Injectable hydrogel composition for treating joint diseases and manufacturing method therefor

Abstract

The present invention relates to a hydrogel composition for joint cavity administration, which is easy to inject into the human body, and uses thereof for preventing or treating lesions in joints, and regenerating cartilage. The hydrogel composition according to the present invention gels in the body and slowly decomposes at a lesion site for two to six months to supplement a defective site, and slowly releases active ingredients such as stem cells or exosomes to the lesion site, thereby inducing continuous inflammation inhibition and cartilage regeneration effects. Therefore, the composition of the present invention can be variously used in the fields of medical devices and pharmaceuticals for the purpose of regenerating cartilage and treating lesions in joints.

Description

Injectable hydrogel composition for treating joint diseases and method for preparing the same

[0001] The present invention relates to a hydrogel composition for intra-articular administration that is easy to inject into the human body and its use for preventing or treating intra-articular lesions and regenerating cartilage.

[0002] Osteoarthritis, also known as degenerative arthritis or degenerative joint disease, is a disease that causes pain and functional impairment due to deformation of articular cartilage and bones caused by degenerative changes in the joints. Osteoarthritis can be classified as primary, idiopathic arthritis, which occurs without a specific antecedent cause, and secondary, secondary arthritis, which is caused by trauma, disease, or deformity that can damage articular cartilage. The prevalence rate continues to increase with aging.

[0003] Unlike rheumatoid arthritis, which exhibits systemic symptoms, osteoarthritis initially presents with localized pain when the joint is used. However, the pain gradually worsens and, as the disease progresses, becomes persistent, regardless of joint use. Patients experience decreased range of motion, swelling, and tenderness around the joints. Joint surfaces may become irregular, causing a crepitus. Treatment for osteoarthritis includes non-pharmacological treatments such as weight loss, lower extremity muscle strengthening exercises, and physical therapy. Pharmacological treatments include intra-articular injections of acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and steroid medications. Surgical options include joint replacement.

[0004] The meniscus is a vital tissue in the knee joint, essential for absorbing shock and maintaining joint stability. However, meniscus damage can be caused by degenerative diseases (e.g., osteoarthritis) or trauma, leading to decreased knee function and pain. Osteoarthritis is a condition that causes inflammation and cartilage destruction in the joint, and is primarily caused by aging, trauma, and genetic factors. Existing treatments primarily rely on surgery or medication, which have long recovery times and the risk of recurrence.

[0005] Corticosteroids are mainly used along with NSAIDs as initial treatment, but NSAIDs have a mild effect and carry a risk of causing chronic digestive disorders, and corticosteroids are known to have excellent anti-inflammatory effects, but are known to cause swelling and irritation, and damage joint cartilage with chronic use and have a high risk of causing blood circulation problems. On the other hand, intra-articular injection of hyaluronic acid, a component of synovial fluid within the joint, has the advantage of reducing friction within the joint, providing lubrication, protecting the joint surface, absorbing shock, and having no systemic side effects. It is also known to promote the natural production of hyaluronic acid within the joint, so it is widely used to improve and treat osteoarthritis pain.

[0006] However, hyaluronic acid has problems such as insufficient gelation speed and stability, inconvenience of injection due to high viscosity, imbalance in retention and decomposition properties in the body, and reduces the viability of additional substances that can enhance therapeutic effects, such as cells, so its commercialization is limited.

[0007] Therefore, there is a need for a novel hydrogel formulation that is easy to inject and effective in treating various joint lesions by inducing cartilage regeneration and inflammation relief.

[0008] Accordingly, the inventors of the present invention were researching a new material that could improve the shortcomings of existing hyaluronic acid and enhance the therapeutic effect, and confirmed that when a combination of alginate and carboxymethyl cellulose or stem cells or exosomes were added and administered intra-articularly, it could remain in the lesion site for a long time and effectively treat lesions such as cartilage regeneration and osteoarthritis.

[0009] Accordingly, the present invention aims to provide a hydrogel composition for intra-articular injection, comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose; or further comprising stem cells or exosomes, and a use thereof.

[0010]

[0011] To achieve the above purpose, the present invention provides a hydrogel composition for intra-articular administration, comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose.

[0012] The present invention also provides a hydrogel composition for preventing or treating intra-articular lesions comprising the composition.

[0013] The present invention also provides a composition for cartilage regeneration comprising the composition.

[0014] The present invention also provides a method for preventing or treating an intra-articular lesion, comprising administering to the joint space of an individual in need thereof a hydrogel composition comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose.

[0015] The present invention also provides a method for cartilage regeneration, comprising administering a hydrogel composition comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose to the joint cavity of an individual in need thereof.

[0016] The hydrogel composition according to the present invention gels in the body and gradually decomposes at the lesion site over a period of 2 to 6 months, thereby replenishing the damaged area and gradually releasing active ingredients, such as stem cells or exosomes, into the lesion site, thereby inducing sustained inflammation suppression and cartilage regeneration effects. Therefore, the composition of the present invention can be utilized in various fields, including medical devices and pharmaceuticals, for the purpose of cartilage regeneration and the treatment of intra-articular lesions.

[0017]

[0018] Figure 1 is a diagram showing the decomposition patterns of hydrogels of various concentrations produced after treating them with enzymes.

[0019] Figure 2 is a diagram showing the cell viability of stem cells mixed with hydrogel after 24 hours and 48 hours.

[0020] Figure 3 is a diagram showing the macroscopic results showing the regeneration of the meniscus defect area at 8 and 16 weeks.

[0021] Figure 4a is a diagram showing the results of Hematoxylin & Eosin (H&E), Safranin O staining, and immunostaining for Type II collagen, showing the regeneration of the meniscus defect area at 8 weeks.

[0022] Figure 4b is a diagram showing the results of Hematoxylin & Eosin (H&E), Safranin O staining, and immunostaining for Type II collagen, showing the regeneration of the meniscus defect area at 16 weeks.

[0023] Figure 4c is a diagram showing the histological tissue quality score showing the regeneration of the meniscus defect area at 8 and 16 weeks.

[0024] Figure 5 is a diagram showing the results of confirming the anti-inflammatory effect at the gene level after injection of the hydrogel composition according to the present invention in a rat osteoarthritis model (##: p<0.01)

[0025] Figure 6 is a diagram showing the results of confirming the anti-inflammatory effect at the protein level after injection of the hydrogel composition according to the present invention in a rat osteoarthritis model (##: p<0.01).

[0026] The present invention relates to a hydrogel composition for intra-articular administration, comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose; or further comprising stem cells or exosomes.

[0027] The hydrogel composition of the present invention is in an injectable form and can be directly applied to a meniscus defect or osteoarthritis site via intra-articular injection. After injection, the hydrogel rapidly gels in the body, filling the defect, and stem cells and exosomes penetrate the defect to aid tissue regeneration. This is a promising treatment method, particularly for osteoarthritis, as it can simultaneously alleviate inflammation and promote cartilage regeneration.

[0028]

[0029] Hereinafter, the present invention will be described in detail.

[0030] The present invention provides a hydrogel composition for intra-articular administration, comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose.

[0031] When the above alginate and carboxymethyl cellulose are each included in amounts of less than 1 wt%, the viscosity of the hydrogel becomes too low, so that sufficient retention time for cartilage regeneration and defect site supplementation at the injection site is not secured, and the mechanical strength is insufficient, so that the hydrogel is quickly decomposed or its shape is not maintained, so that sufficient effect for cartilage regeneration cannot be expected.

[0032] In addition, when the alginate and carboxymethyl cellulose are each included in an amount exceeding 2 wt%, the viscosity increases, which reduces the convenience of injection, the gelation time becomes longer, and immediate gelation does not occur after intra-articular injection.

[0033] Therefore, an appropriate concentration combination of alginate and carboxymethyl cellulose has a significant effect on the treatment of joint lesions, cartilage regeneration, and anti-inflammation, and it is necessary to manufacture a hydrogel composition with an optimal combination.

[0034] In the present invention, alginate and carboxymethyl cellulose were combined in various amounts of 1, 1.5, and 2%, respectively, and the viscosity, gelation time, mechanical strength, and suitability as a support were confirmed accordingly, and through this, a hydrogel for intra-articular administration comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose was derived. Preferably, the alginate and carboxymethyl cellulose of the present invention can be included in a 1:1 weight ratio.

[0035] In addition, the composition of the present invention may additionally include a substance having anti-inflammatory and cartilage regeneration effects to improve joint lesions and promote cartilage regeneration when administered intra-articularly, and may preferably further include stem cells or exosomes.

[0036] The hydrogel composition of the present invention can act as a scaffold for stem cells or exosomes administered together when administered intra-articularly, thereby allowing the stem cells or exosomes to remain in the lesion site for a long time and exhibit a therapeutic effect.

[0037] When the hydrogel of the present invention is administered together with stem cells, the hydrogel induces stem cell infiltration into the meniscus defect site, promoting fibrosis and regeneration. Furthermore, the co-administered stem cells can differentiate into fibrous tissue, thereby promoting cartilage regeneration and tissue restoration, and simultaneously alleviating inflammation and promoting cartilage regeneration.

[0038] When the hydrogel of the present invention is administered together with exosomes, the hydrogel allows the exosomes to be slowly released to the lesion site to provide a sustained effect, and the cell proliferation, inflammation reduction, and cartilage regeneration effects by the exosomes are sustained.

[0039] In the present invention, a composition mixing hydrogel and stem cells or exosomes was injected into the joint cavity, and it was confirmed that it had superior anti-inflammatory, cartilage regeneration, and pain relief effects compared to the group that administered hydrogel alone.

[0040] The above stem cells may be at least one selected from the group consisting of bone marrow stem cells, mesenchymal stem cells, embryonic stem cells, adult stem cells, and induced-pluripotent stem cells, and may be fat, placental, bone marrow, Wharton's jelly, umbilical cord, or umbilical cord blood stem cells. The stem cells may be present in the composition of the present invention in an amount of 1×10 3 1×10 10 cells / 1mL, 1×10 3 1×10 8 cells / 1mL, 1×10 5 1×10 8 cells / 1mL, 1×10 6 1×10 7 It can be included as cells / 1mL.

[0041] The stem cells of the present invention may preferably be placental-derived stem cells, and more preferably may be placental tissue-derived stem cells.

[0042] More specifically, the stem cell of the present invention is a stem cell derived from the detailed tissue of the trophoblast, and may be a stem cell characterized by a different expression pattern of CD markers compared to the stem cell derived from the entire trophoblast, and by exhibiting an XY karyotype, unlike the stem cell derived from the entire trophoblast in which XX and XY karyotypes are mixed. In the present invention, the detailed tissue of the trophoblast may be characterized by a continuous trophoblast layer portion with a thickness of 1.5 to 4 mm from the trophoblast portion adjacent to the chorionic plate among the entire trophoblast layer, and preferably may be a continuous trophoblast layer portion corresponding to a thickness of 2 to 3 mm. In addition, in the present invention, the detailed tissue of the trophoblast is preferably a tissue corresponding to 1 / 10 portion closest to the chorionic plate, when the entire trophoblast layer is divided into 10 equal parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer. The 1 / 10 portion has a length corresponding to the thickness of 1.5 to 4 mm described above.

[0043] In addition, in the present invention, the trophoblast detailed tissue is characterized in that it does not include tissue derived from the placental septum.

[0044] The above exosomes may be stem cell-derived exosomes, plant-derived exosomes, or yeast-derived exosomes, preferably stem cell-derived exosomes, and in a preferred embodiment of the present invention, exosomes isolated from placental stem cells were used. More preferably, they may be stem cells derived from the detailed tissues of the placenta described above. The exosomes may be present in the composition of the present invention in an amount of 1×10 5 1×10 15 , 1×10 5 1×10 12 , 1×10 8 1×10 10 It can be administered as particles.

[0045] The stem cells or stem cell-derived exosomes included in the composition of the present invention may include, without limitation, allogeneic, syngeneic, or xenogeneic origins.

[0046]

[0047] When stem cells or exosomes are additionally included in the composition of the present invention, the hydrogel and stem cells or exosomes may be divided into one compartment or multiple compartments, and in this case, may be provided in the form of a kit including individual compartments. The kit of the present invention may further include instructions regarding user instructions.

[0048]

[0049] The hydrogel composition of the present invention exists in an injectable liquid form and gels in the body by reacting with calcium ions. The hydrogel of the present invention can exhibit gelation properties at temperatures between 25 and 37°C and can exhibit buffering and cushioning effects when administered intra-articularly.

[0050] In addition, the hydrogel composition of the present invention may be for intra-articular injection, and may be characterized as being for 26 to 29G injection.

[0051] When the hydrogel composition of the present invention is injected into the body, it can be gradually decomposed and maintained in the body for 2 to 6 months, thereby providing a long-term joint regeneration effect and mechanical support at the site of defects and lesions.

[0052] The hydrogel composition of the present invention can be used for anti-inflammatory and cartilage regeneration purposes. When the hydrogel of the present invention is administered to a lesion, inflammation is reduced and tissue regeneration is activated at the lesion site. Over a maintenance period of 2 to 6 months, the mechanical strength of the lesion site can be restored to a level similar to that of normal cartilage.

[0053] In a preferred embodiment of the present invention, a hydrogel composition for meniscus regeneration is provided, comprising 1.2 to 1.8 wt% of alginate and 1.2 to 1.8 wt% of carboxymethyl cellulose. The mixed composition of alginate and carboxymethyl cellulose is at a concentration that can well balance mechanical strength and cell compatibility, and the hydrogel can remain appropriately hard while being injectable, making it suitable for the purpose of meniscus regeneration. The concentration may be preferably 1.3 to 1.6 wt% of alginate and 1.3 to 1.6 wt% of carboxymethyl cellulose, more preferably 1.4 to 1.6 wt% of alginate and 1.4 to 1.6 wt% of carboxymethyl cellulose, or 1.5 wt% of alginate and 1.5 wt% of carboxymethyl cellulose.

[0054] In addition, the present invention provides a hydrogel composition for treating osteoarthritis, comprising, as a preferred embodiment, 0.7 to 1.2 wt% of alginate and 0.7 to 1.2 wt% of carboxymethyl cellulose. The mixed composition of alginate and carboxymethyl cellulose provides flexibility and soft gelation to minimize pain upon injection, does not interfere with natural movement within the joint space, and can provide an effective environment for cartilage regeneration and inflammation suppression when combined with cell-based therapy, and is therefore suitable for the purpose of treating osteoarthritis. The concentration may be preferably 0.8 to 1.2 wt% of alginate and 0.8 to 1.2 wt% of carboxymethyl cellulose, more preferably 0.9 to 1.1 wt% of alginate and 0.9 to 1.1 wt% of carboxymethyl cellulose, or 1.0 wt% of alginate and 1.0 wt% of carboxymethyl cellulose.

[0055]

[0056] The hydrogel composition of the present invention can be used as a synovial fluid supplement classified as a biomaterial (medical device) for tissue repair, and can be used by injecting it into the joint cavity after surgery to replace synovial fluid lost after arthroscopic surgery.

[0057] The above arthroscopic surgery may include surgery related to ligament reconstruction and suturing, surgery related to meniscus resection and suturing, and preferably knee arthroscopic surgery. Therefore, the present invention may be administered before or after partial or total meniscectomy, meniscal repair, removal of loose body, synovectomy, removal or fixation of osteochondritis dissecans, repair or reconstruction of cruciate ligament, and fracture fixation, and may be effectively used to supplement intra-articular viscosity, reduce friction, supplement intra-articular synovial fluid, and regenerate cartilage.

[0058] Accordingly, the present invention provides a hydrogel composition for preventing or treating intra-articular lesions, comprising the composition.

[0059] The hydrogel composition for preventing or treating intra-articular lesions according to the present invention can be used in the form of a pharmaceutical composition or medical device by direct injection or implantation into a target area, such as a joint.

[0060] The above-mentioned intra-articular lesion may be a fibrocartilage or elastic cartilage defect, which refers to a fibrocartilage injury caused by mechanical stimulation or inflammatory reaction of fibrocartilage, fibrocartilaginous tissue and / or joint tissue (synovium, joint capsule, subchondral bone, etc.), or an elastic cartilage injury or defect caused by other congenital or acquired factors. It may be caused by one or more diseases selected from the group consisting of osteoarthritis, degenerative arthritis, rheumatoid arthritis, meniscus injury, intervertebral disc herniation, pubic disc injury, temporomandibular joint injury, articular disc injury of the sternoclavicular joint, triangular fibrocartilage complex rupture of the wrist joint, ulnar impingement syndrome, external ear defect, and epiglottis or laryngeal cartilage defect.

[0061] Therefore, the hydrogel composition of the present invention is used for any disease selected from the group consisting of osteoarthritis, degenerative arthritis, inflammatory arthritis, rheumatoid arthritis, osteochondromatosis, distortion, bursitis, menstruation, meniscal tear, meniscus cyst, collateral ligament rupture, anterior cruciate ligament injury, posterior cruciate ligament injury, intra-articular vitreous, exfoliation osteochondritis, plica syndrome, genu varum, knock-knee, and snapping knee. It can be administered to more than one disease.

[0062] Administration of the pharmaceutical composition or medical device according to the present invention may be performed frequently, daily, weekly, several times a week, bimonthly, several times a month, monthly, or as frequently as necessary to provide symptom relief. For intra-articular use, the amount of composition administered may be adjusted depending on the size of the joint and the severity of the condition. The frequency of subsequent intra-articular administrations is spaced according to the time of recurrence of symptoms in the joint.

[0063] The specific dosage level for any particular patient may be appropriately adjusted depending on various factors, including the activity of the composition employed, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, and the severity of the particular disease being treated. The pharmaceutical composition may be prepared and administered in dosage units. However, in special circumstances, higher or lower dosage units may be appropriate. Administration of the dosage unit may be carried out by both a single administration of the composition and multiple administrations of divided doses at specific intervals, or administration may be carried out in several smaller dosage units.

[0064] For example, individuals with osteoarthritis or meniscus damage may receive 1, 2, or 3 injections of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 ml or more per knee. For other joints, the administered volume may be adjusted based on the size of the joint. The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable carrier in addition to the hydrogel composition included as an active ingredient.

[0065]

[0066] The present invention also provides a composition for cartilage regeneration comprising the hydrogel composition.

[0067] The cartilage described above may be fibrocartilage or elastic cartilage, and can be effectively administered to the area requiring minimally invasive treatment of damaged joints using a syringe or the like. It can also be usefully utilized in various environments within the body.

[0068]

[0069] The hydrogel composition for intra-articular administration of the present invention, the hydrogel composition for preventing or treating intra-articular lesions containing the same, or the composition for cartilage regeneration may be administered to individuals including humans, and preferably, may be administered to mammals including humans without limitation. For example, mammals to which the composition for tissue regeneration of the present invention can be administered include companion animals such as dogs and cats, and livestock animals such as horses, cows, and pigs, and the desired effect can be equally achieved by administration to these animals.

[0070] Accordingly, the composition of the present invention may be a veterinary composition. Furthermore, if the composition is a veterinary composition, the stem cells may be of allogeneic or xenogeneic origin, and for example, stem cells isolated from humans may be administered to non-human animals for veterinary purposes.

[0071]

[0072] The present invention also provides a method for preventing or treating an intra-articular lesion, or a method for regenerating cartilage, comprising a step of administering a hydrogel composition comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose to the joint space of an individual in need thereof.

[0073] The above hydrogel composition may further comprise stem cells or exosomes.

[0074] When used in the above method, the stem cells may be of allogeneic origin, syngeneic origin, or xenogeneic origin, and may be characterized by administering allogeneic or xenogeneic stem cells to an individual in need thereof.

[0075] The hydrogel that can be used in the above method can have the same composition as that described in the hydrogel composition for intra-articular administration, and is omitted to avoid complexity in the description.

[0076] In addition, the subject to which the method of the present invention is applied may be a human or a non-human mammal, and in the case of a non-human mammal, it may be used for veterinary purposes and may be administered without limitation to companion animals such as dogs and cats and livestock animals such as horses, cows, and pigs.

[0077] When the method of the present invention is used for veterinary purposes, the stem cells may be of allogeneic or xenogeneic origin, for example, stem cells isolated from humans may be administered to non-human animals for veterinary purposes.

[0078]

[0079] The hydrogel composition for intra-articular administration of the present invention can be prepared by comprising the step of (a) preparing a composition by dissolving 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose in a solvent.

[0080] The above step (a) can be performed at 20 to 30°C for 15 minutes to 1 hour, and the solvent of the above step (a) can be physiological saline and / or PBS.

[0081] Additionally, the manufacturing method of the present invention may further include the steps of freeze-drying and rehydrating the composition. The hydrogel composition manufactured by the method of the present invention can maintain its physical properties identical to its original state even after rehydration after freeze-drying.

[0082]

[0083] Additionally, the manufacturing method of the present invention may further include the steps of freeze-drying and rehydrating the mixture. The hydrogel composition manufactured by the method of the present invention can maintain its original physical properties even after rehydration following freeze-drying.

[0084]

[0085] The contents of the present invention described above are equally applicable to each other as long as they are not mutually contradictory, and it is also included in the scope of the present invention for a person skilled in the art to make appropriate changes and implement the present invention.

[0086]

[0087] Hereinafter, the present invention will be described in more detail through the following examples. These examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited by these examples.

[0088] Example 1. Method for preparing a hydrogel composition

[0089] After preparing hydrogel manufacturing materials, alginate and carboxymethyl cellulose (CMC), as water-soluble polymers, they were mixed with distilled water in ratios appropriate for each concentration and stirred at room temperature (25°C) for approximately 30 minutes until a homogeneous solution was formed. After gradually adding 0.1 M calcium ions (CaCl₂) to the prepared alginate and CMC solutions, they were mixed by stirring at low speed for approximately 10 minutes. The stirring temperature was maintained between 25°C and 30°C, and the mixture was stirred for approximately 10 to 20 minutes to induce mixing, and the concentration was adjusted according to each concentration combination to manufacture the hydrogel. The manufactured experimental groups are shown in Table 1.

[0090] Alginate CMC Experimental Example 11.0% 1.0% Experimental Example 21.5% 1.5% Experimental Example 32.0% 2.0% Experimental Example 41.5% 1.0% Experimental Example 51.5% 1.5% Experimental Example 61.5% 2.0% Experimental Example 72.0% 1.0% Experimental Example 82.0% 1.5% Experimental Example 92.0% 2.0%

[0091] Hydrogels are prepared as injectable liquids and rapidly gel in the body by calcium ions.

[0092]

[0093] Example 2. Confirmation of hydrogel properties

[0094] The viscosity, gelation rate, and mechanical strength of the hydrogels at each concentration prepared in Example 1 were measured. Specifically, the viscosity of the hydrogels at each concentration was measured at 25°C and 37°C using a viscometer.

[0095] Since viscosity affects injectability and the speed of gelation in the body, the viscosity of each concentration was compared to determine whether gelation could be achieved quickly after injection. After adding calcium ions at each concentration, the time to start gelation and the time to complete gelation were visually measured to evaluate how quickly gelation occurred after injection. In addition, the mechanical strength of the hydrogels at each concentration was measured using a compression tester to evaluate whether they provided sufficient mechanical support for meniscus regeneration and whether the strength was sufficient. The results are shown in Table 2.

[0096]

[0097] Experimental Example Concentration 25℃ Viscosity (cP) 37℃ Viscosity (cP) Gelation start time (min) Complete gelation time (min) Mechanical strength (Pa) 1 Alginate 1% + CMC 1% 27.50 ± 22.50 30.50 ± 24.50 1.10 ± 0.90 2.75 ± 2.25 17.50 ± 12.50 2 Alginate 1.5% + CMC 1.5% 55.00 ± 45.00 61.00 ± 49.00 1.65 ± 1.35 3.30 ± 2.70 34.00 ± 26.00 3 Alginate 2% + CMC 2% 82.50 ± 67.50 88.00 ± 72.00 2.20 ± 1.80 3.85 ± 3.15 50.00 ± 40.00 4 Alginate 1.5%+CMC 1% 32.50±27.50 36.00±29.00 1.10±0.90 2.75±2.25 20.00±15.00 5Alginate 1.5%+CMC 1.5% 67.50±52.50 74.00±56.00 1.65±1.35 3.30±2.70 39.50±30.50 6Alginate 1.5%+CMC 2% 100.00±80.00 106.00±84.00 2.20±1.80 3.85±3.15 56.00±44.00 7Alginate 2%+CMC 1%92.50±77.5098.50±81.501.65±1.353.30±2.7046.50±38.508Alginate 2%+CMC 1.5%120.00±100.00126.00±104.002.20±1.803.85±3.1560.00±50.009Alginate 2%+CMC 2%162.50±137.50168.50±141.502.75±2.254.40±3.6077.00±63.00

[0098] As shown in Table 2, the viscosity at 25℃ and 37℃ was the lowest in Experimental Example 1 and the highest in Experimental Example 9, and showed a tendency to increase directly with the concentration. As the concentration of alginate and CMC increased, the viscosity increased, the hydrogel became thicker, and the gelation occurred more strongly. On the other hand, the gelation start time and the complete gelation time progressed faster as the concentration decreased. Experimental Example 1 showed the fastest gelation speed, and it was confirmed that the gelation time increased as the concentration increased. The mechanical strength increased with the concentration, and it was confirmed that the mechanical strength was the strongest in Experimental Example 9. In the treatment of osteoarthritis, hydrogels provide mechanical support within the joint cavity after injection and play an important role in helping cartilage regeneration. In the case of osteoarthritis, inflammation relief, promotion of tissue regeneration, flexibility, and ease of injection are more important factors than mechanical support for hydrogels within the joint cavity.

[0099] The concentration of Experimental Example 1 provides appropriate viscosity and flexibility, allowing sufficient distribution within the joint cavity. This concentration enables rapid gelation while maintaining fluidity within the body, making it smoothly distributed within the joint and effective in suppressing inflammation and promoting regeneration. Furthermore, rapid gelation while maintaining flexibility after injection is important in the treatment of osteoarthritis. The hydrogel of the present invention is prepared in an injectable liquid state and gels within the body. Considering that excessively high viscosity may slow gelation after injection into the joint cavity or hinder intra-articular movement, the 1% concentration of Experimental Example 1 provides optimal viscosity and flexibility. Furthermore, alginate and CMC have excellent biocompatibility and cell affinity, helping therapeutic factors such as stem cells and exosomes to be well maintained within the hydrogel and promote effective regeneration within the body. In particular, the 1% concentration of the hydrogel allows easy cell penetration and provides an effective environment for cartilage regeneration and inflammation relief. Therefore, this concentration can be expected to produce effective therapeutic results in an osteoarthritis model. In conclusion, a 1% alginate + 1% CMC concentration is highly suitable for the treatment of osteoarthritis. This concentration provides a balanced combination of mechanical strength and flexibility, while simultaneously considering both ease of injection and cell compatibility. Therefore, it can be considered the optimal hydrogel concentration for the treatment of osteoarthritis, and is suitable for promoting rapid gelation and effective tissue regeneration within the joint space.

[0100]

[0101] Example 3. Hydrogel resolution

[0102] Hydrogels of various concentrations were prepared as described in Example 1. For each concentration (Experimental Examples 1 to 9), small amounts of hydrogels were prepared in centrifuge tubes and shaped into circles to enable uniform degradation rates to be measured. Degradation experiments were conducted in an environment simulating the digestive environment in the body by adding 0.1% pepsin to PBS (Phosphate Buffered Saline, pH 7.4) or RPMI-1640 medium. This environment is used to reproduce the conditions under which hydrogels decompose in the body. The body temperature was maintained in an incubator at 37°C, and samples were taken out every week to measure weight loss and observe the degree of degradation, and the degradation rate was calculated by calculating the amount of decomposed and the amount of remaining gel. The measured degradation results are shown in Figure 1.

[0103] As shown in Figure 1, the lower the concentration, the faster the hydrogel decomposed, and the higher the concentration, the slower the decomposition. That is, Experimental Example 1 (1% alginate, 1% CMC) decomposed the fastest, and Experimental Example 9 (2% alginate, 2% CMC) decomposed the slowest.

[0104] The difference in degradation rate depending on concentration depends on the mechanical strength and density of the hydrogel, with higher concentrations leading to slower degradation. These results provide important information for determining the appropriate degradation characteristics of hydrogels for meniscus regeneration and osteoarthritis treatment.

[0105]

[0106] Example 4. Isolation of placental-derived mesenchymal stem cells and exosomes

[0107] Placentas were collected from mothers who consented to donation after normal deliveries by cesarean section at Chung-Ang University Gwangmyeong Hospital in accordance with the Institutional Review Board (IRB) guidelines. The collected placentas were transferred to sterile containers, and the amniotic membrane was stripped from the placental tissue. The chorion and the trophoblast layer, approximately 10% of the thickness adjacent to the chorion, were separated using sterile scissors. The separated trophoblast layer was transferred to a 150-mm dish, and the chorion was removed using a scalpel. The tissue was then washed at least five times with phosphate-buffered saline (PBS) to remove blood and blood cells.

[0108] After transferring the washed trophoblast tissue to a 50 ml tube, α-MEM medium supplemented with 0.2% collagenase was added and reacted for 2-3 hours using a shaker at 37℃ to obtain cells derived from the trophoblast layer. The cells derived from the obtained trophoblast layer were filtered through a 100 ㎛ mesh to remove undigested tissue, and α-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25℃ and 1200 rpm for 4 minutes. The supernatant was removed, and α-MEM medium supplemented with fetal bovine serum and antibiotics but not containing growth factors was added to the remaining precipitated cells, and cultured under 37℃ and 5% CO2 conditions. Cells attached to the bottom of the culture vessel were selected from the above culture to obtain stem cells derived from the detailed trophoblast layer. To isolate exosomes, placental stem cells were cultured in α-MEM at 37°C and 5% CO₂. The medium was supplemented with FBS (exosome-depleted FBS, Thermo Fisher Scientific or System Biosciences) and 1% Penicillin-Streptomycin (Gibco, Cat# 15140-122). Exosome secretion was induced for 48 hours when the cell density reached approximately 70%. The supernatant containing exosomes was aseptically collected and centrifuged at 300 × g for 10 min (using Eppendorf 5810R) to remove floating cells. The supernatant was centrifuged at 2,000 × g for 20 min to remove cell debris, and then centrifuged at 10,000 × g for 30 min to remove fine particles and cell debris.

[0109] The pretreated supernatant was centrifuged at 100,000 × g for 70 min using an ultracentrifuge (Beckman Coulter Optima XPN-100 Ultracentrifuge), and the precipitated pellet was resuspended in 1× PBS (Welgene, Cat# LB001-02) and centrifuged again under the same conditions to purify exosomes. The finally obtained exosomes were resuspended in PBS and stored at -80°C.

[0110] Isolated stem cells and exosomes were used together with the hydrogel of the present invention in the examples below.

[0111]

[0112] Example 5. Viability when mixing hydrogels and cells

[0113] The hydrogel manufactured in Example 1 was used to incubate stem cells (1×10 7 Cell viability was measured using the MTT Cell Proliferation Assay Kit (R&D Systems) by mixing with 100 cells / mL of hydrogel. Specifically, the hydrogel and cell mixture at each concentration was dispensed into a 96-well plate, and cultured to ensure that the cells adhere well to the hydrogel and become activated.

[0114] After 24 and 48 hours, MTT reagent was added to each well and incubated at 37°C for 4 hours. DMSO was added to dissolve the MTT converting substance and the absorbance was measured at 570 nm using a microplate reader (VersaMax™ Microplate Reader). The cell viability (absorbance value / control absorbance value × 100) was calculated, and the results are shown in Fig. 2.

[0115] As shown in Fig. 2, after 24 hours, the stem cells showed a higher survival rate as the hydrogel concentration decreased, and the survival rate within 24 hours was confirmed to be high at over 90% at concentration 1, and after 48 hours, the stem cell survival rate decreased overall over time, but maintained a survival rate of over 90% at concentration 1, and showed a relatively low survival rate of about 60% at concentration 9.

[0116] As a result of conducting experiments on the hydrogel concentration combinations of Examples 1 to 4 above, it was confirmed that if the alginate and CMC concentrations are too high, the viscosity increases and injection may become difficult, and since the mechanical strength may vary greatly depending on each concentration combination, it was confirmed that it is important to select a combination that can exhibit appropriate mechanical strength according to the purpose.

[0117] For example, the combination of 1.5% alginate and 1.5% CMC in Experimental Example 5 was judged to be suitable for meniscus regeneration because it was a concentration that could well balance mechanical strength and cell compatibility, and the hydrogel could remain appropriately firm while being injectable.

[0118] In addition, in the osteoarthritis model, the concentration of 1% alginate and 1% CMC in Experimental Example 1 was confirmed to be optimal. This is because the concentration combination in Experimental Example 1 provides flexibility and soft gelation to minimize pain upon injection, does not interfere with natural movement within the joint cavity, and provides an effective environment for cartilage regeneration and inflammation suppression when combined with cell-based therapy.

[0119] Therefore, in Examples 5 and 6 below, a hydrogel concentration suitable for each model was used.

[0120]

[0121] Example 6: Efficacy verification using a meniscus defect model

[0122] In order to verify whether the gel manufactured in the above example (Experimental Example 5) exhibits a meniscus regeneration effect in an animal model of meniscus injury, the following experiments were performed. More specifically, to create an animal model of rabbit joint cartilage injury, healthy rabbits were selected and anesthetized by injection with appropriate amounts of ketamine and lumpun according to their body weight. After confirming that the rabbits were sufficiently anesthetized, the knee joint area of ​​both lower extremities was shaved and secured with a bandage while maintaining the posture. The knee joint area on both sides was disinfected with povidone-alpha, and the patella was palpated to confirm its position. Then, the knee joint was reached through a paramedian approach along an incision line passing above, below, and on the medial side of the patella, and the knee joint was flexed while the patella was turned outward to observe the inside of the joint. After confirming that there were no specific pathological findings, more than two-thirds of the meniscus was removed (meniscectomy). As described above, meniscus damage was induced, and after the patella was returned to its original position, the soft tissue around the patella was sutured with absorbable thread, and the skin was sutured with non-absorbable thread. After confirming that the rabbits were awake from anesthesia, they were allowed to move freely, and analgesics and antibiotics were administered for 5 days after surgery to prevent infection. One week later, without making an incision in the skin of the knee joint of the animal model, saline, experimental example 5 hydrogel alone, hydrogel and stem cells (1 × 10 7 cells / mL) or exosomes (1 × 10 9After 8 and 16 weeks, the damaged and treated menisci were removed from each rabbit and macroscopically evaluated. In addition, after fixation of the tissue, H&E, Safranin O staining, and immunostaining for Type II collagen were performed, and the regenerated menisci were analyzed through quantification using the histological tissue quality score.

[0123] The effects in the meniscus defect model are shown in Figures 3, 4a, b, and c.

[0124] As shown in Figures 3 and 4a, b, and c, compared to when saline or hydrogel was injected alone, in the group injected with a mixture of hydrogel and stem cells or exosomes, fibrous tissue began to form from 8 weeks, and tissue similar to meniscus tissue was formed at 12 weeks. These results demonstrate that meniscus can be regenerated by injecting a mixture of hydrogel and stem cells or exosomes without skin incision one week after meniscus injury in rabbits.

[0125] Meanwhile, a key property of the meniscus scaffold is its compressive strength, which allows it to withstand external forces. After 16 weeks, the regenerated meniscus was cut into two sections using a 4-mm punch. The compressive strength was measured using a tensile tester. The strength of the regenerated meniscus was confirmed by injecting the gel of the present invention. The results are shown in Table 3.

[0126] ConditionMean Compression Strength (N)Standard Deviation (N)p-value(vs Saline)p-value(vs Hydrogel only)Saline1.00.5Hydrogel only3.01.0p < 0.05Hydrogel + MSCs5.01.1p < 0.05p < 0.05Hydrogel + Exosomes5.51.1p < 0.05p < 0.05Rabbit Normal Meniscus4.50.5

[0127] As shown in Table 3, the strength of the regenerated meniscus in the groups injected with hydrogel alone and a mixture of hydrogel and stem cells or exosomes was similar to that of the normal meniscus, compared to the group treated with saline alone. Therefore, it was confirmed that the hydrogel presented in the present invention can be useful for meniscus treatment, either alone or in combination with stem cells or exosomes.

[0128]

[0129] Example 7: Efficacy verification using an osteoarthritis model

[0130] In order to verify whether the hydrogel of Experimental Example 1, which was judged to be optimal for osteoarthritis, exhibits a therapeutic effect in an animal model of osteoarthritis, the following experiment was performed.

[0131] More specifically, an osteoarthritis model was induced in Sprague-Dawley rats (male, 8 weeks old, n=6 / group) by injecting MIA (monosodium iodoacetate, 2 mg, Sigma-Aldrich) into the knee joint. Seven days after induction, the joint cavity was injected with saline, hydrogel alone, hydrogel and stem cells (1 × 10 7 cells / mL) or exosomes (1 × 10 9 The mixture of particles was injected at a single dose of 50 μL.

[0132] Fourteen days after administration, the anti-inflammatory effect was evaluated using RT-qPCR and ELISA methods as follows. For RT-qPCR analysis, RNA was extracted from knee joint tissue (RNeasy Mini Kit, Qiagen), cDNA was synthesized, and the relative expression levels of IL-6 and TNF-α genes were quantified using a Bio-Rad CFX96 system. The results are shown in Figures 5 and 6.

[0133] As shown in Figure 5, the RT-qPCR analysis results showed that the Hydrogel+MSCs group showed the most significant decrease in inflammatory gene expression (IL-6 and TNF-α), and the Hydrogel+Exosomes group and the Hydrogel alone group also showed an anti-inflammatory effect.

[0134] As shown in Figure 6, ELISA protein analysis results showed a similar trend to the mRNA results, with the lowest inflammatory cytokine expression observed in the Hydrogel+MSCs group. All experimental groups showed a statistically significant decrease (p<0.01) compared to the saline group.

[0135]

[0136] To evaluate the pain relief and functional recovery effects, mechanical pain sensitivity and gait indices for each group in the osteoarthritis model were measured 14 days after administration, and the results are shown in Table 4.

[0137] ConditionMechanical Pain Sensitivity (g)Walking Distance (cm)Walking Speed ​​(cm / s)Weight Distribution(Load on Damaged Leg, %)Saline4.0±1.050±100.5±0.140±5Hydrogel only6.0±1.270±150.7±0.145±4Hydrogel + MSCs10.0±1.5120±201.0±0.260±5Hydrogel + Exosomes8.5±1.3110±180.9±0.255±5

[0138] As shown in Table 4, improvements in walking distance and walking speed were confirmed in all hydrogel-containing experimental groups. Specifically, the hydrogel-only group showed improved mechanical pain sensitivity, and walking distance and walking speed were improved compared to the saline group (p<0.05). The hydrogel+MSCs group showed the most improved results in mechanical pain sensitivity, gait index, and weight distribution (p<0.05), followed by the hydrogel+exosomes group (p<0.05). In addition, the treatment effect on osteoarthritis was further confirmed through the degree of improvement in cartilage damage using the OARSI Score, and the results are shown in Table 5.

[0139] ConditionOARSI Score (Mean ± SD)Collagen II Expression (Relative)Sox9 Expression (Relative)Saline5.5±1.01.0±0.21.0±0.1Hydrogel only4.0±1.21.5±0.31.4±0.2Hydrogel + MSCs2.0±0.83.0±0.42.8±0.3Hydrogel + Exosomes2.0±0.82.5±0.32.2±0.2

[0140] As shown in Table 5, the results showed that cartilage damage was significantly improved in the Hydrogel + MSCs group at 2.0±0.8 and the Hydrogel + Exosomes group at 2.0±0.8. As a result of the expression analysis of Collagen II and Sox9, the Hydrogel only group showed Collagen II expression of 1.5, which was 50% higher than the Saline group, the Hydrogel + MSCs group showed Collagen II expression of 3.0, which was 200% higher than the Saline group, and Sox9 expression was also 2.8, which was significantly higher than the Saline group. The Hydrogel + Exosomes group showed Collagen II expression of 2.5, which was 150% higher than the Saline group.

[0141] In summary, these results confirm that the hydrogel composition of the present invention is effective in suppressing inflammation, effectively relieving pain, and restoring gait function, and thus can be useful in the fields of meniscus injury treatment, osteoarthritis treatment, and cartilage regeneration. In particular, the pain relief and gait function restoration effects can be enhanced by additionally including stem cells and exosomes in the hydrogel composition of the present invention.

Claims

1. A hydrogel composition for intra-articular administration, comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose.

2. A hydrogel composition for intraarticular administration, wherein the alginate and carboxymethyl cellulose are included in a weight ratio of 1:1 in the first paragraph.

3. A hydrogel composition for intra-articular administration, wherein the composition further comprises stem cells or exosomes in the first paragraph.

4. A hydrogel composition for intra-articular administration, wherein the hydrogel composition in the first paragraph gels at 25 to 37°C.

5. In the first paragraph, the hydrogel composition is for intra-articular injection, a hydrogel composition for intra-articular administration.

6. A hydrogel composition for intra-articular administration, wherein the hydrogel composition is maintained in the body for 2 to 6 months in the first paragraph.

7. In the first paragraph, the hydrogel composition is a hydrogel composition for intra-articular administration for anti-inflammation and cartilage regeneration.

8. A hydrogel composition for intra-articular administration, wherein the composition is a veterinary composition in the first paragraph.

9. In the first paragraph, the hydrogel composition comprises 1.2 to 1.8 wt% of alginate and 1.2 to 1.8 wt% of carboxymethyl cellulose, and is a hydrogel composition for intra-articular injection for meniscus regeneration.

10. A hydrogel composition for intra-articular administration for the treatment of osteoarthritis, wherein the hydrogel composition comprises 0.7 to 1.2 wt% of alginate and 0.7 to 1.2 wt% of carboxymethyl cellulose in the first paragraph.

11. A hydrogel composition for preventing or treating intra-articular lesions, comprising the composition of any one of claims 1 to 8.

12. A hydrogel composition according to claim 11, wherein the intra-articular lesion is caused by one or more diseases selected from the group consisting of osteoarthritis, degenerative arthritis, rheumatoid arthritis, meniscus damage, intervertebral disc herniation, pubic disc damage, temporomandibular joint damage, articular disc damage of the sternoclavicular joint, triangular fibrocartilage complex rupture of the wrist joint, ulnar impingement syndrome, external ear defect, and epiglottis or laryngeal cartilage defect.

13. A composition for cartilage regeneration, comprising the composition of any one of claims 1 to 8.

14. A method for preventing or treating an intra-articular lesion, comprising administering to the joint space of an individual in need thereof a hydrogel composition comprising 1 to 2 wt% of alginate and 1 to 2 wt% of carboxymethyl cellulose.

15. A method for preventing or treating intra-articular lesions, wherein the composition further comprises stem cells or exosomes in claim 14.

16. A method for preventing or treating an intra-articular lesion in claim 14, wherein the subject is a human or non-human mammal.

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