WO2020036465A1 - Pharmaceutical composition for treating cartilage damage, comprising nasal septum chondrocytes - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cartilage damage, the composition comprising nasal septum chondrocytes (NSCs) as an active ingredient, and a method for producing the NSCs into a spheroidal shape. The NSCs enable the expression of type II collagen which is a constituent component of cartilage, and SOX9 which is involved in chondrogenic differentiation, and an excellent cartilage treatment effect was shown as a result of administrating spheroidal NSCs to an animal model of cartilage damage, and thus the pharmaceutical composition and the method for producing the NSCs, according to the present invention, may be usefully employed in the field of autologous chondrocyte implantation.

Description

Pharmaceutical composition for treating cartilage injury, including nasal septal chondrocytes

The present invention relates to a pharmaceutical composition for treating cartilage damage comprising nasal septum chondrocytes (NSC) as an active ingredient, and a method for producing the nasal septum chondrocytes in the form of a spheroid.

Articular cartilage is composed of dense, resilient connective tissue and is located at several connection points in the skeleton. Cartilage is connected to the bone and the surface is in contact with other cartilage. Cartilage is an avascular tissue and has no nerves, and is usually composed of single cell types of basic chondrocytes and synthesized by an extracellular matrix (ECM).

Articular cartilage treatment still remains an unsolved problem in modern medicine, and while treatment exists, many problems remain. One big problem is that articular cartilage has very low self-repair capacity. To solve this problem, regenerative medicine has begun to be developed, and cartilage tissue engineering can recover tissue through biological replacement.

Autologous chondrocyte implantation (ACI) technique is used when a large area of articular cartilage surface is damaged. The autologous chondrocytes to be transplanted are obtained by enzymatic separation of healthy joint cartilage (biopsy). However, generally known naturally occurring autologous chondrocytes have limitations.

Chondrocyte redifferentiation is limited by in vitro expansion culture, and the chondrogenic potential decreases with age of the donor and uses a large amount of cells. Proliferation of cells is important due to the characteristics of autologous chondrocyte transplantation. Articular cartilage cells are known to have a low proliferative power. Human nasal septal chondrocytes can compensate for this problem in human articular chondrocytes. Human nasal septal chondrocytes have the advantages of higher proliferation rate than human articular chondrocytes, high chondrogenic capacity both in vitro and in vivo, and do not age-dependantly on donors. .

However, while the development of therapeutic drugs for joint damage using nasal septum chondrocytes with such excellent effects is insufficient, the clinical demand for the complete regeneration of damaged cartilage is rapidly increasing, and this demand is increasing at the time of aging society. There is a trend.

The present inventors expressed collagen type 2 and sox 9 in the case of nasal septal chondrocytes and articular cartilage as a result of administering nasal septal chondrocytes cultured in a spheroid form to a cartilage injured animal model. It was confirmed that there is an excellent cartilage treatment ability compared to the cell was completed the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating cartilage damage, comprising nasal septum chondrocytes (NSC) as an active ingredient.

Another object of the invention is a) separating the nasal septum chondrocyte (nasal septum chondrocyte, NSC) from the nasal septum tissue;

b) culturing the nasal septal chondrocytes isolated in step a) using a cell culture medium containing BSA (Bovine serum albumin) in a cell culture vessel capable of culturing the cells in the form of a spheroid. ; And

c) to provide a method for producing nasal septum chondrocytes for treating cartilage damage, comprising recovering the spheroid-type nasal septum chondrocytes cultured in the cell culture vessel.

Still another object of the present invention is to provide a method for treating cartilage damage, comprising administering to a subject a pharmaceutical composition comprising nasal septum chondrocytes (NSC) as an active ingredient.

Still another object of the present invention is to provide a therapeutic composition for cartilage damage of a pharmaceutical composition comprising nasal septum chondrocytes (NSC) as an active ingredient.

Still another object of the present invention is to provide a use for the manufacture of a medicament used for the treatment of cartilage damage of nasal septum chondrocytes (NSC).

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for treating cartilage damage, comprising nasal septum chondrocyte (NSC) as an active ingredient.

In one embodiment of the present invention, the nasal septum chondrocytes may be in the form of a spheroid.

In another embodiment of the present invention, the nasal septal chondrocytes may express collagen type 2 (collagen type 2) or sox 9 (SOX9).

In addition, the present invention comprises the steps of: a) separating the nasal septum chondrocyte (nasal septum chondrocyte, NSC) from the nasal septum tissue;

b) culturing the nasal septal chondrocytes isolated in step a) using a cell culture medium containing BSA (Bovine serum albumin) in a cell culture vessel capable of culturing the cells in the form of a spheroid. ; And

c) providing a method for producing nasal septum chondrocytes for treating cartilage damage, comprising recovering the spheroid-type nasal septum chondrocytes cultured in the cell culture vessel.

In one embodiment of the present invention, nasal septal chondrocytes in step a) can be obtained after filtering in a 40 ~ 50nm filter.

In another embodiment of the present invention, the method may further include a step of mixing the support with the spheroid-type nasal septal chondrocytes recovered after the step c).

In addition, the present invention provides a method for treating cartilage damage, comprising administering to a subject a pharmaceutical composition comprising nasal septum chondrocytes (NSC) as an active ingredient.

The present invention also provides a therapeutic composition for cartilage damage of the pharmaceutical composition comprising nasal septum chondrocytes (NSC) as an active ingredient.

The present invention also provides a use for producing a medicament for use in the treatment of cartilage damage of nasal septum chondrocytes (NSC).

The present invention relates to a pharmaceutical composition for treating cartilage damage comprising nasal septum chondrocytes (NSC) as an active ingredient and a method for producing the nasal septum chondrocytes in the form of a spheroid. The cells express collagen type 2, which is a component of cartilage, and Sox 9 (SOX9), which is involved in chondrocyte differentiation, and spheroid-type nasal septal chondrocytes are administered to cartilage injured animals. Compared with the results of the administration of articular chondrocytes, the cartilage treatment effect was better. The pharmaceutical composition of the present invention and the method for producing nasal septal chondrocytes in the form of spheroids are useful in the field of autologous chondrocyte implantation. Can be used.

1 is a collagen type 2 comparison of 8 types of human nasal septum chondrocytes (hNSCs) and human turbinate derived mesenchymal stem cells (hTMSCs) using Western blot. And Sox9 expression results.

2 is cultured using hematoxylin and Eosin, Alcian blue and Masson trichrome to compare the morphological differences between 2D cell culture and 3D cell culture. This is the result of staining the cells.

Figure 3 shows the results of culturing human nasal septum chondrocytes (hNSCs) and articular chondrocytes (AC) in spheroid culture through live & dead staining.

Figure 4 shows a schematic experimental procedure for confirming the cartilage regeneration ability in the cartilage damaged animal model.

Figure 5 shows a method for making a cartilage damaged animal model and cartilage transplantation method.

Figure 6 stained cartilage defects of normal cartilage and cartilage damage animal model with hematoxylin and Eosin, Alcian blue Safranin O and Trichrome One result is shown.

Figure 7 shows the difference in cartilage regeneration ability when transplanted human nasal septal chondrocytes (hNSCs) and articular chondrocytes (AC) in a cell state without spheroid pelleting or pelleting in a cartilage injury animal model.

8 is a result of confirming that cartilage was engrafted through staining after transplanting human nasal septal chondrocytes into a spheroid form in a cartilage injury animal model.

Figure 9 is a result of verifying cell engraftment after administration of the injection nasal septum chondrocyte treatment in a cartilage injury animal model.

FIG. 10 shows the results of histological analysis of knee cartilage after transplanting human nasal septal chondrocyte spheroids and human articular chondrocyte spheroids mixed with collagen in a cartilage injury animal model.

11 shows the results of morphological differences between knee cartilage after transplanting human nasal septal chondrocytes and human nasal septal chondrocytes spheroids in the cartilage injury animal model.

The present inventors expressed collagen type 2 and sox 9 in the case of nasal septal chondrocytes, and when the spheroid-type nasal septal chondrocytes were administered to a cartilage injured animal model, the cartilage was superior to articular chondrocytes. It was confirmed that there is a therapeutic ability to complete the present invention.

In one embodiment of the present invention was confirmed for the method and culture method of separating human nasal septum chondrocytes from nasal septum tissue (see Example 1).

In another embodiment of the present invention, it was confirmed by Western blot that human nasal septal chondrocytes express collagen type 2, which is a component of cartilage, and Sox 9 (SOX9), which are involved in chondrocyte differentiation (see Example 2).

In another embodiment of the present invention was confirmed for the method for producing human nasal septum chondrocytes in the spheroid form (see Example 3).

In another embodiment of the present invention, it was confirmed that the cells form a sphere in the case of spheroid culture, which is a 3D culture (see Example 4).

In another embodiment of the present invention, it was confirmed that the viability of the spheroid-type nasal septum chondrocytes was superior to that of the spheroid-type articular chondrocytes (see Example 5).

In another embodiment of the present invention was established a cartilage damage animal model and confirmed the engraftment of the pellets as a result of administering the spheroid pellets made using nasal septum cartilage cells to the joint injury site of the cartilage injury animal model (Example 6 and 7).

In another embodiment of the present invention, when the spheroid-type nasal septal chondrocytes were administered to the joint injury site in the cartilage injury animal model, the cells were well grown (see Example 8).

In another embodiment of the present invention, when the spheroid-type human nasal septum chondrocytes are administered in a cartilage-damaged animal model, a smoother cartilage regeneration effect is observed compared to when the spheroid-type human articular chondrocytes are administered. (See Example 9).

In another embodiment of the present invention, when the spheroid-type human nasal septal chondrocytes were administered in the cartilage-damaged animal model, it was confirmed that a smoother cartilage regeneration effect was observed as compared with the administration of human nasal septal chondrocytes. (See Example 10).

As a result of the above embodiment, it was confirmed that the spheroid-type nasal septum chondrocytes according to the present invention can heal cartilage damage. The spheroid-type nasal septal chondrocytes may be used for the treatment of cartilage damage. Can be.

Thus, the present invention provides a pharmaceutical composition for treating cartilage damage, comprising nasal septum chondrocyte (NSC) as an active ingredient.

As used herein, the term "treatment" refers to any action in which cartilage damage is improved or beneficially altered by administration of a pharmaceutical composition according to the present invention.

As used herein, the term "chondrocyte" is a cell present in the cartilage of the cartilage substrate and synthesizing and secreting the cartilage substrate. Rough endoplasmic reticulum, Golgi apparatus is well developed. Appearance coincides with the appearance of cartilage cavity and exists in the form of long oval or flat in subcartilage and articular cartilage surface and semicircular or polygonal in deep layer. Complexes of polysaccharides and proteins bind to the cell membranes of chondrocytes. Because this complex binds in three dimensions to the polysaccharides and fibers of the matrix, chondrocytes are suspended in the matrix. In the present invention, chondrocytes are a concept including even chondrocyte precursor cells, which have already been determined to differentiate into chondrocytes.

The term "nasal septum chondrocytes" used in the present invention is chondrocytes forming the front part of the nasal septum, which is a partition wall dividing the nasal cavity from side to side. Nasal septal chondrocytes of the present invention can be isolated from nasal septal cartilage tissue discarded during one of the most frequent surgery, nasal septal cartilage tissue or nasal septum cartilage tissue under local anesthesia, and with local anesthesia as a donor without weight load. There is no complication. In addition, there is no report on the number of chondrocytes separated from nasal septum tissue, but more numbers can be obtained than articular chondrocytes. Chondrocytes isolated from nasal septum cartilage, which is a supernatural bone, have higher cell proliferation capacity than articular chondrocytes. Excellent ability to produce cartilage specific extracellular matrix.

As used herein, the term "damage" means any phenomenon in which the normal structure of a tissue is morphologically destroyed whatever the cause.

The pharmaceutical composition according to the present invention includes nasal septum chondrocytes as an active ingredient, and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, or oral ingestion, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The composition according to the invention can be administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type of disease, the severity, and the activity of the drug. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex and weight of the patient. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

In the present invention, the nasal septum chondrocytes may be in the form of a spheroid.

As used herein, the term “spheroid” generally refers to a three-dimensional structure in which cells are aggregated such that a cross section may be circular or elliptical, and the shape should be determined in consideration of the characteristics of the cell or cell aggregate, Obviously, it does not mean spheroid or spherical.

In the present invention, the nasal septal chondrocytes may express collagen type 2 (collagen type 2) or sox 9 (SOX9).

In connection with the term "collagen type 2" used in the present invention, collagen is a light protein which is mainly present in the bones and skin of animals and is also distributed in cartilage, organ membranes, hair, etc. exist. In electron microscope, it has a complicated horizontal pattern structure, and it is insoluble in water, dilute acid, and dilute alkali, but when boiled, it becomes gelatin and dissolves. There are six types of such collagen, and collagen type 2 of the present invention is the main component of cartilage.

The term "SOX9" as used in the present invention, together with other members of the HMG-box class DNA binding protein, is known to recognize the CCTTGAG sequence and is involved in chondrocyte differentiation.

In another aspect of the invention, the present invention comprises the steps of: a) separating nasal septum chondrocytes (NSC) from nasal septum tissue;

b) culturing the nasal septal chondrocytes isolated in step a) using a cell culture medium containing BSA (Bovine serum albumin) in a cell culture vessel capable of culturing the cells in the form of a spheroid. ; And

c) providing a method for producing nasal septum chondrocytes for treating cartilage damage, comprising recovering the spheroid-type nasal septum chondrocytes cultured in the cell culture vessel.

Cell culture vessel in step b) of the present invention is not particularly limited as long as it is a container capable of culturing cells in the spheroid form, but in the following examples used StemFIT 3D of Microfit, but is not limited thereto.

Nasal septal chondrocytes in step a) of the present invention may be obtained after filtering in a 30 ~ 50nm filter, but is not limited thereto, preferably 40nm filter.

In step b) of the present invention, the BSA may be 1-5% concentration, but is not limited thereto. Preferably, the BSA is 3% concentration.

In step b) of the present invention, the medium for cell culture may be mixed with the medium and the BSA at a ratio of 2: 0.5 to 1.5, but is not limited thereto. Preferably, the medium and the BSA have a 2: 1 ratio.

After the step c) of the present invention may further comprise the step of mixing the spheroid-type nasal septal chondrocytes and the support recovered.

As used herein, the term "support" mimics the properties of the extracellular matrix (ECM) as it is, and the biological tissue is modified in its shape and function by the interaction of various cells with extracellular substances. The extracellular matrix, which is retained and is mainly composed of organic polymers among extracellular materials, serves as a structural supporter and cell adhesion inducer of tissues. In other words, the cells must be adhered to the ECM to be fused to the tissue to perform the basic functions, and various biological control is possible outside the cell. In the present invention, the support is preferably a porous sponge, nanofibers, hydrogels, collagen and the like, but is not limited thereto and may be any ECM that can be applied clinically.

In another aspect, the present invention provides a method for treating cartilage damage, comprising administering to a subject a pharmaceutical composition comprising nasal septum chondrocytes (NSC) as an active ingredient.

In still another aspect of the present invention, a pharmaceutical composition comprising nasal septum chondrocytes (NSC) as an active ingredient is provided for treating cartilage damage.

In still another aspect of the present invention, there is provided a use for producing a medicament for use in treating cartilage damage of nasal septum chondrocytes (NSC).

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Isolation and Culture of Nasal Septal Chondrocytes

The nasal septal cartilage tissue used in this study was obtained during the process of septal correction and was used with the consent of the patient. Immediately after collecting the nasal septum cartilage tissue, cartilage cells were isolated by washing 3-5 times with saline containing gentamicin (gentamicin).

Biopsied tissues were stored in a 4 ° C. refrigerator for the separation of human nasal septal chondrocytes, and washed twice in PBS (phosphate buffered saline) before separating chondrocytes using the tissues. After washing, the nasal septum cartilage was chopped into 1 mm ³ on a non-coating dish, and the chopped tissue was treated with a type 2 collagenase in a 37 ° C., 5% CO₂ incubator in a non-coated dish. Reaction overnight. (Type 2 collagenase, 0.01g in 10mL low glucose DMEM media, 10% FBS, 1% Antibiotic-Antimycotic) The isolated chondrocytes were filtered using a 40nm filter and harvested.

The harvested chondrocytes were spun down to remove the media and washed with PBS. Chondrocytes were seeded in culture dishes and cultured in a 37 ° C., 5% CO 2 incubator.

Example 2. Expression of Collagen Type 2 and Sox9 in Nasal Septal Chondrocytes

Human Nasal Septal Chondrocytes (hNSCs) and Mesenchymal Stem-derived Mesenchymal Stem Cells (hTMSc) were cultured to investigate the expression of collagen type 2 and SOX9 in nasal septal chondrocytes. Western blot was performed by the method.

First, chondrocytes were harvested using RIPA buffer. About 20 minutes After reacting on ice and pelleting down for 20 minutes in a 4 ° C centrifuge, only the supernatant was used. Protein quantification using BCA assay and SDS The mixture was denatured for 5 minutes at 100 ° C. with a buffer. Quantified protein samples are electrophoresis at 80V on 6% polyacrylamide gel and transferred to PVDF membrane. After blocking the PVDF membrane using 5% skim milk (Skim milk) and then attached to the antibody to confirm it was confirmed.

As a result, collagen type 2 (collagen type 2) and Sox 9 (SOX9) proteins were not expressed in human nasal nasal humerus-derived mesenchymal stem cells as shown in FIG. 1, but in the case of human nasal septal chondrocytes, collagen type 2 (collagen type) 2) and Sox 9 (SOX9) was confirmed that the protein expression is high (Fig. 1).

Example 3. Preparation of Nasal Septal Chondrocytes in Spheroid Form

In the present invention, a cell culture vessel for culturing spheroid cells was used StemFIT 3D (Microfit) and all the addition and replacement of the medium was carried out in the inner corner of StemFIT 3D (Microfit). First, StemFIT 3D (Microfit) was placed on the dish to be cultured, filled with 70% ethanol (EtOH), and then bubbled by pipetting. After all of the bubbles were removed, 70% ethanol was suctioned at the corner of StemFIT 3D (Microfit) with a pipette. At this time, the 70% ethanol in the well (well) was careful not to fall out, so as not to bubble again. Cell culture medium or 1XPBS (Welgene) was filled with 70% ethanol in each well to allow cell culture in the wells without foaming the wells. After observing under a microscope, a single cell prepared by suction was seeded on StemFIT 3D (Microfit), and 3% BSA (Bovine serum albumin) filtered to a ratio of 2: 1 in the medium in which the single cell was released. ) Were added together and seeded, and then waited until the cells had all settled into the wells of StemFIT 3D (Microfit). At this time, if you place it on a microscope and shake it slightly, the medium is shaken and the floating cells are observed. If you wash the cells without all the sinking, the cells are lost so that the cells are not embryonic stem cells. As the size of the Lloyd's got smaller, it waited until all the floating cells had sunk. After 5 minutes, if there were a lot of cells between the wells, the medium was slowly suctioned with a pipette at the inner edge of StemFIT 3D and the cell culture medium was filled to generate surface tension inside StemFIT 3D. Incubation was performed as with conventional cell culture, and the first medium exchange was performed within 4 to 24 hours. When the cells were shown to be aggregated, except for BSA, only the cell culture medium was filled, and after 2 to 3 days, the microscopic spheroid was observed and the 3D spheroid was harvested and mixed with the support for transplantation.

Example 4. Morphology of Nasal Septal Chondrocytes in Spheroid Form

In order to compare the morphological differences between conventional 2D cell culture and spheroidal 3D cell culture, hematoxylin and Eosin, Alcian blue and trichrome were cultured. Cells were stained.

As a result, as shown in FIG. 2, the cells were generally spread in the 2D culture and did not show high density, whereas in the 3D culture, the spheres were formed at high density (FIG. 2).

Example 5 Viability of Nasal Septal Chondrocytes in Spheroid Form

In order to analyze the viability of spheroid nasal septal chondrocytes, spheroid culture was performed for 14 days, and live & dead staining was performed in which live cells were green and dead cells were red.

As a result, both human nasal septum chondrocyte (hNSCs) and human articular chondrocytes (hACs) showed high cell viability and showed over 90% cell viability by 14 days. However, in terms of spheroid structure, human nasal septal chondrocytes and human articular chondrocytes showed a slightly different pattern. After 7 days, human nasal septal chondrocytes spheroids were found to have a very large number of cells. Compared with human nasal septal chondrocytes, it was confirmed that the cells gathered in the spheroid structure somewhat less (FIG. 3).

Example 6. Cartilage Damage Animal Model Establishment

Rat rats were SD rats, 12 weeks old, male, and the experiment was conducted two weeks after the 10-week-old order because the animal room must undergo internal period of acclimatization.

In order to establish a cartilage damage animal model, as shown in Figure 5, the knee of the rat (knee) was incised to expose the femur (femer), and then using a drill (2mm) to make a defect site in the rat's knee. To check for cartilage damage the next day, hematoxylin and Eosin, Alcian blue, Safranin O and trichrome were used as shown in FIG. 6. The defect site was stained.

As a result, it was found that staining of defect sites was not confirmed in all staining reagents such as B, D, F, and H of FIG. 6, so that the cartilage damaged animal model was correctly established.

Example 7 Confirmation of Nasal Septal Chondrocyte Transplantation Effect in Spheroid Form

In order to confirm the effect of nasal septum chondrocyte transplantation of the spheroid form, the experiment was carried out in the steps shown in FIG. First, spheroid pellets made from human nasal septum cartilage cells and human articular chondrocytes were transplanted into cartilage injury model rats, harvested two weeks later, staining cartilage sections, and Change was observed. Alcian blue and Safranin-O staining confirmed glycosaminoglycan (GAG) and proteoglycan levels and cartilage. Trichrom ) Collagen (Collagen) was confirmed by staining.

As a result, as shown in Figures 7 and 8 pellets in the human nasal septal chondrocytes pellet group engrafted, and cartilage specific staining confirmed the therapeutic effect of damaged cartilage.

Example 8 Verification of Cell Engraftment of Injectable Nasal Septal Chondrocyte Treatment Agents in Cartilage Injured Animal Models

After preparing a cartilage injured animal model as described in Example 6, the spheroid-type human nasal septal chondrocytes and collagen were mixed, and the cartilage injured model rats had ⁴ × 10 ⁴ cartilage cells ⁴ × ¹⁰ and collagen 20 μl. To be administered. After 4 or 8 weeks, the cartilage tissue of the knee was collected at the expense of each individual, and the nasal septal chondrocytes administered with a human nuclei antibody were confirmed to engraft well. It was.

As a result, cells were observed at 4 and 8 weeks as shown in FIG.

Example 9 Comparison of Cartilage Regeneration Effects of Human Nasal Septal Chondrocytes and Human Articular Chondrocytes in Spheroid Form

After producing a cartilage injured animal model as described in Example 6, spheroid-type human nasal septal chondrocytes, or spheroid-type human articular cartilage cells were mixed with collagen, respectively, and cartilage injury model rat 1 ⁴ × 10 ⁴ chondrocytes per collagen and 20μl collagen was administered. For histological analysis of knee cartilage, the knee cartilage tissue was sacrificed at 4 weeks after each individual, and the cells were stained with hematoxylin, eosin, alcyan blue, safranin o and horseson trichrome. .

As a result, as shown in FIG. 10, the smoother form of cartilage regeneration at the injury site of the individual transplanted with the spheroid-type human nasal septum chondrocytes than the injury site of the individual transplanted with the spheroid-type human articular chondrocytes. It was confirmed that the effect appeared.

Example 10. Comparison of Cartilage Regeneration Effects after Transplantation of Human Nasal Septal Chondrocytes and Spheroid Human Nasal Septal Chondrocytes

After preparing a cartilage injured animal model as described in Example 6, human nasal septal chondrocytes, or spheroid-type human nasal septal chondrocytes were mixed with collagen, and cartilage per model cartilage injury model rat, respectively. ⁴ × 10 ⁴ cells and 20 μl of collagen were administered. Eight weeks later, each subject was sacrificed for morphological analysis of knee cartilage tissue.

As a result, as shown in Figure 11 it was confirmed that the cartilage regeneration effect of the smoother form in the individual implanted with the spheroid-type human nasal septum chondrocytes than human nasal septum chondrocytes.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

According to the present invention, nasal septum chondrocyte (NSC) expresses collagen type 2, which is a component of cartilage, and Sox 9 (SOX9), which are involved in chondrocyte differentiation, and spheroid-type nasal septum in a cartilage injury animal model. The administration of chondrocytes resulted in excellent cartilage treatment effect on the injured area. The pharmaceutical composition of the present invention and the method for producing nasal septal chondrocytes in the form of spheroids are useful in the field of autologous chondrocyte implantation. It is considered to be of great industrial use in that it can be used.

Claims (8)

1. Nasal septum chondrocytes (nasal septum chondrocyte, NSC) comprising as an active ingredient, cartilage damage treatment pharmaceutical composition.
2. The method of claim 1,

   The nasal septum chondrocytes are characterized in that the spheroid (spheroid) form.
3. The method of claim 1,

   The nasal septal chondrocytes are characterized in that to express collagen type 2 (collagen type 2) or Sox 9 (SOX9), pharmaceutical composition.
4. a) separating nasal septum chondrocytes (NSC) from nasal septum tissue;

   b) culturing the nasal septal chondrocytes isolated in step a) using a cell culture medium containing BSA (Bovine serum albumin) in a cell culture vessel capable of culturing the cells in the form of a spheroid. ; And

   c) recovering the nasal septal chondrocytes of the spheroid form cultured in the cell culture vessel, cartilage damage treatment nasal septum chondrocytes manufacturing method.
5. The method of claim 4, wherein

   Nasal septal chondrocytes in step a) characterized in that obtained after filtering in a filter 40 ~ 50nm, cartilage damage treatment nasal septum chondrocytes manufacturing method.
6. The method of claim 4, wherein

   Method for producing nasal septum chondrocytes for treating cartilage damage, further comprising the step of mixing the support and nasal septum chondrocytes recovered in step c) and the support.
7. A method of treating cartilage damage, comprising administering to a subject a pharmaceutical composition comprising nasal septum chondrocytes (NSC) as an active ingredient.
8. Nasal septum chondrocyte (NSC) of the pharmaceutical composition comprising as an active ingredient, cartilage damage treatment.

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