KR102586165B1 - Tissue consolidant composition with increased adhesion and binding force to biological tissues and preparing method thereof - Google Patents

Abstract

The present invention relates to a tissue fusion agent with increased biological tissue adhesion and cohesion using adhesion-related genes, and more specifically, to a tissue fusion agent produced from fetal cartilage tissue-derived stem cells with increased expression by inserting VCAN, CTGF or EXT1. As it was confirmed that the cartilage tissue fusion composition exhibits significantly superior adhesive strength compared to the cartilage tissue fusion composition produced from stem cells derived from fetal cartilage tissue in which the gene was not inserted, the expression of VCAN, CTGF or EXT1 was increased. The cell composition can be prepared as a tissue fusion agent composition with increased adhesion and binding force to living tissue, and VCAN, CTGF or EXT1 can be provided as a composition for adding a tissue fusion agent.

Description

Tissue consolidant composition with increased adhesion and binding force to biological tissues and preparing method thereof}

The present invention relates to a tissue fusing agent with increased biological tissue adhesion and bonding force using cells with increased expression of adhesion-related genes.

Bioadhesives and sealants are used to suture or apply tissue during surgery, or as anti-bleeding agents or blockers for body fluids and blood. Since they come in contact with the skin, they require biocompatibility and must not be toxic or hazardous in vivo. It should not interfere with the healing of the living body.

Medical adhesive materials currently being commercialized include cyanoacrylate-based, skin glue-based, gelatin glue-based, and polyurethane-based. Closure Medical in the United States commercialized an octylcyanoacrylate medical tissue adhesive called Dermabond in 1997. It received approval for marketing in the European Community in August 2008, and then from the U.S. FDA in 1998.

However, cyanoacrylate-based adhesives sometimes interfere with wound healing because the solidified product lacks flexibility and is hard. Additionally, since it is difficult to decompose in vivo, there is a problem that it is easily encapsulated and becomes a foreign substance. Fibrin glue has a very low adhesive strength, so the resulting lump of skin may fall off from the tissue, and since it is a blood product, there is a risk of viral infection.

In addition, gelatin glue is a bio-derived tissue adhesive that has been developed by mixing gelatin-resorcinol-formalin. In addition, tissue adhesives such as gelatin-glutaraldehyde have been developed, but formalin or glutaraldehyde used as a cross-linking agent have been developed. It has the disadvantage of causing tissue toxicity by causing a cross-linking reaction with proteins in the body.

To overcome these problems, there is a need to develop a biological tissue adhesive that has excellent biocompatibility, excellent mechanical strength in the presence of moisture, and fast and strong adhesion.

Republic of Korea Patent Publication No. 10-2013-0093769 (published on August 23, 2013)

The present invention provides adhesion-related genes with tissue adhesion and bonding ability, and the genes can be used to develop adhesives for regenerative treatment of damaged tissues or organs after surgical operations on tissue damage or organs.

The present invention contains as an active ingredient cells with increased expression of one or more genes selected from the group consisting of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2, and KRT19, and increases tissue adhesion and binding force. Provides a tissue fusion composition.

The present invention provides a composition for adding a tissue healing agent containing as an active ingredient one or more genes selected from the group consisting of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2 and KRT19.

The present invention includes the steps of inserting one or more genes from the group consisting of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2 and KRT19 into cells; Primary culturing the cells into which the gene has been inserted for 5 to 10 days in a medium containing chondrogenic differentiation medium; Secondary monolayer culture of the primary cultured cells; and obtaining a cell membrane in which the secondary monolayer cultured cells and the extracellular matrix are combined.

According to the present invention, the cartilage tissue fusion composition made from stem cells derived from fetal cartilage tissue whose expression is increased by inserting VCAN, CTGF or EXT1 is used for cartilage tissue fusion made from stem cells derived from fetal cartilage tissue without inserting the above genes. As it was confirmed that it exhibits significantly superior adhesive strength compared to the previous composition, the cell composition with increased expression of VCAN, CTGF or EXT1 can be prepared as a tissue fusion composition with increased biological tissue adhesion and bonding power, VCAN , CTGF or EXT1 can be provided as a composition for adding a tissue fusion agent.

Figure 1 is a micrograph comparing genes related to the adhesion control mechanism expressed in an adhesive sheet-cultured using cells derived from human fetal cartilage tissue and genes related to the adhesion control mechanism expressed in an adhesive sheet cultured using cells derived from human adult cartilage tissue. This is the result of array analysis.
Figure 2 is a microarray analysis comparing the adhesion control mechanism-related genes expressed in adhesive sheet cultured using cells derived from human fetal cartilage tissue and the adhesion-related genes expressed when cells derived from human adult cartilage tissue were cultured using a general cell culture method. It is a result.
Figure 3 is a microarray comparing the adhesion control mechanism-related genes expressed in the adhesive sheet cultured using cells derived from human fetal cartilage tissue and the adhesion-related genes expressed when human bone marrow-derived mesenchymal stem cells were cultured using a general cell culture method. This is the result of analysis.
Figure 4 shows the results of gene cloning (DNA cloning) after binding CTGF, EXT1, or VCAN, which are target genes related to adhesion, to the pCMV-HA vector, respectively.
Figure 5 shows the image results of human chondrocytes inserted with the target gene and cultured under the same conditions.
Figure 6 shows the results of inserting the target gene into human cartilage cells and confirming the gene expression level by RT-PCR (Real Time Polymerase Chain Reaction).
Figure 7 shows the image results of inserting the target gene into stem cells derived from fetal cartilage tissue and culturing them under the same conditions.
Figure 8 shows the results of RT-PCR (Real Time Polymerase Chain Reaction) experiment after inserting the target gene into stem cells derived from fetal cartilage tissue.
Figure 9 shows the image results of stem cells derived from fetal cartilage tissue of the present invention treated through siRNA insertion of the target gene and then cultured under the same conditions.
Figure 10 shows the results of confirming the gene expression level by performing RT-PCR (Real Time Polymerase Chain Reaction) after culturing the stem cells derived from fetal cartilage tissue of the present invention by inserting siRNA of the target gene.
Figure 11 shows the process of confirming the adhesive strength of the cartilage tissue fusion composition (TAP-C) made using stem cells derived from fetal cartilage tissue.
Figure 12 shows the results of confirming the adhesive strength of cartilage tissue fusion compositions made using human chondrocytes and fetal cartilage tissue-derived stem cells (FCPC) with and without insertion of the CTGF or EXT1 gene, respectively.
Figure 13 shows the results of confirming the tissue adhesion ability of a biological tissue conjugate prepared using the adhesion-related gene VCAN.

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

The present invention contains genes derived from human fetal cartilage tissue and extracellular matrix derived from fetal cartilage tissue as active ingredients, and genes related to the adhesion control mechanism whose expression is increased in a tissue adhesive in the form of a gel-type cell sheet. The purpose of this study is to identify and provide a conjugate for bonding damaged tissues using the same.

The present invention contains as an active ingredient cells with increased expression of one or more genes selected from the group consisting of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2, and KRT19, and increases tissue adhesion and binding force. A tissue fusion composition can be provided.

The cells may be selected from the group consisting of human cartilage tissue cells and human fetal cartilage tissue-derived stem cells.

The tissue helding agent composition may be used as a tissue adhesive, tissue sealant, anti-adhesion agent, hemostatic agent, or wound coating agent.

The present invention can provide a composition for adding a tissue healing agent containing as an active ingredient one or more genes selected from the group consisting of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2 and KRT19.

According to an embodiment of the present invention, a cartilage damage model made from cartilage tissue of a patient who received a tissue fusion composition (TAP) made from stem cells derived from human fetal cartilage tissue whose expression was increased by inserting the VCAN, CTGF or EXT1 gene. After insertion, a jig with a diameter of 5 mm was contacted and attached to the inserted TAP, and the resistance until the jig was separated from the TAP was checked by pulling at a speed of 1.3 mm/min. Stem cells derived from fetal cartilage tissue were tested. As a result of comparison with the cartilage tissue fusion composition (TAP-C) produced using the same, no clear differences were confirmed in the 1st and 2nd weeks of culture as shown in Figure 9, but compared to the TAP-C group in the 3rd week of culture, the gene It was confirmed that the adhesive strength was very excellent in the insertion group, and the adhesive strength of the cartilage tissue fusion composition made from cells gene knocked down through siRNA insertion was significantly reduced compared to the TAP-C group. there was.

From the above results, the cell composition that increased the expression of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2 and KRT19 can be provided as a tissue fusion composition with increased biological tissue adhesion and binding force, and the gene It was confirmed that it can be provided as a composition for adding a tissue fusion agent.

In addition, the present invention includes the steps of inserting one or more genes from the group consisting of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2 and KRT19 into cells; Primary culturing the cells into which the gene has been inserted for 5 to 10 days in a medium containing chondrogenic differentiation medium; Secondary monolayer culture of the primary cultured cells; and obtaining a cell membrane in which the secondary monolayer cultured cells and the extracellular matrix are combined.

The cells may be selected from the group consisting of human cartilage tissue cells and human fetal cartilage tissue-derived stem cells.

The step of culturing the primary cultured cells into a secondary monolayer may be culturing the primary cultured cells as a monolayer in a growth medium for 15 to 20 days.

The cell membrane includes cells with increased expression of one or more genes selected from the group consisting of CTGF, EXT1, NDST1, ITGA3, CYTL1, TFGB1, SOCS3, VCAN, CHI3L2, and KRT19, and may have increased tissue adhesion and binding force. .

The term “alloying agent” of the present invention refers to a drug that promotes healing and adhesion of skin or muscle tissue.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Isolation and culture of human cartilage tissue cells

Cartilage tissue isolated from the knee joint was washed with phosphate buffered saline (PBS) and then washed in DMEM (DMEM) containing 0.2% (w/v) collagenase (Worthington Biochemical Corp., Lakewood, NJ). Dulbecco's Modified Egle Medium, Gibco, Grand Island, NY) was added and cultured at 37°C in a 5% CO ₂ incubator for 4 hours. The cartilage tissue was completely digested and the released cartilage tissue-derived stem cells were centrifuged at 1700 rpm for 10 minutes to obtain precipitated cartilage tissue cells, which were placed in a tissue culture dish [150 mm (dia.) × 20 mm (h)]. Inoculated at a density of 1 × 10 ⁶ cells.

<Example 2> Isolation and culture of human fetal cartilage tissue-derived stem cells (FCPC)

Cartilage tissue-derived stem cells 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 isolated from the knee joint was washed with phosphate-buffered saline (PBS) and then added with 0.2% (w/v) collagenase (Worthington Biochemical Corp., Lakewood, NJ). DMEM (Dulbecco's Modified Egle Medium, Gibco, Grand Island, NY) medium was added and cultured in an incubator at 37°C and 5% CO ₂ 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 stem cells (fetal cartilage derived progenitor cells; FCPC), which were cultured in a tissue culture dish [150 mm (150 mm)]. dia.) × 20 mm (h)] was inoculated at a density of 1 × 10 ⁶ cells.

<Example 3> Preparation of a composition for tissue union with tissue adhesion and differentiation properties (TAP)

The chondrocytes obtained in Examples 1 and 2 were diluted to a cell density of 1 × 10 ⁶ and then added to cartilage differentiation medium [1% antibiotic-antimycotic, 1.0 mg/mL insulin, 0.55 mg/mL 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. (dexamethasone), 40 ㎍/mL proline, and 10 ng/mL TGF-β were used in a medium containing [DMEM-HG] and cultured in 6 wells for one week at 37°C in a 5% CO ₂ incubator. , monolayer culture was performed for 15 to 18 days in DMEM medium supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin, and 50 μg/mL streptomycin. After culturing, the medium was removed, and PBS was added to obtain a cell membrane in the form of a sheet combined with the extracellular matrix.

<Example 4> Microarray analysis

The gene expression profiles of TAP prepared in Example 3 were compared. Genes whose expression was highly increased in the two TAPs prepared in Example 3 were selected using the whole human genome Agilent 4 × 44K Oligonucleotide Microarray (Macrogen, Korea).

Briefly, total RNA was extracted from TAP, human chondrocytes, or bone marrow stromal cells and subjected to microarray chip hybridization. To compare the gene expression profiles of healthy human chondrocytes and TAP, representative genes for histocompatibility were selected and microarray analysis was performed.

Afterwards, genes expressed when cells derived from human fetal cartilage tissue were cultured on sheets with excellent bioadhesiveness were compared with genes related to electrodeposition expressed when cells derived from human adult cartilage tissue were cultured on sheets, and the gene expression shown in Figure 1 was compared. Confirmed.

In addition, the results of comparing the expression of adhesion-related genes expressed when cells derived from human fetal cartilage tissue were cultured on a sheet with excellent bioadhesiveness and those expressed when cells derived from human adult cartilage tissue were cultured using a general cell culture method are shown in Figure 2 The same gene expression results were confirmed.

Finally, by comparing the expression of genes expressed when human fetal cartilage tissue-derived cells are cultured on sheets with excellent bioadhesiveness and the expression of adhesion-related genes expressed when human bone marrow-derived mesenchymal stem cells are cultured using general cell culture methods, Figure 3 and The same gene expression results were confirmed.

From the above results, genes expressed when cultured in sheet form using cells derived from human fetal cartilage tissue were identified, and adhesion-related genes specifically expressed were confirmed as shown in Table 1 below.

gene Gene Number Sequence CTGF CCN2 or connective tissue growth factor HM015591 NDST1 Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1 U17970 EXT1 Exostosin-1 Q16394 ITGA3 Integrin alpha-3 AC002401 CYTL1 Cytokine-like protein 1 BC031391 TGFB1 Transforming growth factor beta-1 proprotein BT007245 SOCS3 Suppressor of cytokine signaling 3 AF159854 VCAN Versican U26555 CHI3L2 Chitinase-3-like protein 2 U49835 KRT19 Keratin, type I cytoskeletal 19 AF202321

<Example 5> Preparation of a composition for tissue fusion with tissue adhesion and differentiation properties treated with target gene (TAP)

CTGF (CCN2) and EXT1 identified in Example 4 were combined with a vector as shown in FIG. 4, and then an insert gene was prepared through cloning. Gene insertion (gene knock-in) was performed using Gene Transfection Reagent (Lipofectamine 3000 Thermo). A gene knock-down experiment was performed by treating each gene with siRNA (bionics) under the same conditions.

As shown in Figures 5 and 7, human chondrocytes and fetal cartilage tissue-derived stem cells (FCPC) were treated with the gene for 3 days, inserted into the cells, and cell membranes were obtained in the same manner as in Example 3.

In order to confirm gene expression in the composition for tissue fusion prepared through the above process, real-time PCR (Real Time Polymerase Chain) was performed using cell membranes obtained from cartilage cells cultured by treating each gene and fetal cartilage tissue-derived stem cells (FCPC). Reaction, RT-PCR) was performed.

As a result, it was confirmed that EXT1 and CTGF expression was increased in cartilage cells into which the target gene was inserted, as shown in Figure 6, and EXT1 and CTGF expression was also increased in fetal cartilage tissue-derived stem cells into which the target gene was inserted, as shown in Figure 8. could be confirmed. On the other hand, as shown in Figure 7, it was confirmed that EXT1 and CTGF expression was suppressed in fetal cartilage tissue-derived stem cells into which siRNA of the target gene was inserted.

From the above results, it was confirmed that EXT1 and CTGF were effectively inserted into chondrocytes and fetal cartilage tissue-derived stem cells, thereby obtaining cell membranes with increased expression of EXT1 and CTGF genes.

<Example 6> Confirmation of adhesive strength of composition for tissue union

To confirm the adhesive strength of the TAP prepared in Examples 3 and 5 and the composition for tissue union treated with each gene, the adhesive strength was compared.

First, the patient's cartilage tissue discarded after artificial joint surgery was donated along with a consent form. A cartilage damage model was created on the surface of the donated patient's cartilage tissue using a 6mm biopsy punch, and the prepared composition for fusion was inserted.

Next, a jig with a diameter of 5 mm was contacted and attached to the inserted TAP in the same process as shown in FIG. 11, and then pulled at a speed of 1.3 mm/min to check the resistance until the jig was separated from the TAP.

As a result of comparing the cartilage tissue fusion composition (TAP-C) produced using stem cells derived from fetal cartilage tissue and the cell membrane treated with each gene, no clear differences were confirmed in the 1st and 2nd weeks of culture, as shown in Figure 12. However, in the 3rd week of culture, it was confirmed that the adhesion strength was very excellent in the gene insertion group compared to the TAP-C group, and the adhesion of the cartilage tissue fusion composition made from cells with gene knock-down through siRNA insertion was confirmed. It was confirmed that the intensity was significantly reduced compared to the TAP-C group.

In addition, after inserting VCAN into cartilage cells using the same method, a biological tissue conjugate was manufactured and the adhesive ability was confirmed. As a result, it was confirmed that the cartilage-bone adhesion of the biological tissue conjugate was increased, as shown in FIG. 13.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

Claims (8)

Contains cells with increased gene expression consisting of CTGF, EXT1, and VCAN as active ingredients,
A pharmaceutical composition for cartilage tissue synthesis with increased adhesion and binding force to living tissue, wherein the cells are human cartilage tissue cells. delete The cartilage tissue fusion agent pharmaceutical composition according to claim 1, wherein the cartilage tissue fusion agent composition is used as a cartilage tissue adhesive, a cartilage tissue sealant, or an anti-cartilage adhesion agent. delete Inserting genes consisting of CTGF, EXT1, and VCAN into cells;
Primary culturing the cells into which the gene has been inserted for 5 to 10 days in a medium containing chondrogenic differentiation medium;
Secondary monolayer culture of the primary cultured cells; and
Comprising the step of obtaining a cell membrane in which the secondary monolayer cultured cells and the extracellular matrix are combined,
A method of producing a cartilage tissue fusion agent, characterized in that the cells are human cartilage tissue cells. delete The method of claim 5, wherein the step of culturing the primary cultured cells into a secondary monolayer is performed by culturing the primary cultured cells as a monolayer in a growth medium for 15 to 20 days. The method of claim 5, wherein the cell membrane includes cells with increased gene expression consisting of CTGF, EXT1, and VCAN, and tissue adhesion and bonding force are increased.