Method for preparing injectable injection composition derived from animal cartilage, and use thereof

Abstract

To provide a method of preparing an injectable injection composition derived from animal cartilage.SOLUTION: A method for preparing an injection composition for preventing or treating osteoarthritis comprises: a) solubilizing a composition comprising a cartilage acellular matrix (CAM) derived from animal cartilage by stirring at a temperature exceeding 30°C and lower than 55°C; and b) centrifuging the solution of a), collecting the solution, and preparing the injection composition.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] The present invention relates to a method for producing an injectable animal cartilage-derived injectable composition, an injectable composition produced by the said method, and its use. [Background technology]

[0002] Conventional methods for treating damaged cartilage include debridement, bone marrow stimulating technique, osteochondrol autograft, and autologous chondrocyte transplantation. These methods are primarily invasive and performed when cartilage damage is advanced. For early-stage cartilage damage, the injection of hyaluronic acid products into the joint cavity is the most common treatment. Most invasive treatments have problems such as surgical excision, periosteal harvesting, complexity of use, high treatment costs, leakage of adult stem cells induced during bone marrow stimulation, and the formation of abnormal fibrous cartilage due to hemostatic problems. Furthermore, hyaluronic acid injections only serve a simple role in relieving pain through lubrication (Frizziero L, et al.). Clinical and Experimental Rheumatology, 01 Jul 1998, 16(4):441-449).

[0003] Therefore, in order to improve the aforementioned problems, there is an urgent need to develop therapeutic agents that allow for minimally invasive procedures while possessing therapeutic effects beyond simple lubrication.

[0004] On the other hand, the extracellular matrix (ECM) is the remaining part of a tissue after the cells have been removed. It is an aggregate of biomolecules that fills the spaces between cells and physically supports the tissue, creating an environment that can maintain the original structure of living tissues. In particular, collagen, the main component of the extracellular matrix in cartilage tissue, is known as a major component that creates the microenvironment for cell growth and differentiation, along with the tissue structure in cartilage. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Frizziero L, et al., Clinical and Experimental Rheumatology, 01 Jul 1998, 16(4):441-449 [Overview of the project] [Problems that the invention aims to solve]

[0006] As a result of our diligent efforts to treat damaged cartilage, we have completed the present invention by developing an injectable animal cartilage-derived injectable agent that can induce cartilage tissue regeneration by directly injecting an animal cartilage-derived collagen and extracellular matrix-containing biomaterial in an injectable dosage form into the application site without surgical incision. [Means for solving the problem]

[0007] One object of the present invention is to provide a method for producing an injectable composition for the prevention or treatment of osteoarthritis.

[0008] Another object of the present invention is to provide an injectable composition for the prevention or treatment of osteoarthritis, manufactured by the method described above.

[0009] Another object of the present invention is to provide a method for preventing or treating osteoarthritis, comprising the step of administering an injectable composition prepared by the above method into the joint cavity of an individual. [Effects of the Invention]

[0010] The injectable agent according to the present invention is a collagen-containing biomaterial in a dosage form that can be injected into the joint cavity. By directly injecting it into the application site with a needle without surgical incision, it can promote tissue repair and induce cartilage tissue regeneration. Furthermore, the extracellular matrix derived from animal cartilage contained in the injectable agent not only protects articular cartilage tissue but also stimulates an environment in which damaged joint tissue can regenerate by inducing intra-articular stem cells to differentiate into chondrocytes, thereby treating osteoarthritis. Thus, it can be applied as an arthritis treatment agent. [Brief explanation of the drawing]

[0011] [Figure 1] This diagram shows the injectable raw materials and the conditions for water solubility. [Figure 2] These are photographs of CAM solutions prepared over 6 hours under three different water solubility temperature conditions (4°C, 25°C, and 37°C). [Figure 3] This diagram shows the sterile conditions required for injection. [Figure 4] This is a schematic diagram illustrating a method for producing an injectable porcine cartilage-derived injectable agent (CAM solution) according to the present invention. [Figure 5] This figure shows the protein content of injectable porcine cartilage-derived injectable drugs before and after sterilization, as confirmed by SDS-page. [Figure 6] This is the result of confirming the collagen content under different water-solubilization process conditions. [Figure 7] This result demonstrates the cartilage differentiation ability under different water-solubilization process conditions. [Figure 8] This study examined the cartilage tissue regeneration capacity in a rat osteoarthritis model induced by anterior cruciate ligament amputation, comparing administration of umbilical cord blood-derived mesenchymal stem cells for 8 weeks (stem cell administration group) with administration of stem cells and CAM solution prepared using the process shown in Figure 5 for 8 weeks (stem cell + CAM solution administration group). Scale bar = 100 μm. DESCRIPTION OF EMBODIMENTS

[0012] A specific description is given as follows. On the other hand, each description and embodiment disclosed in the present invention can also be applied to other different descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, the scope of the present invention is not considered to be limited by the following specific description.

[0013] One aspect of the present invention for achieving the above object provides a method for producing an injectable composition for preventing or treating osteoarthritis.

[0014] Specifically, the method a) stirring and solubilizing a composition containing animal cartilage-derived acellular extracellular matrix (cartilage acellular marix; CAM) at a temperature higher than 30°C and lower than 55°C; and

[0015] b) centrifuging the aqueous solution of step a), collecting the solution and preparing the injectable composition; but it is not limited thereto.

[0016] In the present invention, step a) may be a step of stirring and solubilizing the composition containing CAM at a temperature higher than 30°C and lower than 55°C.

[0017] In the present invention, the term "acellular extracellular matrix (cartilage acellular marix; CAM)" can be used as a raw material for the injectable composition of the present invention. This can be produced by isolating cartilage from an animal through a known process and decellularizing it via enzymatic or physicochemical methods, or a commercially available product can be used. For example, a product from ATEMS can be used, but it is not limited thereto.

[0018] The CAM may take various forms, such as powder or sponge, and is not particularly limited thereto.

[0019] Furthermore, the animal from which the CAM can be obtained is not particularly limited as long as it is an animal that has cartilage tissue, but it may specifically be a pig.

[0020] In the present invention, the CAM may be sterilized.

[0021] The sterilization method may be one well known in the industry, such as dry heat sterilization, moist heat sterilization, EO (Ethylene oxide) gas sterilization, plasma sterilization, filtration sterilization, or radiation sterilization methods such as X-ray, electron beam, or gamma ray. Specifically, in the present invention, sterilization may be performed by electron beam (E-beam) or gamma ray sterilization method, and more specifically, when sterilization is performed by electron beam sterilization method, sterilization may be performed by irradiating with an E-beam of about 15 to 25 kGy, and the E-beam of about 15 to 25 kGy may be a VDmax 15 E-beam. Also, when sterilization is performed by gamma ray sterilization method, sterilization may be performed by irradiating with gamma rays of about 25 to 40 kGy, and the gamma rays of about 25 to 40 kGy may be a VDmax 25 gamma ray. However, it is not limited thereto.

[0022] In one embodiment of the present invention, it was confirmed that CAM sterilized by irradiation with gamma rays at a VDmax of 25, indicating 25-40 kGy, exhibits an easily injectable dosage form when solubilized under specific temperature and time conditions, while not sterilized, it does not become solubilized and is not injectable (Figures 1 and 2).

[0023] In this invention, the term "about" may be presented before a specific numerical value. As used in this application, the term "about" includes not only the exact number that follows the term, but also all numerical values ​​within an equivalent or similar range. The context in which the number is presented can be considered to determine whether it is similar to or approximately the specific number mentioned. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. Another example is that the term "about" may refer to a range of -5% to +5% of a given numerical value. However, it is not limited to this.

[0024] In the present invention, "water solubilization" means the process of manufacturing a substance into a form that is soluble in water in order to change a non-injectable dosage form into an injectable dosage form. This water solubilization may be carried out by stirring under specific temperature and time conditions.

[0025] The aforementioned stirring method can be any method known in the industry, and is not limited to, for example, stirring can be performed using a stirring incubator.

[0026] In the present invention, the temperature conditions in step a) may be greater than approximately 30°C and less than 55°C, approximately 33°C to 41°C, specifically around 37°C.

[0027] Under temperature conditions lower than those mentioned above, the viscosity of the manufactured solution may increase, preventing the production of an injectable dosage form. Under higher temperature conditions, the collagen content in the manufactured solution may decrease significantly, preventing differentiation of stem cells into chondrocytes and potentially reducing the preventive or therapeutic effect on osteoarthritis.

[0028] In the present invention, the solubilization in step a) may be carried out in less than approximately 24 hours, less than approximately 20 hours, specifically in about 6 to 18 hours.

[0029] If the process is carried out in a shorter time than the aforementioned conditions, water solubility may not occur, resulting in a high viscosity of the manufactured solution and preventing the creation of an injectable dosage form. If the process exceeds the aforementioned time, the collagen content, which is the active ingredient in the injectable drug, may decrease significantly, potentially reducing the osteoarthritis-improving effect of the injectable drug composition.

[0030] In one embodiment of the present invention, the collagen content in an injectable composition solubilized under various temperature (37°C, 55°C) and time (6 hours, 24 hours) conditions was confirmed. The results showed that under 6-hour and 24-hour solubilization conditions, the collagen content was measured lower at 55°C (87.3 μg / mg under 55°C and 6 hours, 61.8 μg / mg under 55°C and 24 hours) compared to 37°C (138.3 μg / mg under 37°C and 6 hours, 117.7 μg / mg under 37°C and 24 hours). This confirmed that the collagen content decreases when the solubilization time is 24 hours or longer and the solubilization temperature is less than 55°C (Figure 6).

[0031] In the present invention, step b) may be a step in which the aqueous solution of a) is centrifuged and the solution is collected to produce an injectable composition.

[0032] The centrifugation may be performed with approximately 100 to 500 g for 1 to 10 minutes, and more specifically, it may be performed with approximately 300 g for 5 minutes, but is not limited thereto.

[0033] The injectable composition of the present invention manufactured as described above can be mixed with a CAM solution or a CAM- solution.

[0034] The method of the present invention may further include a step of freezing the injectable composition of the present invention manufactured after step b) above. Such a freezing step may facilitate storage and handling.

[0035] The freezing method described above can be used without limitation, provided it is a method that is publicly known in this industry.

[0036] The method of the present invention may further include a step of sterilizing the injectable composition of the present invention manufactured after step b) above.

[0037] The sterilization method described above is as stated above, and in the above stage, sterilization may be performed by irradiating with an E-beam of about 15 to 25 kGy, but is not limited thereto. By the above sterilization method, only impurities can be removed without the loss or destruction of collagen, which is the active ingredient in the injectable composition of the present invention.

[0038] In one embodiment of the present invention, when the injectable composition of the present invention is manufactured under water-solubility temperature conditions of 33°C and 35°C, it was confirmed that both the pre- and post-sterilization solutions using an E-beam with a VDmax of 15 and an intensity of 15-25 kGy are injectable.

[0039] In another embodiment of the present invention, the proteins in the injectable composition of the present invention before and after sterilization were examined using SDS-page. The results showed that the injectable composition in the unsterilized state mainly contained a protein that is collagen type 2 alpha, corresponding to approximately 130 kDa. After the sterilization process using an E-beam with Vmax 15, there was a slight decrease, but the main protein content remained unchanged (Figure 5).

[0040] Another aspect of the present invention provides an injectable composition derived from animal cartilage for the prevention or treatment of osteoarthritis, manufactured by the method of the present invention.

[0041] The terminology used here is as described above.

[0042] The injectable composition of the present invention may contain collagen at a concentration of approximately 120 μg / mg or more relative to the total weight of the composition, but is not limited thereto.

[0043] The injectable composition of the present invention may also induce differentiation of stem cells into chondrocytes.

[0044] The injectable composition of the present invention may also have tissue repair and cartilage tissue regeneration effects.

[0045] In one embodiment of the present invention, injectable compositions solubilized under various temperature (37°C, 55°C) and time (6 hours, 24 hours) conditions were added to the differentiation medium of umbilical cord blood-derived mesenchymal stem cells to induce chondrocyte differentiation for two weeks. The results showed that differentiation from stem cells to chondrocytes was induced when the injectable composition produced by the 37°C and 6-hour solubilization process was added to the differentiation medium. However, differentiation to chondrocytes was not induced when the injectable compositions solubilized under the conditions of 37°C and 24 hours, 55°C and 6 hours, and 55°C and 24 hours were added to the differentiation medium (Figure 7).

[0046] The injectable composition of the present invention may be administered in combination with stem cells.

[0047] The aforementioned mixed administration means administering the pharmaceutical composition of the present invention and stem cells simultaneously, with a time delay, independently, or in combination for the prevention, treatment, reduction, or alleviation of disease symptoms. The aforementioned mixed administration may be used in combination with concomitant administration.

[0048] In this invention, the term "stem cell" refers to a cell that has the ability to differentiate into various tissues, i.e., an undifferentiated cell. The stem cell may be a pluripotent stem cell, an adult stem cell, an induced pluripotent stem cell, an embryonic stem cell, or an adult stem cell.

[0049] The aforementioned stem cells may be of human or animal origin, and may be derived from umbilical cord, umbilical cord blood, cartilage, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta, but are not limited thereto.

[0050] The stem cell of the present invention may specifically be an adult stem cell. Adult stem cells are undifferentiated cells that differentiate into cells of specific tissues when needed, and can be extracted from already grown body tissues such as bone marrow and brain cells. The adult stem cell may be, but is not limited to, any one or more stem cells selected from the group consisting of mesenchymal stem cells, mesenchymal stem cells, and pluripotent stem cells.

[0051] More specifically, the stem cell of the present invention may be a mesenchymal stem cell, and even more specifically, may be a mesenchymal stem cell derived from umbilical cord blood, but is not limited thereto.

[0052] The co-administered stem cells may be about 1.0×10 5 cells to 1.0×10 8 cells, specifically about 2.5×10 5 cells to 1.0×10 8 cells, about 1.0×10 7 cells to 1.0×10 8 cells, or about 1.0×10 7 cells to 5.0×10 7 cells, more specifically about 2.5×10 7 cells, but is not limited thereto.

[0053] As described above, when the injectable composition of the present invention is co-administered with stem cells, the injectable composition may be contained in an amount of about 1 to 5% by weight relative to the total weight of the mixed composition to be administered, specifically about 1.5 to 3% by weight, more specifically about 2% by weight, but is not limited thereto.

[0054] In one embodiment of the present invention, 2.5×10 umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) were injected into the joint cavity of an osteoarthritis model induced by anterior cruciate ligament transection in rats 5 cells / group was administered for 8 weeks (stem cell administration group), hUCB-MSC (2.5×10 5When cells (group) and the CAM solution (2% by weight, 1 mg / group), which is the injectable composition of the present invention, were administered together for 8 weeks (stem cell + CAM solution administration group), the cartilage tissue regeneration ability was confirmed to be significantly superior in the group in which stem cells were administered mixed with the injectable composition of the present invention compared to the stem cell administration group in which umbilical cord blood-derived mesenchymal stem cells were treated alone (Figure 8).

[0055] The injectable composition of the present invention may be a pharmaceutical composition.

[0056] The pharmaceutical compositions of the present invention have use for the "prevention" and / or "treatment" of osteoarthritis. In preventive use, the pharmaceutical compositions of the present invention may be administered to individuals who have or are suspected of having the disease or symptoms described in the present invention. In therapeutic use, the pharmaceutical compositions of the present invention are administered in an amount sufficient to treat, or at least partially cessate, the disease or symptoms described in the present invention to individuals such as patients who already have the disease described in the present invention. The amount effective for such use depends on the severity and course of the disease or symptoms, previous treatments, the individual's health status and responsiveness to the drug, and the judgment of a physician or veterinarian.

[0057] The pharmaceutical composition of the present invention may further contain a suitable carrier, excipient, or diluent, which is typically used. In this case, the content of the injectable composition of the present invention, which is the active ingredient in the pharmaceutical composition, is not particularly limited, but may be about 1 to 5% by weight, specifically about 1.5 to 3% by weight, relative to the total weight of the pharmaceutical composition.

[0058] The pharmaceutical composition may have one dosage form selected from the group consisting of sterile aqueous solutions, non-aqueous solvents, suspensions, tablets, pills, powders, granules, capsules, oral solutions, emulsions, syrups, oils, freeze-dries, and suppositories, and may be in various parenteral or oral dosage forms. When compounding, it is prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and such solid dosage forms are prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[0059] In the present invention, the pharmaceutical composition of the present invention is a sterile aqueous solution for parenteral administration, non-aqueous solution It may be formulated as a solvent or suspension, and more specifically, it may be formulated as an injectable preparation.

[0060] The injectable composition of the present invention can be administered to an individual in a pharmaceutically effective amount.

[0061] In this invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective dose may be determined by factors including the individual's species and severity, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0062] The injectable composition of the present invention may be administered as an individual therapeutic agent, mixed with other therapeutic agents, administered sequentially or simultaneously with conventional therapeutic agents, or administered as a single or multiple dose. It is important to administer the amount that provides the greatest effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by those skilled in the art. The preferred dosage of the injectable composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the duration, and the administration may be once a day or divided into several doses.

[0063] In the present invention, the injectable composition of the present invention may be administered in combination with stem cells, as described above.

[0064] The injectable compositions of the present invention can be administered without particular limitation to individuals for the prevention or treatment of osteoarthritis. The method of administration is not limited to any method commonly used in the art. The injectable compositions may be administered intra-articular (e.g., intracartilage), subcutaneously, intraperitoneally, intrapulmonaryly, and intranasally, and for local treatment, they may be administered by a suitable method, if necessary, including administration to the lesion, i.e., intra-articular (e.g., intracartilage). For example, they may be administered (injected) by intra-articular injection.

[0065] The injectable composition of the present invention may generally be administered at a concentration of about 1 to 5% by weight, specifically, about 1.5 to 3% by weight, but is not limited thereto.

[0066] Another aspect of the present invention provides a method for preventing or treating osteoarthritis, comprising the step of administering the injectable composition of the present invention into the joint cavity of an individual.

[0067] The terminology used here is as described above.

[0068] In the present invention, the term "individual" refers to any animal that has or may have osteoarthritis, and the injectable composition of the present invention can be administered to an individual suspected of having osteoarthritis to treat the individual efficiently. The individual is not particularly limited and may be, but is not limited to, animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs, goats, birds, etc.

[0069] In the present invention, the term "administration" means introducing the injectable composition of the present invention into an individual suspected of having osteoarthritis by any appropriate method, and the route of administration can be through a variety of parenteral routes, as long as it can reach the target tissue. The injectable composition of the present invention can be administered in a pharmaceutically effective amount, which is as described above.

[0070] Furthermore, in the present invention, the injectable composition of the present invention may be administered in mixture with stem cells, as described above.

[0071] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples, which would be obvious to those with ordinary skill in the art to which the present invention pertains.

[0072] Example 1. Confirmation of injectable raw materials and water-solubilization conditions. To ensure a process for producing injectable CAM solutions, three types of raw materials (sterilized CAM-WS powder, unsterilized CAM-WS sponge, and sterile CAM-WS sponge) (ATEMS, South Korea) were eluted in a stirring incubator under three different water solubilization temperatures (4°C, 25°C, 37°C) and three different water solubilization times (3 hours, 6 hours, 18 hours). The CAM solutions were then prepared by centrifuging 300g for 5 minutes, and the injectability of each solution was analyzed. Sterilization was performed by irradiation with gamma rays at a VDmax of 25, indicating a dose of 25-40 kGy.

[0073] As a result, as shown in Figure 1 and Figure 2, which shows CAM solutions produced for 6 hours under three different water-solubility temperature conditions (4°C, 25°C, and 37°C), it was confirmed that an injectable CAM solution could not be produced using unsterilized CAM-WS sponge raw materials. However, when produced using sterilized CAM-WS powder and sterilized CAM-WS sponge raw materials, an injectable form of CAM solution could be produced under the water-solubility temperature condition of 37°C.

[0074] Example 2. Confirmation of sterile conditions for injection To ensure a manufacturing process for CAM solutions that are injectable after sterilization, CAM solutions were prepared using sterile CAM-WS sponges under three different water-solubility temperature conditions (30°C, 33°C, 35°C) and two different water-solubility time conditions (6-hour elution, 18-hour elution). After sterilization by irradiation with an E-beam with a VDmax of 15 (15-25 kGy), the injectability was analyzed.

[0075] As a result, as can be seen in Figure 3, when the CAM solution was produced at 30°C, it was found to be difficult to inject both before and after sterilization. When the CAM solution was produced at 33°C and 35°C, it was found to be injectable both before and after sterilization.

[0076] Through this process, we confirmed that when a CAM solution is manufactured including a step of solubilization under temperature conditions exceeding 30°C, the manufactured CAM solution has sterilization stability.

[0077] Based on the results of Example 2 described above, a manufacturing process for a CAM solution that can be injected even after sterilization was established, and a representative example of this manufacturing process is shown in Figure 4.

[0078] Example 3. Confirmation of collagen content before and after sterilization in the injection process. The proteins in the CAM solution produced in the process shown in Figure 4, both before and after sterilization, were examined using SDS-page.

[0079] As a result, as can be seen in Figure 5, it was confirmed that the unsterilized CAM solution mainly contains a protein that is collagen type 2 alpha, corresponding to approximately 130 kDa. Although there was a slight decrease after the E-beam sterilization process with VDmax 15, it was confirmed that the main protein content remained unchanged.

[0080] Through this process, the optimal temperature for water solubility in the CAM solution manufacturing process for injectable dosage forms was selected as being above 30°C and below 55°C, and the optimal time as less than 24 hours.

[0081] Example 4. Confirmation of collagen content under water-solubilization process conditions To confirm the difference in collagen content depending on the water solubilization process conditions, sterile CAM-WS sponges were eluted in a stirring incubator under two different water solubilization temperatures (37°C and 55°C) and two different water solubilization times (6 hours and 24 hours). CAM solutions were then prepared by centrifuging 300g for 5 minutes, and the collagen content of each solution was checked.

[0082] As a result, as can be seen in Figure 6, under water solubilization conditions of 6 hours and 24 hours, the measured collagen content was lower at 55°C (87.3 μg / mg under 55°C and 6 hours conditions, and 61.8 μg / mg under 55°C and 24 hours conditions) compared to 37°C (138.3 μg / mg under 37°C and 6 hours conditions, and 117.7 μg / mg under 37°C and 24 hours conditions). This confirmed that collagen content decreases when the water solubilization time is 24 hours or longer and the water solubilization temperature is 55°C or higher.

[0083] Example 5. Comparison of cartilage differentiation ability under water-solubilization process conditions. A CAM solution prepared under the same water-solubilizing conditions as in Example 4 was added to the differentiation medium for umbilical cord blood-derived mesenchymal stem cells, and chondrocyte differentiation was induced for two weeks. The cells were then observed through Safranin-O staining.

[0084] As a result, as shown in Figure 7, differentiation from stem cells to chondrocytes was induced when the CAM solution produced by a water-solubilization process at 37°C for 6 hours was added to the differentiation medium. On the other hand, CAM solutions solubilized under conditions of 37°C for 24 hours, 55°C for 6 hours, and 55°C for 24 hours did not induce differentiation to chondrocytes when added to the differentiation medium.

[0085] As can be seen from the results above, the injectable agent produced through the process derived in the above-described example has cartilage differentiation efficacy and was confirmed to be suitable for use as a treatment for osteoarthritis.

[0086] Example 6. Effect of enhancing cartilage regeneration ability To confirm the cartilage regeneration-enhancing effect of the CAM solution produced under the same water-solubilizing conditions as in Example 4, a rat osteoarthritis model induced by anterior cruciate ligament amputation was created.

[0087] Specifically, 65 rats that were determined to be free of quarantine abnormalities during the purification period were anesthetized by intraperitoneal injection of a mixture of ketamine 20 mg / kg (Yuhan Corp., Seoul, Korea) and xylazine 3 mg / kg (Rompun; Bayer Korea Corp., Seoul, Korea). Hair was removed from the right knee joint and proximal tibia of each individual using a depilator, and after disinfection with betadine, a longitudinal incision of approximately 1 cm was made at the medial aspect of the knee joint, centering on the tibial tuberosity (medial parapatellar approach). The joint capsule was exposed below the incision and incised, and after positioning the patella laterally, the knee was flexed to expose the anterior cruciate ligament and meniscus in the surgical field. The intermediate parenchyma of the anterior cruciate ligament was completely incised using a micro-scissor, and the ligament's support to the tibial bone was confirmed to be loose through the positive anterior drawer's test. Subsequently, absorbable sutures 4-0 were used. Monosyn(R)(B. Braun Surgical SA, Rubi, S The joint capsule and subcutaneous tissue were continuously sealed using pain (pain), and the skin was simply knotted with non-absorbable suture 4-0 Nylon (Blue nylon, Ailee Co., Ltd., South Korea). To prevent infection after the surgery, the surgical site was disinfected with betadine for 3-4 days, and cefazolin (Cefazol®, Ailee Co., Ltd.) was used. Glubet (Korea) was administered intramuscularly once daily for three days at a dose of 100 mg / kg. All experimental animals were allowed to move freely in their cages for eight weeks without any separate bandage treatment at the surgical site.

[0088] The intraarticular cavity of the rat anterior cruciate ligament amputation-induced osteoarthritis model prepared as described above. 2.5 x 10⁻¹⁴ umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) 5 When cells / group were administered for 8 weeks (stem cell administration group), hUCB-MSC (2.5 x 10) 5The regenerative capacity of cartilage tissue was confirmed when both cells (per group) and a CAM solution (2% by weight, 1 mg / group) prepared in the process shown in Figure 4 were administered for 8 weeks (stem cell + CAM solution administration group). Cartilage tissue was observed through Safranin-O staining at 8 weeks after administration.

[0089] As a result, as shown in Figure 8, we confirmed that the group in which hUCB-MSC was administered mixed with CAM solution showed significantly superior cartilage tissue regeneration ability compared to the group in which hUCB-MSC was administered alone.

[0090] Based on the results of the above-mentioned examples, the injectable agent according to the present invention not only protects articular cartilage tissue by containing extracellular matrix and collagen derived from porcine cartilage, but also induces intra-articular stem cells to differentiate into chondrocytes and stimulates an environment in which damaged joint tissue can regenerate, thereby improving osteoarthritis. Therefore, it can be applied as a therapeutic agent for arthritis.

[0091] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. a) A step of solubilizing a composition containing an animal cartilage-derived cell-free extracellular matrix (cartilage acetular marix; CAM) by stirring it at a temperature above 30°C and below 55°C; and b) A method for producing an injectable composition for the prevention or treatment of osteoarthritis, comprising the step of centrifuging the aqueous solution of a) and collecting the solution to produce an injectable composition.

2. The method according to claim 1, wherein step a) is performed in less than 24 hours.

3. The method according to claim 1, wherein the CAM derived from animal cartilage is sterilized.

4. The method according to claim 1, further comprising the step of sterilizing the manufactured injectable composition.

5. The method according to claim 1, wherein the animal is a pig.

6. An injectable composition for the prevention or treatment of osteoarthritis, manufactured by the method described in claim 1.

7. The injectable composition according to claim 6, wherein the injectable composition contains collagen at a concentration of 120 μg / mg or more based on the total weight of the composition.

8. The injectable composition according to claim 6, wherein the injectable composition induces the differentiation of stem cells into chondrocytes.

9. The injectable composition according to claim 6, wherein the injectable composition has tissue repair and cartilage tissue regeneration effects.

10. The injectable composition according to claim 6, wherein the injectable composition is administered in mixture with stem cells.

11. The stem cells administered in the aforementioned mixture are 1.0 × 10 ５ ce11s~1.0×10 ８ The injectable composition according to claim 10, wherein the cells are cells.

12. A method for preventing or treating osteoarthritis, comprising the step of administering the injectable composition according to claim 6 into the joint cavity of an individual.

13. The method according to claim 12, wherein the injectable composition is administered in mixture with stem cells.

14. The stem cells administered in the aforementioned mixture are 1.0 × 10 ５ cells~1.0×10 ８ The method according to claim 13, wherein cells.

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