KR20200115293A - Composition for tissue integration possessing tissue adhesive and differentiation property, and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a tissue fusion composition having tissue adhesion and differentiation characteristics, and a preparation method therefor. In the present invention, a tissue fusion composition in the form of a gel or sheet is prepared using stem cells and a stem cell-derived extracellular matrix, and it has been confirmed that the composition adheres and binds superbly to biological tissues and can be differentiated into cartilage, bone, cornea, growth plate, and the like after transplantation, and thus the composition can be used as an adhesive and a differentiation agent in regenerative therapy of damaged tissues or organs, therefore being ultimately and effectively usable as an agent for tissue fusion.

Description

BACKGROUND OF THE INVENTION Field of the Invention: Composition for tissue integration possessing tissue adhesive and differentiation property, and manufacturing method thereof

The present invention relates to a composition for fusion of tissues having tissue adhesion and differentiation properties, and a method of manufacturing the same.

As fibrin sealants were approved by the US FDA in 1998, new tissue adhesives are constantly being developed every year. These tissue adhesives are in the spotlight as a material that can replace techniques such as suture, clip surgery, and moxibustion, which are conventionally used in surgical or medical surgery.

Conventional surgical techniques such as sutures have strong tensile strength, but have disadvantages such as causing pain and removal after the procedure, whereas tissue adhesives have a fast adhesion time, simple use, and no need to remove after the procedure. Although it has an advantage, there is a limitation in that the adhesion is remarkably deteriorated in the presence of low adhesion and elongation and moisture. Accordingly, research is ongoing to overcome the limitations of the tissue adhesive as described above.

Medical tissue adhesives are in direct contact with tissues, so biocompatibility is required.Because they are usually used in vivo, in case the adhesive flows into body fluids and blood, it must be free from toxicity and harm to the human body under stricter conditions and is a biodegradable material. It should be.

Tissue adhesives that are currently commercialized and/or put into practical use are largely cyanoacrylate adhesives, fibrin adhesives, gelatin adhesives, and polyurethane adhesives. Cyanoacrylate adhesives have recently been in the spotlight for research on instant adhesives having high functionality and high performance. The cyanoacrylate-based tissue adhesive is a single material that is cured by moisture without an initiator at room temperature in a short time, has a transparent appearance, and has a large adhesive strength, but has a disadvantage of being weak against impact and poor heat resistance. In addition, due to its severe toxicity, it is rarely used now, and it is partially used in other countries except the United States, and its use is restricted in some cases due to tissue toxicity and vulnerability.

Fibrin adhesive has the advantage of being able to adhere quickly without affecting the moisture present in the bonding site, has no platelet and coagulation disorder, and has excellent biocompatibility. However, there are disadvantages in that the adhesion is weak, the biodegradation rate is fast, and there is a risk of blood infection.

In addition, although gelatin adhesive has high tissue adhesion, formalin or glutaaldehyde used as a crosslinking agent has a disadvantage that it causes a crosslinking reaction with proteins in the living body, causing tissue toxicity, and polyurethane-based adhesives absorb water on the surface of living tissues It has the advantage of increasing adhesion with water, curing within minutes by reacting with water, and being biodegraded, but there is a disadvantage in that aromatic diisocyanate, a synthetic raw material, has biotoxicity.

On the other hand, the adhesives developed to date have hardly contributed to the fusion between tissue and tissue or between tissue and implant. That is, since the tissue is not permanently dropped by the adhesive, the two tissues or implants fall apart again after the biodegradation of the adhesive. To overcome this shortcoming, cell therapy products that supply therapeutic cells from outside are being studied. That is, by injecting cells between tissues and tissues or between tissues and implants, the transplanted cells secrete extracellular matrix to attach adhesive substances. However, cell therapy products do not have any adhesiveness, so a separate adhesive or material is required while cells generate tissue.

As described above, tissue adhesives require adhesion between the two tissues, and in order to permanently attach the two tissues, the tissue adhesive functions as a cell therapy that can differentiate into cells of the target adherent tissue and ultimately secrete the extracellular matrix. Ideal. Such cell-mounted tissue adhesives can be used for the treatment of many tissue damage in the body. Examples include muscle rupture, ligament damage, bone attachment, and cartilage regeneration. Therefore, the development of tissue adhesives loaded with tissue adhesion and differentiated cells is expected to bring breakthrough advances in transplant therapy.

Korean Patent Registration No. 10-0775958 (registered on November 6, 2007)

An object of the present invention is to provide a tissue adhesive product (TAP) composition having tissue adhesion and differentiation properties, and a method of manufacturing the same.

In order to achieve the above object, the present invention provides a composition for tissue fusion comprising stem cells and stem cell-derived extracellular matrix as an active ingredient.

In addition, the present invention provides a tissue for transplantation prepared by attaching two or more tissues with the composition.

In addition, the present invention (a) separating and culturing stem cells from fetal cartilage tissue; (b) obtaining a cell membrane comprising the cultured stem cells and their extracellular matrix; (c) centrifuging the obtained cell membrane to obtain a cell pellet; And (d) culturing the obtained cell pellet in a medium; it provides a method for producing a composition for fusion comprising a.

In the present invention, a composition for fusion of a gel-like or sheet-like tissue was prepared using stem cells and stem cell-derived extracellular matrix, and the composition has excellent adhesion and bonding power to living tissues, and after transplantation, cartilage, bone, and cornea , As it was confirmed that differentiation is possible with growth plates, etc., it can be used as an adhesive and differentiating agent for the treatment of damaged tissue or organ regeneration, and ultimately, it can be effectively used as a tissue fusion agent.

1 is a result of confirming the adhesive strength of the cartilage tissue bonding agent composition (TAP) of the present invention.
2 is a result of confirming the attachment to the damaged site after implantation of TAP labeled with the fluorescence expression factor PKH-26 in a cartilage injury model.
3 is a result of safranin O staining and hematoxylin & eosin staining of TAP prepared by culturing for 1 week, 2 weeks, and 3 weeks.
4 is a result of confirming regeneration of cartilage damage after transplantation through histological analysis after TAP is transplanted into a cartilage damage model.
5 is a result of confirming the differentiation into corneal cells using stem cells derived from fetal cartilage tissue.
6 is a result of confirming the differentiation into sheet-shaped corneal cells using TAP.
7 is a diagram showing a method of manufacturing a growth plate damage model and implanting a TAP.
8 is a result of confirming differentiation into cartilage tissue and growth plate tissue by TAP in the growth plate injury model.
9 is a result of confirming the formation of collagen and glycoprotein by TAP in the growth plate damage model.
10 is a result of confirming the effect of improving length growth and angular deformation by TAP in the growth plate damage model.
11 to 13 are results confirming the formation of cartilage tissue by TAP in the growth plate damage model.
14 is a result of confirming the regeneration of the lumbar intervertebral disc by TAP.
FIG. 15 is a result of a gross image image confirming cell differentiation 7 days after culture of a fetal cartilage-derived stem cell sheet.
16 is a Western blotting analysis result confirming the expression levels of Myf5 and MyoD in order to confirm the differentiation into myoblasts 7 days after culture of the fetal cartilage-derived stem cell sheet.
17 is a result of confirming the expression of Myf5 and MyoD by performing immunocytochemistry (ICC) to confirm the differentiation into myoblasts 7 days after culture of the embryonic cartilage-derived stem cell sheet.

The present invention provides a composition for tissue fusion having tissue adhesion and differentiation properties comprising stem cells and stem cell-derived extracellular matrix as an active ingredient.

The term “stem cells” used in the present invention may be stem cells isolated from fetal cartilage tissue, preferably cartilage progenitor cells separated after completely digesting cartilage tissue using collagenase or the like (fetal cartilage derived progenitor cell). ; FCPC).

The term “stem cell-derived extracellular matrix” as used in the present invention is a collection of biopolymers composed of molecules synthesized by cells from stem cells derived from fetal cartilage tissue and secreted and accumulated extracellularly. Collagen, elasticity They include fibrous proteins such as cattle, complex proteins such as glycosaminoglycans, and cell adhesion proteins such as fibronectin and laminin.

The term "cartilage" used in the present invention includes, but is not particularly limited, supernatural bone (hyaline cartilage), fibrocartilage (fibrocartilage), or elastic cartilage (elastic cartilage). Articular cartilage, ear cartilage, non-cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, occipital cartilage, and vertebral cartilage are included without limitation.

The composition may be differentiated into cornea, epithelium, growth plate, bone, cartilage, ligaments, muscle or skin tissue, but is not limited thereto.

It should be noted that the composition may be prepared in a gel shape or a sheet shape, but is not limited thereto.

The term "gel" as used in the present invention is a jelly-like substance that has physical properties ranging from a soft and weak range to a strong and coarse range, and means a solid that does not flow in a steady state, and most of the weight of the gel Because of the liquid or three-dimensional network structure, it behaves like a solid as a whole.

The composition may be maintained at 25 to 40° C. for 20 to 30 hours, but is not limited thereto.

In addition, the present invention provides a tissue for transplantation prepared by attaching two or more tissues with the composition.

The term “transplantation” used in the present invention generally refers to a process of transferring cells, tissues, or organs of a donor to damaged tissues or organs of a recipient, and in the present invention, tissue defects and damage of the composition for tissue fusion , Means application to the defective area. Transplantation can be carried out by methods known in the art. For example, it can be performed surgically, and can be injected directly into the affected area.

In addition, the present invention (a) separating and culturing stem cells from fetal cartilage tissue; (b) obtaining a cell membrane comprising the cultured stem cells and their extracellular matrix; (c) centrifuging the obtained cell membrane to obtain a cell pellet; And (d) culturing the obtained cell pellet in a medium; it provides a method for producing a composition for fusion comprising a.

The cell membrane obtained in step (b) may be obtained by including all of the extracellular matrix together with the cells attached to the bottom without the step of separating the cells.

Compressive strength, adhesion, and applicability of the composition may be adjusted according to the cultivation period of step (d).

The term “applicability (spreadability)” used in the present invention refers to the property of spreading among physical properties, and refers to the property of spreading smoothly over the entire surface without becoming a lump when applied to the affected area. In the present invention, spreadability refers to the degree of spreading per unit weight of the sample when a force of 5 N is applied to the sample vertically for 1 second at a rate of 1 mm/minute.

The term “adhesion” as used in the present invention refers to the property of attaching other materials to a material, and refers to the property that the material does not fall off when applied to the affected area. In the present invention, adhesion refers to the resistance force until the material adhered to the jig and the affected part is separated from each other while being pulled at a rate of 1.3 mm/min after contacting and attaching a material to the affected part with a jig having a diameter of 5 mm.

The composition may be differentiated into cornea, epithelium, growth plate, bone, cartilage, ligaments, muscle or skin tissue, but is not limited thereto.

In addition, the present invention can provide a composition for tissue fusion having tissue adhesion properties, including stem cells and stem cell-derived extracellular matrix as an active ingredient.

The term “regeneration” used in the present invention generally refers to an action to restore a tissue or organ to its original state or restore its function when a part of the body or its function is lost in an organism. This regenerative capacity is stronger the simpler the system and the lower the degree of systematic evolution.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Isolation and culture of stem cells derived from human fetal cartilage tissue

Stem cells derived from cartilage tissue were isolated from the knee joint of a 12-15 week old fetus (Source: IRB NO. AJIRB-CRO-07-139 approved by the Ethics Committee of Ajou University Hospital). Briefly, cartilage tissue separated from the knee joint was washed with phosphated buffered saline (PBS), and then 0.2% (w/v) collagenase (collagenase, Worthington Biochemical Corp., Lakewood, NJ) was contained. DMEM (Dulbecco's Modified Egle Medium, Gibco, Grand Island, NY) medium was added, followed by incubation in a 37°C, 5% CO ₂ incubator for 4 hours. The cartilage tissue-derived stem cells released after the cartilage tissue was completely digested were centrifuged at 1700 rpm for 10 minutes to obtain precipitated cartilage-derived progenitor cells (FCPC), and a tissue culture dish [150 mm ( dia.) X 20 mm (h)] was inoculated at a cell density of 1 X 10 ⁶ .

Example 2: Preparation of a composition for tissue fusion having tissue adhesion and differentiation properties (TAP)

After diluting the cartilage stem cells obtained in Example 1 to a cell density of 2 X 10 ⁵ , 10% fetal bovine serum (FBS), 50 units/mL penicillin, and 50 μg/mL streptomycin were added. Monolayer culture was performed in DMEM medium for 15 to 18 days. After culture, the medium was removed, and 0.05% trypsin-EDTA (Gibco) was added to obtain a cell membrane bound to the extracellular matrix. To obtain a cell membrane to which the cells and the extracellular matrix were bound, after 0.05% trypsin-EDTA treatment, the entire cell membrane including the cells and the extracellular matrix was obtained at once without separating the cells with a pipette.

Cell membranes containing the obtained cells and extracellular matrix are 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 DMEM-HG to which 10 ng/ml TGF-β is added] and centrifuged at 250 xg for 20 minutes to prepare a pellet-shaped structure.

The prepared cell pellet was put in a culture dish, and cultured for 1 week, 2 weeks and 3 weeks in a 37°C, 5% CO ₂ incubator using the same cartilage differentiation medium as described above to prepare a composition for tissue fusion.

Example 3: Measurement of adhesive strength of composition for tissue fusion

In Example 2, in order to check the adhesive strength of the TAP, a Universal Testing Machine (Model H5K-T, H.T.E, UK) was used to compare the adhesive strength with the fibrin adhesive.

The patient's cartilage tissue, which was discarded after artificial joint surgery, was donated along with consent. A cartilage damage model was made using a 6 mm biopsy punch on the patient's cartilage tissue surface, and the prepared TAP was inserted. Then, after attaching a jig with a diameter of 5 mm in contact with the inserted TAP, the resistance force until the jig was separated from the TAP was measured while pulling at a speed of 1.3 mm/min.

As a result, referring to FIG. 1, the adhesive strength of the TAP of the present invention was initially 2.52 kPa, which was weaker than the fibrin adhesive, but after 3 weeks, as the tissues were differentiated, the adhesive strength of 34.47 kPa was far superior to that of the fibrin adhesive. Confirmed.

Example 4: Confirmation of in vivo adhesion of TAP labeled with fluorescence expression factor PKH-26

PKH-26, a fluorescent expression factor labeled on the cell surface, was attached to the TAP to determine whether it was expressed in vitro or in vivo. A TAP labeled with the fluorescence expression factor PKH-26 was prepared, incubated in vitro for a week, and then fluorescence was confirmed to be well expressed, and then transplanted into a rabbit partial cartilage injury model. One week after transplantation, the knee at the implantation site was separated and sectioned to a thickness of 4 μm using a frozen sectioning machine, and then a slide was prepared.

As a result of observation using an optical microscope and a fluorescence microscope, as shown in FIG. 2, it was confirmed that the TAP remained at the damaged part of the cartilage and the fluorescence expression factor was attached. From the above results, it was confirmed that the TAP of the present invention can be applied and attached to the affected area.

Example 5: Confirmation of differentiation into cartilage tissue

From the cell pellet of Example 2, every 1 week in the process of preparing TAP, it was fixed with 4% formalin, embedded in paraffin, cut to a thickness of 4 μm, and a cross-section was taken to detect accumulated sulfated proteoglycans. It was stained with Safranin O and hematoxylin & eosin (H&E).

As a result, as shown in FIG. 3, it was confirmed that, as time passed from 1 week to 3 weeks, in hematoxylin & eosin staining, the cell spacing increased and the cell shape became similar to chondrocytes, and in safranin O staining, 3 weeks It was confirmed that the amount of protein sugar increased and lacuna, which can be seen in cartilage, was formed.

In addition, after the TAP of the present invention was implanted into a rabbit partial cartilage injury model, the knee at the implantation site was separated and sectioned to a thickness of 4 μm using a frozen sectioning machine, and then a slide was prepared, followed by safranin O staining. .

As a result, as shown in FIG. 4, when looking at the border between the donor cartilage and the recipient cartilage and the border between the cartilage and bone 4 weeks after transplantation, the integration of cartilage and cartilage, cartilage and bone is superior compared to the control group. After 8 weeks of transplantation, it was confirmed that the cartilage and cartilage, and the cartilage and bone merged almost completely, and the cartilage was recovered similar to the normal tissue to the extent that the damaged area was hardly identified.

Example 6: Confirmation of differentiation into corneal epithelial cells

6-1. Corneal epithelial cell differentiation

Fetal cartilage stem cells (3 X 10 ⁵ cells/cm ² ) are 2% KnockOut ^TM Serum Replacement (KnockOut ^TM SR, Thermo Fisher Scientific, Waltham, MA, USA), 10 ng/ml keratinocyte growth factor (KGF, Wako Pure). Chemical, Japan), 10 ng/ml hepatocyte growth factor (HGF, Wako Pure Chemical, Japan), 20 ng/ml epithelial growth factor (EGF, Wako Pure Chemical, Japan), 0.5 μg/ml hydrocortisone (Sigma-Aldrich, St.Louis, USA), 5 μM retinoic acid (Sigma-Aldrich, St.Louis, USA) in a low-glucose DMEM medium (HyClone, Logan, UT, USA) at 37°C, 5% CO ₂ incubator. Culture was carried out, and the medium was changed every 2 days.

6-2. Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 20 minutes, and incubated with 0.1% Triton X-100 for 15 minutes and 5% BSA for 1 hour. Thereafter, the cells were reacted with the primary antibody [anti-PAX6, anti-BCRP/ABCG2, anti-p63, anti-CK3/12 (1:200 dilution, Abcam, Cambridge, UK)] at room temperature for 2 hours, Washed with PBS and reacted with secondary antibodies [goat anti-mouse IgG H & L and goat anti-rabbit IgG H & L (1:1000 dilution, Alexa Fluor® 488, Abcam, Cambridge, UK)] at room temperature for 1 hour Made it. Thereafter, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus, and the cells were observed using a fluorescence microscope (Mi8, Leica Microsystems, Wetzlar, Germany).

6-3. FACS

Cells (passage 5) were analyzed using a corneal epithelial stem cell marker and a corneal epithelial differentiation marker. Cells were treated with primary antibodies (anti-CD34-FITC (BD Biosciences, San Jose, CA, USA), anti-CD105 (BD Biosciences, San Jose, CA, USA), anti-PAX6, anti-BCRP/ABCG2, anti- p63, anti-CK3/12 (1:500 dilution, Abcam, Cambridge, UK)] and reacted at room temperature for 1 hour, washed with PBS, and secondary antibodies [goat anti-mouse IgG H & L and goat anti -Rabbit IgG H & L (1:1000 dilution, Alexa Fluor® 488, Abcam, Cambridge, UK)] and reacted at room temperature for 1 hour. Stained cells were analyzed by flow cytometry (Becton Dickinson FACSavantage).

6-4. Western Blot

After collecting the cells, the cells were lysed by reacting at 4° C. for 30 minutes using RIPA buffer to which a protease inhibitor (Rockland Immunochemicals, Pennsylvania, USA) was added. After the cell lysate was centrifuged at 4° C. and 12,000 g for 15 minutes, the protein was quantified. SDS-PAGE was performed using 20 μg of protein and transferred to a PVDF membrane. Thereafter, the PVDF membrane was subjected to primary antibodies [actin (1:1000) (GeneTex Inc., California, USA), anti-PAX6, anti-BCRP/ABCG2, anti-p63 and antiCK3 (1:200, Abcam, Cambridge, UK). )] and reacted for 2 hours at room temperature, and blocked with skim milk to prevent non-specific binding. Then, after reacting for 1 hour with a secondary antibody [HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (1:1000, GeneTex Inc., California, USA)], a chemiluminescence kit (ECL kit, Bio-Rad, Hercules, CA, USA) was used to visualize the band, and image analysis was performed using a Chemiluminescence system (Fusion SL2, VILBER LOURMAT, France) and Image J (NIH, USA).

6-5. Histological and immunohistochemistry

After fixing the sample with 4% formaldehyde (Duksan Chemical, Korea), it was embedded in paraffin wax (Merck, Darmstadt, Germany). After making a 4 mm thick slide section, it was stained with hematoxylin & eosin to confirm fetal cartilage stem cell sheet and cell morphology in vivo.

For immunohistochemical analysis of CK3 and Human nucleus, react with methanol added with 3% hydrogen peroxide (Duksan Chemical, Korea) for 10 minutes and then with pepsin solution (Golden Bridge International, Inc., Mukilteo, WA, USA) for 10 minutes. Reacted. After blocking with PBS to which 1% BSA was added, at room temperature with a primary antibody [anti-CK3 (1:100, Abcam, Cambridge, UK), anti-human nucleus (1:100, Millipore, Massachusetts, USA)] After reacting for 1 hour and 30 minutes, reacted with a secondary antibody (biotinylated-anti mouse IgG (SPlink HRP Detection Kit; Golden Bridge International, Inc., Mukilteo, WA, USA) for 30 minutes, and then with HRP-conjugated streptavidin solution for 30 minutes. Finally, before mounting, react with 3,3'-diaminobenzidine (DAB) solution (Golden Bridge International, Inc., Mukilteo, WA, USA), and then Mayer's hematoxylin (YD Diagnostics, Seoul, Korea). ) Was counter-stained.

As a result, as shown in Fig. 5A, the morphology of fetal cartilage stem cells cultured in the differentiation medium was compared with that of SV40 corneal epithelial cells. SV40 corneal epithelial cells were cultured in Cnt-Pr medium. As a result, no morphological difference was observed between the two cells, and it was confirmed that the morphology of fetal cartilage stem cells was similar to that of corneal epithelial cells.

As shown in FIG. 5B, the result of comparing fetal cartilage stem cells cultured in differentiation medium and SV40 corneal epithelial cells using corneal epithelial stem cell markers (ABCG2, p63) and corneal epithelial differentiation markers (PAX6, CK3/12), SV40 It was confirmed that ABCG2, p63, PAX6 and CK3/12 were expressed in corneal epithelial cells, and PAX6 and CK3/12 were not expressed in fetal cartilage stem cells cultured in a general medium, and ABCG2 and p63 were low, but differentiation medium In the case of fetal cartilage stem cells cultured in, it was confirmed that ABCG2, p63, PAX6 and CK3/12 were all highly expressed, thereby confirming that the expression of corneal epithelial stem cell markers and corneal epithelial differentiation markers in fetal cartilage stem cells was induced. I did.

5C and 5D, fetal cartilage stems cultured in differentiation medium using stem cell markers (CD34, CD105), corneal epithelial stem cell markers (ABCG2, p63), and corneal epithelial differentiation markers (PAX6, CK3/12) As a result of comparing the cells and SV40 corneal epithelial cells, it was confirmed that the expression of corneal epithelial stem cell markers and corneal epithelial differentiation markers was induced in fetal cartilage stem cells cultured in differentiation medium similar to SV40 corneal epithelial cells.

Example 7: Chemically burned corneal epithelial animal model

7-1. Preparation of fetal cartilage stem cell sheet

Fetal cartilage stem cells were subcultured through trypsin treatment (passage 4-5). Non-adherent cells were washed by replacing them with fresh medium 2-3 times. Thereafter, fetal cartilage stem cells (2 X 10 ⁵ cells/cm ² ) were 100 U/ml penicillin G and 100 μg/ml streptomycin (HyClone), insulin-transferrin-selenium (ITS, Gibco BRL, NY, USA), 50 μg/ml ascorbate-2 phosphate, 100 nM dexamethasone, 40 μg/ml proline, 1.25 mg/ml bovine serum albumin, 100 μg/ml sodium pyruvate (Sigma-Aldrich, St. Louis, USA) It was cultured in a sheet-medium containing high-glucose DMEM medium, and cultured for 3-4 days in a 37°C, 5% CO ₂ incubator until the fetal cartilage stem cells were removed from the culture dish.

7-2. Corneal epithelial animal model with chemical burns

Animal experiments were conducted after obtaining approval from Ajou University Animal Experiment Ethics Committee. Corneal limbal stem cell deficiency (LSCD) was induced in the right eye of each rabbit (18 animals). After intramuscular injection of a mixture of ketamine and zoletyl to anesthetize, a filter paper having a diameter of 8 mm was saturated with 1 M NaOH, placed on the corneal limbus for 30 seconds, washed with saline solution for 1 minute, and this was repeated 3 times. The fetal cartilage stem cell sheet and the PKH26-labeled fetal cartilage stem cell sheet were placed on the cornea with forceps, and were sutured with 6-0 black silk sutures at the top, bottom, left and right sides.

As a result, as shown in Fig. 6B, as a result of analyzing the effect of the fetal cartilage stem cell sheet in the rabbit animal model with chemical burns on the right eye, normal corneal epithelium was observed on days 0 and 7 in the normal group (A, D). , In the control group, damaged corneal epithelium was observed on days 0 and 7 (B, E), whereas when the fetal cartilage stem cell sheet was treated, corneal epithelium was observed on days 0 and 7 (C, F), It was confirmed that the corneal epithelium damaged by the fetal cartilage stem cell sheet was treated.

In addition, as shown in Fig. 6C, as a result of analyzing the sheet position and viability of fetal cartilage stem cells on the 7th day after transplantation, a fetal cartilage stem cell sheet labeled with PKH26 was observed at the damaged site (A), and fetal cartilage at the damaged site It was confirmed that the stem cell sheet survived (B).

6D, as a result of analyzing the healing ability using CK3, which is a corneal epithelial differentiation marker, on the 7th day of transplantation, it was confirmed that the fetal cartilage stem cell sheet was located at the damaged site and showed a healing effect on corneal damage when treated. .

Example 8: Confirmation of differentiation into growth plate tissue

As shown in FIG. 7, the growth plate portion of the rabbit was drilled at an angle with a 2 mm punch twice, and the TAP of the present invention was implanted. After that, the implantation site was separated and sectioned to a thickness of 4 μm using a frozen sectioning machine, and then a slide was manufactured. The negative control group only damaged the growth plate, and as the positive control group, bone wax, which is currently most widely used in clinical practice, was used.

As a result, as shown in Fig. 8, it was confirmed that cartilage was formed in the group transplanted with the TAP of the present invention after 8 weeks. The warning agent used as a positive control has the effect of filling the graft space, but there is a limitation in that the graft material is maintained as bone tissue, and angular deformation of the bone occurs, whereas the TAP of the present invention forms cartilage and is transplanted even when observed with the naked eye. It was confirmed that the cartilage was regenerated so that the part was not visible.

In addition, bone tissue is formed in the negative control group and the positive control group at the damaged graft site through safranin O and hematoxylin & eosin staining, whereas in the group transplanted with the TAP of the present invention, the cartilage tissue is clearly located at the growth plate position. I could confirm that there is.

In addition, as shown in FIG. 9, as a result of performing immunostaining, it was confirmed that collagen and glycoproteins were formed in the group to which TAP was transplanted similarly to the normal group, and collagen X, which is the stage of bone formation, was formed after 8 weeks. As a result, it is expected that ossification will occur late or be prevented.

Example 9: Confirmation of cartilage formation ability in growth plate

The TAP of the present invention was implanted into the growth plate injury model of rabbits, and ossification capacity was confirmed in the cartilage in the growth plate (Group 1: negative control, Group 2: positive control (warning agent), Group 3: TAP treatment group) (left bar: Average length, right bar: Example results)

As a result, as shown in FIG. 10, a difference in length growth was observed only in the negative control group, and angular deformation was most observed, and angular deformation was also observed in the group implanted with a warning agent, which is a positive control.

In addition, as shown in FIG. 11, bone tissue was formed at the damaged site in the negative control group and the positive control group through safranin O, hematoxylin & eosin staining, but it was confirmed that the TAP of the present invention was maintained as cartilage tissue. .

Overall, in the results of 4 weeks, it was confirmed that cartilage tissue was filled in the damaged growth plate area only in the TAP-grafted group, unlike the negative control group and the positive control group, whereas a hypertrophic marker was expressed in the growing area, confirming the possibility of length growth.

In addition, as shown in FIG. 12, the group to which TAP was transplanted was stained with HuNA to confirm the transplanted tissue, and the proliferating cells were confirmed by staining with PCNA.

In addition, as shown in FIG. 13, as a result of performing safranin O and uCT at 4, 14, 21, and 28 weeks after transplantation, at the results of 4, 14, 21, and 28 weeks, almost the same trend was confirmed in the group transplanted with TAP. I did. In the negative control group and the positive control group, no cartilage tissue was found in the damaged area of the growth plate, and bone tissue was formed in the u-CT result. In particular, in the case of the negative control group, angular deformation occurred in all states.

In addition, as shown in Figure 14, the result of comparing the regeneration of the lumbar intervertebral discs through histological examination (lumbar 2-3: normal control, lumbar 3-4: TAP treatment group, lumbar 4-5: negative control, lumbar 5-6: TAP treatment group), it was confirmed that the fibrous rim defects of the intervertebral discs between the lumbar spine 3-4 and 5-6 were restored.

Example 10: Confirmation of muscle differentiation effect using fetal cartilage-derived stem cells sheet (TAP-C)

In order to make artificial muscle tissue that can be used for muscle regeneration by using a fetal cartilage-derived stem cell sheet in the laboratory, the muscle differentiation effect was confirmed by using TAP-C.

Fetal cartilage-derived stem cells (FCSC) were dispensed into a 6-well plate, and in order to confirm the difference in differentiation according to the density and thickness of the cell sheet, the sheet group was 3 × 10 ⁵ /well group and 2 × It consisted of 10 ⁶ /well groups. As a medium, High-glucose DMEM (Hyclone) to which 10% FBS was added was used.

After 1 day of cultivation, the medium was replaced with a Myoblast-differentiation induction medium. The medium was 1 ng/ml transforming growth factor-β1 (TGF-β1; R&D systems), non-essential amino acids (NEAA; Invitrogen), insulin-transferrin-selenium (ITS; Gibco) in DMEM/nutrient mixture F-12 (Invitrogen). ) Was added and cultured for 7 days to differentiate FCSCs into myoblasts.

After 7 days of culture, in order to confirm differentiation into myoblasts, western blotting and immunocytochemistry (ICC) were performed to confirm the expression levels of Myf5 and MyoD.

As a result, as shown in Figs. 15 to 17, the expression of Myf5 and MyoD, which act as major factors at the initial stage of muscle differentiation, was shown, indicating that the FCSC sheet was differentiated into myoblasts.

As the specific parts of the present invention have been described in detail above, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (9)

A composition for tissue fusion having tissue adhesion and differentiation properties, comprising stem cells and stem cell-derived extracellular matrix as an active ingredient. The composition of claim 1, wherein the tissue is selected from the group consisting of cornea, epithelium, growth plate, bone, cartilage, ligaments, muscle and skin tissue. The composition of claim 1, wherein the composition is in a gel shape or a sheet shape. A tissue for transplantation prepared by attaching two or more tissues with the composition according to claim 1. (a) separating and culturing stem cells from fetal cartilage tissue;
(b) obtaining a cell membrane comprising the cultured stem cells and their extracellular matrix;
(c) centrifuging the obtained cell membrane to obtain a cell pellet; And
(D) culturing the obtained cell pellet in a medium; a method for producing a composition for fusion tissue according to claim 1 comprising. [6] The method of claim 5, wherein the cell membrane obtained in step (b) is obtained by including all of the extracellular matrix together with the cells attached to the bottom without separating the cells. [6] The method of claim 5, wherein the compressive strength, adhesion, and coating properties of the composition are adjusted according to the culture period in step (d). The method of claim 5, wherein the tissue is selected from the group consisting of cornea, epithelium, growth plate, bone, cartilage, ligaments, muscle and skin tissue. A composition for tissue fusion having tissue adhesion properties, comprising stem cells and stem cell-derived extracellular matrix as an active ingredient.

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