Composition for cartilage regeneration and preparing thereof

Abstract

The present invention provides a cartilage regenerating composition including a fetal cartilage tissue-derived cell and an extracellular matrix derived from a fetal cartilage tissue, and a preparing method thereof. According to the present invention, the cartilage-regenerating composition may produce a three-dimensional tissue of a size suitable for use as a cartilage without a scaffold, may be easily transplantable regardless of the size and shape of the cartilage defect at the site of administration since it can be administered in the form of a gel, but has high application and adhesion, may exhibit a high binding ability to the host tissue, and may have a phenotype of mature cartilage tissue, thereby exhibiting an excellent cartilage regeneration effect.

Claims

1. A method of preparing a cartilage regenerating composition comprising a fetal cartilage tissue-derived cell and extracellular matrix derived from the fetal cartilage tissue-derived cell, the method comprising: (a) incubating a fetal cartilage tissue-derived cell in a 2-dimensional (2D) culture using a culture medium containing 10% serum to form a cell sheet; (b) obtaining a cell sheet including the cultured fetal cartilage tissue-derived cell and extracellular matrix expressed from the cultured fetal cartilage tissue-derived cell; (c) centrifuging the obtained cell sheet to obtain a 3-dimensional (3D) construct; and (d) incubating the 3D construct in a serum free-cartilage differentiation medium.

2. The cartilage-regenerating composition prepared by the method of claim 1, wherein the composition is gel-like and has the following properties in vitro: compressive strength at Young's modulus of less than 20 kPa when pressed at 1 mm/min speed; spreadability having a coverage of 0.1 to 2.0 mm.sup.2/mg when a force of 5 N was applied to a sample for 1 second at a speed of 1 mm/min; and adhesiveness of 0.5 to 5.0 kPa when a material was in contact with a jig having a diameter of 5 mm and an affected part to be attached thereto, to be pulled at a rate of 1.3 mm/min to be separated therefrom.

3. The cartilage-regenerating composition prepared by the method of claim 2, wherein the cartilage-regenerating composition has the following properties: compressive strength at Young's modulus of less than 0.2 to 20 kPa when pressed at 1 mm/min speed; spreadability having a coverage of 0.2 to 1.7 mm.sup.2/mg when a force of 5 N was applied to a sample for 1 second at a speed of 1 mm/min; and adhesiveness of 0.9 to 4.5 kPa when a material was in contact with a jig having a diameter of 5 mm and an affected part to be attached thereto, to be pulled at a rate of 1.3 mm/min to be separated therefrom.

4. The cartilage-regenerating composition of claim 2, wherein the cartilage-regenerating composition exhibits a characteristic of mature cartilage by enhancing expression of glycoprotein and collagen under an in-vivo condition.

5. A pharmaceutical composition for treating a cartilage defect disease comprising the cartilage-regenerating composition according to claim 2 as an active ingredient.

6. The pharmaceutical composition of claim 5, wherein the cartilage defect diseases include at least one of degenerative arthritis, rheumatoid arthritis, fractures, muscle tissue damage, plantar fasciitis, humerus ulcer, calcified myositis, and joint damage caused by fracture nonunion and trauma.

7. A method for treating a cartilage defect disease by administering to a patient a pharmaceutically effective amount of the cartilage regenerating composition according to claim 2.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a schematic view showing a preparing method of a cartilage-regenerating composition according to an exemplary embodiment of the present invention.

(2) FIG. 2 illustrates a photograph showing the appearance of a gel-like cartilage-regenerating composition prepared by incubating for one week, two weeks, or three weeks, and a volume of the tissue according to an exemplary embodiment of the present invention.

(3) FIG. 3 illustrates results of a gel-type cartilage regeneration composition prepared by incubating for one week, two weeks, or three weeks and comparing the results with Safranin-O staining and hematoxylin & & Eosin) according to an exemplary embodiment of the present invention.

(4) FIG. 4 illustrates moisture content, DNA content, glucosamine glycans, and hydroxyproline content of a cartilage-regenerating composition prepared according to an exemplary embodiment of the present invention by incubating for one week, two weeks, or three weeks.

(5) FIG. 5 illustrates results of confirming the young's modulus (kPa) of the cartilage-regenerating composition prepared according to an embodiment of the present invention and the cartilage-regenerating composition administered ex VIVO.

(6) FIG. 6 illustrates checked results of a histological staining (Safranin O) performed for visualizing a cartilage-regenerating composition prepared according to an exemplary embodiment of the present invention and the amount of protein sugars in the cartilage-regenerating composition prepared with infant cartilage cells.

(7) FIG. 7 shows results of checking the coating property (spreadability) of a gel-like cartilage-regenerating composition prepared by incubating for one week, two weeks, or three weeks according to an exemplary embodiment of the present invention.

(8) FIG. 8 shows results of checking the adherence of a gel-like cartilage-regenerating composition prepared by incubating for one week, two weeks, or three weeks according to an exemplary embodiment of the present invention.

(9) FIG. 9 illustrates results of checking a formation degree of a cartilage-regenerating composition depending on a medium composition.

(10) FIG. 10 illustrates results of incubating a cartilage-regenerating composition prepared according to an exemplary embodiment of the present invention, transplanting it in a human cartilage block, incubating it in a nude mouse subcutaneously, and then performing histological staining and immunostaining.

(11) FIG. 11 illustrates results of checking the fluorescence expression of a cartilage-regenerating composition according to an exemplary embodiment of the present invention, labeled with a fluorescent expression factor PKH-26.

(12) FIG. 12 illustrates results of checking the attachment of a cartilage damage site by transplanting a cartilage-regenerating composition prepared according to an exemplary embodiment of the present invention, labeled with a fluorescent expression factor PKH-26 to the damaged cartilage site.

(13) FIG. 13 illustrates results of checking regeneration of cartilage damage after transplanting a cartilage-regenerating composition for according to an exemplary embodiment of the present invention into a rabbit knee cartilage damage model through histological analysis.

(14) FIG. 14 illustrates results of checking regeneration of cartilage damage after transplanting a cartilage-regenerating composition for according to an exemplary embodiment of the present invention into a monkey knee cartilage damage model through MRI.

(15) FIG. 15 illustrates results of checking regeneration of cartilage damage after transplanting a cartilage-regenerating composition for according to an exemplary embodiment of the present invention into a monkey knee cartilage damage model through tissue-dry.

MODE FOR INVENTION

(16) Hereinafter, exemplary embodiments of the present invention will be described in more detail. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

<Exemplary Embodiment 1> Preparing of Cartilage-Regenerating Composition

(17) A schematic diagram of steps for producing a cartilage-regenerating composition is shown in FIG. 1, and the preparing method is as follows.

(18) For the preparation of a cartilage-regenerating composition containing fetal cartilage tissue-derived cells and an extracellular matrix derived from fetal cartilage tissue, a fetus of 10 to 15 weeks (source: IRB NO. AJIRB-MED-SMP-10-268) from the knee joints.

(19) Specifically, the cartilage tissues separated from the knee joints were washed with PBS (phosphated buffered saline), and then incubated with 0.2% (w/v) collagenase (Worthington Biochemical Corp., Lakewood, N.J.) in DMEM (Dulbecco's Modified Egle Medium, Gibco, Grand Island, N.Y.) for 4 hours. Chondrocytes released by completely digesting the cartilage tissues were centrifuged at 1700 rpm for 10 minutes, and then precipitated chondrocytes were resuspended in a tissue culture dish (density of 1×106 cells per 150 mm (dia.)×20 mm (h) per culture dish).

(20) The chondrocytes were diluted in DMEM supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 μg/mL streptomycin, and then incubated for 15-18 days in monolayers. After the incubation, the medium was removed, and 0.05% trypsin-EDTA (Gibco) was added to obtain a cell membrane bound to the extracellular matrix. When the cell membrane bound with the cells and the extracellular matrix was obtained, the cell membrane including the cells and the extracellular matrix were obtained at one time without pipetting the cells after 0.05% trypsin-EDTA (Gibco) treatment.

(21) The obtained cell membrane bound with the cells and the extracellular matrix was placed in a tube of 50 ml including cartilage differentiation medium (1% antibiotic-antimycotic, 1.0 mg/mL insulin, 0.55 mg/mL human transferrin, 0.5 mg/mL sodium selenite, 50 μg/mL ascorbic acid, 1.25 mg/mL bovine serum albumin (BSA), 100 nM dexamethasone, 40 μg/mL proline) and 10 μg/ml TGF-β (Dulbecco's Modified Egle Medium-High Glucose; DMEM-HG) and centrifuged at 250×g for 20 minutes to prepare a pellet-shaped structure.

(22) The prepared cell pellets were placed in an incubator dish containing the same cartilage differentiation medium as that of the above composition and incubated for one week, two weeks, and three weeks in an incubator with 5% carbon dioxide at 37° C. to prepare a cartilage-regenerating composition.

(23) The photographs and tissue volume of the cartilage-regenerating composition prepared by the above-described method are illustrated in FIG. 2. FIG. 2(A) illustrates the naked eye (1 mm per gradation) of the tissue, and FIG. 2(B) illustrates the volume of the tissue. As shown in FIG. 2, a spherical gel-type composition was prepared according to the preparation of the cartilage-regenerating composition, and it was seen that the size and volume of the gel-type composition increased with the incubation period. It was prepared from cell pellet by culturing cells and extracellular matrix in a spherical form, and it was seen that shape deformation was easily occurred since it corresponded to a gel type.

<Exemplary Embodiment 2> Histological Analysis of Cartilage-Regenerating Composition

(24) The cells were fixed in 4% formalin for 1 week in the course of preparing the composition for cartilage-regenerating from the cell pellet of the exemplary embodiment 1, and then embedded in paraffin and cut to a thickness of 4 μM. Then, for detection of accumulated sulfated proteoglycan, the cross-sections were subjected to Sarfanin-O staining and hematoxylin (H & E).

(25) The result is illustrated in FIG. 3.

(26) As checked in FIG. 3, it was seen that the cell interval became wider and the cell shape became similar to chondrocytes in hematoxylin (H & E) as the time elapsed from one week to three weeks. In addition, it was seen that Sarfanin-O staining, which is a method of staining protein sugar, increases an amount of protein sugar at two weeks and three weeks to form lacuna, which can be seen in cartilage.

<Exemplary Embodiment 3> Total Content of Glycosaminoglycan (GAG) and Analysis of Cartilage-Regenerating Composition

(27) The moisture content, DNA content, glycosaminoglycan content, and hydroxyproline content of the cartilage-regenerating composition, which were incubated for one week, two weeks, and three weeks, were measured.

(28) To that end, the moisture content of the cartilage-regenerating composition was measured in weight after the incubation, and lyophilized to be expressed as a percentage when compared it with a dry weight thereof. For the DNA content, the amount of DNA contained in 1 mg of dry weight was measured using PicoGreen Kit. For the glycosaminoglycan content, it was decomposed for 16 hours in a 60° C. papain solution (5 mM L-cysteine, 100 mM Na2HPO4, 5 mM EDTA, papain type III 125 μg/mL, pH 7.5) and then centrifuged at 12,000×g for 10 minutes, and absorbance was measured at 550 nm wavelength using ELISA Reader (BIO-TEK, Instruments, INC., USA) by the centrifuged supernatant and DMB (dimethylmethylene-blue) colorimetric analysis (colorimetric assay, Heide, T. R. and Gernot, J., Histochem. Cell Biol., 112:271, 1999). For total collagen content, it was dissolved in an HCl solution, treated at 121° C. for 10 minutes, and centrifuged at 12,000×g for 10 minutes, and absorbance was measured at 480 nm wavelength using ELISA Reader by mixing the centrifuged supernatant, chloramine T, and dimethylamino-benzaldehyde. Normal cartilage was used as a control group.

(29) The result is illustrated in FIG. 4.

(30) As checked in FIG. 4, it was seen that the moisture content was 95% on average, the amount of DNA was 6.88±1.01 μg/mg for one week, 5.74±0.40 μg/mg per week, and 5.05±0.77 μg/mg for three week, and the DNA amount was reduced as the time elapsed, but there is no change in the DNA amount when compared with the increase in size. The biochemically analyzed total GAG contents were increased to 16.76±2.8 μg/mg (dry weight) for one week, 35.87±5.1 μg/mg (dry weight) for two weeks, and to 48.98±8.0 μg/mg as the incubation period is increased. In particular, it was seen that the total GAG content of the cartilage-regenerating composition has become closer to the natural cartilage tissue as the incubation period becomes longer, considering that the GAG content in the natural cartilage tissue is about 62.8±5.1 μg/mg (dry weight). The amount of hydroxyproline was increased to 7.78±1.89 μg/mg for one week, 40±11.74 μg/mg for two weeks, and 87.2±3.57 μg/mg at 3 weeks, respectively as the incubation period is increased.

<Exemplary Embodiment 4> Measuring Physical Strength of Cartilage-Regenerating Composition

(31) The compressive strength of the cartilage-regenerating composition was checked using a universal testing machine (Model H5K-T, HTE, UK).

(32) The cartilage-regenerating composition prepared by performing the incubation for one week, two weeks, and three weeks in the exemplary embodiment 1 was first measured for compressive strength under an in vitro condition, and the cartilage-regenerating composition, which was incubated in vitro for two weeks, was incubated in ex vivo model for two weeks, four weeks, and eight weeks, and then the compressive strength was measured to compare the changes in compressive strength by in vivo incubation. The Ex vivo model was created as follows. First, cartilage tissue (Ajou University Hospital, IRB No. AJIRB-MED-SMP-11-205), which was discarded after knee arthroplasty for osteoarthritis patients, was collected to make defects similar in shape to actual cartilage damage, and then the cartilage-regenerating composition was inserted into the defect site and transplanted into the rat subcutaneously to be incubated for two, four, and eight weeks.

(33) Each sample (n=6) was photographed, and then the cross-section and the height were calculated by using an image J program, in order to measure the compressive strength. Each sample was pressed at a rate of 1 mm/min until the strain of the tissue reached 20%, and then the value of Young's modulus was measured at a strain of 10 to 16%, and the in vitro and ex vivo results were illustrated in FIG. 5.

(34) As checked in FIG. 5, the cartilage-regenerating composition showed 5.21 kPa for one week, 10.62 kPa for two weeks, and 15.83 kPa for three weeks in vitro, maintaining the gel shape. However, the intensity increased to 50.81 kPa for two weeks, 155.58 kPa for four weeks, and 602.04 kPa for eight weeks in the ex vivo state, and the intensity increased to a level similar to normal cartilage tissue over time in an in vivo environment.

(35) It was seen that from the above results that the cartilage-regenerating composition according to the present invention was able to have a compressive strength similar to that of cartilage tissue when administered to human body.

<Exemplary Embodiment 5> Checking Whether or not a Cartilage-Regenerating Composition was Produced Depending on Cell Source Difference

(36) The availability of a cartilage-regenerating composition including a fetal cartilage tissue-derived cell and an extracellular matrix derived from a fetal cartilage tissue according to the present invention was checked by varying cell sources.

(37) The cartilage-regenerating composition was prepared using the cell source as a human infant cartilage in the same manner as in the exemplary embodiment 1, in order to compare it with the cartilage-regenerating composition according to the exemplary embodiment 1.

(38) The two cartilage-regenerating compositions were incubated in a cartilage medium for three weeks to be used. The cartilage-regenerating compositions were fixed in 4% formalin and then embedded in paraffin, cleaved to 4 μm thickness, to perform Sarfanin-O staining on the cross-section.

(39) The result is illustrated in FIG. 6.

(40) As checked in FIG. 6, it was seen that in the case of human infant chondrocyte artificial cartilage tissue, proteoglycan was expressed only outside the tissue, whereas in the case of human fetal chondrocyte artificial cartilage tissue, the protein sugar is distributed evenly throughout the tissue, resulting in confirming the excellent effect of the cartilage-regenerating composition according to the present invention. That is, it was seen that the cartilage-regenerating composition according to the present invention may be prepared by using fetal fatal chondrocyte and the extracellular matrix thereof as the cell sources.

<Exemplary Embodiment 6> Analysis of Coating Property of Cartilage-Regenerating Composition

(41) The coating property (spreadability) of the cartilage-regeneration composition prepared in the exemplary embodiment 1 was measured using a universal testing machine (Model H5K-T, HTE, UK).

(42) An experimental method for measuring the coating property was set up considering the physically characteristic of the cartilage-regeneration composition. The cartilage-regeneration composition (n=6) was weighed and placed on a flat floor, and a force of 5 N for 1 second at a rate of 1 mm/min was applied vertically to the sample using a jig. After photographing the sample, the image was analyzed with an image J program to calculate the area of the sample spread on the floor.

(43) The result is illustrated in FIG. 7.

(44) FIG. 7(A) illustrates a result of checking the coating property, and FIG. 7(B) illustrates numerical values thereof. As checked in FIG. 7, it was seen that as a results of analyzing the coating property of the sample, the coating property showed 1.09±0.062 mm.sup.2 for one week, 0.77±0.001 mm.sup.2/mg for two weeks, and 0.48±0.004 mm.sup.2/mg for three weeks, and the coating property per unit weight as the time elapsed. This seemed to be related to the result that the cartilage-regenerating composition is increased in strength and tissue becomes harder as the incubation period elapses in the exemplary embodiment 4.

(45) Techniques of generally known cartilage-regenerating materials have been developed with emphasis only on the strength to withstand loads, and thus there has been a problem that a cartilage-regenerating material cannot be suitably applied on the cartilage damage site.

(46) That is, it was seen that the cartilage-regenerating composition according to the present invention exhibits a characteristic of being spread and applied onto the damaged area when inserted into the cartilage damage site unlike the known cartilage regeneration materials.

<Exemplary Embodiment 7> Analysis of Adherence of Cartilage-Regenerating Composition

(47) The adherence of the cartilage-regeneration composition prepared in the exemplary embodiment 1 was measured using a universal testing machine (Model H5K-T, HTE, UK).

(48) The cartilage tissue of the patient to be discarded after surgery was donated with consent. A cartilage damage model was prepared using a 6 mm biopsy punch on the surface of the cartilage tissue of the patient, and the prepared cartilage-regenerating composition was inserted. Then, a jig with a diameter of 5 mm was placed in contact with the inserted cartilage-regenerating composition and pulled at a rate of 1.3 mm/min to measure the resistance until the jig was separated from the cartilage-regenerating composition. Alginate, which is a gel-like biomaterial, was inserted into the cartilage damage model in the same manner to compare its adherence.

(49) The result is illustrated in FIG. 8.

(50) FIG. 8(A) illustrates a photograph showing results of testing the adherence of the chondral defect by using an adult cartilage tissue in the above experimental model, and FIG. 8B illustrates results of checking the change in the adherence of the cartilage-regenerating composition according to an incubation period.

(51) As checking in FIG. 8B, it was seen that as a results of analyzing the adherence of the sample depending on the incubation period, the adherence showed 2.624±0.154 kPa for one week, 1.799±0.146 kPa for two weeks, and 1.058±0.067 kPa for three weeks, and as the time elapsed, the cartilage-regenerating composition and the adhesion of patient cartilage tissue was slightly decreased, and the adhesion was significantly higher than that of the alginate (0.094 0.014 kPa) used as the control group.

(52) From the above results, it was seen that the cartilage-regenerating composition according to the present invention has a very high adhesion in cartilage tissue compared to the conventional gel-type sample, and the adhesion may be adjusted to an appropriate level depending on the incubation period.

<Exemplary Embodiment 8> Checking Cartilage-Regenerating Composition Depending on Medium Composition

(53) The cartilage-regenerating composition were prepared in the same manner as in the method of the exemplary embodiment 1, while changing the medium composition in order to check generation change of the cartilage-regenerating composition depending on the change in the medium composition.

(54) A composition prepared by the 3-week incubation using the cartilage-regenerating composition prepared in the exemplary embodiment 1 and the differentiation medium containing fetal bovine serum (Medium composition: 1% antibiotic-antimycotic, 1.0 mg/mL insulin, 0.55 mg/mL human transferrin, 0.5 mg/mL sodium selenite, Dulbecco's Modified Egle Medium-High Glucose (DMEM-HG) containing Ascorbic acid, 100 nM dexamethasone, Dulbecco's Modified Egle Medium-High Glucose containing 40 μg/mL proline and 10 ng/ml TGF-β; DMEM-HG) as a medium was analyzed through Sarfanin-O staining.

(55) Each of the above compositions were fixed in 4% formalin and then embedded in paraffin, cleaved to 4 μm thickness, to perform Sarfanin-O staining on the cross-section.

(56) The result is illustrated in FIG. 9.

(57) As checked in FIG. 9, it was seen that the cartilage-regeneration composition prepared in the cartilage differentiation medium (medium 1) showed GAG throughout the general cells, but in the composition prepared in the differentiation medium containing the fetal bovine serum (medium 2), cartilage GAG remained and the cells in the center were killed.

(58) It was seen from the result that the cartilage medium corresponded to a suitable medium composition in the preparation of the cartilage-regeneration composition according to the present invention.

<Exemplary Embodiment 9> Checking Histological and Immunological Characteristics of Cartilage-Regenerating Composition

(59) The cartilage-regenerating composition prepared for two weeks in the cartilage medium according to the exemplary embodiment 1 was transplanted into a block of a same shape as a cartilage damage model, and then incubated in the nude mouse hypodermically for two, four, eight, twelve weeks (w), and osteochondral autologous transplantation (OAT) which is generally used in clinical practice was used as a control group. The tissues were taken out, each block was fixed with 4% formalin, embedded in paraffin and cleaved to a thickness of 4 μm, and imunohistochemical staining was performed on the cross-section for visual confirmation of Safranin-O staining and amount of collagen. Immunostaining checked first collagen and second collagen.

(60) The result is illustrated in FIG. 10.

(61) As checked in FIG. 10, it was seen that a shape similar to that of normal cartilage was observed over time after the cartilage-regenerating composition was transplanted. Specifically, safranin-O, which may detect the amount of protein sugars, showed little expression after two weeks of transplantation, but it was seen to be similar to normal cartilage over time. When the cartilage is demineralized, the amount of first collagen increases, but when it is differentiated into cartilage and differentiated into normal cartilage, the amount of second collagen increases. In ex vivo, the first and second collagens were not stained at two weeks, but the amount of second collagen increased with the passage of four weeks and eight weeks. After 12 weeks, the amount of collagen was similar to that of normal cartilage. Particularly, the expression of type II collagen, which is the most collagen in cartilage, reached to the level of normal cartilage at 12 weeks.

<Exemplary Embodiment 10> Checking In Vivo Attachment of Cartilage-Regenerating Composition Labeled with Fluorescence Expression Factor PKH-26

(62) A cartilage-regenerating composition was prepared by incubating a cell labeled with the fluorescence expression factor PKH-26 on a cell surface thereof under the cartilage medium according to the method of the exemplary embodiment 1 to check whether the fluorescent expression factor PKH-26 was expressed on days 1 and 7 after the incubation.

(63) The result is illustrated in FIG. 11. As illustrated FIG. 11, it was seen that the expression of the fluorescent element was well performed in the cartilage-regenerating composition in vitro.

(64) The prepared cartilage-regenerating composition was then transplanted into a partial cartilage damage model of the rat.

(65) Specifically, an 8-week-old rat knee was incised, the cartilage of the femur was scratched with a curette of No. 12, and a cartilage regeneration composition with the fluorescent expression factor PKH-26 was transplanted.

(66) On 3 and 7 days after the transplantation, the knees of the transplanted area were separated and slices were prepared with a 4 μm thickness using a freezing machine. The tissue and fluorescence expression of the injured area of the partial cartilage were checked using an optical microscope and a fluorescence microscope to examine whether the transplanted cartilage-regenerating composition remained. The result is illustrated in FIG. 12.

(67) As checked in FIG. 12, it was seen that the cartilage-regenerating composition adhered to the cartilage damage site on both 3 days and 7 days after the cartilage-regenerating composition was coated.

(68) It was seen from the above result that the cartilage-regenerating composition was able to be coated and adhered to the affected part in vivo.

<Exemplary Embodiment 11> Checking Cartilage-Regenerating Effect Depending on Transplantation of Cartilage-Regenerating Composition in Partial Cartilage Damage Model of Rabbit

(69) The cartilage-regenerating composition incubated in a cartilage medium in vitro according to the exemplary embodiment 1 of the present invention is transplanted into a partial layer cartilage defect model of a rabbit produced in the exemplary embodiment 9.

(70) At 6 and 12 weeks after transplantation, the regeneration of cartilage tissue was visually checked and the recovery of damage was checked by the histological staining, i.e., the saranin-O staining method.

(71) The result is illustrated in FIG. 13.

(72) In FIG. 13, ACI indicates an autologous chondrocyte implantation group, Defect indicates an untreated group, and in vitro 2w indicates a result of an experiment using the cartilage-regenerating composition incubated in the cartilage differentiation medium for two weeks.

(73) As checked in FIG. 13, it was seen that when six and twelve weeks elapsed after the transplantation of the cartilage-regenerating composition according to the present invention, the normal tissue was restored to such an extent that the cartilage damage site was hardly observed, to be similar to be the normal tissue, and this effect was confirmed to be almost the same level as that of the autologous chondrocyte implantation group, which is a positive control group.

<Exemplary Embodiment 12> Checking Cartilage-Regenerating Effect Depending on Transplantation of Cartilage-Regenerating Composition in Knee Cartilage Damage Model of Monkey

(74) The cartilage damage model was constructed using a 3 mm biopsy punch at a femur medial condyle portion of the monkey knee. The cartilage-regeneration compositing was incubated in the damaged cartilage defect site for 2 weeks in vitro, and the cartilage regeneration degree was checked by imaging MRI for 8 weeks, 16 weeks, and 24 weeks. An untreated group was used as the control group.

(75) The cartilage regeneration effect in the animal model was checked by MRI, safranin-O staining and hematoxylin & eosin staining.

(76) FIG. 14 illustrates a result of an experiment analyzed through MRI. As checked in FIG. 14, it was seen that cartilage was formed in the group transplanted with the cartilage-regenerating composition over time in the MRI result for 24 weeks, and it was regenerated to normal cartilage such that a transplanted region was invisible when the animal was sacrificed after 24 weeks

(77) In addition, in FIG. 15, it was seen through results of safranin-O staining and Hematoxylin & Eosin staining that the cartilage collapsed and the amount of protein sugars was reduced in the control group where no treatment was performed, but the cartilage damage site was normally recovered in the experiment group to which the cartilage-regenerating composition was transplanted. In addition, it was seen that, in the case of the control group, the cartilage collapsed by damage even in the trochlea portion and the lateral condyle portion as well as the femur portion, i.e., the transplanted site, while the cartilage was formed in the transplanted site and the surrounding tissues were not affected in the group to which the cartilage-regenerating composition was transplanted.

(78) While this invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.