Hydrogel composition for intra-articular administration and use thereof

Abstract

The present invention relates to a hydrogel composition for intra-articular administration, which is easily injected into the human body, and use thereof for supplementing joint synovial fluid, supplementing viscosity, and preventing or treating intra-articular lesions. The hydrogel composition according to the present invention can improve the gelation temperature, viscosity, biodegradability, and tissue support capability of hyaluronic acid and, at the same time, can improve practitioner convenience and patient compliance due to the reduced injection pressure thereof. In addition, the hydrogel composition can increase the effect of ameliorating intra-articular lesions when co-administered with stem cells or exosomes, and thus can have various applications in medical devices and pharmaceutical fields for the purpose of cartilage regeneration and intra-articular lesion treatment.

Description

Hydrogel composition for intra-articular administration and use thereof

[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 supplementing joint synovial fluid, supplementing viscosity, and preventing or treating intra-articular lesions.

[0002] Osteoarthritis, also known as degenerative arthritis or degenerative joint disease, is a disease characterized by degenerative changes in the joints, leading to deformation of articular cartilage and bone, resulting in pain and functional impairment. Osteoarthritis can be categorized as primary / idiopathic arthritis, which occurs without a specific predisposing cause, and secondary / secondary arthritis, which is caused by trauma, disease, or deformity that can damage articular cartilage. The prevalence of osteoarthritis continues to increase with aging.

[0003] Unlike rheumatoid arthritis, which exhibits systemic symptoms, osteoarthritis initially presents with localized pain when the joint is used. The pain gradually worsens, and as the disease progresses, it 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] Corticosteroids are mainly used along with NSAIDs as initial treatment, but NSAIDs have a mild effect and carry a risk of causing chronic digestive problems, 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 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, and is widely used to improve and treat osteoarthritis pain.

[0005] However, despite these advantages of hyaluronic acid, hyaluronic acid has problems such as lack of gelation speed and stability, inconvenience of injection due to high viscosity, imbalance of retention and decomposition properties in the body, and it reduces the survival rate of additional substances that can increase the therapeutic effect, such as cells, so its commercialization is limited.

[0006] Therefore, there is an urgent need to develop a next-generation hydrogel composition that possesses both gel properties and bioresponsiveness while maintaining the intra-articular viscosity supplement effect, which is the advantage of hyaluronic acid.

[0007] Accordingly, the inventors of the present invention, while researching a new material that can improve the shortcomings of existing hyaluronic acid and increase the therapeutic effect, confirmed that by using an optimal combination of hyaluronic acid (HA), collagen, and Pluronic, the gelation temperature, viscosity, biodegradability, and tissue support of hyaluronic acid can be improved, while reducing the injection pressure, thereby improving the convenience of the practitioner and patient compliance, and completed the present invention.

[0008] Accordingly, the present invention aims to provide a hydrogel composition for intra-articular administration containing hyaluronic acid, collagen, and pluronic acid, and its use for supplementing joint synovial fluid, supplementing intra-articular viscosity, and preventing or treating intra-articular lesions.

[0009] To achieve the above purpose, the present invention provides a hydrogel composition for intra-articular administration comprising hyaluronic acid, collagen, and pluronic in a weight ratio of 1: 0.3 to 0.7: 6 to 10.

[0010] The present invention also provides a hydrogel composition for intra-articular administration, comprising 0.1 to 3.0 wt% of hyaluronic acid, 0.1 to 2.0 wt% of collagen, and 1 to 20 wt% of pluronic acid.

[0011] The present invention also provides a composition for supplementing joint synovial fluid comprising the hydrogel composition.

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

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

[0014] The present invention also provides a method for supplementing joint synovial fluid, comprising administering to the joint space of an individual in need thereof a hydrogel composition comprising hyaluronic acid, collagen, and pluronic acid in a weight ratio of 1:0.3 to 0.7:6 to 10.

[0015] 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 hyaluronic acid, collagen, and pluronic acid in a weight ratio of 1:0.3 to 0.7:6 to 10.

[0016] The present invention also provides a method for cartilage regeneration, comprising a step of administering a hydrogel composition comprising hyaluronic acid, collagen, and pluronic acid in a weight ratio of 1:0.3 to 0.7:6 to 10 to a joint space of an individual in need thereof.

[0017] The hydrogel composition according to the present invention can improve the gelation temperature, viscosity, biodegradability, and tissue support of hyaluronic acid, while simultaneously reducing injection pressure, thereby improving practitioner convenience and patient compliance. Furthermore, it can be co-administered with stem cells or exosomes to enhance the joint lesion improvement effect, making it suitable for various applications in medical devices and pharmaceuticals for the purposes of cartilage regeneration and the treatment of intra-articular lesions.

[0018] Figure 1 is a diagram showing the results of confirming the survival rate of mesenchymal stem cells on the 7th day according to the use of the composition of Experimental Example 2 using a fluorescence microscope (X40).

[0019] Figure 2 is a diagram showing the results of confirming the anti-inflammatory effect at the gene level after injection of a hydrogel composition according to the present invention into an osteoarthritis rat model.

[0020] Figure 3 is a diagram showing the results of confirming inflammation inhibition at the protein level after injecting a hydrogel composition according to the present invention into an osteoarthritis rat model.

[0021] The present invention relates to a hydrogel composition for intra-articular administration comprising hyaluronic acid, collagen and pluronic acid, and its use.

[0022] The hydrogel composition of the present invention can improve the gelation temperature, viscosity, biodegradability, and tissue support of hyaluronic acid, while reducing the injection pressure, thereby improving operator convenience and patient compliance.

[0023] Hereinafter, the present invention will be described in detail.

[0024] The present invention provides a hydrogel composition for intra-articular administration comprising hyaluronic acid, collagen, and pluronic acid in a weight ratio of 1:0.3 to 0.7:6 to 10.

[0025] The hydrogel composition of the present invention utilizes a triple combination of hyaluronic acid, collagen, and pluronic acid, and exhibits significantly superior physical properties compared to a hydrogel containing hyaluronic acid alone. In the present invention, it was confirmed that the physical properties of the hydrogel according to the triple combination do not simply increase in proportion to the increase in their contents, but that the effect may vary depending on their optimal combination. Therefore, the hyaluronic acid, collagen, and pluronic acid may be included in a weight ratio of 1:0.3 to 0.7:6 to 10, preferably 1:0.5 to 0.6:7.5 to 10, and most preferably 1:0.5:10.

[0026] In addition, the hydrogel composition of the present invention may be a hydrogel composition for joint administration comprising 0.1 to 3.0 wt% of hyaluronic acid, 0.1 to 2.0 wt% of collagen, and 1 to 20 wt% of pluronic.

[0027] The hyaluronic acid may be included in the total composition in an amount of 0.1 to 3.0 wt%, 0.1 to 2.5 wt%, 0.2 to 2.5 wt%, 0.3 to 2.5 wt%, 0.4 to 2.5 wt%, 0.5 to 2.5 wt%, 0.5 to 2.0 wt%, for example, 0.5 wt%, 1 wt%, or 2 wt%.

[0028] In the present invention, the hyaluronic acid may be hyaluronic acid itself or a hyaluronic acid salt, and the hyaluronic acid salt may be one or more selected from the group consisting of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and tetrabutylammonium hyaluronate, but is not limited thereto.

[0029] The hyaluronic acid of the present invention may be hyaluronic acid having an average molecular weight of 800 to 1500 kDa, 800 to 1200 kDa, or 800 to 1000 kDa. In a preferred embodiment of the present invention, hyaluronic acid having an average molecular weight of 1000 kDa is used, but is not limited thereto.

[0030] Additionally, the collagen may be included in the total composition in an amount of 0.1 to 2.0 wt%, 0.2 to 2.0 wt%, 0.2 to 1.5 wt%, 0.2 to 1.0 wt%, 0.3 to 1.0 wt%, for example, 0.3 wt%, 0.5 wt%, or 1 wt%.

[0031] The collagen of the present invention may include various isoforms known in the art without limitation, and may include, for example, collagen types 1, 3, 4, 6, 7, 13, 14, and 17 present in human tissues, and preferably collagen types 1, 3, or 7. In a preferred embodiment of the present invention, collagen type 1 is used.

[0032] In addition, the pluronic may be included in the total composition in an amount of 1 to 20 wt%, 3 to 20 wt%, 4 to 20 wt%, 4 to 18 wt%, 4 to 17 wt%, 5 to 16 wt%, 5 to 15 wt%, for example, 5 wt%, 10 wt%, or 15 wt%. The pluronic of the present invention may include, without limitation, pluronics known in the art, for example, pluronic F127, pluronic P123, or pluronic L44, and in a preferred embodiment of the present invention, pluronic F127 is used.

[0033] The hydrogel composition of the present invention comprises a triple combination of hyaluronic acid, collagen, and pluronic acid, and exhibits gelation properties at 25 to 37°C. Therefore, the hydrogel composition of the present invention can rapidly transition into a gel state under body temperature conditions when administered intra-articularly, thereby exhibiting a buffering and cushioning effect within the joint cavity. In addition, the hydrogel composition of the present invention exhibited excellent retention capacity in a degradation rate test using collagenase, with a minimum of 48% and a maximum of 78% remaining on day 7 compared to the hyaluronic acid-only comparative experimental group.

[0034] Meanwhile, fillers, supports, and injections containing hyaluronic acid have the disadvantage of low syringe penetration power due to the high viscosity of hyaluronic acid, and patients complain of discomfort due to high injection pressure. However, the hyaluronic acid composition containing the triple combination of the present invention, when measured with 26G and 29G syringes, showed a viscosity reduction of approximately 30% compared to the existing hyaluronic acid-only comparative experimental group, and accordingly, the injection pressure was also reduced by 30 to 40%, thereby improving the shortcomings of the existing injection composition.

[0035] Accordingly, the composition of the present invention may be for intra-articular injection, and may be characterized as being for 26 to 29G injection. When the composition of the present invention is injected with a 26 to 29G syringe, the injection pressure is 150 kPa or less.

[0036] 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.

[0037] 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 at the lesion site and exhibit a therapeutic effect.

[0038] 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.

[0039] 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.

[0040] The stem cells of the present invention may preferably be placental-derived stem cells, and more preferably may be placental tissue-derived stem cells.

[0041] 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.

[0042] In addition, in the present invention, the trophoblast detailed tissue is characterized in that it does not include tissue derived from the placental septum.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] The present invention also provides a composition for supplementing joint synovial fluid or an intra-articular viscous supplement comprising the hydrogel composition.

[0047] Synovial fluid is a type of fluid that surrounds the cartilage within the joint cavity. It exists in small amounts in all joints, providing lubrication and nourishment to the articular cartilage. The composition of the present invention can act as a lubricant and shock absorber by reducing friction within the joint by supplementing intra-articular viscosity, thereby alleviating joint pain, inhibiting cartilage degeneration, and normalizing synovial membrane function.

[0048] Therefore, the joint synovial fluid supplement 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.

[0049] 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.

[0050] Accordingly, the present invention provides a hydrogel composition for preventing or treating intra-articular lesions, comprising the composition.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057]

[0058] The present invention also provides a composition for cartilage regeneration comprising the hydrogel composition.

[0059] 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.

[0060]

[0061] The hydrogel composition for intra-articular administration of the present invention, the composition for replenishing joint fluid comprising the same, the hydrogel composition for preventing or treating intra-articular lesions, 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 them.

[0062] 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.

[0063]

[0064] The present invention also provides a method for supplementing joint synovial fluid, a method for preventing or treating an intra-articular lesion, or a method for regenerating cartilage, comprising a step of administering to the joint space of an individual in need thereof a hydrogel composition comprising hyaluronic acid, collagen, and pluronic acid in a weight ratio of 1:0.3 to 0.7:6 to 10.

[0065] The above hydrogel composition may further comprise stem cells or exosomes.

[0066] 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 stem cells of allogeneic or xenogeneic origin to an individual in need thereof.

[0067] 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.

[0068] 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.

[0069] 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.

[0070]

[0071] The hydrogel composition for intra-articular administration of the present invention can be prepared by the following method.

[0072] (a) a step of preparing a composition in which hyaluronic acid is dissolved in a solvent;

[0073] (b) adding collagen to the composition of step (a) and stirring at low temperature; and

[0074] (c) A step of adding pluronic to the stirred solution in step (b) and mixing.

[0075] The hyaluronic acid, collagen, and pluronic may be included in a weight ratio of 1:0.3 to 0.7:6 to 10, or may be included in a weight ratio of 0.1 to 3.0% of hyaluronic acid, 0.1 to 2.0% of collagen, and 1 to 20% of pluronic.

[0076] The above step (a) can be performed at 3 to 10°C for 1 to 3 hours, the above step (b) can be performed for 7 to 15 hours, and the above step (c) can be performed for 20 to 25 hours.

[0077] The solvent in step (a) above may be physiological saline and / or PBS.

[0078] In addition, the manufacturing method of the present invention may further include a step (d) of freeze-drying and rehydrating the mixture of step (c). 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.

[0079]

[0080] 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.

[0081]

[0082] 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.

[0083]

[0084]

[0085] Example 1. Preparation of hydrogel composition and confirmation of gelation properties

[0086] To optimize the hydrogel composition ratio, three combination groups were designed, and the weight % of each composition is shown in Table 1.

[0087] Hyaluronic acid (HA) Collagen F127 Experimental example 10.5% 0.3% 5% Experimental example 21.0% 0.5% 10% Experimental example 32.0% 1.0% 15%

[0088] Each composition was prepared by mixing hyaluronic acid (Ildong Pharmaceutical, MW 1,000 kDa), Collagen Type I (Koken Atelocollagen), and F127 (BASF Pluronic®F127) in sterile saline solution (SJW, 0.9% NaCl). First, HA was stirred at 4°C for 2 hours to completely dissolve, then collagen was added and stirred for 12 hours, and then F127 was added and mixed at low speed for 24 hours to prepare the composition. The hydrogel composition of the present invention was prepared by selectively using sterile PBS or saline solution (0.9% NaCl) as a solvent. Each composition was prepared in the form of a fluid, translucent gel and was stably maintained for more than 1 week under refrigerated storage.

[0089]

[0090] Example 2. Freeze-drying and rehydration conditions

[0091] Experimental Examples 1 to 3 manufactured in Example 1 were filled into sterile vials (Schott AG) at 1 mL each, and then pre-frozen at -80°C for 12 hours. Freeze-drying was performed using IlShin BioBase FD8508 equipment for primary drying (-30°C, 150 mTorr, 24 hours) and secondary drying (+20°C, 50 mTorr, 6 hours). 1 mL of physiological saline was added to the obtained freeze-dried powder during rehydration. The freeze-dried powder gelled within approximately 2 minutes, and the physical properties were confirmed to show that the transparency and viscosity were maintained similar to the original state even after freeze-drying and rehydration.

[0092]

[0093] Example 3. Confirmation of physical properties of each composition

[0094] 3.1 Comparison of gelation characteristics and decomposition rates by composition

[0095] In this example, the gelation characteristics and decomposition rates of the compositions of 4% HA (control group) and Experimental Examples 1 to 3 were compared.

[0096] 1) Gelation Transition Temperature: After dispensing 1 mL of each composition into a 2 mL tube, the transition temperature was confirmed by the tube inverting method while heating at 1℃ intervals. As a result, 4% HA did not exhibit gelation, and Experimental Example 1 exhibited gelation transition at approximately 29.8±0.5℃, Experimental Example 2 at 31.2±0.6℃, and Experimental Example 3 at 32.5±0.4℃.

[0097] 2) In vitro degradation rate: 100 mg of each composition was immersed in collagenase II (1 U / mL) or PBS, and the residual weight was measured at 1, 3, 5, and 7 days. 4% HA remained 40% on the 3rd day, less than 10% on the 7th day, 48% on the 7th day for Experimental Example 1, 62% on the 7th day for Experimental Example 2, and more than 78% on the 7th day for Experimental Example 3.

[0098] 3) Comparison of transparency and gel consistency: As a result of comparing transparency and gel consistency by performing light transmittance and visual observation, Experimental Example 2 showed the most transparency and the highest uniformity and stability.

[0099]

[0100] Through the above results, it was confirmed that the composition of the present invention, particularly the composition of Experimental Example 2, is fluid when mixed at low temperature, but quickly transitions to a gel state when reaching body temperature, forming a structure that can function as a viscous supplement and cell support within the joint cavity.

[0101]

[0102] 3.2 Viscosity and injectability evaluation

[0103] In this example, the viscosity and injectability of the compositions of 4% HA (control group) and Experimental Examples 1 to 3 were compared. Viscosity measurements were performed at 37°C and a shear rate of 10 s-¹ using a rheometer (TA Instruments DHR-2, 40 mm plate), and injectability was measured using 26G and 29G syringes via a SensoNor pressure sensor (p75-2015) and an automatic injector (NE-300 Infusion Pump).

[0104] As a result of the measurement, the viscosity of the 4% HA single composition was 2,600±150 cP, and the viscosity of Experimental Example 2 was measured to be 1,850±110 cP, which was reduced by approximately 30% compared to the existing 4% HA. As a result of measuring the injection pressure with 26G and 29G syringes (BD Ultra-Fine™) under the same conditions, the 4% HA was 160-180 kPa, and the present composition was at the level of 100-120 kPa, confirming that the composition of the present invention has improved ease of injection.

[0105] The above results show that, compared to the existing 4% HA single formulation, the composition of Experimental Example 2 of the present invention has a viscosity that is 20% or more lower at the same temperature (37°C) and an injection pressure that is 30 to 40% lower, thereby significantly improving syringe penetration power and patient discomfort.

[0106] In addition, 4% HA can usually be injected using a 22G to 26G syringe, but when using a 29G or thinner gauge, a relatively high injection pressure is required, which may cause fatigue of the operator and discomfort of the patient. In comparison, the composition of the present invention exhibits a reduction in injection pressure of 30% or more at the same gauge, thereby reducing injection resistance and improving injection stability during cell mixing.

[0107] Therefore, it was confirmed that the composition of the present invention, which is suitable for clinical injection with an injection pressure of 100-120 kPa when using a 26G / 29G syringe and a gel viscosity of 1,800-2,000 cP, is a more effective composition for injection into a joint or joint cavity.

[0108]

[0109] 3.3 Confirmation of viscoelasticity according to temperature

[0110] Storage modulus (G') and loss modulus (G") were measured at 5 to 45°C using a rheometer.

[0111] As a result, the G' / G" transition points were confirmed at 29.2±0.4℃ for Experimental Example 1, 31.1±0.5℃ for Experimental Example 2, and 33.4±0.6℃ for Experimental Example 3, confirming that the gel transition temperature increases as the hydrogel composition increases. In particular, the composition of Experimental Example 2 showed the most linear stability. 4% HA confirmed no G' / G" crossing (non-gelation).

[0112] The results of comparative evaluation of gelation temperature, 7-day residual rate, viscosity, injection pressure, and transition temperature for the compositions of Experimental Examples 1 to 3 are shown in Table 2.

[0113] CompositionGelation temperature(℃)7-day residual rate(%)Viscosity(cP)Injection pressure(kPa, 29G)G' / G"Transition temperature(℃)4%HANone<10%2,600±150160±20NoneExperimental example 129.8±0.548±3.5850±9090±529.2±0.4Experimental example 231.2±0.662±4.21,850±110110±831.1±0.5Experimental example 332.5±0.478±4.82,900±140170±1233.4±0.6

[0114]

[0115] As shown in Table 2 above, all of Experimental Examples 1 to 3 showed excellent properties compared to 4% HA, but it was confirmed that Experimental Example 2 (Hyaluronic Acid 1%, Collagen 0.5%, F127 10%) was the most excellent composition in terms of gelation, injectability, stability, etc.

[0116] Therefore, the following experiment was conducted using the composition of Experimental Example 2.

[0117]

[0118] Example 4. Isolation of placental-derived mesenchymal stem cells and exosomes

[0119] 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.

[0120] 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.

[0121] 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.

[0122] Isolated stem cells and exosomes were used together with the hydrogel of the present invention in the examples below.

[0123]

[0124] Example 5. Cell viability evaluation

[0125] In the composition of Experimental Example 2, placental-derived mesenchymal stem cells were added to the hydrogel at a concentration of 1×10 6 After mixing at a concentration of 10 cells / mL, 500 μL was dispensed into a 24-well plate. The cell viability of stem cells was analyzed using a fluorescence microscope (ZEISS LSM980 Confocal Microscope) on days 1, 3, and 7 using the Live / Dead Viability / Cytotoxicity Kit (Invitrogen L3224), and the results are shown in Figure 1.

[0126] As shown in Figure 1, the average survival rate after 7 days was maintained at 87.5±3.4%, and the cells survived within the gel, maintaining a spherical shape. This suggests that the hydrogel composition provides a microenvironment suitable for cell survival.

[0127]

[0128] Example 6. Anti-inflammatory efficacy in osteoarthritis model

[0129] An osteoarthritis model was induced in Sprague-Dawley rats (male, 8-week-old, n=6 / group) by intra-articular injection of MIA (monosodium iodoacetate, 2 mg, Sigma-Aldrich). Seven days after induction, a single 50 μL injection of the composition was administered as follows:

[0130] G1: MIA-induced injury model (untreated injury group)

[0131] G2: 4% HA only

[0132] G3: Experimental Example 2 Hydrogel (HA+Collagen+F127)

[0133] G4: Experimental Example 2 Hydrogel + MSC (1×10 7 cells / 1mL)

[0134] G5: Experimental Example 2 Hydrogel + Exosomes (1×10 9 particles / rat)

[0135]

[0136] Fourteen days after administration, the anti-inflammatory effect was evaluated in an osteoarthritis model 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 Figure 2.

[0137] As shown in Fig. 2, the RT-qPCR analysis results showed that the expression of IL-6 and TNF-α in the HA-only administration group (G2) decreased by approximately 24% and 21%, respectively, compared to the untreated injury control group (G1). On the other hand, the experimental example 2 (G3) administration group showed a decrease of approximately 36% and 32%, respectively, indicating that all inflammatory factors were improved compared to HA alone.

[0138] Further improved anti-inflammatory effects were observed in the G4 (including MSCs) and G5 (including exosomes) experimental groups, which combined stem cells or exosomes. In the G4 treatment group, IL-6 and TNF-α mRNA levels were reduced by approximately 58% and 62%, respectively, while in the G5 group, they were reduced by 64% and 68%, respectively, confirming that the exosome-containing composition exhibited slightly superior effects compared to the stem cell-only group.

[0139] Inflammatory cytokine expression was additionally confirmed through EILSA protein analysis, and the results of measuring changes in protein expression in each experimental group are shown in Figure 3.

[0140] As shown in Figure 3, ELISA protein analysis results showed a similar trend to the mRNA results, with the lowest inflammatory cytokine expression observed in G5. All experimental groups showed statistically significant decreases (p<0.01) compared to G1, and G4 and G5, in particular, demonstrated complex compositions with excellent anti-inflammatory effects.

[0141]

[0142] Example 7. Analysis of cartilage regeneration effects

[0143] Experimental Example 2 To confirm the cartilage regeneration effect of the hydrogel composition, an osteoarthritis model was induced by intra-articular injection of MIA (monosodium iodoacetate, 2 mg, Sigma-Aldrich) into Sprague-Dawley rats (male, 8 weeks old, n=6 / group). Seven days after injury induction, a single intra-articular injection of the composition was performed into each experimental group (G1-G5), and 4 weeks later, knee joint tissues were extracted and subjected to histological and molecular biological analyses.

[0144] Knee joint tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm thickness. Tissue sections were stained with hematoxylin & eosin (H&E) and Safranin O / Fast Green, and the degree of pathological damage was quantitatively assessed based on the OARSI (Observed Osteoarthritis Damage Score) criteria (0-6 scale). In addition, immunohistochemical (IHC) staining for Collagen II and Sox9, markers of cartilage regeneration, was performed, and the relative expression levels were analyzed under a fluorescence microscope. The measurement results for each experimental group are shown in Table 3.

[0145] Group Cartilage thickness (㎛) (Relative) OARSI score Collagen II expression (Relative) Sox9 expression (Relative) G1 (Untreated injury group) 1.00±0.12 5.1±0.3 1.00±0.10 1.00±0.11 G2 (4% HA) 1.21±0.11 4.2±0.4 1.35±0.15 1.38±0.13 G3 (Experimental example 2) 1.30±0.10 3.1±0.2 1.58±0.14 1.61±0.12 G4 (Experimental example 2 + MSC) 1.80±0.13 1.9±0.3 2.30±0.18 2.32±0.16 G5 (Experimental Example 2 + Exosome) 1.85±0.15 1.8±0.2 2.40±0.17 2.42±0.14

[0146]

[0147] As shown in Table 3 above, the G4 (including MSCs) and G5 (including exosomes) groups showed approximately 1.8- and 1.85-fold increases in cartilage thickness, respectively, compared to the damaged control group (G1). The damaged model control group (G1) showed a mean OARSI score of 5.1 ± 0.3, indicating that extensive cartilage loss and subchondral bone exposure were observed, indicating that severe cartilage damage was induced. On the other hand, the 4% hyaluronic acid only treatment group (G2) showed a score of 4.2 ± 0.4, indicating that the damage was somewhat alleviated, but structural recovery of the cartilage layer was limited.

[0148] In Experimental Example 2, the single-administration group (G3) had a score of 3.1 ± 0.2, indicating that the degree of preservation of cartilage tissue was increased, and the extent of inflammatory response and cartilage loss was significantly reduced. In particular, in group G4, where placental-derived stem cells (MSCs) were mixed with the composition of Experimental Example 2, the OARSI score decreased to 1.9 ± 0.3, and group G5, where exosomes were mixed, showed the lowest score at 1.8 ± 0.2, suggesting that the recovery and regeneration of cartilage structure were most effective (p < 0.01, compared to G1).

[0149] These results experimentally demonstrate that the hydrogel composition of the present invention is effective in repairing damaged cartilage tissue, and that its efficacy is significantly enhanced, particularly when combined with stem cells or exosomes. In addition, the relative expression levels of Collagen II and Sox9 significantly increased by approximately 2.3 times in G4 and approximately 2.4 times in G5 compared to G1 (p < 0.01). These results indicate that the hydrogel composition of the present invention, when used in combination with stem cells or exosomes, can induce effective cartilage regeneration through structural restoration of cartilage tissue and induction of expression of chondrocyte-specific markers.

[0150]

[0151] Example 8: Evaluation of pain relief and recovery of walking function (mechanical pain sensitivity and gait analysis)

[0152] Experimental Example 2 To evaluate the pain relief and functional recovery effects of the hydrogel composition, the composition of the present invention was administered to an osteoarthritis model, and 14 days after administration, mechanical pain sensitivity and gait indices were measured for each group (G1 to G5). Pain sensitivity tests were performed using Von Frey filaments, and the results confirming improvements in sensitivity to pain stimuli and gait function are shown in Table 4.

[0153] GroupWithdrawal Threshold (g)Walking distance (cm)Ground contact area (mm²)Weight distribution (%)G1 (Untreated injury group)4.2±0.643.1±5.224.8±4.742.3±6.5G2 (4% HA)6.3±0.758.2±6.134.2±5.151.7±5.9G3 (Experimental example 2)7.4±0.564.5±5.839.3±4.656.9±6.1G4 (Experimental example 2+MSC)8.8±0.471.6±4.946.5±4.062.1±5.4G5 (Experimental example 2+exosomes)9.1±0.373.2±5.148.3±3.763.5±4.8

[0154] As shown in Table 4, the pain sensitivity test results showed that the G4 group containing stem cells (MSCs) and the G5 group containing exosomes showed approximately 1.8- and 2.1-fold increases in pain thresholds, respectively, compared to the injured control group (G1) (p<0.05). This indicates a significant decrease in sensitivity to pain stimuli. Significant improvements were also confirmed in the analysis of walking function. In particular, the G5 group showed an approximately 45% increase in walking distance compared to the G1 group, approaching normal walking ability. In addition, the weight-bearing capacity increased by 38% in the G4 group and 41% in the G5 group, with the load distribution ratio through the injured lower extremity. These results indicate that pain was reduced and functional recovery was achieved when using the joint.

[0155] Additionally, in the analysis of the contact area of ​​the sole, the G5 group showed a contact area that was approximately 35% wider than the control group due to the reduction in pain, which shows that ground contact was more stable during walking.

[0156] In summary, these results show that the hydrogel composition of the present invention is effective in alleviating pain and restoring walking function in an animal model induced with osteoarthritis, and in particular, it was confirmed that the pain alleviation and walking function restoration effects can be enhanced by additionally including stem cells and exosomes.

Claims

1. A hydrogel composition for intra-articular injection comprising hyaluronic acid, collagen, and pluronic acid in a weight ratio of 1:0.3 to 0.7:6 to 10.

2. A hydrogel composition according to claim 1, wherein the hydrogel composition gels at 25 to 37°C.

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

4. A composition in accordance with claim 3, wherein the hydrogel composition is for injection of 26 to 29G.

5. A hydrogel composition according to claim 1, wherein the composition further comprises stem cells or exosomes.

6. 0.1 to 3.0 wt% of hyaluronic acid, 0.1 to 2.0 wt% collagen, and A hydrogel composition for intra-articular administration, comprising 1 to 20 wt% of pluronic acid.

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

8. A composition for supplementing joint synovial fluid, comprising a composition of any one of claims 1 to 7.

9. A hydrogel composition for preventing or treating intra-articular lesions, comprising a composition according to any one of claims 1 to 7.

10. A hydrogel composition according to claim 9, 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.

11. A composition for cartilage regeneration, comprising the composition of any one of claims 1 to 7.

12. A method for supplementing joint synovial fluid, comprising the step of administering a hydrogel composition comprising hyaluronic acid, collagen, and pluronic acid in a weight ratio of 1:0.3 to 0.7:6 to 10 to a joint space of an individual in need thereof.

13. A method for supplementing joint synovial fluid in claim 12, wherein the composition further comprises stem cells or exosomes.

14. A method for supplementing synovial fluid in accordance with claim 13, wherein the subject is a human or non-human mammal.

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