KR102167443B1 - In vitro osteoarthritis condition model and drug screening method for osteoarthritis using the same - Google Patents

Abstract

The present invention relates to an in vitro osteoarthritis experimental model and a method for screening a therapeutic agent for osteoarthritis using the same, and more particularly, to chondroitin sulfate or a derivative thereof; Chondrocyte; Cell culture medium; And an in vitro osteoarthritis model composition comprising chondroitinase, an in vitro three-dimensional osteoarthritis model using the same, and an osteoarthritis treatment screening method using the same.
The in vitro osteoarthritis model composition and the three-dimensional osteoarthritis model of the present invention very similarly simulate various morphological or molecular physiological changes occurring in the cartilage of an actual osteoarthritis patient, so the study of the condition of osteoarthritis and the efficacy of the treatment for osteoarthritis It can be used very usefully.

Description

In vitro osteoarthritis condition model and drug screening method for osteoarthritis using the same}

The present invention relates to an in vitro osteoarthritis model and a method for screening a therapeutic agent for arthritis using the same, and more particularly, to chondroitin sulfate or a derivative thereof; Chondrocyte; Cell culture medium; And it relates to an in vitro ( in vitro ) arthritis model composition comprising chondroitinase, an in vitro three-dimensional arthritis model using the same, and an arthritis treatment screening method using the same.

Osteoarthritis (OA) is the most common characterized by hypertrophic differentiation of chondrocytes followed by chondrocyte death and damage to the extracellular matrix (ECM) of the cartilage in response to excessive mechanical stress. It is a degenerative joint disease. Proteolytic enzymes such as MMP-13 and ADAMTS5, which are secreted from enlarged chondrocytes, play a key role in the development of osteoarthritis. This indicates that nitric oxide and reactive oxygen species produced in chondrocytes by mechanical stress increase the decomposition of chondroitin sulfate, a matrix around chondrocytes, and this action plays an important role in the mechanism of osteoarthritis development. Suggests.

The extracellular matrix of hyaline cartilage is mainly composed of type 2 collagen, proteoglycans and water molecules. Type 2 collagen, the most abundant collagen protein in the cartilage extracellular matrix, serves as a skeleton that provides tensile strength to joint cartilage. The most abundant proteoglycans are aggrecans, which are composed of chondroitin sulfate and keratin sulfate bound to a linear protein stem. The major protease involved in the destruction of cartilage extracellular matrix during osteoarthritis is MMP-13 for collagen and ADAMTS5 for aggrecan. The destruction of the extracellular matrix of cartilage by these proteases can generate collagen and glycosaminoglycan fragments that can act as damage-associated molecular patterns (DAMPs). Molecules that can act as DAMPs include biglycan, fibronectin, low molecular weight hyaluronic acid, and tenascin C in osteoarthritis. DAMPs are involved in OA progression by inducing the production of low-grade inflammation and proteases through activation of Toll-like receptors.

On the other hand, chondroitin sulfate (CS) is a kind of sulfated GAG, a long branched polysaccharide, and is the main component of cartilage proteoglycans. According to spectroscopic analysis, CS content accounts for 35% weight/dry weight (w/dw) of young articular cartilage and 20% w/dw of adult human articular cartilage. In addition, among the cartilage extracellular matrix GAG, it interacts most closely with chondrocytes. Chondroitin sulfate was 5-7 times more associated with chondrocytes than hyaluronic acid and keratin sulfate. The half-life of proteroglycans far from cartilage extracellular matrix to chondrocytes is very long, 100-800 days, whereas proteroglycans in contact with chondrocytes have a relatively short half-life of 4-30 days. Therefore, the decomposition of chondroitin sulfate, which is abundant in cartilage and directly in contact with chondrocytes, has the potential to play an important role in the physiology of chondrocytes, and may be critical for the progression of osteoarthritis.

As such, it has been reported that chondroitin sulfate, which is a major component of the extracellular matrix of articular cartilage, is less expressed in the cartilage of osteoarthritis patients and the length of the chain is shortened. How these changes affect the morphological changes of joint cells or the expression of proteases has not been reported yet.

Accordingly, the present inventors were examining the effect of the decomposition of chondroitin sulfate, which constitutes the extracellular matrix of articular cartilage, on chondrocytes, while the decomposition product of chondroitin sulfate acts as DAMPs and itself alone can induce osteoarthritis-like pathology. It was found that 1) chondroitin sulfate-based three-dimensional hydrogel culture provided a cartilage-like environment through chondrocyte culture, and treated with chondroitinase to treat arthritis in vitro. The present invention was completed by confirming that a model composition can be provided and that 2) an in vitro screening system for osteoarthritis treatment drugs can be constructed using this.

Accordingly, an object of the present invention is chondroitin sulfate or a derivative thereof; Chondrocyte; Cell culture medium; And it is to provide an in vitro ( in vitro ) arthritis model composition comprising chondroitinase (chondroitinase).

Another object of the present invention is (a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;

(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);

(c) culturing the three-dimensional structure in a cell culture medium; And

(d) to provide an in vitro three-dimensional arthritis model prepared by a method comprising the step of treating the cell culture medium with chondroitinase.

Another object of the present invention is (a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;

(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);

(c) culturing the three-dimensional structure in a cell culture medium;

(d) treating the cell culture medium with chondroitinase and an arthritis drug candidate; And

(e) morphological characteristics of chondrocytes in the three-dimensional structure; Expression of oxidative stress markers, inducible nitrogen oxide synthase (iNOS), or extracellular matrix degrading enzymes in chondrocytes; And comparing the difference between the untreated control group and the arthritis treatment candidate treatment group with respect to at least one evaluation item selected from the group consisting of the amount of NO production in the culture medium to provide an arthritis treatment screening method.

In order to achieve the object of the present invention, the present invention is chondroitin sulfate (chondroitin sulfate) or a derivative thereof; Chondrocyte; Cell culture medium; And it provides an in vitro ( in vitro ) arthritis model composition comprising chondroitinase (chondroitinase).

In order to achieve another object of the present invention, the present invention includes (a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;

(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);

(c) culturing the three-dimensional structure in a cell culture medium; And

(d) Provides an in vitro three-dimensional osteoarthritis model prepared by a method comprising the step of treating the cell culture medium with chondroitinase.

In order to achieve another object of the present invention, the present invention includes (a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;

(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);

(c) culturing the three-dimensional structure in a cell culture medium;

(d) treating the cell culture medium with chondroitinase and an osteoarthritis therapeutic agent; And

(e) morphological characteristics of chondrocytes in the three-dimensional structure; Expression of oxidative stress markers, inducible nitrogen oxide synthase (iNOS), or extracellular matrix degrading enzymes in chondrocytes; And comparing the difference between the untreated control group and the osteoarthritis treatment candidate treatment group for at least one evaluation item selected from the group consisting of the amount of NO production in the culture medium.

Hereinafter, the present invention will be described in more detail.

According to an embodiment of the present invention, as a result of decomposing CS by treating chondroitinase in chondroitinase cultured with chondroitin sulfate (hereinafter referred to as “CS”), osteoarthritis (hereinafter referred to as “OA”) ) Hypertrophic morphological change of chondrocytes was observed, as in the patient's cartilage. In addition, as a result of evaluating the change in the expression of genes in the chondrocytes, the expression of MMP-13 and ADAMTS5, which are responsible for decomposing the extracellular matrix (hereinafter referred to as “ECM”), which is a key characteristic of OA, and oxidative stress. The expression of the markers Fth1, Hmox1 and Txn was significantly increased, and it was confirmed that the NO level in the culture supernatant was also significantly increased. Through these results, it was confirmed that under the condition that CS is decomposed into chondroitinase, the morphological and molecular physiological changes similar to those in actual osteoarthritis appear in chondrocytes.

Conventionally, it was known that the expression level of CS in the cartilage of osteoarthritis patients was reduced and the chain length of CS was shortened, but there was no report on how the decomposition product of CS affects chondrocytes. In particular, it has not been previously reported that the CS decomposition product by chondroitanase itself can induce a pathological change similar to osteoarthritis in chondrocytes, and this discovery is the first report of the present inventors through the present invention.

Accordingly, the present inventors established a three-dimensional chondrocyte hydrogel culture method based on chondroitin sulfate, and treated with chondroitinase to induce damage to the extracellular matrix occurring in osteoarthritis situations. Under these conditions, a candidate substance for osteoarthritis treatment was treated and the effect on the expression of an important proteolytic enzyme in the development of osteoarthritis was confirmed to develop a method that can be used for screening of the candidate substance as a treatment for osteoarthritis.

Thus, the present invention is chondroitin sulfate (chondroitin sulfate) or a derivative thereof; Chondrocyte; Cell culture medium; And it provides an in vitro ( in vitro ) osteoarthritis model composition comprising chondroitinase (chondroitinase).

In the present invention, the chondroitin sulfate is a sulfated glycosaminoglycan composed of D-gluronic acid, N-acetyl galactosamine, and sulfate, and is often contained in cartilage and connective tissue. In particular, CS has been reported to have anti-inflammatory effects in animal models and is used as a component of the dermal layer of an FDA-approved skin substitute for burn treatment.

In the present invention, the type and molecular weight of the chondroitin sulfate are not particularly limited, and preferably chondroitin sulfate A (chondroitin-4-sulfate), chondroitin sulfate C (chondroitin-6-sulfate), chondroitin sulfate D (chondroitin-2, 6-sulfate) and chondroitin sulfate E (chondroitin-4,6-sulfate) may be one or more selected from the group consisting of, cartilage has the most chondroitin sulfate C is the most preferred (A & R 1988; 31:1028).

In the present invention, the chondroitin sulfate derivative refers to chondroitin sulfate into which a functional group capable of forming a crosslink by itself is introduced, and the type of the functional group capable of forming a crosslink is not particularly limited, but for example For example, it may include a methacryl group, an acrylic group, and a thiol group.

It should be clearly understood that a method for preparing a chondroitin sulfate derivative into which a functional group capable of forming a crosslink is introduced is well known in the art, and that a person skilled in the art will be able to apply it to the present invention by utilizing these known techniques. .

In the present invention, the cell culture medium refers to a mixture for growth and proliferation of cells in vitro containing essential elements such as growth and proliferation of cells. Preferably, the basic medium may be a chemically defined medium whose constituents and contents are clearly identified. The basic medium of the present invention may be artificially synthesized and used, or a commercially available chemically defined medium may be used. Commercially prepared media include, for example, Dulbecco's buffered saline, Earl balanced salt, Hank balanced salt, Tyrode salt, Alciber solution, Gay balanced salt solution, Crab-Hengelite denaturation buffer, Crab-Ringer bicarbonate buffer, Puck Saline, Dulbecco's Modified Eagle's Medium, Dulbecco's Modified Eagle's Medium/Nutrients F-12 Ham, Nutrient Mixture F-10Ham (Ham's F-10), Medium 199, Eagle Minimum Essential Medium, RPMI-1640 Medium, Ames Medium, BGJb Medium (Fitton-JacksonModification), Click medium, CMRL-1066 medium, Fisher medium, Glascow minimal essential medium (GMEM), Iscove modified Dulbecco medium (IMDM), L-15 medium (Leibovitz), McCoy 5A modified medium, NCTC Medium, Swim S-77 medium, Weymouth medium, William medium E, MEM medium, and combinations thereof.

The cell culture medium of the present invention may further contain any additives added to the medium for cell culture in the art. As other components in the culture medium, specifically, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine , L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, L-valine, and the like, preferably L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-tryptophan Amino acids such as L-valine; i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, ascorbic acid, etc., preferably biotin, folic acid, lipoic acid , Nicotinic acid amide, calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and vitamins such as ascorbic acid; Lipid factors such as choline chloride, choline tartrate, linoleic acid, oleic acid, cholesterol, and the like, preferably choline chloride; Energy sources such as glucose, galactose, mannose, and fructose, preferably glucose; Sodium chloride, potassium chloride, potassium nitrate, and the like, preferably a penetration pressure regulator such as sodium chloride); Iron sources such as EDTA iron, iron citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate and the like, preferably ferric chloride, iron EDTA, and iron citrate; PH buffering agents such as sodium hydrogen carbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, and the like, preferably sodium hydrogen carbonate, can be exemplified.

In addition to the above components, for example, trace metal elements such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride and sodium sulfite, preferably copper sulfate, zinc sulfate, and magnesium sulfate; Surfactants such as Tween 80 and Pluronic F68; And recombinant insulin, recombinant IGF, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-, ethanolamine hydrochloride, sodium selenate, retinoic acid, putresin hydrochloride, and the like, preferably Growth cofactors such as sodium selenate, ethanolamine hydrochloride, recombinant IGF, and putrescine hydrochloride; Nucleosides, such as deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine, uridine, etc. may be added. Further, in the preferred examples of the present invention, antibiotics such as streptomycin, penicillin G potassium, and gentamicin, and pH indicators such as phenol red may be included.

In the present invention, the chondroitinase refers to an enzyme that degrades chondroitin sulfate, and its type is not particularly limited, and includes all of the chondroitinases known in the art or new chondroitinases to be discovered in the future. do. Preferably, the chondroitinase may be at least one selected from the group consisting of chondroitinase B, chondroitinase C, chondroitinase AC, and chondroitinase ABC, and most preferably chondroitinase It could be the first ABC.

In the osteoarthritis model composition of the present invention, the chondroitin sulfate or derivative thereof is contained in an amount of 1 to 10% (w/v), and the chondrocytes are 1x10 ⁵ to 1x10 ⁹ cells/mL and the chondroitinas are 0.001 to 1 It can be included in unit/ml.

Preferably, the chondroitin sulfate or a derivative thereof is contained in 1 to 7% (w/v), and the chondrocytes contain 1x10 ⁶ to 1x10 ⁸ cells/mL and the chondroitinase at 0.01 to 0.5 unit/ml Can be

Most preferably, the chondroitin sulfate or a derivative thereof is contained in an amount of 1 to 5% (w/v), and the chondrocytes are 1x10 ⁷ to 1x10 ⁸ cells/mL and the chondroitinase is 0.05 to 0.3 unit/ml Can be included as.

The present invention also includes (a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;

(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);

(c) culturing the three-dimensional structure in a cell culture medium; And

(d) Provides an in vitro three-dimensional osteoarthritis model prepared by a method comprising the step of treating the cell culture medium with chondroitinase.

In the present invention, the steps (a) to (d) will be described in detail as follows.

(a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;

The step (a) is a step of mixing the raw materials of the in vitro three-dimensional osteoarthritis model of the present invention, and the above-described same can be applied to the chondroitin sulfate or its derivatives, and cartilage cells.

In the present invention, the crosslinking agent chemically reacts with the molecular sieve of the thermoplastic polymer to connect the molecular sieves to each other. In the present invention, in the step (a), chondroitin sulfate or a derivative thereof; Between the biocompatible polymers; And/or the chondroitin sulfate or a derivative thereof and the biocompatible polymer.

In the present invention, the type of the crosslinking agent is not particularly limited, but aluminum hydroxide, aluminum hydroxide gel, hydrous aluminum silicate, kaolin, aluminum acetate, aluminum lactate, aluminum stearate, calcium chloride, magnesium chloride, aluminum chloride, aluminum metasilicate It may be a polyvalent metal salt such as magnesium and magnesium aluminate, or an organic acid such as tartaric acid, glycolic acid, glutamic acid, lactic acid, maleic acid, and citric acid.

In the present invention, the photoinitiator refers to a material that causes rapid crosslinking upon exposure to light. Non-limiting examples of suitable optical initiators include acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide and 1-hydroxycyclohexyl phenyl ketone, lithium phenyl-2,4,6-trimethylbenzoyl Phosphinate, benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide, 1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-4'-(2-hydroxyethoxy )-2-methylpropane, etc. are mentioned. The amount of the optical initiator added may vary depending on the wavelength and time of the exposed light. The wavelength of the preferred ultraviolet ray for crosslinking may be 300 nm to 400 nm, more preferably 350 nm to 400 nm, 350 nm to 390 nm, 355 nm to 380 nm, 360 nm to 370 nm, most preferably May be 365 nm.

In the present invention, the biocompatible polymer refers to a polymer material that can form a hydrogel that becomes a skeleton of a three-dimensional structure and does not negatively affect the growth of chondrocytes. In the present invention, the type of the biocompatible polymer is not particularly limited as long as it is a biocompatible polymer that is used in cell culture and medical product manufacturing in the art. For example, alginic acid, gelatin, collagen, hyaluronic acid, chitosan, fibrinogen, Fibrin, fibronectin, laminin, elastin, proteoglycan, agarose, silk protein, glycosaminoglycan, cellulose, pectin, polyethylene glycol, and derivatives thereof.

In the present invention, the derivative of the biocompatible polymer may be interpreted as having the same meaning as the derivative of chondroitin sulfate described above.

In the composition of step (a) of the present invention, chondroitin sulfate or a derivative thereof is 1 to 10% (w/v), the chondrocytes are 1x10 ⁵ to 1x10 ⁹ cells/mL, the crosslinking agent or photoinitiator is 0.01 to 10% ( w/v) and the biocompatible polymer may be included in an amount of 1 to 10% (w/v).

Preferably, the chondroitin sulfate or derivative thereof is 1 to 7% (w/v), the chondrocytes are 1x10 ⁶ to 1x10 ⁸ cell/mL, the crosslinking agent or photoinitiator is 0.01 to 5% (w/v) and the living body The compatible polymer may be contained in an amount of 1 to 8% (w/v).

More preferably, the chondroitin sulfate or derivative thereof is contained in 1 to 5% (w/v), the chondrocytes are 1x10 ⁷ to 1x10 ⁸ cells/mL, the crosslinking agent or photoinitiator is 0.01 to 1% (w/ v) and the biocompatible polymer may be included in an amount of 3 to 8% (w/v).

Most preferably, the chondroitin sulfate or a derivative thereof (CS-MA; chondroitin sulfate-methacrylate) is contained in 3% (w/v), the chondrocytes are 1.5 x 10 ⁷ cells/mL, and the photoinitiator is 0.05% ( w/v) and the biocompatible polymer may be included in an amount of 5% (w/v).

(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);

The step (b) is a step of inducing crosslinking of a polymer material through a method such as temperature change, chemical change, or light irradiation in the composition in step (a), and the polymer included in the composition in step (a) A specific crosslinking method according to the type of material, the type of crosslinking agent, the type of photoinitiator, etc. can be performed by a person skilled in the art by selecting appropriately.

In the present invention, the shape and size of the 3D structure, and the manufacturing method are not particularly limited, and for example, the composition prepared in step (a) is filled in a 3D printer, and then crosslinked after 3D printing in a predetermined shape. It may be prepared by inducing bonding, or may be prepared by inducing crosslinking by filling in a mold having a certain shape.

(c) culturing the three-dimensional structure in a cell culture medium;

In the step (c), the three-dimensional structure prepared in step (b) is placed in a cell culture medium to provide an environment in which chondrocytes contained in the three-dimensional structure can normally grow.

According to an embodiment of the present invention, it was confirmed that the chondrocytes cultured in the step (c) matured normally and were not transformed into an abnormal form.

In the present invention, the cell culture medium may be applied in the same manner as described above.

(d) treating the cell culture medium with chondroitinase;

The step (d) is a step of creating an environment similar to osteoarthritis to chondrocytes growing in a three-dimensional structure based on chondroitin sulfate, specifically, by treating the three-dimensional structure with chondroitinase, chondroitin sulfate is decomposed. This is the step of inducing a pathological reaction similar to osteoarthritis in chondrocytes.

In the present invention, the chondroitinase may be applied in the same manner as described above.

Briefly, the chondroitinase may be treated in a cell culture medium at 0.001 to 1 unit/ml, preferably 0.01 to 0.5 unit/ml, most preferably 0.05 to 0.3 unit/ml. I can.

In addition, the chondroitinase-treated cell culture medium may be exchanged according to a certain cycle, for example, chondroitinase once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. The cell culture medium treated with rotinase can be exchanged.

According to an embodiment of the present invention, after culturing the three-dimensional structure in a culture medium in step (c) for 1 to 2 weeks, as a result of treatment with chondroitinase for 1 to 2 weeks, pathological phenomena similar to osteoarthritis It has been confirmed to be observed.

Accordingly, the step (c) of the present invention is maintained for 1 to 5 weeks, and the step (d) may preferably be started after 1 day to 2 weeks from the start date of step (c).

More preferably, the step (c) of the present invention is maintained for 1 to 4 weeks, and the step (d) may be started after 1 day to 2 weeks from the start date of step (c).

Most preferably, the step (c) of the present invention is maintained for 2 to 3 weeks, and the step (d) may be started after 1 day to 2 weeks from the start date of step (c).

According to another embodiment of the present invention, the three-dimensional osteoarthritis model cultured for a total of 3 weeks through steps (a) to (d) above is the hypertrophic form of chondrocytes, as shown in the cartilage of a patient with osteoarthritis at the end of the experiment. Hypertrophic morphological change was observed, and as a result of evaluating the change in gene expression in the chondrocytes, MMP- which is responsible for decomposing the extracellular matrix (hereinafter referred to as “ECM”), a key feature of osteoarthritis. 13 and ADAMTS5 expression, the expression of Fth1, Hmox1 and Txn, which are markers of oxidative stress, were significantly increased, and the NO level in the culture supernatant was also found to be significantly increased. Through these results, it was found that the in vitro three-dimensional osteoarthritis model of the present invention, which provides conditions for the degradation of chondroitin sulfate into chondroitinase, can induce morphological and molecular physiological changes similar to those in actual osteoarthritis. Could know.

The present invention also includes (a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;

(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);

(c) culturing the three-dimensional structure in a cell culture medium;

(d) treating the cell culture medium with chondroitinase and an osteoarthritis therapeutic agent; And

(e) morphological characteristics of chondrocytes in the three-dimensional structure; Expression of oxidative stress markers, inducible nitrogen oxide synthase (iNOS), or extracellular matrix degrading enzymes in chondrocytes; And comparing the difference between the untreated control group and the osteoarthritis treatment candidate treatment group for at least one evaluation item selected from the group consisting of the amount of NO production in the culture medium.

Since the above-described in vitro three-dimensional osteoarthritis model of the present invention simulates the morphological and molecular physiological changes occurring in actual osteoarthritis almost as it is, it can be very usefully used for screening of candidates for osteoarthritis treatment.

Steps (a) to (d) of the screening method of the present invention may be applied in the same manner as described in the three-dimensional osteoarthritis model.

On the other hand, the method of treating the candidate osteoarthritis drug in step (d) of the present invention may be changed according to the effect of the drug candidate, for example, it may proceed simultaneously with the chondroitinase treatment, or the treatment The candidate substance may be treated first and then chondroitinase may be treated, or the candidate therapeutic substance may be treated after chondroitinase treatment.

In the present invention, the kind of the candidate osteoarthritis therapeutic agent is not particularly limited, and is preferably a natural product, synthetic compound, RNA, DNA, polypeptide, enzyme, protein, ligand, antibody, metabolite of bacteria or fungi, and bioactive molecules. It may be selected from the group consisting of.

In the present invention, step (e) is a step of comparing various physiological or pathological changes observed in the treatment group for osteoarthritis treatment candidates and the untreated control group, wherein the physiological or pathological change refers to the shape of chondrocytes in the three-dimensional structure. Academic characteristics; Expression of oxidative stress markers, inducible nitrogen oxide synthase (iNOS), or extracellular matrix degrading enzymes in chondrocytes; And it may be one or more selected from the group consisting of the amount of NO production in the culture medium.

More specifically, the morphological characteristics of the chondrocytes are hypertrophic changes; The oxidative stress marker is at least one selected from the group consisting of Rth1, Hmox1 and Txn; The extracellular matrix degrading enzyme may be MMP-13 or ADAMTS5.

On the other hand, in step (e), when a result that satisfies at least one selected from the group consisting of the following (i) to (iv) in the osteoarthritis treatment candidate material treatment group compared to the untreated control group is shown, the candidate material is effective in treating osteoarthritis It may further include determining to represent:

(i) inhibition of hypertrophic change of chondrocytes in the three-dimensional structure;

(ii) decreased expression of oxidative stress markers in chondrocytes, decreased expression of inducible nitrogen oxide synthase (iNOS), or decreased expression of extracellular matrix degrading enzymes; And

(iii) Reduction of NO production in the culture medium.

The in vitro osteoarthritis model composition and the three-dimensional osteoarthritis model of the present invention very similarly simulate various morphological or molecular physiological changes that appear in the cartilage of an actual osteoarthritis patient. It can be used very usefully.

1 including FIGS. 1A to 1C shows the results of confirming the morphological changes of chondrocytes and the expression of chondrocyte differentiation marker genes that appear while culturing a chondroitin sulfate (CS)-based three-dimensional hydrogel containing chondrocytes for 7 weeks. Show.
1A shows a three-dimensional hydrogel solution consisting of 3% (w/v) of CS and 5% (w/v) of polyethylene glycol (PEG) to cultivate chondrocytes in a CS-based three-dimensional hydrogel. Is mixed with the chondrocytes and photoinitiator at a concentration of 1.5 X 10 ⁷ cells/mL. After sealing the lower end of the self-made mold with a PCR film sterilized with an autoclave, the mixture was each added in an amount of 100 ul/well and exposed to UV of 365 nm for 5 to 20 minutes to confirm the most suitable UV exposure time.
Figure 1b is a result of confirming the morphological change and the degree of production of cartilage substrates of chondrocytes cultured in a CS-based three-dimensional hydrogel made by the method presented in 1a through H&E and Safranin-O staining.
Figure 1c is a result of confirming the expression change of the differentiation marker gene of chondrocytes through RT-qPCR.
Figure 2, including Figures 2a to 2f, shows hypertrophy-like changes in chondrocytes only by decomposition of the extracellular matrix (ECM) consisting of chondroitin sulfate (CS), MMP-13, ADAMTS5, and oxidative stress in chondrocytes. The results of a series of experiments showing that it is sufficient to cause an increase in
Figure 2a is a schematic diagram schematically showing a schedule for treating chondroitinase ABC (ChABC) in a CS-based three-dimensional hydrogel culture system. E15.5 Primary chondrocytes isolated from long bones of mice were added to a three-dimensional hydrogel culture system consisting of 3% (w/v) CS and 5% (w/v) polyethylene glycol (PEG) at 1.5 X. Incubate chondrocytes at a concentration of 10 ⁷ cells/mL for 3 weeks. ChABC is treated in a three-dimensional hydrogel culture system for 1 week or 2 weeks as shown in FIG. 1A.
FIG. 2B is a diagram illustrating the overall morphology of chondrocytes in a three-dimensional hydrogel culture system through hematoxylin & eosin staining.
Figure 2c is a result of analyzing the expression level of the marker gene for chondrogenic differentiation (chondrogenic differentiation) by qRT-PCR.
2D is a result of analyzing the RNA expression levels of target genes included in the major signaling systems related to chondrocytes.
2E is a result of observation of MMP-13 and 8-oxo-dG, which are markers for hypertrophy of chondrocytes and oxidative stress, by immunofluorescence staining.
2F is a diagram showing quantification of the ratio of cells stained with MMP-13 or 8-oxo-dG relative to all cells.
3 including FIGS. 3A to 3D is a result of confirming that CS-based ECM degradation in a three-dimensional chondrocyte culture system increases MMP-13, ADAMTS5, oxidative stress and NO production through TLR2 and TLR4.
3A is a view showing the results of H&E staining for observing the overall morphological change of chondrocytes. Primary chondrocytes cultured in a CS-based three-dimensional hydrogel culture system were treated with chondroitinase ABC (ChABC) in the presence of OxPAPC (TLR2 and TLR4 inhibitor) or LPS-RS (TLR4-MD2 inhibitor) for 2 weeks. . In the upper drawing, the black square is enlarged and shown in the lower drawing.
3B is a result of analyzing the RNA expression of the indicated target genes by real-time qRT-PCR.
3C is a result of measuring the NO concentration in the culture supernatant of the three-dimensional hydrogel culture system.
Figure 3d is a diagram observed by immunofluorescence staining of 8-oxo-dG, MMP-13 and ADAMTS5 in a frozen section of a three-dimensional hydrogel culture system, and a graph showing the quantification of the proportion of cells whose staining is determined to be positive.
Figure 4, including Figures 4a to 4e, is a result of confirming that the catabolic changes of chondrocytes due to CS decomposition are dependent on MAPK, NF-kB, STAT4 and NO signaling systems.
4A is a result of observation by staining a frozen section of a three-dimensional hydrogel culture system treated with ChABC in the presence of various signal transduction inhibitors with H&E.
4B is a result of real-time qRT-PCR analysis of the expression levels of genes indicated in chondrocytes cultured in a three-dimensional hydrogel culture system treated with ChABC in the presence of various signal transduction inhibitors.
Figure 4c is a result of measuring the NO concentration in the culture supernatant of the three-dimensional hydrogel culture system cultured in the presence of various signaling inhibitors.
4D is a result of observation by immunofluorescence staining of MMP-13 (red staining) or 8-oxo-dG (yellow green color). DAPI (blue) was used as a nuclear counterstain.
4E is a result of quantifying the ratio of target (MMP-13 or 8-oxo-dG)-positive cells/DAPI-positive cells in the immunofluorescence staining experiment.
5, including FIGS. 5A and 5B, is a result of confirming that inhibition of NO mitigates phosphorylation of MAPK and NF-kB signaling systems, but not STAT3 signaling systems.
5A is a Western blot result of observing the phosphorylation of MAPK, IkBa and STAT3 by LPS in the presence or absence of 1400w, an iNOS inhibitor, over time.
5B is a schematic diagram showing the mechanism of catabolic action of chondrocytes by decomposition of CS-based ECM.

Hereinafter, the present invention will be described in detail.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

1. Experiment method

(1) primary chondrocyte culture

Primary chondrocytes were isolated from long bones of E15.5 mice. Bone (humeral radius, femur and tibia) in α-MEM-based organ culture medium supplemented with 0.2% BSA, 0.25 mM ascorbic acid, 1 mM β-glycerophosphate, 0.25% L-glutamine and 0.25% penicillin streptomycin. Isolate and equilibrate. Incubated overnight at 37°C and 5% CO ₂ . These cultured cells were incubated for 15 minutes by gently shaking trypsin-EDTA at 37°C, and then digested for 2 hours in DMEM medium containing 3 mg/ml of collagenase P (Worthington, Lakewood, NJ) and 10% FBS. Made it. Fragmented long bones were filtered through a 40-μm nylon mesh (BD Bioscience, San Jose, CA) and cells were collected by centrifugation. Cells were cultured in 2:3 DMEM:F12 medium containing 10% FBS and 0.25% L-glutamine. In order to minimize dedifferentiation, primary chondrocytes of passage 0 were used in the experiment.

(2) 3D hydrogel culture filled with primary chondrocytes and treatment of chondroitinase ABC

The chondroitin sulfate-methacrylate (CS-MA) polymer was synthesized according to a conventionally reported method. Briefly, 2-morpholino ethanesulfonic acid (MES, Sigma-Aldrich, St. Louis, MO) and sodium chloride were completely dissolved by stirring in distilled water. Chondroitin sulfate sodium (Sigma-Aldrich), N-hydroxysuccinimide (NHS, Sigma-Aldrich) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1-ethyl- 3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, EDC, Sigma-Aldrich) was added to the solution, stirred for 5 minutes, and then 2-aminoethyl methacrylate (AEMA, Sigma-Aldrich) was added. The reaction mixture was kept blocked from light for 24 hours at room temperature, dialyzed for 4 days in distilled water, and then filtered through a 0.22 μm syringe filter. CS-MA was dried in a vacuum oven and at room temperature overnight and then stored at -20°C.

Poly(ethylene glycol diacrylate), PEGDA, Laysan Bio) 5% (w/v), CS-MA polymer 3% (w/v) and photoinitiator (2-hydroxyl) to encapsulate chondrocytes -4'-(2-hydroxyethoxy)-2-methylpropane (Sigma-Aldrich) 0.05% (w/v) was added to PBS and vortexed to completely dissolve, and then primary chondrocytes were It was added to the mixture at a density of 1.5 x 10 ⁷ cells/ml. After thoroughly mixing the hydrogel solution containing primary chondrocytes 30 times, 100 μl of the cell-hydrogel suspension was sealed with a PCR film. The gel mold was put into a gel mold, and then polymerized by exposure to UV (365 nm wavelength) of 3 mW/cm ² for 10 minutes using a long wave ultraviolet lamp (UVP, LLC, California, USA). After removing the cells, the gel was placed on a 100 mm plate containing PBS, and the hydrogel filled with cells was cultured in a 24-well plate containing 1.5 ml of primary chondrocyte growth medium for 3 weeks.

Chondroitinase ABC (ChABC, 0.1 unit/ml, Sigma-Aldrich) is a variety of inhibitors (30 μg/ml of OxPAPC for TLR2/4, and 10 μg/ml LPS-RS for TLR4-MD2 from Invitrogen, 10 μM SB203580 for p38 MAPK from Sigma-Aldrich, 20nM PD98059 for ERK1/2 MAPK from Cell Signaling Technology, and 20μM SP600125 for JNK MAP kinase, 10μM JSH-23 for NF-kB, 50μM 1400w for NOS, 3mM N-acetyl-cysteine (NAC) for ROS and 5 μM Stattic for Stat3 from Sigma-Aldrich) for 1 or 2 weeks. The medium was changed every 3 days.

(3) Real-time quantitative polymerase chain reaction analysis (Real-time qPCR analysis)

Total RNA was isolated from a 3D hydrogel containing primary chondrocytes cultured using Easy Blue reagent (iNtRON biotechnology, Korea). cDNA was synthesized from 2 μg of total RNA using a random hexameter, oligod(T) primer and reverse transcription kit (ELPIS Biotech, Korea). Performed. Expression of the target gene was normalized to Gapdh. The sequence of the primers used for RT-qPCR is shown in Table 1 below.

Gene GenBank Acc. No. Sequences Amplicon (bp) Sox9 NM_011448 Forward: 5'-CAGCCCCTTCAACCTTCCTC-3'
Reverse: 5'-TGATGGTCAGCG- TAGTCGTATT-3' 94 Col2 NM_031163 Forward: 5'-CGGTCCTACGGTGTCAGG-3'
Reverse: 5'-TTATACCTCTGCCCATTCTGC-3' 70 Agc NM_007424 Forward: 5'-CCAGCCTACACCCCAGTG-3'
Reverse: 5'-GAGGGTGGGAAGCCATGT-3' 66 Ihh NM_010544 Forward: 5'-TGCATTGCTCTGTCAAGTCTG-3'
Reverse: 5'-GCTCCCCGTTCTCTAGGC-3' 93 Col10 NM_009925 Forward: 5'-GCATCTCCCAGCACCAGA-3'
Reverse: 5'-CCATGAACCAGGGTCAAGAA-3' 85 Mmp13 NM_008607 Forward, 5'-GCCAGAACTTCCCAACCAT-3'
Reverse: 5'-TCAGAGCCCAGAATTTTCTCC-3' 92 Runx2 NM_001146038 Forward: 5'-GCCCAGGCGTATTTCAGA-3'Reverse: 5'-TGCCTGGCTCTTCTTACTGAG-3' 82 Col1a1 NM_007742 Forward: 5'-CTTCACCTACAGCACCCTTGTG-3'Reverse: 5'-TTGGTGGTTTTGTATTCGATGACT-3' 80 Atf4 NM_009716 Forward: 5'-TCGATGCTCTGTTTCGAATGG-3'Reverse: 5'-AAGATCACATGTGTCATCCAACGT-3' 80 Gadd45b NM_008655 Forward: 5'-GAGGATGATATCGCTCTGCAGAT-3'Reverse: 5'-ACCCGGACGATGTCAATGTC-3' 80 Axin2 NM_015732 Forward: 5'-GACGCACTGACCGACGATT-3'Reverse: 5'-TTCTTACTCCCCATGCGGTAA-3' 80 Wisp1 NM_018865 Forward: 5'-GACCTGACCGAGTTGCCTAATG-3'
Reverse: 5'-GCGAAGTCTTCCCCATCGT-3' 80 Wnt5a NM_009524 Forward: 5'-CATTGGAGAAGGTGCGAAGAC-3'
Reverse: 5'-CCACTGTGCTGCAGTTCCAT-3' 80 Csf1 NM_007778 Forward: 5'-TCCTCATGAGCAGGAGTATTGC-3'
Reverse: 5'-CAGGACCTTCAGGTGTCCATTC-3' 80 Stat1 NM_001205313 Forward: 5'-TGAGATGTCCCGGATAGTGG-3'
Reverse: 5'-CGCCAGAGAGAAATTCGTGT-3' 80 Irf1 NM_008390 Forward: 5'-GGAAGGGAAGATAGCCGAAGA-3'
Reverse: 5'-ATCCCTTGCCATCGATGTGT-3' 80 Socs3 NM_007707 Forward: 5'-CCGCTTCGACTGTGTACTCAAG-3'Reverse: 5'-CCGTGGGTGGCAAAGAAA-3' 80 Gata3a NM_008091 Forward: 5'-CGGAAGAGGTGGACGTACTTTT-3'Reverse: 5'-CGTAGCCCTGACGGAGTTTC-3' 80 Bax NM_007527 Forward: 5'-GTGAGCGGCTGCTTGTCT-3'
Reverse: 5'-GGTCCCGAAGTAGGAGAGGA-3' 68 Ccnd1 NM_007631 Forward: 5'-GCGTACCCTGACACCAATCTC-3'
Reverse: 5'-CTCCTCTTCGCACTTCTGCTC-3' 80 Pcna NM_011045 Forward: 5'-TTTGAGGCACGCCTGATCC-3'
Reverse: 5'-GGAGACGTGAGACGAGTCCAT-3' 80 Hes1 NM_008235 Forward: 5'-CCAGCCAGTGTCAACACGA-3'Reverse: 5'-AATGCCGGGAGCTATCTTTCT-3' 80 Hey1 NM_010423 Forward: 5'-GCGCGGACGAGAATGGAAA-3'
Reverse: 5'-TCAGGTGATCCACAGTCATCTG-3' 80 Ptch1 NM_008957 Forward: 5'-GGAAGGGGCAAAGCTACAGT-3'
Reverse: 5'-TCCACCGTAAAGGAGGCTTA-3' 62 Gli1 NM_010296 Forward: 5'-CTGACTGTGCCCGAGAGTG-3'Reverse: 5'-CGCTGCTGCAAGAGGACT-3' 64 Hhip NM_020259 Forward: 5'-CCCAGACTCACAATGGAAAACTCT-3'
Reverse: 5'-GGTGTAGTAGCCGTTTCGACAGA-3' 80 Fth1 NM_010239 Forward: 5'-TGACCACGTGACCAACTTACG-3'
Reverse: 5'-CCAGGGTGTGCTTGTCAAAGA-3' 80 Hmox1 NM_010442 Forward: 5'-TGACCACGTGACCAACTTACG-3'
Reverse: 5'-CCAGGGTGTGCTTGTCAAAGA-3' 80 Txn NM_011660 Forward: 5'-ATCAAGCCCTTCTTCCATTCC-3'
Reverse: 5'-TCCTGGCAGTCATCCACATC-3' 80 Arnt NM_001037737 Forward: 5'-CCTGGCTCGAAAACCAGACA-3'Reverse: 5'-TGTTGCCAGTTCCCCTCAAG-3' 80 Serpine1 NM_008871 Forward: 5'-ATGACTGGGTGGAAAGGCATAC-3'
Reverse: 5'-CAGGCGTGTCAGCTCGTCTA-3' 80 Vegfa NM_001025250 Forward: 5'-GCAGCTTGAGTTAAACGAACG-3'
Reverse: 5'-GGTTCCCGAAACCCTGAG-3' 94 Cycs NM_007808 Forward: 5'-GGTGATGTTGAAAAAGGCAAGAA-3'
Reverse: 5'-TTATGCTTGCCTCCCTTTTCC-3' 80 AtpsynF1 NM_007505 Forward: 5'-CCTGGAATTATCCCCCGAAT-3'Reverse: 5'-CCAATGGGCACCAGGCTAT-3' 80 Cox4i NM_009941 Forward: 5'-TCACTGCGCTCGTTCTGAT-3'
Reverse: 5'-CGATCGAAAGTATGAGGGATG-3' 80 Pepck NM_011044 Forward: 5'-CTGGCCAAGATTGGTATTGAACT-3'
Reverse: 5'-GATATGCCCATCCGAGTCATG-3' 80 G6pase NM_008061 Forward: 5'-CATGTACCGGAAGAGCTGCAA-3'
Reverse: 5'-GGACCAAGGAAGCCACAATG-3' 80 Sod1 NM_011434 Forward: 5'-GCGGTGAACCAGTTGTGTTG-3'
Reverse: 5'-CCCATACTGATGGACGTGGAA-3' 80 Sod2 NM_013671 Forward: 5'-CCACACATTAACGCGCAGAT-3'Reverse: 5'-TCGGTGGCGTTGAGATTGT-3' 80 Gpx1 NM_008160 Forward: 5'-AAGTGCGAAGTGAATGGTGAGA-3'
Reverse: 5'-GGGTCGTCACTGGGTGTTG-3' 80 Catalase NM_009804 Forward: 5'-GGCCTCGCAGAGACCTGAT-3'
Reverse: 5'-ACCCCGCGGTCATGATATTA-3' 80 Hk2 NM_013820 Forward: 5'-CACGGAGCTCAACCAAAACC-3'
Reverse: 5'-CCGGAACCGCCTAGAAATCT-3' 100 Ldha NM_010699 Forward: 5'-CTCAAGGACCAGCTGATTGTGA-3'
Reverse: 5'GCACCAACCCCAACAACTGT-3' 80 Pgk1 NM_008828 Forward: 5'-TGGACAAGCTGGACGTGAAG-3'Reverse: 5'-GCCTTGATCCTTTGGTTGTTTG-3' 100 iNos NM_010927 Forward: 5'-GCAAGCATGACTTCCGAGTGT-3'
Reverse: 5'-GCCCAAGGTAGAGCCATCTG-3' 100 Gapdh NM_008084 Forward: 5'-TGTCCGTCGTGGATCTGAC-3'
Reverse: 5'-CCTGCTTCACCACCTTCTTG-3' 75

(4) Frozen section and H&E staining

The cultured chondrocyte-containing hydrogel was washed with PBS and fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany). The entire hydrogel fixed with OCT compound (Thermo Fisher Scientific, Waltham, MA) was covered on the tissue-based mold and then frozen at low temperature with 2-methyl-butane (Thermo Fisher Scientific) cooled with dry ice. The 5-μm section was prepared by Cryostat (Micron HM350, Thermo Fisher Scientific). Sections were air-dried overnight at room temperature and then stained with hematoxylin and eosin.

(5) Immunofluorescence staining

Immunofluorescence staining for detecting the expression of Mof13, Adamts5, and 8-oxo-dG was performed on the frozen sections of the hydrogel containing cells. Briefly, the portion dried in an oven at 37° C. for 1 hour was fixed with cold acetone for 20 minutes and washed twice with PBS. After 1 hour blocking with 1% BSA, antibodies to Mmp13 (ab39012), Adamts5 (ab41037, Abcam, Cambridge, UK) were used to detect chondrocyte differentiation status, or the degree of DNA oxidation to analyze oxidative stress. For analysis, 8-oxo-deoxyguanosine (4354-MC-050, Trevigen, Gaithersburg, MD) was treated with the fragment at 4°C overnight. The sections were washed with PBS, incubated with Alexa Fluor 488 or 594-conjugated secondary antibody (Thermo Fisher Scientific) for 1 hour, and then counterstained with DAPI. The section was treated with an anti-fade mounting solution (Invitrogen, Carlsbad, CA), and imaged and quantified under a Nikon ECLIPSE Ni fluorescence microscope (Nikon, Japan).

(6) Nitric oxide assay

The nitric oxide assay was performed according to the previously reported method. Briefly, the primary chondrocyte-containing hydrogel was cultured for 3 weeks, and the amount of nitrite, a stable metabolite of NO, in the culture medium was measured as an indicator of NO production. 100 μl of the culture solution was mixed with the same amount of Griess reagent (Cell Signaling Technology, Danvers, MA) in a 96 well plate, incubated at room temperature for 10 minutes, and absorbance at 540 nm was measured with a microplate reader (BioTek, Winooski). Fresh medium was used as a blank in all experiments. The amount of nitrite was determined from the sodium nitrite standard curve.

(7) TLR4 signal activation and Western blot using LPS

Primary chondrocytes were treated with 1 μg/ml of LPS (Sigma-Aldrich) for the indicated time point (0, 5, 15, 30, 60 and 120 minutes) in the presence or absence of 50 μM of 1400 W, and then RIPA protease and It was dissolved in a buffer to which a phosphatase inhibitor (Roche Diagnostics, Indianapolis, IN) was added. Equal amounts of cell lysates from each sample were separated by 10% SDS-PAGE and transferred to PVDF membrane (Amersham Biosciences, Buckinghamshire, UK). The membrane was blocked with 5% skim milk for 1 hour at room temperature and then incubated with the primary antibodies listed below. After washing the membrane with TBS-T, the membrane was incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 hour and washed again. The signal on the membrane was visualized using a chemiluminescent solution (Amersham Biosciences) and detected on FluorChem FC2 (Alpha Inotech). Primary antibodies against p-Erk1/2 (Thr202/Tyr204), p-Ikb (Ser32/36), p-Stat3 (Tyr705) were purchased from Cell Signaling Technology, and β-Actin was purchased from Sigma-Aldrich.

(8) statistical analysis

Differences between mean values were analyzed using the Mann-Whitney U test or the Kruskal-Wallis ANOVA test. All analyzes were performed using SPSS version 14.0 software (SPSS, Chicago, IL). The results were expressed as mean±standard deviation S.Es, and statistical significance was recognized when the p value was less than 0.05.

2. Experiment result

(1) CS-based ECM degradation causes hypertrophic changes in chondrocytes accompanied by expression of MMP-13 and oxidative stress.

The present invention investigated the phenotypic characteristics of primary chondrocytes under CS-based 3D hydrogel scaffold culture conditions. The contents of GAG stained with Safranin-O increased from the 3rd week and decreased from the 5th week. The decrease in GAG was associated with morphological changes in chondrocytes in H&E staining. Chondrogenic genes such as Sox9, Col2, and Aggrecan (Agc) decreased until the 5th week, but increased until the 7th week. Ihh, a pre-hypertrophic marker of chondrocytes, increased at 3 weeks. Hypertrophic markers of type X collagen (Col10), MMP-13 and Runx2, osteoblast markers, and type I collagen a1 (Col1a1) increased at 7 weeks (FIGS. 1A to 1C ).

These results confirmed that the three-dimensional hydrogel culture system used in the present invention is suitable for inducing maturation of chondrocytes, and that it does not induce hypertrophy of chondrocytes for at least three weeks of culture.

In order to confirm the effect of CS degradation on chondrocytes, chondroitinase ABC (ChABC) was treated in a CS-based three-dimensional hydrogel culture system for 1 to 2 weeks (FIG. 2A). As a result, hypertrophy of chondrocytes, which looks like an increase in the volume of cytoplasm due to the decomposition of CS, was observed (Fig. 2b).

As a result of evaluating the expression of the chondrocyte marker gene, the expression of MMP-13 was remarkably increased by ChABC treatment, and the expression of ADAMTS5 was increased by treatment with ChABC for 1 week and decreased by treatment for 2 weeks (Fig. 2c).

In order to evaluate the effect of the signaling pathway according to CS degradation, the present inventors evaluated the expression of target molecules regulated by major signaling pathways in chondrocyte physiology.

As a result, it was confirmed that Socs3 RNA expression was remarkably increased and STAT3 signaling was activated, and the expression of oxidative stress markers such as Fth1, Hmox1 and Txn all increased. However, the target molecules of Wnt, Notch, and Hedgehog signals decreased, and cyclinD1, a marker of cell proliferation, decreased. There was no difference in the expression of genes related to TGFβ, NFkB and hypoxia (Fig. 2D).

As a result of immunofluorescence staining for quantifying the relative level of protein, CS degradation by ChABC increased the expression of 8-oxo-dG, a marker for oxidative stress as well as MMP-13 (FIGS. 2e and 2f).

(2) The decomposition product of CS by chondroitinase acts as DAMPs (damage associated molecular patterns) through TLR2 and TLR4.

Next, the inventors tested whether CS degradation products could function as DAMPs that could lead to increased proteases and oxidative stress. In previous studies, the present inventors reported that ligands for TLR2 and TLR4 are significantly related to the expression of MMP-3 and MMP-13 in chondrocytes (Sci Rep. 2018;8(1):487). Based on this, the function of TLR2 and TLR4 was inhibited using OxPAPC, a double inhibitor of TLR2 and TLR4, or LPS-RS, which functionally inhibits TLR4 through MD2 inhibition, and then chondroitinase treatment of chondrocytes The phenotype was observed.

Morphologically, OxPAPC and LPS-RS partially inhibited the hypertrophic change of chondrocytes caused by CS degradation induced by chondroitinase (Fig. 3A). The increase of MMP-13 and ADAMTS5 due to CS degradation was significantly reduced by OxPAPC and LPS-RS. In addition, OxPAPC and LPS-RS significantly inhibited the increase in oxidative stress markers such as Fth1, Hmox1, and Txn (Fig. 3b).

Well-known inducible nitrogen oxide synthase (iNOS), which is known to have an important effect on cartilage degeneration, was significantly increased in RNA levels by CS degradation by chondroitinase, but this increase was observed in TLR2 and by OxPAPC and LPS-RS. It was greatly suppressed by TLR4 inhibition (Fig. 3b).

NO levels in the culture supernatant were significantly reduced by TLR2 and TLR4 inhibitors (Fig. 3c). As a result of immunofluorescence staining, it was confirmed that 8-oxo-dG, MMP-13 and ADAMTS5, which were significantly induced by CS degradation, were significantly suppressed by OxPAPC and LPS-RS (Fig. 3D).

These results indicate that the degradation products of CS act as DAMPs, which are closely involved in oxidative stress and increase of proteases such as MMP-13 and ADMATS5 through TLR2 and TLR4.

(3) The effect of CS degradation on the phenotype of chondrocytes is dependent on MAP kinase, NF-kB, STAT3 and NO signaling systems.

Next, an inhibitor assay was performed to confirm whether TLR downstream signaling is involved in the phenotypic change of chondrocytes related to CS degradation. MAP kinase and NF-kB signaling, the major downstream signals of TLR2 and TLR4, were inhibited with SB203580 for p38 MAPK, PD98059 for MEK1/ERK, SP600125 for JNK, and JSH-23 for NFkB. NO production by iNOS was 1400W, oxidative stress was N-acetyl-cysteine (NAC), and STAT3 activity was inhibited by Stattic (Fig. 4a). The inhibitors except for JSH-23 and NAC inhibited the hypertrophic morphological change of chondrocytes caused by CS degradation (FIG. 4A).

In addition, MMP-13 RNA expression by ChABC-mediated CS degradation was inhibited by these inhibitors. ADAMTS5 expression was reduced by inhibitors of p38 and JNK MAP kinase, NF-kB and NO were inhibited at the RNA level, while they were increased by ERK1/2 and STAT3 inhibitors. The expression of Fth1 and Hmox1 and markers of oxidative stress were decreased by the above inhibitors, but were not affected by JSH-23, but rather increased by NAC. Another oxidative stressor marker, Txn, was reduced only by ERK1/2 MAP kinase inhibitors. iNOS expression was reduced by all inhibitors except NAC (FIG. 4B ).

These experimental results were similarly reproduced in the NO concentration in the culture supernatant of the three-dimensional hydrogel culture system (Fig. 4c).

The expression of MMP-13 evaluated by immunofluorescence staining was attenuated by p38, ERK1/2, JNK MAP kinase, NF-kB, iNOS and STAT3 inhibitors, whereas it was not affected by the antioxidant NAC. All of the above inhibitors inhibited the expression of 8-oxo-dG, a marker of oxidative stress (Fig. 4d).

These results suggest that MAP kinase, NF-kB, NO, and STAT3-related signaling systems play a very important role in the catabolic phenotype change of chondrocytes related to CS degradation.

(4) NO enhances activation of MAP kinase and NF-kB signaling system

To elucidate the interaction between NO and other catabolic pathways such as MAPKase, NFkB and STAT3 in TLR4 activation, 1400w was used to investigate the inhibitory effect of iNOS on LPS-mediated phosphorylation of each signal in monolayer culture conditions of primary chondrocytes. And evaluated. LPS treatment of primary chondrocytes phosphorylated p38 and JNK MAP kinase within about 30 minutes, but inhibition of the iNOS-NO system delayed this activation. In addition, NO inhibition did not affect the initial activation of EK1/2 MAP kinase until within 5 minutes, but delayed the second activation within 30 minutes. Phosphorylation of IkB constituting the Canonical NF-kB signaling system was significantly inhibited by NO inhibition. However, STAT3 phosphorylation was not affected by NO inhibition (Fig. 5A).

These results suggest that not only MAP kinase and NF-kB signaling increase iNOS-mediated NO production, but NO has a positive feedback loop with NO signaling that increases activation of MAP kinase and NF-kB signaling system.

As discussed above, decomposition of CS in a CS-based hydrogel culture system not only causes hypertrophic morphological changes in chondrocytes by itself, but also has a sufficient effect to induce the production of MMP-13 and ADAMTS5 in chondrocytes. Confirmed to exert. In the CS-based hydrogel culture system according to the present invention, the degradation products of CS act as DAMPs through TLR2 and TLR4 to activate MAP kinase, NF-kB and STAT3, which forms a positive feedback loop with the NO signaling system. do.

In fact, in the cartilage tissue of osteoarthritis patients, hypertrophy of chondrocytes and destruction of extracellular matrix were observed, and the increase in proteolytic enzymes such as MMP-13 and ADMATS-5 in chondrocytes is important for the occurrence of osteoarthritis (Lancet. 2015; 386(9991):376, Arthritis Rheum. 2009;60(12):3723, Nature 2005;434:644). In the present invention, as a result of inducing destruction of the extracellular matrix by treatment with chondroitinase, morphological hypertrophy of chondrocytes was induced, and it was confirmed that this is not a true hypertrophy of chondrocytes, but a pseudo hypertrophy due to water increase. According to a recent study, the hypertrophy of chondrocytes goes through two stages. The first is intrinsic hypertrophy in which the expression of hypertrophy-related genes and proteins is increased together, which occurs in the early stages of cartilage differentiation (Nature. 2013;495(7441):375). The second is pseudohypertrophy that occurs due to the influx of water from outside without an increase in genes or proteins, and the hypertrophy accompanied by destruction of the extracellular matrix of the present invention is largely attributed to this. In addition, the special point of this study is that proteolytic enzymes such as MMP-13 and ADMATS-5 are increased without an increase in hypertrophy-related signals such as Runx2, Ihh, and Wnt signals in the process of pseudohypertrophy, which is the most characteristic of osteoarthritis pathophysiology. It is consistent with the normal part.

These results suggest that the model in which the three-dimensional hydrogel structure of this study is treated with chondroitinase to destroy the extracellular matrix of cartilage can reproduce the characteristics of human osteoarthritis cartilage as it is.

Therefore, the experimental model of the present invention that treats CS-degrading enzyme in a CS-based hydrogel culture system including chondrocytes almost identically simulates the pathological phenomena occurring in the cartilage of osteoarthritis patients. , It is expected to be very useful in screening and evaluating the efficacy of osteoarthritis treatments.

The in vitro osteoarthritis model composition and the three-dimensional osteoarthritis model of the present invention very similarly simulate various morphological or molecular physiological changes occurring in the cartilage of an actual osteoarthritis patient, so the study of the condition of osteoarthritis and the efficacy of the treatment for osteoarthritis It can be used very usefully, so it has excellent industrial applicability.

Claims (12)

delete delete delete delete (a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;
(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);
(c) culturing the three-dimensional structure in a cell culture medium; And
(d) an in vitro three-dimensional osteoarthritis model prepared by a method comprising the step of treating the cell culture medium with chondroitinase.
The method of claim 5, wherein the biocompatible polymer is alginic acid, gelatin, collagen, hyaluronic acid, chitosan, fibrinogen, fibrin, fibronectin, laminin, elastin, proteoglycan, agarose, silk protein, glycosaminoglycan, cellulose, pectin , In vitro ( in vitro ) three-dimensional osteoarthritis model, characterized in that at least one selected from the group consisting of polyethylene glycol and derivatives thereof.
The method of claim 5, wherein in the composition of step (a), chondroitin sulfate or a derivative thereof is 1 to 10% (w/v), the chondrocytes are 1x10 ⁵ to 1x10 ⁹ cells/mL, the crosslinking agent or photoinitiator is 0.01 to In vitro ( in vitro ) three-dimensional osteoarthritis model, characterized in that 10% (w / v) and the biocompatible polymer is contained in 1 to 10% (w / v).
The method according to claim 5, wherein step (c) is 1, and held there for to 5 weeks, wherein the step (d) in vitro (in vitro, characterized in that, starting 1 day to 2 weeks elapsed from the step (c) date ) Three-dimensional osteoarthritis model.
(a) chondroitin sulfate or a derivative thereof; Chondrocyte; Crosslinking agents or photoinitiators; And preparing a composition comprising a biocompatible polymer;
(b) preparing a three-dimensional structure by inducing crosslinking of the composition of (a);
(c) culturing the three-dimensional structure in a cell culture medium;
(d) treating the cell culture medium with chondroitinase and an osteoarthritis therapeutic agent; And
(e) morphological characteristics of chondrocytes in the three-dimensional structure; Expression of oxidative stress markers, inducible nitrogen oxide synthase (iNOS), or extracellular matrix degrading enzymes in chondrocytes; And comparing the difference between the untreated control group and the osteoarthritis treatment candidate treatment group for at least one evaluation item selected from the group consisting of the amount of NO production in the culture medium.
The method of claim 9, wherein in step (d), the chondroitinase and the osteoarthritis drug candidate are treated at the same time or separately.
The method of claim 9, wherein the morphological characteristics of the chondrocytes are hypertrophic change; The oxidative stress marker is at least one selected from the group consisting of Rth1, Hmox1 and Txn; And the extracellular matrix degrading enzyme is MMP-13 or ADAMTS5.
The method of claim 9, wherein in step (e), when a result that satisfies at least one selected from the group consisting of the following (i) to (iv) in the osteoarthritis treatment candidate material treatment group is shown compared to the untreated control group, the candidate material Osteoarthritis treatment screening method, characterized in that it further comprises the step of determining to exhibit the osteoarthritis treatment effect:
(i) inhibition of hypertrophic change of chondrocytes in the three-dimensional structure;
(ii) decreased expression of oxidative stress markers in chondrocytes, decreased expression of inducible nitrogen oxide synthase (iNOS), or decreased expression of extracellular matrix degrading enzymes; And
(iii) Reduction of NO production in the culture medium.

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