KR101340458B1 - Composition Comprising Hydrogel for Transplant to Cartilage - Google Patents

Abstract

The present invention relates to a composition for cartilage transplantation comprising a hydrogel exhibiting excellent cartilage regeneration ability and a method for producing the same.
The composition for cartilage transplantation including the hydrogel of the present invention can be easily applied to cartilage defects or damaged areas with minimally invasive treatment, and the regeneration of cartilage effectively occurs due to the penetration and growth of synovial membrane tissue after transplantation, and the protective effect of cartilage is excellent. Exert.

Description

Composition for cartilage transplantation including hydrogels {Composition Comprising Hydrogel for Transplant to Cartilage}

The present invention relates to a composition for cartilage transplantation comprising a hydrogel.

More specifically, the present invention relates to a syringe-injectable hydrogel cartilage transplant composition containing synovial membrane cells and elastic components.

Degenerative arthritis is a disease in which local degenerative changes occur as the articular cartilage wears down, also called osteoarthritis or osteoarthritis. Degenerative arthritis is one of chronic arthritis, which is a disease caused by overgrowth of bones in the joint surface due to degenerative changes in the joint cartilage which receives a lot of weight. In the disease, the cartilage cells that make up the cartilage composition are deteriorated due to aging, and the cartilage loses its elasticity, and as time passes, the surface of the cartilage becomes rough, and various kinds of substances enter the inside of the joint cavity wrapped with the joint membrane. It becomes inflamed. Clinically, recurrent pain, joint stiffness, and gradual dyskinesia of the joints are present.

The causes of degenerative arthritis are deeply related to aging and excessive weight, and the more and more severely in women as they age. Early symptoms include one or two joints with stiff pain, such as stiffness, and prolonged prolongation of the cartilage, hardening of the cartilage, excessive formation of bone around the joint, and deformation of the joint.

The cause of degenerative changes in articular cartilage has not yet been identified, but an absolute decrease in the number of chondrocytes and imbalances in the synthesis and degradation of chondrocytes in chondrocytes is known as one cause. Thus, degenerative changes in articular cartilage reduce cartilage strength and ability as a cushion due to degradation of cartilage substrate.

Hyaluronic acid (hyaluronic acid) is a substance that is contained in cartilage tissue in vivo, it plays a big role in increasing the elasticity of the cartilage tissue, has a high moisture content, and has excellent viscoelasticity. In general, acceleration of hyaluronic acid is observed in osteoarthritis patients.

Treatment of degenerative arthritis currently used clinically includes drug therapy (analgesics, steroids, nonsteroidal anti-inflammatory drugs, etc.), cartilage protection agents (hyaluronic acid, glucosamine, chondroitin, etc.) or surgical treatment (arthroscopic surgery, Proximal tibia osteotomy, joint replacement, total knee arthroplasty, etc.). However, drug therapy has only the effect of non-specifically alleviating the pain or inflammatory response itself, and cartilage protector only serves to temporarily protect joints by nourishing chondrocytes or alleviating shock. In addition, in the case of surgical treatment such as resection, partial or total resection can improve the acute pain by eliminating mechanical symptoms mainly, but there is a limitation that it does not prevent the progression of arthritis due to long-term degenerative changes. Indications are very limited and many failure rates have been reported. In order to overcome the limitations of the existing treatment for degenerative arthritis, cartilage treatment using tissue engineering techniques has emerged, and clinical studies on the human body are currently under way after various animal experiments. However, animal experiments using these various trials have not yet demonstrated satisfactory cartilage regeneration and articular cartilage protection.

It is well known that synovial membrane cells play an important role in healing in intra-articular injuries, such as cartilage damage, and have demonstrated the ability to differentiate into articular cartilage or fibrocartilage. In addition, clinically, minimally invasive arthroscopic surgery can be easily collected, so much expectation for its application and therapeutic application.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made intensive studies to develop a composition for cartilage transplantation that can be easily injected into a cartilage defect or damaged area by syringe. As a result, the present invention was completed by preparing a composition for hydrogel cartilage transplantation containing a synovial membrane cell and an elastic component, and confirming its effective cartilage regeneration effect.

Therefore, an object of the present invention is to provide a composition for cartilage transplantation comprising a hydrogel.

Another object of the present invention to provide a method for producing the composition for cartilage transplantation.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the present invention (a) a hydrogel; And (b) provides a composition for cartilage transplant comprising a synovial membrane cell seeded on the hydrogel.

The present inventors have made diligent research efforts to develop a composition for cartilage transplantation that is injectable with minimally invasive treatment to a cartilage defect or injury site. As a result, the present invention was completed by preparing a composition for hydrogel cartilage transplantation containing a synovial membrane cell and an elastic component, and confirming its effective cartilage regeneration effect.

As used herein, the term “gel” refers to a jelly-like material that has physical properties ranging from soft and weak to strong and coarse, and does not show flow in a steady state. Silver behaves entirely like a solid due to its liquid or three-dimensional network structure.

The term “hydrogel” refers to a hydrophilic gel with a three-dimensional network structure, also called aquagel. Hydrogels exhibit elasticity that is almost similar to natural tissue because of their water content. As a backbone in engineering, any cell that can be seeded can be used without limitation, for example, Smart Gel, silicone hydrogel, polyacrylamide hydrogel that detects pH, temperature or metabolite concentration. , Agarose hydrogel, methylcellulose hydrogel, polyvinyl alcohol hydrogel, sodium polyacrylate hydrogel, acrylate hydrogel, chondroitin hydrogel, glucosamine hydrogel, glycosaminoglycan hydrogel, small intestine mucosa intestinal submucosa) hydrogel, periost eum) hydrogel, perichondrium hydrogel, fibrin hydrogel, fibrinogen hydrogel, thrombin hydrogel, hyaluronic acid hydrogel and collagen hydrogel, but are not necessarily limited thereto.

In one embodiment, the hydrogel is chondroitin, glucosamine, glycosaminoglycans, porcine small intestinal submucosa, periosteum, perichondrium, fibrin, fibrinogen, thrombin, hyaluronic acid and collagen It is a hydrogel containing at least one selected from the group consisting of, and preferably includes hyaluronic acid, collagen and fibrinogen.

According to a preferred embodiment of the present invention, the collagen included in the hydrogel is type 1 collagen. Collagen type 1 plays an important role in the attachment and proliferation of seeded synovial cells as a support.

In one embodiment, the hydrogel of the present invention has a biodegradation rate of 0.1 to 6 wt% / day in an in vivo environment, and is injectable into a cartilage defect site by a syringe, thereby enabling cartilage transplantation with minimally invasive treatment.

The term "synovial membrane cell" refers to a group of cells present in the synovial membrane, and is interchangeable with the same meaning as synovium-derived cells, unless specifically noted otherwise.

Synovial membrane cells used in the present invention are cells extracted from a subject or allogenous synovial membrane cells. As used herein, the term “homogenous synovial membrane cell” refers to a synovial membrane cell derived from the same species, and has the meaning of encompassing autologous synovial membrane cells and other homologous synovial membrane cells. Preferably the density of the synovial cells that are seeded on a support include, but are not particularly limited ^{particularly, 1 x 10 3 - 1 x} 10 7 cells / cell density of the gel (㎖), more preferably from 1 x 10 ⁵ - 1 x 10 ^It is seeded at a cell density of ⁸ cells / gel (ml).

Hydrogels to which the synovial membrane cells are sown can be transplanted immediately to the cartilage site, but preferably after proliferation of the synovial membrane cells. The growth period is not particularly limited.

The medium that can be used in the proliferation process may be any medium conventionally used for culturing animal cells, for example, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), α-MEM (Stanner, CP et al., Nat . New Biol . 230: 52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp . Med . 147: 923 (1978)), 199 medium (Morgan et al., Proc . Soc . Exp . Bio. Med) ., 73: 1 (1950) ), CMRL1066, RPMI1640 (Moore et al, J. Amer Med Assoc 199:.... 519 (1967)....), F12 (Ham, Proc Natl Acad Sci USA 53: 288 (1965)), F10 (Ham, RG Exp . Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)) , A mixture of DMEM and F12 (Barnes, D. et al., Anal . Biochem . 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, C. J. Natl . Cancer Inst . 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc . Soc . Exp . Biol . Med . 100: 115 (1959)) and the MCDB series (Ham, RG et al., In Vitro 14:11 (1978)).

In one embodiment, the hydrogel seeded with the synovial membrane cells of the present invention is cultured in a chondrogenic medium, and the chondrogenic medium preferably includes DMEM, F12, a mixture of DMEM and F12 and RPMI 1640, most preferably DMEM. Medium can be used. A description of the medium can be found in R. Ian Freshney, Culture of Animal Cells , A Manual of Basic Technique , Alan R. Liss, Inc., New York, which is incorporated herein by reference. The medium preferably contains serum (eg fetal calf serum).

The composition for cartilage transplantation comprising the hydrogel of the present invention is implanted in a cartilage defect or damaged part to exhibit excellent cartilage regeneration ability. When using the implantable composition of the present invention, the regeneration of cartilage effectively occurs due to infiltration and growth of synovial membrane tissue, and a protective effect of cartilage occurs.

Cartilage to which the composition for cartilage transplantation of the present invention can be applied includes free cartilage, hyaline cartilage, fibrocartilage or elastic cartilage, and is not particularly limited. For example, the composition for cartilage transplantation of the present invention is articular cartilage (articular Cartilage), ear cartilage, non-cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage and spinal cartilage, etc. It can be effectively used in the areas of defects and damage of the cartilage without limitation to the cartilage area.

According to another aspect of the invention, the invention comprises the steps of (a) acquiring synovial membrane cells; And (b) mixing the obtained synovial membrane cells with a hydrogel.

Since the preparation method of the present invention relates to a method for preparing and preparing a composition for cartilage transplantation comprising the hydrogel described above, common contents in relation to the composition for cartilage transplantation including the hydrogel are to avoid excessive complexity of the present specification. For the sake of brevity, the description is omitted.

The synovial membrane cells are capable of differentiating into articular cartilage or fibrocartilage and can be easily collected clinically with minimally invasive treatment, which can be extracted from a subject or obtained from allogeneic synovial cells. have. Prior to seeding, the synovial membrane cells thus obtained may be cultured with a sufficient cell number to be sown on the hydrogel, and the obtained synovial membrane cells may be seeded in the hydrogel by mixing with the hydrogel. In addition, in one embodiment of the present invention, the method may further comprise culturing the synovial membrane cells and hydrogel mixture.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating cartilage defects comprising the composition for cartilage transplantation.

According to a preferred embodiment of the present invention, the cartilage defect, which is a disease to which the composition of the present invention is applied, is meant to encompass the case where there is damage, defect or deficiency in the cartilage included in the body, such as cartilage necrosis, osteochondritis, Cartilage rupture, cartilage trauma, arthritis, cartilage deficiency and congenital tracheal softening, but are not necessarily limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to the composition for cartilage transplantation included as an active ingredient.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered parenterally, and in the case of parenteral administration, it can be administered directly to the site of cartilage damage or defect.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably from 0.001 mg / kg to 1000 g / kg (body weight) per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a composition for cartilage transplantation comprising a hydrogel exhibiting excellent cartilage regeneration ability and a method for producing the same.

(Ii) The composition for cartilage transplantation comprising the hydrogel of the present invention can be easily applied to cartilage defects or damaged areas with minimally invasive treatment, and cartilage regeneration effectively occurs due to penetration and growth of synovial membrane tissue after transplantation, and excellent cartilage. Exerts a protective effect.

1 is a photograph of gels of various mixing ratios prepared for enzymatic digestion of gels.
2 is an enzymatic degradation profile of gels with various composition ratios over time.
Figure 3 is a photograph showing the results of 28 days culture of gel / cell constructs.
Figure 4 shows the results of Live / Dead Viability analysis after 1 day and 3 days of gel / cell construct culture. The viable cells in the gel are green and the dead cells are red.
5 shows the results of the Live / Dead Viability assay after 14 days of gel / cell construct culture. The viable cells in the gel are green and the dead cells are red.
Figure 6 shows the viable cells (green) and dead cells (red) in (a) 1 day culture and (c) 2 weeks culture of gel ① and (b) 1 day culture and (d) 2 weeks culture of gel ②. ) Shows an enlarged image.
7 is a graph showing the results of the MTS analysis.
8 is a graph showing the average amount of synthesized total GAG relative to the amount of DNA.
9 is a photograph showing proteoglycan expression stained with safranin O after 3 and 4 weeks of culture.
10 is a confocal micrograph showing collagen expression of type 1 after 3 and 4 weeks of culture.
11 is a confocal micrograph showing type 2 collagen expression after 3 and 4 weeks of culture.
12 shows the results of RT-PCR analysis showing cartilage specific ECM gene expression.
Figure 13 shows the morphology and H & E staining results of the rabbit knee joint trochlear groove 1 week after the cartilage formation experiment.
Figure 14 shows the morphological structure and H & E staining results of the rabbit knee joint protuberance after 1 week of cartilage formation experiment.
Figure 15 shows the morphology, H & E staining results and safranin O staining results of the rabbit knee joint trochlear groove 4 weeks after the cartilage formation experiment. Red fluorescence is synovial membrane cells injected with the gel.
Figure 16 shows the morphological structure, H & E staining results and safranin O staining results of the rabbit knee joint protuberance after 4 weeks of cartilage formation experiment. Red fluorescence is synovial membrane cells injected with the gel.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Materials and Methods

Isolation and Culture of Rabbit Synovial Cells

A synovial membrane was obtained from a rabbit 12 weeks old and weighing 2.6-3 kg. The synovial membrane was ground in phosphate-buffered saline (PBS) and digested with 0.02% collagenase (Sigma, Saint Louis, Mo, USA) overnight at 37 ° C., 5% CO 2. Cells were separated using a 70 μm sieve, then centrifuged at 1500 rpm, 4 ° C. for 5 minutes and washed twice with PBS. The cells were suspended in medium. The medium used was low glucose Dulbecco's modified Eagle's medium, LG-DMEM, GIBCO, Grand, containing 10% fetal bovine serum (FBS, Life Technologies, Rockville, MD, USA) and antibiotic-antifungal solution (GIBCO). Island, NY, USA). Plated at a density of 2 x 10 ⁴ in a 150 mm culture dish containing the medium. Cells were attached to dishes for 48 hours, then medium was exchanged and grown at 37 ° C., 5% CO ₂ for about 14 days (P0). The medium was replaced after 2 days to induce cell adhesion and non-adherent cells were removed.

Cells were cultured in a single layer in medium and incubated at 37 ° C. under 5% CO ₂ atmosphere. Medium was changed every three days. After about 10-14 days of culture (passage 0, P0) cells were cultured until they reached 80-90% confluence.

Preparation and Enzymatic Degradation of Gels

To prepare gels at various mixing ratios of Table 1 below and simulate the in vivo degradation profiles, gels of various mixing ratios were 37 ° C. in PBS containing 1 mg / ml lysozyme (Sigma, USA) at 95% relative humidity. And 5% carbon dioxide conditions. Degradation of the gel was assessed by measuring the weight percentage of remaining gel (FIG. 2).

Cell encapsulation

The isolated rabbit synovial membrane cells were treated with 2 x 10 ⁷ cells / ml in a solution containing fibrinogen (Green Cross, Korea), 10 mg / ml HA (LG CI, Korea) and 3% type 1 collagen (RMS innovations, UK). Suspended. The cells were uniformly mixed with the gel prepared above and thrombin / CaCl ₂ was added to capture the cells in the gel at a density of about 4 × 10 ⁶ cells / gel construct.

Cartilage formation Chondrogenic ) Differentiation

The gel / cell constructs formed above were cultured in chemically purified cartilage medium to determine cell differentiation capacity differences in gels and the results are shown in FIG. 3. Cartilage producing medium was 100 μM ascorbate-2-phosphate (Sigma), proline (Sigma) 40 μg / ml, and ITS ^™ + Premix Universal Medium Supplement (BD Biosciences, Bedford, MA, USA) (6.25 μg / ml insulin Low glucose DMEM (GIBCO) supplemented with 6.25 μg / ml transferrin, 6.25 μg / ml aselenoic acid, 1.25 mg / ml bovine serum albumin, 5.35 mg / ml linoleic acid). 100 nM dexamethasone (Sigma) was initially added to the medium.

Cell proliferation assay

Viable cells in the gel were analyzed using the Live / Dead Viability Kit (Molecular Probes, USA) and MTS [3 (4,5-dimethylthiazol-2yl) -5- (3-carboxymethoxy-phenyl) -2- ( 4-sulfophenyl) -2H-tetrazolium] assay was performed to determine viable cell numbers. MTS analysis provides a color optical means for determining the number of viable cells in proliferation or cytotoxicity assays. 100 μl 0.1% in low glucose DMEM with 20 μl CellTiter AQueous One Solution reagent Reagent (Promega Corporation, Madison, Wis.) Containing 1% antibiotic-antifungal solution and MTS tetrazolium compound and electronic coupling reagent (phenazine ethosulfate) Cells were treated with collagenase (Sigma, Saint Louis, Mo.) at 37 ° C. for 3 hours. Absorbance at 490 nm was then recorded using a 96-well plate reader (microplate spectrophotometer, VersaMax; Molecular Devices Corp, Sunnyvale, Calif.), Using a standard dilution of serial dilutions of synovial membrane-derived cells of the same passage. Plotted) to determine the number of viable cells (FIGS. 4-7).

GAG  And DNA  Quantitative analysis

Synthesized glycosinoglycans (GAG) were measured by binding to DMB dye and the amount of total GAG was averaged against the amount of DNA by indole analysis. Specifically, the total glycosinoglycan (GAG) amount was determined using the dimethylmethylene blue method. To analyze the glycosinoglycan amount, fractions were overnight at 55 ° C. using paraffin / buffer solution (0.1 M sodium formate, 200 μg / ml paraffin in 5 mM EDTA, 5 mM L-cysteine, pH 3.0). Digested. After centrifugation at 6000 rpm for 5 minutes, 50 μl of supernatant was plated in a 96 well plate and 250 μl of DMB staining solution was added. GAG levels were determined from absorbance at 530 and 590 nm using an ELISA reader. Chondroitin-6-sulfate was dissolved and used as a standard, and the total amount of GAG was averaged against the amount of DNA (FIG. 8).

Tissue Observation after Cartilage Differentiation

Samples at each time point were stained with H & E and safranin O for proteoglycan detection and the results are shown in FIG. 9. In addition, the expression of type 1 and type 2 collagen was analyzed by immunocytochemical staining as follows. Samples were fixed in 4% paraformaldehyde at room temperature for 4 hours and embedded in tissue tech embedding medium (Sakura Finetek, Torrance, Calif., USA) for -70 ° C. for 20 minutes. A 5 μm thick cut slide was made at −20 ° C. After drying overnight at room temperature, washed with PBS (phosphate buffered saline) and treated with slides at 37 ° C. for 30 minutes at 50 units / ml chondroitinase ABC (0.1 M sodium acetate, 0.1 M Tris, pH 7.3). Then washed three times for 5 minutes. Treatment with blocking solution (Zymed Laboratories Inc., San Francisco, Calif., USA) for 10 minutes at room temperature blocked the non-specific reaction. Mouse monoclonal anti-collagen 2 antibody (2 ug / ml Lab Vision-Neomarkers, Fremont, CA, USA) was treated for 1 hour at room temperature. After washing three times with PBS, the cleavage site was treated for 1 hour at room temperature with FITC-linked goat anti-mouse Ig antibody (Zymed Laboratories Inc., San Francisco, CA, USA) diluted 1: 100 ratio, and PBS Washed again with. The slides were placed on a medium containing DAPI, and then observed by confocal microscopy (LSM 510, Carl Zeiss, Germany) (FIGS. 10 and 11).

Reverse transcriptase Polymerase chain reaction

Cartilage specific ECM gene expression was analyzed by RT-PCR. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA; phenol + guanine isothionate). CDNA was synthesized using a primary cDNA chain synthesis kit (Fermentas, Life sciences, EU). Briefly, mix 1 ug of RNA, 1 ul of oligo dT primer, 5X reaction buffer, RNase inhibitor, 10 mM dNTP mixture with M-MuLV reverse transcriptase and reverse transcription for 15 minutes at 25 ° C and 60 minutes at 37 ° C. After incubation for 10 minutes at 70˚C to deactivate the reverse transcriptase. PCR amplification of cDNA results was performed using PCR PreMix (Bioneer, South Korea). Collagen type 1 (COL I), type 2 collagen (COL II), type 10 collagen (COL X), Aggrecan, Sox 9 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) The expression of was investigated. Primers and reaction conditions used to amplify are shown in Table 2 below. The reaction product was digested electrically on 1% agarose gel and visualized with ethidium bromide (EtBr) (FIG. 12).

gene direction order cycle Annealing Temperature (℃) Amplicon
(bp) COL I (F) 5'-CGTGGTGACAAGGGTGAGAC-3 ' 25 52 827 (R) 5'-TAGGTGATGTTCTGGGAGGC-3 ' COL II (F) 5'-TTCAGCTATGGAGATGACAATC-3 ' 35 58 472 (R) 5'-AGAGTCCTAGAGTGACTGAG-3 ' COL X
(F) 5'-CCCTTTTTGCTGCTAGTATCC-3 '   28 58 468 (R) 5'-CTGTTGTCCAGGTTTTCCTGGCAC-3 ' Aggrecan (F) 5'-TGAGGAGGGCTGGAACAAGTACC-3 ' 32 57 350 (R) 5'-GGAGGTGGTAATTGCAGGGAACA-3 ' Sox 9 (F) 5'-CCCGATCTGAAGAAGGAGAGC-3 ' 32 58 381 (R) 5'-GTTCTTCACCGACTTCCTCCG-3 ' GAPDH (F) 5'-ATTGTTGCCATCAATGACCC-3 ' 25 55 579 (R) 5'-AGTAGAGGCAGGGATGATGTT-3 '

COL I: Collagen Type 1

COL II: Collagen Type 2

COL X: Collagen Type 10

GAPDH: glyceraldehyde-3-phosphate dehydrogenase

(F): tablets, sense primer

(R): reverse, antisense primer

bp: base pair

In Vivo  Cartilage Formation Experiment

Osteochondral defects were formed in the trochlear groove and the medial projection of the rabbit knee joint (Ø: 4 mm, depth: 3 mm) using a conventionally manufactured surgical tool. The gel and thrombin solution was injected using a double syringe. Only osteochondral defects were added to the control group, and the experimental group was divided into two groups and one group was injected with gel ② and thrombin solution containing no synovial membrane cells, and the other group was injected with gel ② and thrombin solution containing synovial membrane cells. At 1, 4 and 12 weeks after injection, the animals were sacrificed and the results of cartilage formation were observed (FIGS. 13 to 16).

Statistical analysis

All experiments were performed three times and the results were analyzed by a paired t -test. P values less than 0.005 were considered significantly.

Experiment result

In the degradation experiments, hydrogels with higher fibrinogen ratios degraded more slowly. Gels containing 0.75: 0.75: 1.5 (gel ①) and 0.5: 0.5: 2 (gel ②) of HA, type 1 collagen and fibrinogen remained at least 40% of the original gel after 3 weeks of culture (FIG. 2). . The enzymatic degradation profile of the gel over time is shown in FIG. 2. Therefore, the present inventors conducted further experiments by selecting gel ① and gel ② from gels having various composition ratios.

Most synovial cells planted in the gel survived and proliferated for 2 weeks (FIGS. 6 and 7). GAG synthesis from cells planted in gel ① and gel ② was shown to increase up to 3 weeks (FIG. 8). Cartilage differentiation resulted in abundant proteoglycans in ECM (FIG. 9) and cartilage-like cells planted therein. Positive staining for collagen type 1 (FIG. 10) and type 2 collagen (FIG. 11). The results were shown.

RT-PCR results also showed cartilage specific ECM gene expression (FIG. 12). In the rabbit defect model, the cartilage defect site injected with the gel and cells was filled by the regenerated cartilaginous tissue at 4 weeks of injection, whereas the majority of the controls were not filled with defects (FIGS. 13-16). Cells injected with the gel were observed with red fluorescence in the defect site (FIGS. 15 and 16 bottom). The defect edges were fully integrated with the regenerated tissue.

The purpose of this study was to develop and prepare a novel biocompatible hydrogel that can be used as an injectable cell carrier for cartilage regeneration comprising HA, collagen type 1 and fibrin glue. The present inventors confirmed that hydrogel planted with synovial membrane cells adheres to cartilage defects and promotes accumulation of articular cartilage such as ECM, and thus may be a good candidate for cartilage regeneration.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

(a) a hydrogel comprising hyaluronic acid, collagen and fibrinogen; And (b) cartilage graft composition comprising a synovial membrane cell seeded on the hydrogel.
The composition of claim 1, wherein the hydrogel is injectable by syringe.
delete The composition of claim 1, wherein the hydrogel comprises hyaluronic acid, collagen, and fibrinogen having a weight ratio of 1: 1: 1.25: 0.5: 1.25, 0.75: 0.75: 1.5, or 0.5: 0.5: 2. .
5. The composition of claim 4 wherein the collagen is type 1 collagen.
The composition of claim 1, wherein the synovial membrane cells are seeded onto the hydrogel at a density of 1 × 10 ⁵ to 1 × 10 ⁸ cells / gel (ml).
The composition of claim 1, wherein the hydrogel has a biodegradation rate of 0.1 to 6% by weight.
The composition of claim 1, wherein the cartilage is hyaline cartilage, fibrocartilage or elastic cartilage.
According to claim 1, wherein the cartilage is composed of articular cartilage (articular Cartilage), ear cartilage, non-cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage and spinal cartilage Compositions selected from the group.
Method of producing a composition for cartilage transplantation comprising the step of mixing the isolated synovial membrane cells with a hydrogel containing hyaluronic acid, collagen and fibrinogen.
The method of claim 10, wherein the synovial membrane cells are extracted from a cartilage defect treatment subject or allogeneic synovial membrane cells.
The method of claim 10, wherein the method further comprises culturing the synovial cell and hydrogel mixture.
10. A pharmaceutical composition for treating cartilage defects comprising the composition of any one of claims 1, 2 and 4-9.
14. The composition of claim 13, wherein the cartilage defect is selected from the group consisting of cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma, arthritis, cartilage deficiency and congenital organ softening.

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