KR102156602B1 - Composition for Gellan-gum Hydrogels Containing miRNA-140-5p and Composition for treatment of Cartilage Regeneration using it - Google Patents

Abstract

The present invention relates to a gellan gum hydrogel composition containing miRNA-140-5p and a composition for treating cartilage damage using the same, and by differentiating bone marrow-derived stem cells into chondrocytes, miRNA-140 capable of supplying insufficient chondrocyte sources It relates to a novel gellan gum hydrogel composition containing -5p and a composition for treating cartilage damage that can be applied to regeneration of damaged cartilage and treatment of osteoarthritis using the same. When the composition for treating cartilage damage of the present invention is injected into the body, it is specifically expressed in cartilage and is involved in cartilage cell differentiation, ECM decomposition, and intracellular differentiation. It is suitable as an injection for cartilage damage treatment due to its high elasticity and durability. In addition, it does not have cytotoxicity, and has the advantage of not being dissolved by inflammatory reactions or toxic substances. In addition, in order to enhance the cartilage regeneration effect, autologous knee cartilage may be collected, thereby minimizing the disadvantages of existing autologous chondrocyte transplantation.

Description

Composition for Gellan-gum Hydrogels Containing miRNA-140-5p and Composition for treatment of Cartilage Regeneration using it}

The present invention relates to a gellan gum hydrogel composition containing miRNA-140-5p and a composition for treating cartilage damage using the same, and by differentiating bone marrow-derived stem cells into chondrocytes, miRNA-140 capable of supplying insufficient chondrocyte sources It relates to a novel gellan gum hydrogel composition containing -5p and a composition for treating cartilage damage that can be applied to regeneration of damaged cartilage and treatment of osteoarthritis using the same.

Osteoarthritis (OA), an age-related joint disease, does not currently have an effective treatment for the disease, and its cause is not fully identified. It is known that the causes of osteoarthritis include a decrease in the number of chondrocytes and decomposition of the extracellular matrix (ECM) of cartilage. Chondrocytes that form cartilage are derived from mesenchymal progenitor cells. During embryonic development, mesenchymal progenitor cells proliferate, aggregate in mesenchymal condensation, and then differentiate into chondrocytes, which later form cartilage templates for bone and cartilage. Chondrocytes secrete unique extracellular matrix (ECM) proteins such as type II collagen and Aggrecan to produce the cartilage matrix.

Currently, when cartilage is damaged, various methods are used for treatment depending on the degree of damage. Representative treatment methods include autologous cartilage cell transplantation, bone marrow stimulation using bone marrow stem cells, cartilage regeneration therapy using adult stem cells and embryonic stem cells, and make bone marrow cells flow out of damaged areas to treat damaged areas. Such as micro-fracture. [Z. Lin, C. Willers, J. Xu, and M. H Zheng, Tissue Eng., 12, 1971 (2006)]

However, the above-mentioned treatments for cartilage damage have a disadvantage in that they require a difficult surgical method and several problems occur during the treatment process. Therefore, tissue engineering methods that can effectively treat these various disadvantages have been proposed.

As a biomaterial used in tissue engineering, cartilage regeneration materials include collagen, hyaluronic acid, gelatin, and gellan gum. Among them, gellan gum (GG) is a complex polysaccharide in which gluconic acid, rhamnose, and glucose (1:1:2) are regularly arranged, and has excellent odorless, colorless, heat resistance, and enzyme resistance. . In addition, it is possible to control the mechanical properties of hydrogels through ion addition and pH control, and there is no cytotoxicity, so it has been studied variously as a biomaterial. (JT Oliveira, L. Martins, R. Picciochi, IB Malafaya, RA Sousa , NM Neves, JF Mano and RL Reis, Journal of Biomedical Materials Research Part A, 93A, 852 (2010)).

Micro RNA (miRNA) is an evolutionarily conserved single-stranded, non-coding RNA molecule containing about 22 nucleotides capable of regulating gene expression by binding to specific sequences in the 3'-untranslated region of messenger RNA (mRNA). As such, mammalian miRNAs exhibit tissue-specific or developmental patterns of expression and are involved in a variety of biological processes including cell differentiation, tumorigenesis, and immune responses. Previous studies also found reduced miR-140 expression in human osteoarthritis (Oliopoulos et al., 2008; Miyaki et al., 2009), which may contribute to the abnormal gene expression pattern of OA arthritis. (Iliopoulos D, Malizos KN, Oikonomou P, Tsezou A PLoS One. 3, 11, e3740, (2008))

miRNA-140-5p (hereinafter, miRNA-140) was specifically expressed in cartilage and was shown to be involved in chondrocyte differentiation, ECM degradation, and intracellular differentiation. Recent studies have revealed a cartilage-specific correlation with miRNA-140 expression in mouse and human OA. (Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Genes Dev. 24, 117385 (2010)).

As a related art, U.S. Patent Publication No. 2015-0231274, WO 2015-174934 A9, WO 2015-034436 A1, and the like, propose techniques for combining miRNA with polysaccharides and applying them to cartilage-related treatment.

However, there are studies on the polysaccharide technology or the effect of miRNA on cartilage damage treatment as described above, but a formulation that can be applied to cartilage regeneration treatment has not been proposed, but only theoretical studies have been proposed.

As such, studies on an effective agent for cartilage regeneration treatment using the above-mentioned miRNA-140-5p are important, but studies on compositions that can be easily applied to cartilage damage treatment have not been properly conducted. There is no effective approach.

US Patent Publication No. 2015-0231274 WO 2015-174934 A9 WO 2015-034436 A1

J. T. Oliveira, L. Martins, R. Picciochi, I. B. Malafaya, R. A. Sousa, N. M. Neves, J. F. Mano and R. L. Reis, Journal of Biomedical Materials Research Part A, 93A, 852 (2010) Oliopoulos et al., 2008; Miyaki et al., 2009) found that miR-140 expression was reduced. (Iliopoulos D, Malizos KN, Oikonomou P, Tsezou A PLoS One. 3, 11, e3740, (2008) Iliopoulos D, Malizos KN, Oikonomou P, Tsezou A PLoS One. 3, 11, e3740, (2008) Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Genes Dev. 24, 117385 (2010)

In order to improve the problems not solved in the prior art as described above, the present invention solves the development of a new composition for cartilage regeneration and osteoarthritis treatment only by local injection of the damaged area without incision in the surgical site to minimize the surgical site. To

As a result of long research and review for solving the problem, the present inventors developed a gellan gum hydrogel containing miRNA-140-5p for the treatment of osteoarthritis, confirmed that there is a practical cartilage regeneration effect, and completed the present invention.

Accordingly, an object of the present invention is to provide a gellan gum hydrogel composition containing miRNA-140-5p, which has a cartilage regeneration effect by engaging in cartilage cell differentiation, ECM degradation and intracellular differentiation for the treatment of not only osteoarthritis but also damaged cartilage. have.

In addition, another object of the present invention is to provide a composition for cartilage regeneration and osteoarthritis treatment using a gellan gum hydrogel composition containing cells containing miRNA-140-5p that can be directly injected and applied to the joints of the human body. have.

In addition, another object of the present invention is to provide a medical kit containing a composition for cartilage regeneration and osteoarthritis treatment and prevention comprising a gellan gum hydrogel composition containing cells containing miRNA-140-5p.

In order to solve the problems of the present invention as described above, the present invention provides a gellan gum hydrogel composition in the form of a hydrogel containing miRNA-140-5p in gellan gum.

In addition, the present invention provides a composition for treating cartilage damage comprising a gellan gum hydrogel composition containing cells containing miRNA-140-5p as described above as an active ingredient.

In addition, the present invention provides a kit for treating arthritis for injection containing the composition for treating cartilage damage as described above as an active ingredient.

The advantages and effects of the gellan gum hydrogel composition containing miRNA-140-5p according to the present invention are summarized as follows.

(i) The hydrogel composition of the present invention has the effect of miRNA-140-5p, which has the ability to differentiate from bone marrow-derived stem cells into chondrocytes, and is highly effective by selectively combining and using natural polymer materials suitable for living organisms unique to gellan gum. It is suitable as an injection for cartilage treatment due to its elasticity and durability. Therefore, it can be used as a treatment for cartilage regeneration and osteoarthritis that is safe and exhibits excellent synergistic effects.

(ii) In the present invention, a gellan-gum hydrogel composition containing miRNA-140-5p can be prepared by a simple manufacturing method and can be conveniently used as an injection for human injection. It has the effect of being very easy to apply.

(iii) According to the present invention, there is an excellent effect of minimizing a surgical site, shortening an operation time, and enabling rapid treatment after surgery by manufacturing a histo-engineered injection-type hydrogel.

1 is a photo of a scaffold for a gellan gum hydrogel composition containing cells containing miRNA-140-5p prepared according to the present invention, and a gellan gum hydrogel composition not containing cells containing miRNA-140-5p. This is a picture of the support body.
2 is a SEM photograph taken to confirm the roughness and porosity of the surface of a support after lyophilizing the gellan gum hydrogel composition prepared according to the present invention.
3 is a photograph confirming the efficiency of miRAN transfected into cells 3 days after seeding and hardening bone marrow stem cells containing miRNA-140-5p in an aqueous gellan gum solution prepared according to the present invention with Fam siRNA negative control.
Figure 4 is a gellan gum hydrogel composition, respectively, bone marrow-derived stem cells, bone marrow-derived stem cells containing miRNA-140-5p, and chondrocytes were freeze-dried for each day (1, 3, 5, 7 days) after seeding. This is a picture confirming the morphology of
5 is a gellan gum hydrogel composition, respectively, bone marrow-derived stem cells, bone marrow-derived stem cells containing miRNA-140-5p, and chondrocytes were seeded and analyzed for cell proliferation by day (1, 3, 5, 7 days). It is a graph.
6 is a view of bone marrow-derived stem cells and chondrocytes by time (12, 24 hours) after seeding of bone marrow-derived stem cells and chondrocytes containing bone marrow-derived stem cells and miRNA-140-5p in a cell culture plate according to the present invention. This is a graph that generalizes and quantifies the gene expression of Sox9, Aggrecan, and Col II with GAPDH.
Figure 7a is a gellan gum hydrogel composition according to the present invention, bone marrow-derived stem cells and bone marrow-derived stem cells and chondrocytes containing miRNA-140-5p are uniformly mixed with an aqueous gellan gum solution for each day (1, 3 days) This is a band photo after electrophoresis of gene expression in bone marrow-derived stem cells and chondrocytes.
7b is a graph that was quantified by generalizing the electrophoresis result to GAPDH, and is a graph showing the result of the phenotype of SOX9, Aggrecan, and Col II.

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

The present invention is for the treatment of cartilage damage by combining a gellan gum with excellent biocompatibility and miRNA-140-5p involved in cartilage differentiation and an aqueous gellan gum solution for arthritis and cartilage regeneration, which has yet to be studied. It provides an invention for use as a composition.

The present invention has tried to invent a method of minimizing additional reoperation, side effects, and surgical range unlike the existing osteoarthritis surgery by changing the paradigm for the existing osteoarthritis treatment. As a result, an injectable hydrogel capable of effectively regenerating a damaged cartilage area was prepared using a biocompatible material, excellent in cartilage cell proliferation, and effective maintenance of function.

The present invention is characterized in that it is a preferred composition, basically comprising gellan gum as a main component, and containing miRNA-140-5p therein.

According to a preferred embodiment of the present invention, miRNA-140-5p contained in gellan gum may consist of a mixture of cells containing miRNA-140-5p as an auxiliary component.

According to a preferred embodiment of the present invention, gellan gum used as a main component in the present invention has a high modulus of elasticity when prepared from a hydrogel, and mechanical properties can be adjusted according to a crosslinking method or content. Gellan gum hydrogel forms a secondary coil helix structure when the temperature falls below a certain temperature to gel. Because gellan gum has a low toxicity rate and is easy to process, it can be used as an injection-type therapeutic agent that can be injected into the human body without surgical intervention if a hydrogel is prepared by mixing it with cells containing miRNA-140-5p.

According to a preferred embodiment of the present invention, miRNA-140-5p is specifically expressed in cartilage and has been shown to be involved in chondrocyte differentiation, ECM degradation and intracellular differentiation. Therefore, according to the present invention, when a gellan gum hydrogel containing miRNA-140-5p is injected into an arthritis site, it can have a positive effect.

In a preferred embodiment of the present invention, a gellan gum hydrogel composition is characterized in that cells containing miRNA-140-5p involved in chondrocyte differentiation, ECM degradation and intracellular differentiation are blended. In addition, the hydrogel composition according to the present invention can be used as a composition for treating cartilage damage.

According to a preferred embodiment of the present invention, the concentration of the gellan gum hydrogel is preferably 1-3% by weight, more preferably 1-2% by weight.

According to a preferred embodiment of the present invention, the gellan gum hydrogel used in the present invention may be composed of a hydrogel obtained by mixing pure gellan gum powder with water, preferably tertiary distilled water, and crosslinking with CaCl ₂ .

According to a preferred embodiment of the present invention, when bone marrow-derived stem cells transfected with miRNA-140-5p are added to the hydrogel and uniformly mixed, a gellan gum hydrogel containing cells containing miRNA-140-5p is mixed. It can be composed of a composition.

According to a preferred embodiment of the present invention, the concentration of miRNA-140-5p is preferably 10-50pmol, more preferably 25-40pmol, per 7ml of medium contained in the cell culture flask. If the content is too low, there is no addition effect, and if the content is too high, the amount wasted is large, which is not preferable.

The present invention comprises the steps of preparing a bone marrow-derived stem cell transfected with miRNA-140-5p;

Preparing a cell suspension in which the amount of the bone marrow-derived stem cells is 1×10 ⁶ cells/ml;

Diluting 200-300 μl of medium in the cell suspension and mixing it with an aqueous gellan gum solution to obtain a mixture;

Solidifying the mixture to prepare a gellan gum hydrogel containing cells transfected with miRNA-140-5p

It includes a method for preparing a gellan gum hydrogel composition containing miRNA-140-5p comprising a.

According to a preferred embodiment of the present invention, the amount of bone marrow-derived stem cells transfected with miRNA-140-5p was diluted 200-300 μl in a cell suspension of 1×10 ⁶ cells/ml and mixed with an aqueous gellan gum solution. Then, it can be solidified at room temperature and prepared as a gellan gum hydrogel composition in which cells transfected with miRNA-140-5p are mixed.

According to a preferred embodiment of the present invention, in order to obtain cells containing miRNA-140-5p, miRNA-140-5p is transfected into bone marrow-derived stem cells for 1-2 days, usually one day, and then, for example, Trypsine is used. After removing the cells, the mixture may be mixed with a cell suspension containing 5-30, more preferably 5-15% of the weight of the gellan gum aqueous solution.

According to a preferred embodiment of the present invention, one or more of collagen or growth factors TGF-β1, BMPs, Dexamethasone, and the like may be added as an additive to the gellan gum hydrogel composition containing miRNA-140-5p.

According to a preferred embodiment of the present invention, the hydrogel composition of the present invention may be used as an injection when applied to a damaged area of the knee cartilage, and may be a formulation that can be directly injected around the treatment area.

According to a preferred embodiment of the present invention, an example of such a dosage form may be prepared as an injectable arthritis treatment kit filled with a composition for treating cartilage damage comprising a gellan gum hydrogel composition containing miRNA-140-5p as an active ingredient. have.

Therefore, the composition for treating cartilage damage in the form of a hydrogel of the present invention has a remarkably convenient effect in its use compared to the conventional one.

Therefore, the present invention provides a composition for treating cartilage damage comprising the gellan gum hydrogel composition containing miRNA-140-5p as described above as an active ingredient.

According to a preferred embodiment of the present invention, when the composition for treating cartilage damage is injected into the body, it is very suitable as an injection for cartilage treatment due to the high elasticity and durability peculiar to gellan gum as well as cartilage differentiation effect. This result seems to be due to the synergistic effect that appears due to the selective binding of a specific cell, miRNA-140-5p-containing cell, and a specific polysaccharide, gellan gum according to the present invention.

According to a preferred embodiment of the present invention, in the case of using the composition for treating cartilage damage of the present invention, in order to enhance the cartilage regeneration effect in the past, autologous knee cartilage is collected and treated by a method resulting from autologous chondrocyte transplantation. It is possible to minimize the various disadvantages that appear.

In addition, according to the present invention, the composition for treating cartilage damage as described above may be provided as a therapeutic agent for injectable arthritis by having a mechanical strength suitable for injection. That is, when the hydrogel composition according to the present invention is injected into a cartilage injury site, for example, a gellan gum hydrogel composition containing bone marrow-derived stem cells containing miRNA-140-5p can be applied as an injection.

Therefore, the present invention includes a therapeutic agent for arthritis for injection comprising the composition for treating cartilage damage as described above.

According to a preferred embodiment of the present invention, in order to prepare a gellan gum hydrogel composition in which cells containing miRNA-140-5p are formulated, it may be prepared by the following method.

First, prepare lipofectamin and miRNA-140-5p mimics to transfect stem cells with bone marrow-derived stem cells and miRNA-140-5p cultured in an appropriate cell culture solution.

According to a preferred embodiment of the present invention, in the step of transfecting bone marrow-derived stem cells with miRNA-140-5p, miRNA-140-5p is used in a state that is maintained at a low temperature, and the bone marrow-derived stem cells are used in a cell culture flask. When -80% is filled, miRNA-140-5p is transfected.

According to a preferred embodiment of the present invention, a gellan gum aqueous solution is prepared and heated. Preferably, gellan gum powder is added to distilled water heated to a temperature of 90-100° C. and heated for 20 to 30 minutes under stirring to prepare an aqueous gellan gum solution.

According to the present invention, the heated gellan gum aqueous solution may be cooled to 35-45° C., and then bone marrow-derived stem cells transfected with miRNA-140-5p may be added and mixed.

Since miRNA-140-5p is slowly degraded after 1 to 3 days after transfection into cells with a small molecule substance, the decomposition time of miRNA-140-5p in gellan gum hydrogel is delayed, and long-term cartilage differentiation effect can be expected. have.

According to a preferred embodiment of the present invention, when the composition for treating cartilage damage of the present invention is injected in the form of injection, the dosage can be adjusted within the range of 0.1 to 1 ml depending on the cartilage damage site. It is preferable to administer within the range of 0.1 to 1 ml for knee arthritis patients, preferably based on an adult with a weight of 60 Kg.

According to a preferred embodiment of the present invention, the composition for administering cartilage damage of the present invention may be administered repeatedly on a weekly or monthly basis depending on the degree of cartilage damage and regeneration.

When the composition for treating cartilage damage of the present invention is injected into the body, it is suitable as an injection for cartilage treatment due to the effect of helping differentiation into cartilage cells and the high elasticity and durability of gellan gum.

In addition, according to the present invention, there is no specific toxicity or side effects when applying the composition for treating cartilage damage.

In particular, in order to enhance the cartilage regeneration effect, autologous knee cartilage may be collected, thereby minimizing the disadvantages of existing autologous chondrocyte transplantation.

In addition, according to the present invention, the gellan gum composition containing cells containing miRNA-140-5p is naturally biodegraded and absorbed by the body after cell growth is completed, so it has the advantage of being biocompatible over other materials.

Hereinafter, the present invention will be described in detail by examples, and the following examples are intended to illustrate the present invention, and the contents of the present invention are not limited by the following examples.

Preparation Example 1: Collection and culture of knee cartilage cells

Chondrocytes were isolated and collected from the knee cartilage of a 6-week-old female New Zealand white rabbit (Hanil laboratory animal center, Wanju, Korea). The collected knee cartilage is immediately washed with phosphate buffer solution (PBS, pH 7.4, Gibco, NY, USA) at least 3 times, then cut into pieces of 1 mm2 or less, put in PBS, and centrifuged for 3 minutes at 12000 rpm. . The chopped cartilage was dissolved in a tissue in an incubator (37° C. 5% CO ₂ ) for 24 hours with a 0.25 wt% collagenase A (Roche, Applied Science, Germany) solution.

Dissolved tissue was re-centrifuged, and Dulbecco's modified eagle medium (DMEM, Gibco) and F-12 nutrient medium mixture (Ham's F-12, Gibco), 10% fetal calf serum (FBS, Gibco) and 1% antibiotics (100) units/mL penicillin and 100 μg/mL straptomycin), 200 mM L-glutamine (Gibco), 50 μg/mL ascorbic acid (Sigma), and 15 mM HEPES buffer 1 M (Gibco). It was dispensed into a cell culture flask and cultured under conditions of 37° C. 5% CO ₂ .

Preparation Example 2: Isolation and culture of bone marrow-derived stem cells

Bone marrow-derived stem cells were extracted from the leg bones of a 6-week-old female New Zealand white rabbit (Hanil laboratory animal center, Wanju, Korea). The extracted bone marrow-derived stem cells were immediately washed three or more times with a phosphate buffer solution (PBS, pH 7.4, Gibco, NY, USA). Then, it was filtered with a 90 µm nylon mesh and centrifuged to obtain alpha-minimum essential culture medium (α-MEM) (Lonza, Walkersville, MD, USA), 20% fetal bovine serum (FBS, Gibco) and 1% antibiotics (100 units). The culture solution mixed with /mL penicillin was dispensed into a cell culture flask, and cultured under the condition of 5% CO ₂ at 37° C. After 5 days, the suspensions floating in the cell culture flask were washed and washed at 37° C. and 5% CO. Cultured under ₂ conditions.

Example 1: Preparation of gellan gum hydrogel containing miRNA-140-5p

When the bone marrow-derived stem culture flask is filled with 40-70% of cells, mix the cells with Lipofectamin and miRNA-140-5p at a weight ratio of 1:1, and then sprinkle about 10% of the medium contained in the cell culture flask dish at 37°C for 24 hours. Incubate under 5% CO ₂ conditions. After 24 hours, the cells were removed using Trypsine, and the cells were removed from the chondrocyte culture flask as a control using Trypsine.

Add 1wt% gellan gum (0.8g) to distilled water (80ml) heated to 90°C, stir, and heat for 20 minutes. When the gellan gum powder is completely dissolved, add 0.03% calcium chloride (0.03g) and dissolve for 10 minutes. After lowering the gellan gum aqueous solution to 37-40°C, add 300 μl of medium to the separated cells, and then add the gellan gum aqueous solution, bone marrow mesenchymal cells, cells transfected with miRNA-140-5p, and cartilage cells, respectively. After mixing, pour into a petri dish and harden at room temperature. The hardened gel is cut into 6mm in diameter and 3mm in thickness using a bioxy sponge.

1 is a photograph of a support body of a gellan gum hydrogel prepared according to the manufacturing method of Example 1 of the present invention.

1 shows a photo of a scaffold for a gellan gum hydrogel composition containing cells containing miRNA-140-5p prepared as described above, and a gellan gum hydrogel composition not containing cells containing miRNA-140-5p. The supports are compared and shown.

Experimental Example 1: Confirmation of the morphology of the support

SEM observation was performed to confirm the porosity of the prepared 1 wt% gellan gum hydrogel and the surface roughness of the support. First, the hydrogel was lyophilized and the morphology was checked in a sponge state. As a result, the pore size of the support was 125-201 μm. SEM observation pictures for each support are shown in FIG. 2.

In FIG. 2, after lyophilizing the gellan gum hydrogel composition prepared according to the above, the roughness and porosity of the surface of the support can be confirmed.

Experimental Example 2: Confirmation of cell injection efficiency of miRNA-140-5p

To confirm the efficiency of miRNA-140-5p transfection of rabbit bone marrow-derived stem cells, using Fam siRNA negative control, as in the method of manufacturing a gellan gum hydrogel containing cells containing miRNA-140-5p. After transfecting the derived stem cells with siRNA, the efficiency was confirmed by confocal microscopy. The green part was confirmed that the siRNA injected into the cell appeared in fluorescence, and the blue part was the nucleus of DAPI-stained rabbit bone marrow-derived stem cells.

3 is an experimental result confirming the efficiency of miRNA-140-5p injected into the cells after injecting miRNA-140-5p into rabbit bone marrow-derived stem cells in an aqueous gellan gum solution prepared according to the present invention.

Here, 3 days after seeding of bone marrow stem cells containing miRNA-140-5p in the prepared gellan gum aqueous solution, 3 days later, the efficiency of miRAN transfected into cells was confirmed by Fam siRNA negative control.

Experimental Example 3: Confirmation of Cell Morphology

After lyophilizing the prepared hydrogel support, the surface porosity of the support was observed using SEM. Rabbit bone marrow bone marrow-derived stem cells and chondrocytes were collected, cultured on a scaffold for 3, 7, 14 days, fixed with 2.5% glutaraldehyde, and the morphology of chondrocytes was confirmed. In the bone marrow-derived stem cell group, the bone marrow-derived stem cell group containing miRNA-140-5p, and the chondrocyte group, there was no significant difference in cell proliferation, and it was confirmed that all three groups of extracellular matrix were formed.

Figure 4 is a photograph confirmed through SEM 3, 7, and 14 days after mixing the aqueous gellan gum solution prepared according to the present invention and rabbit chondrocytes, respectively.

In Figure 4, bone marrow-derived stem cells, bone marrow-derived stem cells containing miRNA-140-5p, and chondrocytes, respectively, in a gellan gum hydrogel composition are freeze-dried for each day (1, 3, 5, 7 days) after seeding. Cell morphology can be confirmed.

Experimental Example 4: Evaluation of cell proliferation rate

MTT (dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay to determine the proliferation rate of bone marrow-derived stem cells, bone marrow-derived stem cells containing miRNA-140-5p, and chondrocytes on a hydrogel scaffold As a result of conducting, it was confirmed that there was no significant difference in the proliferation rate as time passed.

5 is a graph confirming the proliferation rate of bone marrow-derived stem cells, bone marrow-derived stem cells containing miRNA-140-5p, and chondrocytes seeded on the gellan gum hydrogel scaffold prepared according to the present invention.

5, bone marrow-derived stem cells, bone marrow-derived stem cells and chondrocytes containing miRNA-140-5p, respectively, are seeded in the gellan gum hydrogel composition prepared above, and then the cells are seeded by day (1, 3, 5, 7 days). Proliferation can be confirmed.

Experimental Example 5: Analysis of gene expression in cell culture flask

Bone marrow-derived stem cells, bone marrow-derived stem cells containing miRNA-140-5p, and chondrocytes were cultured for 12 hours and 24 hours for gene phenotypes. According to the results of expression of ECM-related genes such as aggrecan and type II collagen, it was confirmed that the expression was highest in bone marrow-derived stem cells containing miRNA-140-5p.

6 is a graph analyzed after confirming the cartilage-related marker gene expression level of rabbit bone marrow-derived stem cells, miRNA-140-5p-containing bone marrow-derived stem cells, and chondrocytes in a cell culture flask according to the present invention by electrophoresis .

In Figure 6, bone marrow-derived stem cells and chondrocytes containing bone marrow-derived stem cells and miRNA-140-5p in a cell culture plate are seeded hourly (12, 24 hours) after seeding, sox9, Aggrecan of bone marrow-derived stem cells and chondrocytes, The result of generalization of the gene expression of Col II to GAPDH and quantification is shown.

Experimental Example 6: Analysis of gene expression in gellan gum hydrogel

When comparing the gene expression levels of chondrocytes in a hydrogel containing gellan gum and miRNA-140-5p, a gellan gum hydrogel transfected with col II, Sox9, Aggrecan, and miRNA-140-5p phenotype related to cartilage genes. It came out highest in Accordingly, it was confirmed that miRNA-140-5p plays a positive role in chondrocyte differentiation.

As such confirmation data, Fig. 7a is a photograph of a band confirmed through electrophoresis for the phenotypes of Col II, Sox9, and Aggrecan.

7B is a graph of an electrophoresis band photograph.

In Figure 7a, after uniformly mixing bone marrow-derived stem cells and bone marrow-derived stem cells and cartilage cells containing the miRNA-140-5p with the gellan gum aqueous solution in the above prepared gellan gum hydrogel composition, each day (1, 3 days) After electrophoresis of gene expression in bone marrow-derived stem cells and chondrocytes, a band photo is shown.

In addition, FIG. 7B is a graph obtained by generalizing the electrophoresis result to GAPDH and quantifying it, and shows the result of the phenotype of SOX9, Aggrecan, and Col II.

Claims (5)

It is made in the form of a hydrogel containing miRNA-140-5p in gellan gum.
miRNA-140-5p is a bone marrow-derived stem cell transfected with miRNA-140-5p. After transfecting the bone marrow-derived stem cells with miRNA-140-5p, remove the cells using Trypsine, and then add 7 ml of cell culture medium. In the bone marrow-derived stem cell suspension transfected with miRNA-140-5p at a concentration of 10 to 50 pmol, 200-300 μl of medium is diluted and mixed,
Gellan gum hydrogel is a hydrogel obtained by mixing distilled water with pure gellan gum powder and crosslinking with CaCl ₂ and has a concentration of 1 to 3% by weight,
Bone marrow-derived stem cells transfected with the miRNA-140-5p are mixed with the gellan gum hydrogel,
A gellan gum hydrogel composition containing miRNA-140-5p, characterized in that, made in the form of an injection for treating cartilage damage.
delete Preparing bone marrow-derived stem cells;
Mixing the bone marrow-derived stem cells with Lipofectamin and miRNA-140-5p at a weight ratio of 1:1, spraying them on a medium contained in a cell culture flask dish, and culturing under conditions of 37° C. and 5% CO ₂ for 24 hours;
To prepare bone marrow-derived stem cells transfected with miRNA-140-5p in a state containing miRNA-140-5p at a concentration of 10-50 pmol for 7 ml of cell culture medium by removing cells from the cultured flask dish using Trypsine. step;
Preparing a cell suspension in which the amount of bone marrow-derived stem cells transfected with the miRNA-140-5p is 1×10 ⁶ cells/ml;
Preparing a bone marrow-derived stem cell suspension transfected with miRNA-140-5p by diluting 200-300 μl of medium in the cell suspension;
Mixing distilled water with pure gellan gum powder and crosslinking with CaCl ₂ to prepare an aqueous gellan gum solution;
The bone marrow-derived stem cells transfected with miRNA-140-5p are mixed with the gellan gum aqueous solution, but the gellan gum concentration is 1 to 3% by weight, and the bone marrow-derived stem cells transfected with miRNA-140-5p are mixed. Obtaining a gellan gum hydrogel;
Method of producing a gellan gum hydrogel composition containing miRNA-140-5p made in the form of an injection for cartilage damage treatment comprising a.
Injectable arthritis treatment comprising a gellan gum hydrogel composition containing miRNA-140-5p in the form of an injection for treating cartilage damage according to claim 1.
Injectable arthritis treatment kit containing a gellan gum hydrogel composition containing miRNA-140-5p in the form of an injection for treating cartilage damage according to claim 1 as an active ingredient.

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