.

[0186] Specifically, the hyaluronic acid (HA) mixture group showed the fastest and most active cell migration to the sub-transwell region as time progressed after treatment. In particular, at the 48-hour mark, it exhibited a cell area of ​​approximately 76%, which was higher than that of the positive control group, demonstrating the most superior ability to induce cell migration among the experimental groups.

[0187] The gelatin mixture also showed steady cell migration over time and exhibited a cell occupancy rate of approximately 49% at 48 hours, demonstrating a significant migration-promoting effect compared to the negative control group.

[0188] On the other hand, in the case of the PN mixture group, some cell migration (approximately 40%) was observed up to the 24-hour mark, but at the 48-hour mark, the cell occupancy rate decreased sharply to a value close to 0%. This is consistent with the toxicity evaluation results of the previous Experimental Example 9, and it is determined that the PN component caused cytotoxicity, resulting in the death or shedding of migrating cells. The CMC mixture group also showed a very low cell occupancy rate across all time points, confirming that it failed to induce or inhibited cell migration at all.

[0189] Therefore, it was reconfirmed that hyaluronic acid (HA) is the optimal biocompatible polymer that, when mixed with the dry-derived extracellular matrix powder of the present invention, most effectively promotes cell migration, which is the initial stage of tissue regeneration, and maintains cell activity even during prolonged treatment.

[0190] Experimental Example 11: Analysis of Cell Secretome of Compositions According to Types of Biocompatible Polymers

[0191] Protein array analysis was performed to determine whether the composition of the present invention acts on cells to promote the secretion (secretome) of various growth factors and cytokines effective for tissue regeneration, angiogenesis, and inflammation control. Specifically, each composition prepared in Experimental Example 8 (Control, HA group, PN group, CMC group, Gelatin group) was mixed with a culture medium (MEM + 10% FBS + 1% AA) at a ratio of 20 ml per 4 g, and eluted at 37°C for 72 hours. Subsequently, the eluent was obtained by collecting only the supernatant without a separate filtering process. The obtained eluent was treated with human-derived fibroblast cells and cultured for a certain period, after which the culture supernatant was recovered. To analyze the effective proteins present in the recovered supernatant, the expression of a total of 55 factors related to angiogenesis and tissue regeneration was confirmed using the Human Angiogenesis Array Kit.

[0192] Figure 15 shows the results of analyzing cell secretory factors (Secretome) secreted from compositions mixed with various biocompatible polymers, where (a) is an array image of the HA mixture group, (b) is an array image of the gelatin mixture group, (c) is an array image of the PN mixture group, (d) is an array image of the CMC mixture group, and (e) is an array image of the control group (cells alone), and (f) is a reference map showing the location of each spot. Here, boxes marked with solid lines indicate detected factors, and boxes marked with dotted lines indicate factors that appear darker because their expression levels are visually significantly increased compared to the control group.

[0193] Referring to Figure 15, it can be seen that there are significant differences in the types and amounts of effective factors secreted by cells depending on the type of biocompatible polymer.

[0194] Specifically, only 7 factors were detected in the control group (e), PN mixed group (c), and CMC mixed group (d), and the intensity of the spots was also relatively weak. This means that when PN or CMC is mixed with ECM powder, it does not have a significant effect on cell activity or the secretion of active substances, or remains at the level of the control group.

[0195] On the other hand, 12 factors were detected in the gelatin mixture group (b), which was more than the control group, and 13 effective factors were detected in the hyaluronic acid (HA) mixture group (a), which was the highest among the experimental groups. In addition, in the case of the HA mixture group, the intensity (expression amount) of each spot was significantly darker compared to the control group, confirming that the secretion amount of effective factors increased significantly.

[0196] The 13 factors strongly expressed in the HA mixture group and their biological functions are shown in Table 3 below.

[0197] [Table 3]

[0198]

[0199] As can be seen in Table 3 above, the HA mixture promotes the secretion of tissue protective factors such as TIMP-1, as well as powerful angiogenic and tissue regeneration factors such as VEGF, HGF, and Angiogenin. This suggests that HA can create a complex biochemical environment necessary for the regeneration of damaged ligament and tendon tissues by creating synergy with tendon-derived extracellular matrix powder. This implies that hyaluronic acid (HA) is the optimal biocompatible polymer that, when mixed with the tendon-derived extracellular matrix powder of the present invention, awakens cell activity and most powerfully induces the secretion of abundant growth factors and cytokines necessary for tissue regeneration.

[0200] Experimental Example 12: Analysis of Gene Expression Related to Fibrosis and Tissue Remodeling According to Type of Biocompatible Polymer

[0201] To confirm the effect of the composition of the present invention on the structural formation and remodeling of tissues during the healing process of damaged tendons / ligaments, changes in related gene expression were analyzed using qPCR (Quantitative Polymerase Chain Reaction).

[0202] Specifically, the HA mixture and gelatin mixture prepared in Experimental Example 8 were eluted into a culture medium and treated with human-derived fibroblast cells, and cultured for 24 hours. Subsequently, the cells were harvested, Total RNA was extracted, and cDNA was synthesized, after which RT-qPCR was performed using a fibrosis-related gene panel. To ensure the reliability of the data, only genes with a Ct (Cycle threshold) value of 35 or less were selected, and the relative fold change of each gene compared to the control group was calculated and analyzed.

[0203] Figures 16 and 17 are graphs showing changes in gene expression related to fibrosis and tissue remodeling following treatment with a hyaluronic acid (HA) mixed composition, and Figures 18 to 23 are graphs showing changes in gene expression related to fibrosis and tissue remodeling following treatment with a gelatin mixed composition.

[0204] Referring to Figures 16 to 23, the difference in tissue regeneration mechanisms according to each polymer characteristic in the gene expression intensity and pattern between the two experimental groups is clearly confirmed.

[0205] Specifically, (b) in the case of the gelatin mixture group, there were 10 genes (ITGB5, GREM1, MMP1, CTGF, THBS1, TGFB2, MMP3, STAT1, ITGB3, IL13RA2) whose expression levels increased by more than four times compared to the control group. In particular, the expression of CTGF (Connective Tissue Growth Factor) and GREM1, growth factors involved in connective tissue formation, was found to be more than four times higher, and tissue remodeling enzymes MMP-1 and MMP-3 were also actively expressed. This suggests that gelatin has the characteristic of strongly promoting the formation of physical tissue structures and metabolism during the initial healing process.

[0206] On the other hand, (a) in the hyaluronic acid (HA) mixture group, three genes (THBS1, ITGB3, ACTA2) were identified with expression levels increasing by more than four times compared to the control group, showing a distinct and stable gene expression pattern. Specifically, the HA mixture group exhibited the following functional regenerative mechanisms.

[0207] First, ITGB3 (Integrin beta-3), which mediates adhesion between cells and the matrix, and ACTA2 (Alpha-smooth muscle actin), which is essential for the contraction and structural repair of the wound site, increased significantly by more than four times, strongly inducing physical tissue regeneration.

[0208] In addition, remodeling enzymes such as MMP-1 and MMP-14 were appropriately expressed at a twofold level, creating a remodeling environment that stably rearranged tissues rather than causing rapid changes.

[0209] In addition, rather than the rapid increase in CTGF (tissue formation factor) expression observed in the gelatin group, the expression of factors necessary for cell adhesion and contraction was prominent, allowing for the expectation of balanced tissue regeneration.

[0210] This suggests that hyaluronic acid (HA) is the most ideal biocompatible polymer that induces stable remodeling by selectively enhancing cell adhesion and functional repair (contraction) without the risk of excessive inflammation or fibrosis when mixed with dry-derived extracellular matrix powder.

[0211] Experimental Example 13: Analysis of Wound Healing-Related Gene Expression According to Type of Biocompatible Polymer

[0212] To compare and evaluate the effect of the composition of the present invention on gene expression throughout wound healing, qPCR analysis was performed using a wound healing-related gene panel. Specifically, RNA extraction and cDNA synthesis were carried out in the same manner as in Experimental Example 12, but RT-qPCR was performed targeting a wound healing-related gene panel. The expression level of each gene was calculated as the relative fold change relative to the control group and displayed as a bar graph.

[0213] FIGS. 24 to 26 are graphs showing changes in wound healing-related gene expression following treatment with a hyaluronic acid (HA) mixed composition, and FIGS. 27 to 29 are graphs showing changes in wound healing-related gene expression following treatment with a gelatin mixed composition.

[0214] Referring to Figures 24 to 29, differences in healing mechanisms according to the characteristics of each polymer were confirmed in the gene expression patterns between the two experimental groups.

[0215] Specifically, (a) in the hyaluronic acid (HA) mixture group, 9 genes that increased by more than twofold and 11 genes that decreased by more than 50% were identified compared to the control group. In the HA mixture group, potent immune cell and regenerative cell attracting factors (chemokines) such as CXCL1 and CCL2 increased by more than twofold. This is a unique characteristic of HA not observed in the gelatin mixture group, indicating that the cell recruitment mechanism, which rapidly attracts necessary cells to the wound site, is operating powerfully; this is consistent with the results of Experimental Example 10 (cell motility). Furthermore, ACTA2, which is involved in wound contraction, and TAGLN, a cytoskeletal protein, increased to promote physical wound closure, while early growth factors such as HGF, FGF7, and TIMP1 showed a pattern of decreasing by more than 50% compared to the control group. This suggests that after undergoing the initial recruitment phase, HA inhibits excessive proliferation and regulates the transition to the tissue maturation and stabilization phases.

[0216] On the other hand, in the (b) gelatin mixed group, 16 genes with expression levels more than doubled compared to the control group were identified, and no genes with decreased expression were observed. The gelatin group showed a significant increase in collagen genes such as COL1A1 and COL1A2, as well as degrading enzymes such as MMP1 and MMP2, and an increase in PTGS2 (COX-2), an inflammation and pain mediator. This demonstrates that gelatin has the characteristic of actively inducing matrix metabolism and inflammatory healing responses necessary in the early stages of wound healing. However, an increase in the expression of key inducing factors such as CXCL1 and CCL2, which were characteristic of the HA group, was not observed.

[0217] In summary, it was confirmed that while gelatin aids in initial structure formation through active metabolism, hyaluronic acid (HA) is an optimal biocompatible polymer that enables efficient tissue regeneration by strongly activating cell recruitment signals and rapidly inducing tissue maturation.

[0218] The foregoing description of this specification is for illustrative purposes only, and those skilled in the art to which one aspect of this specification pertains will understand that other specific forms can be easily modified without altering the technical concept or essential features described in this specification. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0219] The scope of this specification is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of this specification.

Claims

1. Dry-derived extracellular matrix powder; One or more biocompatible polymers selected from the group consisting of hyaluronic acid, gelatin, collagen, alginate, chitosan, elastin, fibronectin, laminin, and fibrinogen; and Pharmaceutically acceptable carrier; A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon disease, comprising 2. In Paragraph 1, The above-mentioned dry tissue-derived extracellular matrix powder is a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, having a crude fat content of less than 5% by weight.

3. In Paragraph 1, The above-mentioned tendon-derived extracellular matrix powder is a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, having a TIMP-1 protein content of 20,000 pg or more per 1 mg of powder.

4. In Paragraph 1, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, wherein the above-mentioned tendon-derived extracellular matrix powder is included in an amount of 0.5 to 15 weight% based on 100 weight% of the total composition.

5. In Paragraph 1, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, wherein the above-mentioned biocompatible polymer is included in an amount of 0.5 to 15 weight percent based on 100 weight percent of the total composition.

6. In Paragraph 1, The above biocompatible polymer is hyaluronic acid, gelatin, or a mixture thereof, and is a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases.

7. In Paragraph 1, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon disease, wherein at least a portion of the above-mentioned biocompatible polymer is crosslinked by a crosslinking agent.

8. In Paragraph 7, The above crosslinking agent is one or more selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and 1,2-(bis(2,3-epoxypropoxy)ethylene), a ligament Or a pharmaceutical composition for the prevention, improvement, or treatment of tendon disease.

9. In Paragraph 7, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, comprising 1 to 99 weight% of a biocompatible polymer crosslinked by the crosslinking agent, based on 100 weight% of the above biocompatible polymer.

10. In Paragraph 2, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, wherein the crude fat content of the above-mentioned extracellular matrix powder derived from the tendon is 3% by weight or less.

11. In Paragraph 3, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, wherein the TIMP-1 protein content contained in 1 mg of the above-mentioned tendon-derived extracellular matrix powder is 38,000 pg or more.

12. In Paragraph 3, The above-mentioned extracellular matrix powder is a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, wherein the biocompatibility index, which is the value obtained by dividing the above-mentioned TIMP-1 protein content (pg / mg) by the above-mentioned crude fat content (weight%), is 10,000 or higher.

13. In Paragraph 12, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, having a biocompatibility index of 30,000 or higher.

14. In Paragraph 1, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, wherein the average particle size of the above-mentioned extracellular matrix powder derived from the tendon is 425 μm or less.

15. In Paragraph 1, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon disease, wherein the above-mentioned tendon-derived extracellular matrix powder is prepared by a method comprising steps (a) to (e) below: (a) Step of collecting tendon tissue; (b) a step of decellularizing the above dry tissue to obtain a dry tissue-derived extracellular matrix; (c) a step of freeze-drying the above-mentioned dry extracellular matrix; (d) a step of degreasing the freeze-dried dry-derived extracellular matrix; and (e) A step of obtaining a dry-derived extracellular matrix powder by degassing the above-mentioned degreased dry-derived extracellular matrix and then freeze-grinding it.

16. In Paragraph 1, A pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, having a pH of 5.0 to 8.

0.

17. In Paragraph 1, The above pharmaceutical composition is an injectable hydrogel formulation and is a pharmaceutical composition for the prevention, improvement, or treatment of ligament or tendon diseases, having a viscosity of 2,000 to 5,000 cP.

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