Pharmaceutical composition for treating cartilage diseases, comprising, as active ingredient, magnetic nanoparticle-embedded nasal septal chondrocytes

Abstract

The present invention relates to a pharmaceutical composition for treating cartilage damage diseases, comprising, as an active ingredient, magnetic nanoparticle-embedded nasal septal chondrocytes , and establishes a method for embedding magnetic nanoparticles into nasal septal chondrocytes and a method for preparing spheroids by culturing magnetic nanoparticle-embedded nasal septal chondrocytes. It was identified that the magnetic nanoparticle-embedded nasal septal chondrocytes prepared by the method of the present invention and spheroids prepared therefrom exhibit cartilage damage treatment activity while maintaining mobility in a magnetic field. In addition, since the activity is maintained even if spheroids are prepared by mixing magnetic nanoparticle-embedded nasal septal chondrocytes and nasal septal chondrocytes, the present invention can be effectively used as a composition for treating cartilage damage diseases that has an excellent therapeutic effect without causing side effects by having a significantly reduced addition ratio of the magnetic nanoparticles.

Description

Pharmaceutical composition for treating cartilage disease comprising nasal septal cartilage cells loaded with magnetic nanoparticles as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for treating cartilage disease, which comprises nasal septal cartilage cells loaded with magnetic nanoparticles as an active ingredient.

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2024-0029917, filed February 29, 2024, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Articular cartilage is a dense, elastic connective tissue that is located at several joints in the skeleton. It is connected to bone, and its surfaces are in contact with other cartilage at joint points. Cartilage is an avascular tissue, non-innervated, and is composed of rudimentary chondrocytes, usually single cells, and is synthesized by an extracellular matrix (ECM).

[0005] Articular cartilage repair remains an unresolved problem in modern medicine. While treatment options exist, many challenges remain. One major issue is the extremely limited self-repair capacity of articular cartilage. To address this issue, regenerative medicine has emerged, and cartilage tissue engineering aims to repair tissue through biological replacement.

[0006] Autologous chondrocyte implantation (ACI) is used to repair damaged articular cartilage surfaces, where the transplanted autologous chondrocytes are obtained by enzymatically isolating a small biopsy of healthy articular cartilage. However, the commonly known naturally occurring autologous chondrocytes have limitations.

[0007] Chondrocyte redifferentiation has limitations when expanded in vitro, and the chondrogenic capacity decreases with the age of the donor. In addition, cell proliferation is important in autologous chondrocyte transplantation that uses a large amount of cells, and articular chondrocytes are known to have a relatively low proliferation capacity. Human nasal septal chondrocytes can complement these problems of human articular chondrocytes. Human nasal septal chondrocytes have a higher proliferation rate than human articular chondrocytes, and their chondrogenic capacity is also high both in vitro and in vivo, and does not decrease depending on the age of the donor.

[0008] However, while the development of a treatment for joint damage using nasal septal cartilage cells with such excellent effects is insufficient, the clinical demand for complete regeneration of damaged cartilage is rapidly increasing, and this demand is expected to increase further as we enter an aging society.

[0009]

[0010] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cartilage damage disease, which comprises nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient.

[0011] Another object of the present invention is to provide a kit for preventing or treating cartilage damage disease, comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles.

[0012] Another object of the present invention is to provide a method for producing nasal septal cartilage cells loaded with magnetic nanoparticles, comprising the following steps:

[0013] (S1) A step of producing nasal septum chondrocytes (NSCs) by treating nasal septum cartilage tissue isolated from nasal septum tissue with type 2 collagenase; and

[0014] (S2) A step of culturing the above nasal septum cartilage cells and further adding magnetic nanoparticles to culture them.

[0015] Another object of the present invention is to provide a method for producing a mixed septal cartilage cell spheroid loaded with magnetic nanoparticles for treating cartilage damage diseases, comprising the following steps:

[0016] (S1) A step of culturing nasal septum chondrocytes (NSCs) isolated from nasal septum tissue and magnetic nanoparticles to produce nasal septum chondrocytes loaded with magnetic nanoparticles; and

[0017] (S2) A step of culturing the nasal septum cartilage cells and the nasal septum cartilage cells loaded with the magnetic nanoparticles by adding them to a medium for culturing them in a spheroid form.

[0018]

[0019] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0020]

[0021] The present invention provides a pharmaceutical composition for preventing or treating cartilage damage disease, comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient.

[0022] In one embodiment of the present invention, the nasal septum cartilage cells containing the magnetic nanoparticles may be in the form of a spheroid, but are not limited thereto.

[0023] In one embodiment of the present invention, the nasal septum chondrocytes loaded with the magnetic nanoparticles may express type 2 collagen, but are not limited thereto.

[0024] In one embodiment of the present invention, in the nasal septum cartilage cells containing the magnetic nanoparticles, the magnetic nanoparticles may be added in an amount of more than 0 to 800 μg based on 1x10^6 nasal septum cartilage cells, but is not limited thereto.

[0025] In one embodiment of the present invention, the spheroid may be a mixture of nasal septum cartilage cells and nasal septum cartilage cells loaded with magnetic nanoparticles, but is not limited thereto.

[0026] In one embodiment of the present invention, the number ratio of the nasal septum chondrocytes: the nasal septum chondrocytes loaded with magnetic nanoparticles may be (5 to 7) : (3 to 5), but is not limited thereto.

[0027] In one embodiment of the present invention, the nasal septal cartilage cells loaded with the magnetic nanoparticles can maintain mobility in a magnetic field regardless of the concentration of the magnetic nanoparticles, but are not limited thereto.

[0028] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0029] a) Inducing a reparative effect of hypoechoic material in the area of ​​cartilage damage;

[0030] b) produces a cartilage regeneration effect; and

[0031] c) Anti-inflammatory effect is shown.

[0032] In one embodiment of the present invention, the composition may be for injection, but is not limited thereto.

[0033] In one embodiment of the present invention, the composition may be administered by any one method selected from the group consisting of arthroscopic injection and intra-articular injection, but is not limited thereto.

[0034] In one embodiment of the present invention, the cartilage damage disease may be at least one selected from the group consisting of osteoarthritis, degenerative arthritis, articular cartilage defect, rheumatoid arthritis, fracture, meniscus damage, muscle damage, ligament damage, plantar fasciitis, lateral epicondylitis, calcific myositis, joint or cartilage damage due to trauma, and nonunion of fracture, but is not limited thereto.

[0035] The present invention provides a kit for preventing or treating cartilage damage disease, comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles.

[0036] The present invention provides a method for producing nasal septum cartilage cells loaded with magnetic nanoparticles, comprising the following steps:

[0037] (S1) A step of producing nasal septum chondrocytes (NSCs) by treating nasal septum cartilage tissue isolated from nasal septum tissue with type 2 collagenase; and

[0038] (S2) A step of culturing the above nasal septum cartilage cells and further adding magnetic nanoparticles to culture them.

[0039] The present invention provides a method for producing a mixed septal cartilage cell spheroid loaded with magnetic nanoparticles for treating cartilage damage diseases, comprising the following steps:

[0040] (S1) A step of culturing nasal septum chondrocytes (NSCs) isolated from nasal septum tissue and magnetic nanoparticles to produce nasal septum chondrocytes loaded with magnetic nanoparticles; and

[0041] (S2) A step of culturing the nasal septum cartilage cells and the nasal septum cartilage cells loaded with the magnetic nanoparticles by adding them to a medium for culturing them in a spheroid form.

[0042]

[0043] In addition, the present invention provides a method for preventing or treating cartilage damage disease, comprising a step of administering a pharmaceutically effective amount of a composition containing nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient to a subject in need thereof.

[0044] In addition, the present invention provides a use of a composition comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient for preventing or treating cartilage damage diseases.

[0045] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating cartilage damage disease, comprising a composition containing nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient.

[0046]

[0047] According to a pharmaceutical composition for treating cartilage damage disease comprising nasal septal chondrocytes loaded with magnetic nanoparticles as an active ingredient, a method for incorporating magnetic nanoparticles into nasal septal chondrocytes and a method for producing spheroids by culturing nasal septal chondrocytes loaded with magnetic nanoparticles were established. It was confirmed that the nasal septal chondrocytes loaded with magnetic nanoparticles produced by the method of the present invention and the spheroids produced therefrom exhibit cartilage damage treatment activity while maintaining mobility in a magnetic field. In addition, since the activity is maintained even when the nasal septal chondrocytes loaded with magnetic nanoparticles and the nasal septal chondrocytes are mixed to produce spheroids, the addition ratio of magnetic nanoparticles can be significantly reduced, so that the composition can be usefully utilized as a composition for treating cartilage damage disease with excellent therapeutic effects without causing side effects.

[0048]

[0049] Figure 1a is a schematic diagram showing an isolation and culture protocol for isolating and culturing nasal septal cartilage cells (hereinafter, hNCs) from the nasal septal cartilage tissue established in the present invention.

[0050] Figure 1b shows the morphological characteristics of hNCs isolated and cultured from nasal septum cartilage tissue, indicating that no morphological changes occur due to the hNCs isolation and culture protocol of the present invention.

[0051] Figure 1c is a graph comparing the amount of type 2 collagen expression in hNCs and hNCs spheroids produced therefrom, showing that the amount of type 2 collagen expression in hNCs spheroids was significantly increased.

[0052] Figures 2a and 2b show the results of microscopic and TEM analyses, respectively, showing that hNCs (hNCs cellbots) containing magnetic nanoparticles were successfully fabricated by incorporating magnetic nanoparticles into hNCs.

[0053] Figure 3 shows whether there is a change in the cell proliferation ability of hNCs loaded with magnetic nanoparticles depending on the concentration of magnetic nanoparticles, and indicates that there is no change in the cell proliferation ability of hNCs.

[0054] Figures 4a and 4b show that when magnetic nanoparticles are incorporated at different concentrations and hNCs spheroids are produced, they can be produced regardless of the concentration and mobility is maintained.

[0055] Figure 5a is a schematic diagram of a method for fabricating mixed hNCs spheroid cellbots to reduce the amount of magnetic nanoparticles.

[0056] Figure 5b shows the mobility of mixed hNCs spheroid cellbots in a magnetic field according to the hNCs spheroid cellbot mixing ratio.

[0057] Figure 6a is an animal model of osteoarthritis created to confirm the cartilage damage treatment effect of hNCs spheroid cellbot.

[0058] Figure 6b shows the results of an analysis of forestry symptoms when hNCs spheroid cellbots were transplanted into an animal model of osteoarthritis, showing changes in feed intake and body weight.

[0059] Figure 6c shows representative Micro CT images of each individual, showing the results of cross-sectional imaging when hNCs spheroid cellbots were transplanted into an animal model of osteoarthritis.

[0060] Figure 6d shows the results of histopathological analysis when hNCs spheroid cellbots were transplanted into an animal model of osteoarthritis, and shows the histopathological analysis photographs of each individual after H&E staining.

[0061] Figure 6e shows the results of a scoring analysis based on the histopathological analysis results.

[0062]

[0063] The present invention provides a pharmaceutical composition for preventing or treating cartilage damage disease, comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient.

[0064] In the present invention, “chondrocytes” are cells that exist within the cartilaginous lamina of the cartilaginous matrix and synthesize and secrete cartilaginous matrix, and have well-developed rough endoplasmic reticulum and Golgi apparatus. Their external shape matches that of the lacunae of the cartilaginous matrix, and they are oblong or flat under the perichondrium and on the surface of the articular cartilage, and semicircular or polygonal in the deeper part. A complex of polysaccharides or proteins is bound to the cell membrane of chondrocytes. Since this complex is three-dimensionally bound to the polysaccharides or fibers of the matrix, chondrocytes float in the matrix. In the present invention, chondrocytes are a concept that also includes chondrogenic precursor cells, which are cells that have already determined the direction of differentiation into chondrocytes.

[0065] In the present invention, “nasal septum chondrocytes” are cartilage cells that form the front part of the nasal septum, which is a partition wall that divides the nasal cavity into left and right. The nasal septum chondrocytes of the present invention can be isolated from nasal septum cartilage tissue discarded during nasal septal correction surgery, one of the most frequent surgeries, or from nasal septum cartilage tissue obtained through a simple biopsy under local anesthesia, and have the advantage of no complications due to local anesthesia as a donor site that does not bear weight. In addition, although there is no report on the number of chondrocytes isolated from nasal septum tissue, a larger number can be secured compared to articular chondrocytes, and chondrocytes isolated from nasal septum cartilage, which is hyaline cartilage, have a higher cell proliferation ability than articular chondrocytes and an excellent ability to produce cartilage-specific extracellular matrix when cultured in vitro.

[0066] In addition, the present invention provides a method for isolating and culturing nasal septal cartilage cells, comprising the following steps:

[0067] (S1) Step of cutting the separated septal cartilage and treating it with type 2 collagenase;

[0068] (S2) A step of culturing the cartilage treated with the above type 2 collagenase and then filtering and harvesting it; and

[0069] (S3) Step of culturing the harvested chondrocytes for three passages.

[0070] In the present invention, “magnetic particles” are magnetic substances that can be applied in various ways due to their structural and magnetic properties, such as MRI contrast agents, bio-diagnostic devices, anti-counterfeiting ink, and device control devices inside speakers. In real life, where there are many devices that use electricity, they can be controlled by magnetism. Types include iron oxide (Fe2O3, Fe3O4), ferrite (a form in which one Fe in Fe3O4 is replaced by another magnetic atom, such as CoFe2O4, MnFe2O4), and alloys (alloyed with precious metals to increase oxidation problems caused by magnetic atoms, conductivity, and stability, such as FePt, CoPt). In practical applications, these magnetic particles are not used in powder form, but are made in a form dispersed in a liquid and used in various fields. This is because when a solution (ferrofluid) is made and a magnetic field is applied, only the part where the magnetic field is present rises sharply along the magnetic field lines. Considering industrial applications, using magnetic fields in this way has the advantage of being very cost-effective and being able to be freely controlled by changing the strength and direction of the magnetic field.

[0071] In the present invention, "magnetic nanoparticles" are magnetic particles exhibiting ferromagnetism as described above, and are nano-sized, ultra-fine materials. They are generally known to exhibit sizes ranging from several nanometers to several hundred nanometers. The term "magnetic nanoparticles" in the present invention has a broad meaning, encompassing magnetic particles that may be referred to as "magnetic nanoparticles" in the art. For example, in the present invention, the magnetic nanoparticles may have a size of 1 to 500 nm, 1 to 400 nm, 1 to 300 nm, 1 to 200 nm, 1 to 100 nm, 30 to 500 nm, 30 to 400 nm, 30 to 300 nm, 30 to 200 nm, 30 to 100 nm, 50 to 500 nm, 50 to 400 nm, 50 to 300 nm, 50 to 200 nm, 50 to 100 nm, 70 to 500 nm, 70 to 400 nm, 70 to 300 nm, 70 to 200 nm, 70 to 100 nm, 90 to 500 nm, 90 to 400 nm, 90 to 300 nm, 90 to It may be 200 nm, 90 to 100 nm, and in one embodiment of the present invention, 100 nm magnetic nanoparticles were used, but the present invention is not limited thereto.

[0072] In the present invention, Chemicell's fluidMAG-DX, 4104-1 (1 ml); 4104-5 (5 ml) were used, but the present invention is not limited thereto. The characteristics of the magnetic nanoparticles used in the present invention are as follows:

[0073] (1) Product description: Aqueous dispersion of magnetic nanoparticles; (2) Weight of Volume: 25 mg / ml; (3) Core: Maghemite; (4) Matrix: Dextran; (5) Size: 100nm; (6) Number of particles: ~1.8 x 10^15 / g; (7) Density: ~1.25g / cm^3; (8) Type of Magnetization: Superparamagnetic; (9) Functional Group: Hydroxyl groups; Autoclaved; (10) Storage Buffer: ddH2O; (11) Storage: 4~8℃, (12) Do not freeze; (13) Expiry date: Two years after production date.

[0074]

[0075] In the present invention, “damage” means any phenomenon in which the normal structure of a tissue is morphologically destroyed, regardless of the cause, and “cartilage damage” may mean a damage phenomenon that appears in cartilage.

[0076] In one embodiment of the present invention, the nasal septum cartilage cells containing the magnetic nanoparticles may be in the form of a spheroid, but are not limited thereto.

[0077] In the present invention, "spheroid" refers to a three-dimensional cell aggregate capable of self-organization. A spheroid is a miniature, simplified, in vitro three-dimensional organ that mimics the anatomical structure of actual tissues and can be used for various drug screening and disease models. A spheroid generally refers to a three-dimensional structure in which cells are aggregated to the extent that their cross-section appears circular or oval. However, it should be noted that this shape should be determined by considering the characteristics of the cell or cell aggregate, and does not necessarily imply a perfect spheroid or sphere.

[0078] In one embodiment of the present invention, the nasal septum chondrocytes loaded with the magnetic nanoparticles may express type 2 collagen, but are not limited thereto.

[0079] In the present invention, with regard to “type 2 collagen,” collagen is a light protein that is mainly present in the bones and skin of animals and is also distributed in cartilage, organ membranes, hair, etc., and is also called collagen and exists as a fibrous solid. When viewed under an electron microscope, it has a complex horizontal stripe structure and is insoluble in water, dilute acid, or dilute alkali, but dissolves when boiled and becomes gelatin. There are six types of collagen, and type 2 collagen is one of them and is known to be the main component of cartilage.

[0080] In the present invention, it was confirmed that type 2 collagen was expressed not only in nasal septal chondrocytes isolated from nasal septum tissue, but also in spheroids produced by culturing nasal septal chondrocytes. Although spheroids of nasal septal chondrocytes may exhibit a higher level of type 2 collagen expression than nasal septal chondrocytes, this is not limited thereto.

[0081] In one embodiment of the present invention, in the nasal septum cartilage cells containing the magnetic nanoparticles, the magnetic nanoparticles may be added in an amount of more than 0 and less than or equal to 800 μg based on 1x10^6 nasal septum cartilage cells, but is not limited thereto.

[0082] In the present invention, the amount of the magnetic nanoparticles added is 0 to 800 μg or less, 0 to 700 μg or less, 0 to 600 μg or less, 0 to 500 μg or less, 0 to 400 μg or less, 30 to 800 μg or less, 30 to 700 μg or less, 30 to 600 μg or less, 30 to 500 μg or less, 30 to 400 μg or less, 60 to 800 μg or less, 60 to 700 μg or less, 60 to 600 μg or less, 60 to 500 μg or less, 60 to 400 μg or less, 80 to 800 μg or less, 80 to 700 μg or less, 80 to 600 μg or less, 80 to 500 μg or less, 80 to It may be, but is not limited to, 400 μg or less, 100 to 800 μg or less, 100 to 700 μg or less, 100 to 600 μg or less, 100 to 500 μg or less, or 100 to 400 μg or less.

[0083] In one embodiment of the present invention, the spheroid may be a mixture of nasal septum cartilage cells and nasal septum cartilage cells loaded with magnetic nanoparticles, but is not limited thereto.

[0084] In one embodiment of the present invention, the number ratio of the nasal septum chondrocytes: the nasal septum chondrocytes loaded with magnetic nanoparticles may be (5 to 7) : (3 to 5), but is not limited thereto.

[0085] In one embodiment of the present invention, the nasal septal cartilage cells loaded with magnetic nanoparticles can maintain mobility in a magnetic field regardless of the concentration of the magnetic nanoparticles, but is not limited thereto. That is, in the present invention, the nasal septal cartilage cells loaded with magnetic nanoparticles can maintain mobility in a magnetic field regardless of whether they are in a spheroid shape or regardless of the concentration of the magnetic nanoparticles.

[0086] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0087] a) Inducing a reparative effect of hypoechoic material in the area of ​​cartilage damage;

[0088] b) produces a cartilage regeneration effect; and

[0089] c) Anti-inflammatory effect is shown.

[0090] In one embodiment of the present invention, the composition may be for injection, but is not limited thereto.

[0091] In one embodiment of the present invention, the composition may be administered by any one method selected from the group consisting of arthroscopic injection and intra-articular injection, but is not limited thereto.

[0092] While the composition of the present invention is primarily intended for injection, the injection method can encompass any method commonly used in the art. Accordingly, the composition of the present invention can be administered via arthroscopic injection, intra-articular injection, or other methods. Furthermore, the composition can be administered not only through injection therapy without arthroscopy, but also through direct administration methods, such as through administration after surgical incision, but is not limited thereto.

[0093] In one embodiment of the present invention, the cartilage damage disease may be at least one selected from the group consisting of osteoarthritis, degenerative arthritis, articular cartilage defect, rheumatoid arthritis, fracture, meniscus damage, muscle damage, ligament damage, plantar fasciitis, lateral epicondylitis, calcific myositis, joint or cartilage damage due to trauma, and nonunion of fracture, but is not limited thereto.

[0094] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0095] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0096] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0097] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0098] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0099] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0100] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0101] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0102] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0103] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0104] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0105] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0106] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0107] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0108] The pharmaceutical composition of the present invention can be administered to a subject via various routes. Any route of administration is conceivable, including subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0109] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0110] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0111] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0112] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0113] In addition, the present invention provides a method for preventing or treating cartilage damage disease, comprising a step of administering a pharmaceutically effective amount of a composition containing nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient to a subject in need thereof.

[0114] In addition, the present invention provides a use of a composition comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient for preventing or treating cartilage damage diseases.

[0115] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating cartilage damage disease, comprising a composition containing nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient.

[0116]

[0117] The present invention provides a kit for preventing or treating cartilage damage disease, comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles.

[0118] In the present invention, the "kit" refers to a tool that can prevent or treat cartilage damage diseases using nasal septum chondrocytes loaded with magnetic nanoparticles of the present invention. In this case, the nasal septum chondrocytes loaded with magnetic nanoparticles may be nasal septum chondrocytes, and in this case, according to one embodiment of the present invention, a culture composition for culturing in a spheroid form may be additionally included, but is not limited thereto.

[0119] In addition to the above-described substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. typically required for their storage and processing methods. Specifically, each component may be applied one or more times without limitation, there is no restriction on the order in which each substance is applied, and the application of each substance may be performed simultaneously or in microseconds.

[0120] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0121]

[0122] The present invention provides a method for producing nasal septum cartilage cells loaded with magnetic nanoparticles, comprising the following steps:

[0123] (S1) A step of producing nasal septum chondrocytes (NSCs) by treating nasal septum cartilage tissue isolated from nasal septum tissue with type 2 collagenase; and

[0124] (S2) A step of culturing the above nasal septum cartilage cells and further adding magnetic nanoparticles to culture them.

[0125] The present invention provides a method for producing a mixed septal cartilage cell spheroid loaded with magnetic nanoparticles for treating cartilage damage diseases, comprising the following steps:

[0126] (S1) A step of culturing nasal septum chondrocytes (NSCs) isolated from nasal septum tissue and magnetic nanoparticles to produce nasal septum chondrocytes loaded with magnetic nanoparticles; and

[0127] (S2) A step of culturing the nasal septum cartilage cells and the nasal septum cartilage cells loaded with the magnetic nanoparticles by adding them to a medium for culturing them in a spheroid form.

[0128] In the step (S1) of the method for producing a mixed septal cartilage cell spheroid loaded with magnetic nanoparticles for treating cartilage damage disease of the present invention, the step of producing the septal cartilage cells loaded with magnetic nanoparticles may include, but is not limited to, the method for producing the septal cartilage cells loaded with magnetic nanoparticles described above in one embodiment of the present invention.

[0129]

[0130] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0131]

[0132] [Example]

[0133]

[0134] Example 1. Isolation of nasal septum-derived chondrocytes and production of spheroids

[0135] Example 1-1. Isolation and culture of nasal septum-derived chondrocytes

[0136] A protocol for isolating nasal septum-derived chondrocytes (hNCs) was established by treating human nasal septum cartilage tissue discarded during septal correction surgery and human nasal septum cartilage tissue obtained during biopsy with type 2 collagenase (Fig. 1a). First, biopsied human nasal septum cartilage was washed three times with phosphate-buffered saline (PBS) and plated on a non-coated dish at a thickness of 1 mm. 3 After finely chopping, the cleaved tissue was treated with 0.2% type II collagenase in growth media (low DMEM, 10% FBS, 1% PS) and reacted overnight in a cell culture incubator. The isolated hNCs were filtered using a 70 μm filter and harvested. When the isolated hNCs were subcultured up to passage 3, the characteristic shape of chondrocytes was observed, and it was confirmed that there was no significant morphological change in the cells during the cell culture process up to passage 3 (Fig. 1b). Through this, the culture process protocol was established.

[0137]

[0138] Example 1-2. Comparison of Type 2 Collagen Expression Levels in hNCs and hNCs Spheroids

[0139] The protein expression levels of collagen type 2 in hNCs isolated and cultured in Example 1-1 and hNCs spheroids prepared therefrom were comparatively analyzed by Western blot. Specifically, hNCs were harvested using RIPA buffer, reacted on ice for approximately 20 minutes, pelleted down in a 4°C centrifuge for 20 minutes, and only the supernatant was used. Proteins were quantified using the BCA quantitative method and denatured with SDS buffer at 100°C for 5 minutes. The quantified protein samples were electrophoresed at 80 V on a 6% polyacrylamide gel and transferred to a PVDF membrane. Afterwards, the PVDF membrane was blocked with 5% skim milk and the antibody of interest was attached for confirmation.

[0140]

[0141] As a result, it was confirmed that the expression level of type II collagen, a cartilage-specific marker, was increased in hNCs spheroids compared to hNCs, which are a cell state (Fig. 1c).

[0142]

[0143] Example 2. Fabrication and verification of hNCs loaded with magnetic nanoparticles.

[0144] Example 2-1. Fabrication of hNCs loaded with magnetic nanoparticles

[0145] hNCs loaded with magnetic nanoparticles (fluidMAG-DX, 4104-1 (1 ml); 4104-5 (5 ml), Aqueous dispersion of magnetic nanoparticles, Chemicell) were fabricated. Specifically, 1x10^6 hNCs separated by the method of Example 1-1 were seeded with 10 ml of culture medium in a 100 mm culture dish and incubated overnight. After confirming that the hNCs were well attached to the culture dish, they were washed twice with DPBS and the concentration of the magnetic nanoparticles to be loaded (0 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml) were diluted in the culture medium and incubated for approximately 3 days.

[0146]

[0147] After three days, observation under a microscope confirmed that magnetic nanoparticles were successfully incorporated into hNCs, as shown in Fig. 2a.

[0148]

[0149] Example 2-2. Precision analysis of hNCs loaded with magnetic nanoparticles

[0150] In order to precisely analyze the hNCs loaded with magnetic nanoparticles produced in Example 2-1, transmission electron microscope (TEM) analysis was performed. At this time, hNCs loaded with magnetic nanoparticles were produced in the same manner as in Example 2-1 using 100 ug / ml of magnetic nanoparticles and used for the analysis. Culture was performed using a confocal-only dish as a culture dish, and transmission electron microscope analysis was performed. Specifically, before cell fixation, 2.5% GA fixative was preheated to 37°C in a water bath or incubator without using a rinse. A volume of warm 2.5% GA fixative equal to the cell culture medium was added along the wall of a 12-well culture plate (fixative vol / medium vol = 1 / 1). After that, the culture plate was rotated 4 to 5 times on the culture plate, and the culture plate was left on the culture plate for 5 minutes. After labeling with a clear sample identification number, the culture plate was sealed well with parafilm and kept at 4°C until the existing TEM sample preparation training / service schedule was scheduled.

[0151]

[0152] As a result, it was confirmed by TEM analysis that hNCs containing magnetic nanoparticles were successfully manufactured, similar to Example 2-1 (Fig. 2b).

[0153]

[0154] Example 3. Confirmation of the effect of magnetic nanoparticle concentration on hNCs cell proliferation ability.

[0155] According to the present invention, we analyzed whether there was an abnormality in the cell proliferation ability of hNCs according to the concentration of magnetic nanoparticles incorporated. To this end, hNCs incorporated with magnetic nanoparticles at each concentration were produced by treating them with 0 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml magnetic nanoparticles, respectively, and the cell proliferation ability of each hNC was confirmed on days 1, 3, and 5. At this time, the cell proliferation ability was analyzed using WST-8. Specifically, cells were prepared in 6-well culture dishes according to the culture conditions, and the WST-8 solution was diluted with the culture medium at a ratio of 10:1 and reacted with the cells at 37°C for 1 h for each time condition, and the wavelength value was confirmed at 450 nm.

[0156]

[0157] As a result, it was confirmed that magnetic nanoparticles did not show any effect on the proliferation ability of hNCs regardless of concentration (Fig. 3).

[0158]

[0159] Example 4. Fabrication of hNCs spheroids loaded with magnetic nanoparticles and confirmation of the independency between mobility and the amount of magnetic nanoparticles.

[0160] Example 4-1. Fabrication of hNCs spheroids using magnetic nanoparticles of different concentrations.

[0161] In Example 3, it was confirmed that the hNCs proliferation ability was maintained regardless of the concentration of magnetic nanoparticles incorporated. In this example, it was analyzed whether the amount of magnetic nanoparticles affected the production of hNCs spheroids. At this time, 0 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml magnetic nanoparticles were used, as in Example 3. Specifically, StemFIT 3D-Plastic (H853400-P) was used as the cell culture vessel for spheroid cell culture, and all medium additions and replacements were performed at the inner corner of StemFIT 3D-Plastic (H853400-P). First, StemFIT 3D-Plastic (H853400-P) was placed on the dish in which culture was to be performed, DPBS was filled, and then bubbles were removed by pipetting. After all bubbles were removed, DPBS was suctioned from the corner of StemFIT 3D-Plastic (H853400-P) using a pipette. Be careful not to drain all the DPBS inside the microwells to prevent bubbles from forming again. To enable cell culture in the wells without forming bubbles, cell culture medium was filled while each well was still filled with DPBS. After pipetting once more to remove the DPBS inside the microwells, suction was performed, and the prepared single cells were seeded on StemFIT 3D-Plastic (H853400-P) and waited for 10 minutes until all the cells settled inside the microwells of StemFIT 3D-Plastic (H853400-P). At this time, if you place it on the microscope and shake it gently, the medium will shake and floating cells will be observed.If you wash the cells before they have all settled, you will lose that many cells, and in the case of cells other than embryonic stem cells, the size of the spheroids will decrease, so we waited until all the floating cells had settled. After 10 minutes, if there were many cells between the wells, we slowly suctioned the medium from the inner corner of the StemFIT 3D with a pipette and filled the cell culture medium so that surface tension was created inside the StemFIT 3D-Plastic (H853400-P).

[0162] The viability of cells cultured in concave molds was assessed using a live / dead assay kit (Invitrogen, Carlsbad, CA, USA). Specifically, 5 μl of calcein AM solution (Invitrogen) and 20 μl of ethidium homodimer-1 solution (Invitrogen) were dissolved in 10 ml of DPBS (Gibco-BRL) and added to the cells. The cells were then incubated at 37°C for 20 minutes.

[0163]

[0164] As a result, it was confirmed that although magnetic nanoparticles were incorporated into hNCs under different concentration conditions and hNCs spheroids were produced, there was no effect on the production of hNCs spheroids (Fig. 4a).

[0165]

[0166] Example 4-2. Analysis of the mobility of hNCs spheroids according to the concentration of magnetic nanoparticles and confirmation of its independency.

[0167] The mobility of hNCs spheroids produced by adding 100 μg / ml, 200 μg / ml, and 400 μg / ml magnetic nanoparticles, respectively, using the method of Example 4-1 was analyzed. Spheroid culture was performed under each culture condition incorporating magnetic nanoparticles, and then the mobility of the spheroids was confirmed using a magnet.

[0168]

[0169] As a result, it was confirmed that hNCs spheroids fabricated from hNCs loaded with magnetic nanoparticles moved in a desired direction in a magnetic field regardless of the concentration of magnetic nanoparticles (Fig. 4b).

[0170]

[0171] Therefore, hNCs spheroids manufactured with hNCs loaded with magnetic nanoparticles were proven to be capable of moving according to magnetism, regardless of the amount of magnetic nanoparticles loaded, and therefore, hNCs spheroids manufactured with magnetic nanoparticles loaded in the present invention are named “hNCs spheroid cellbots.”

[0172]

[0173] Example 5. Establishment of a method for manufacturing hNCs spheroid cellbots to reduce the use concentration of magnetic nanoparticles.

[0174] In the spheroid production step, a new culture method was developed that allows movement within a magnetic field and incorporates less magnetic nanoparticles than the existing production method by utilizing both cells incorporated with and without magnetic nanoparticles (Fig. 5a).

[0175]

[0176] Spheroids were cultured and produced by mixing hNCs without magnetic nanoparticles and hNCs loaded with 100 μg / ml of magnetic nanoparticles at a certain number ratio. At this time, the number ratios of hNCs and hNCs loaded with magnetic nanoparticles (hNCs:MNP_hNCs) were produced as 50% (50:50), 60% (60:40), and 70% (70:30), respectively, and the hNCs spheroid cellbots produced by mixing at each ratio were named 50% mixed hNCs spheroid cellbots, 60% mixed hNCs spheroid cellbots, and 70% mixed hNCs spheroid cellbots.

[0177]

[0178] We analyzed whether the mixed hNCs spheroid cellbots produced at each ratio maintained their mobility in a magnetic field. At this time, the experimental method was applied in the same manner as in Example 4-2.

[0179] As a result, it was confirmed that the mixed hNCs spheroid cellbot moved smoothly under a magnetic field regardless of the mixing ratio.

[0180]

[0181] Example 6. Confirmation of the excellent osteoarthritis treatment activity of hNCs spheroid cellbot.

[0182] Example 6-1. Establishment of an animal model of osteoarthritis

[0183] To evaluate the efficacy of hNCs spheroid cellbots for articular cartilage regeneration, conducted by the Daegu-Gyeongbuk Medical Industry Promotion Foundation, a rabbit model of cartilage damage was first created. Specifically, rabbits were fasted for 1 day, and then anesthesia was induced with 5 mg / kg of Alfaxalone intramuscularly. Anesthesia was maintained with 2–4% isoflurane inhalation. The knee surgical site was shaved, and povidone (10%) and alcohol (70%) were applied three times. After covering the surgical site with a sterile drapery, the skin, subcutis, muscle, and synovium of the knee area were incised. The patella was laterally retracted to expose the distal end of the femur, and a circular defect, 4 mm in diameter, was induced in the cartilage of the intercondyar groove using a high-speed drill to create a rabbit model of osteoarthritis, a cartilage damage animal model (Fig. 6a).

[0184]

[0185] Next, hNCs spheroid cellbots were produced to confirm the therapeutic activity. At this time, hNCs cells: hNCs cells loaded with 100 ug / ml magnetic nanoparticles were used in a ratio of 7:3 (70%:30%) to produce a 70% mixed hNCs spheroid cellbot. Then, the 70% mixed hNCs spheroid cellbots were transplanted into the defect site of an osteoarthritis animal model at a concentration of 1x10^7 cells / ml, 50 to 80 ul per animal, to produce an experimental group. After fixation with a tissue adhesive, the blood film, muscle, and skin were sutured, and the patient was admitted after disinfection of the surgical site. Sham and negative control groups were used as control groups. The following analysis was performed for each group to confirm the osteoarthritis therapeutic activity.

[0186]

[0187] Example 6-2. Analysis of clinical symptoms after hNCs spheroid cellbot transplantation.

[0188] Clinical symptoms were observed for each group produced using the method of Example 6-1. To observe clinical symptoms, feed intake and body weight changes were checked for a total of 4 weeks.

[0189]

[0190] As a result (Fig. 6b), feed intake decreased in the negative control group and experimental group after surgery, but showed a tendency to recover in the second week, and the Sham group showed no significant change. In addition, body weight measurements showed no statistically significant differences between each group, and no unusual findings were observed in clinical symptoms or clinical pathology tests.

[0191]

[0192] Example 6-3. Evaluation of CT and histopathological analysis after hNCs spheroid cellbot transplantation

[0193] Histopathological analysis was performed on each group produced using the method of Example 6-1. At this time, for the tomography, rabbits corresponding to each group were euthanized, and the femurs were extracted and micro CT images were taken.

[0194]

[0195] As a result, it was revealed that hypoechoic material rather than bone partially repaired the defect area around the defect area in the experimental group and the negative control group.

[0196] Additionally, histopathological examination revealed significant differences between the experimental group and the sham group, whereas no significant differences were observed between the experimental group and the negative control group. When only the defect area was examined, a tendency toward higher cartilaginous tissue formation was confirmed in the experimental group compared to the negative control group (Figs. 6c and 6d).

[0197]

[0198] The histopathological analysis results were scored and quantitatively analyzed. The combined results of filing of defects and inflammation confirmed a significant difference in the experimental group compared to the sham group, but no significant difference compared to the negative control group (Fig. 6e).

[0199]

[0200] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0201]

[0202] According to the pharmaceutical composition for treating cartilage damage disease comprising nasal septum chondrocytes loaded with magnetic nanoparticles of the present invention as an active ingredient, a method for incorporating magnetic nanoparticles into nasal septum chondrocytes and a method for producing spheroids by culturing nasal septum chondrocytes loaded with magnetic nanoparticles were established. It was confirmed that the nasal septum chondrocytes loaded with magnetic nanoparticles produced by the method of the present invention and the spheroids produced therefrom exhibit cartilage damage treatment activity while maintaining mobility in a magnetic field. In addition, since the activity is maintained even when the nasal septum chondrocytes loaded with magnetic nanoparticles and the nasal septum chondrocytes are mixed to produce spheroids, the addition ratio of magnetic nanoparticles can be significantly reduced, so that the composition can be usefully utilized as a composition for treating cartilage damage disease with excellent therapeutic effects without causing side effects, and thus has industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating cartilage damage disease, comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient.

2. In paragraph 1, A pharmaceutical composition wherein the nasal septum cartilage cells containing the magnetic nanoparticles are in the form of a spheroid.

3. In paragraph 1, A pharmaceutical composition, wherein the magnetic nanoparticles are added in an amount of more than 800 μg per 1x10^6 nasal septum cartilage cells, in which the magnetic nanoparticles are incorporated.

4. In paragraph 2, The above spheroid is a pharmaceutical composition comprising a mixture of nasal septum cartilage cells and nasal septum cartilage cells loaded with magnetic nanoparticles. A pharmaceutical composition, wherein the number ratio of the above nasal septum cartilage cells: nasal septum cartilage cells loaded with magnetic nanoparticles is (5 to 7) : (3 to 5).

5. In paragraph 1, The above composition is a pharmaceutical composition for injection, A pharmaceutical composition, wherein the injection is administered by any one selected from the group consisting of arthroscopic injection and intra-articular injection.

6. In paragraph 1, A pharmaceutical composition, wherein the above cartilage damage disease is at least one selected from the group consisting of osteoarthritis, degenerative arthritis, articular cartilage defect, rheumatoid arthritis, fracture, meniscus damage, muscle damage, ligament damage, plantar fasciitis, lateral epicondylitis, calcific myositis, joint or cartilage damage due to trauma, and nonunion of fracture.

7. A kit for preventing or treating cartilage damage disease, comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles.

8. A method for manufacturing nasal septum cartilage cells loaded with magnetic nanoparticles, comprising the following steps: (S1) A step of producing nasal septum chondrocytes (NSCs) by treating nasal septum cartilage tissue isolated from nasal septum tissue with type 2 collagenase; and (S2) A step of culturing the above nasal septum cartilage cells and further adding magnetic nanoparticles to culture them.

9. A method for manufacturing a mixed septal cartilage cell spheroid loaded with magnetic nanoparticles for treating cartilage damage disease, comprising the following steps: (S1) A step of culturing nasal septum chondrocytes (NSCs) isolated from nasal septum tissue and magnetic nanoparticles to produce nasal septum chondrocytes loaded with magnetic nanoparticles; and (S2) A step of culturing the nasal septum cartilage cells and the nasal septum cartilage cells loaded with the magnetic nanoparticles by adding them to a medium for culturing them in a spheroid form.

10. A method for preventing or treating cartilage damage disease, comprising administering a pharmaceutically effective amount of a composition containing nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient to a subject in need thereof.

11. Use of a composition comprising nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient for preventing or treating cartilage damage diseases.

12. Use for manufacturing a preparation for preventing or treating cartilage damage disease, comprising a composition containing nasal septum chondrocytes (NSCs) loaded with magnetic nanoparticles as an active ingredient.

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