KR20190012593A - Gellan-gum hydrogels composition containing hesperidin - Google Patents

Abstract

The present invention is characterized by being used as a composition for treating cartilage damage by using an injectable hydrogel composition having a gellan-gum hydrogel, which is a biologically suitable natural material, as a base, and hesperidin is mixed therein. When the composition for treating cartilage damage of the present invention is injected into a body, it is possible to have an effect of hesperidin which alleviates inflammation by collagen production and anti-inflammatory action, and thus have high elasticity and durability unique to gellan gum, thereby being suitable as an injection for cartilage treatment. In addition, the composition for treating cartilage damage has no cytotoxicity, has an advantage of not being dissolved by inflammatory reaction or toxic substances, and thus, can minimize disadvantages which occur from an existing autologous chondrocyte implantation by collecting an autologous knee cartilage in order to enhance a cartilage regeneration effect.

Description

Gellan-gum hydrogels composition containing hesperidin < RTI ID = 0.0 >

The present invention relates to a gelatin gum hydrogel composition containing hesperidin, and more particularly, to a gelatin gum hydrogel composition comprising hesperidin in the form of hydrogels using gellan gum supplemented with biocompatible and injectable hesperidin for the repair of damaged cartilage and osteoarthritis And a method of injecting the same into a composition for cartilage regeneration having cartilage regeneration and bone inflammation relieving effects.

Osteoarthritis, a degenerative joint disease, is characterized by cartilage degeneration and bone hyperplasia. Osteoarthritis is the most common degenerative fracture and elderly disorder seen in clinical practice. The cartilage is a tissue composed of cartilage cells and cartilage tissue. It absorbs shock to protect the joints. The cartilage of the cartilage of the cartilage of the suprasellar cartilage covers the joint surface. Smooth movement.

The cartilage that protects these joints is worn out due to aging, obesity, excessive use of joints, or rupture due to degenerative changes, which causes osteoarthritis. Osteoarthritis can manifest symptoms such as joint inflammation, pain, swelling, edema, and deformity. These symptoms of osteoarthritis reduce the quality of life in patients. However, due to the low self-healing ability of articular cartilage, osteoarthritis is now a challenging disease area because there is no correct treatment. [Bone Res (Wei Zhang, Hongwei Ouyang, Crispin R Dass, and Jiake Xu) 2016; 4; 15040.]

Ultimately, the treatment of osteoarthritis is aimed at reducing patient pain and improving joint function. However, existing non-steroidal anti-inflammatory agents, analgesic antipyretics and synthetic analgesic anesthetics, are effective at alleviating pain and edema, but they can not prevent cartilage damage, which is a fundamental problem, and are difficult to take long-term, with most serious side effects.

Thus, as a result of improving the efficiency of tissue regeneration and treatment by improving cartilage regeneration and repairing damaged cartilage repair, there has been a need for a tissue engineering method for cartilage regeneration.

Gellan Gum is a biodegradable material that is suitable for bioinjection and has excellent properties of heat resistance, acid resistance and resistance to enzymes as well as hydrophilic colloid (hydrocolloid) to form a gel when injected into the body to keep the injured area moist, The application in biomedical engineering is wide to help natural decomposition of tissue. It is an ester with an O-glycosidic bond and contains an acyl group. Depending on the content of the acyl group, a high acyl gellan gum, a low acyl gellan gum, It is divided into gum (low acyl gellan gum). In particular, glucan, which is a component of gellan gum, is known to have an environment similar to that of the actual cartilage glycosaminoglycan, making it suitable for studies of cartilage restoration and regeneration (Balakrishnan B1, Joshi N1, Jayakrishnan A2, Banerjee R3, Self-crosslinked oxidized alginate / gelatin hydrogel as injectable, adhesive biomimetic scaffolds for cartilage regeneration, Acta Biomater, 2014 Aug; 10 (8): 3650-63.)

Hesperidin is a kind of vitamin P, which is known to help collagen production, to be effective in anti-inflammatory and aging-related diseases, and to affect joints and cartilage (Japanese Patent No. 4838772, 2005-255527).

Despite the necessity of injection-type hydrogels using the above-mentioned tissue engineering biomaterials, research has not been conducted.

Japanese Patent No. 4838772 Japanese Patent Application Laid-Open No. 2005-255527

1. Balakrishnan B1, Joshi N1, Jayakrishnan A2, Banerjee R3, Self-crosslinked oxidized alginate / gelatin hydrogel as injectable, adhesive biomimetic scaffolds for cartilage regeneration, Acta Biomater, 2014 Aug; 10 (8): 3650-63.

In order to solve the problems of the prior art as described above, development of a method for efficiently improving cartilage damage and treating osteoarthritis using hesperin and gellan gum is a problem to be solved.

The present inventors therefore sought to develop a method for cartilage regeneration and osteoarthritis treatment through a non-surgical, tissue engineering method capable of minimizing the surgical site. As a result, the inventors of the present invention completed the present invention by developing gellan gum hydrogel-injected arthritis treating agent containing hesperidin for treating cartilage and confirming cartilage repair.

Accordingly, an object of the present invention is to provide a hydrogel composition for cartilage regeneration and osteoarthritis treatment through a non-surgical tissue engineering method capable of minimizing a surgical site.

Another object of the present invention is to provide a composition for treatment of cartilage regeneration and injury, which is capable of treating arthritis and restoring cartilage by using gellan gum hydrogel containing injected hesperidin for cartilage treatment.

Another object of the present invention is to provide an injectable composition for treating cartilage containing hesperidin and gellan gum which has anti-inflammatory, antioxidant and cartilage regeneration effects for treating cartilage defects and arthritis.

In order to solve the above-mentioned problems, the present invention provides a gellan gum hydrogel composition containing hesperidin, characterized in that hesperidin is mixed with gellan gum as an effective ingredient for alleviating cartilage regeneration and bone inflammation, and is formed into a hydrogel form do.

The present invention also provides a composition for treating cartilage damage comprising a gellan gum hydrogel composition containing hesperidin as a cartilage regeneration effective ingredient.

In addition, the present invention provides a therapeutic agent for an injectable arthritis comprising the composition for treating cartilage damage containing hesperidin and gellan gum.

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

(i) The present invention provides a composition for treating cartilage regeneration and arthritis, which comprises hesperidin and gellan-gum as cartilage restoration and osteoarthritis treatment agents using biomaterials, and is very useful for treatment of bioengineered arthritis Do.

(ii) The composition for regenerating cartilage according to the present invention can greatly improve the therapeutic effect of cartilage damage through use of gellan gum hydrogel-type composition for regenerating cartilage damaged with hesperidin, It has an effect that can be directly applied to the site.

(iii) According to the present invention, since the composition for regeneration of cartilage is made into a tissue engineering injection type hydrogel, the side effects and the operation time can be greatly shortened due to the non-surgical treatment.

FIG. 1 is a photograph showing the preparation of a composition for cartilage regeneration containing hesperidin and gellan gum of the present invention in which gelatin gum hydrogel was added with the amounts of hesperidin (0 mg, 1 mg, 2 mg, and 4 mg) to be.
FIG. 2 is a graph showing the proliferation rate of rabbit chondrocytes sown on the hesperidin / gellan gum hydrogel support prepared according to the present invention.
FIG. 3 is a photograph of the surface of the support and the rabbit chondrocytes sown on the leaf after freeze-drying the hesperidin / gellan gum hydrogel support prepared according to the present invention through BIo-LV SEM 2 weeks after sowing.
4 is a graph showing compressive strength of a hesperidin / gellan gum hydrogel support prepared according to the present invention.
5a. 5b and 5c show the expression of the cartilage-related marker gene in the rabbit chondrocytes seeded on the gellan gum hydrogel support by the content of hesperidin in different amounts of hesperidin in the hesperidin / gellan gum hydrogel support according to the present invention, Beta-actin as a control.

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

According to one aspect of the present invention, there is provided a gellan gum hydrogel composition comprising a biocompatible polymer, hesperidin, for the treatment of cartilage damage such as arthritis.

According to a preferred embodiment of the present invention, unlike the conventional cartilage treatment method, a new composition for treatment of a method for minimizing additional surgery and side effects is provided, which uses a tissue-engineered biocompatible material effective for proliferation and function maintenance of cartilage cells , A novel composition of injectable gellan gum hydrogel which can effectively regenerate cartilage damaged parts was prepared.

According to a preferred embodiment of the present invention, it is known that hesperidin used in the present invention is suitable for bioavailability, assists in collagen production, has an anti-inflammatory action, is effective for diseases related to aging, and positively affects articular cartilage have.

The gellan gum hydrogel used in the present invention is a natural material suitable for a living body, and has a high elastic modulus and a rigidity that can be controlled during production. The gellan gum hydrogel has a secondary coiled helix structure that gels when the temperature falls below a certain temperature. In particular, since gellan black has low toxicity and is easy to handle, when hydrogels are mixed with hesperidin, they can be used as an injectable remedy without surgical operation.

According to a preferred embodiment of the present invention, the concentration of the gellan gum hydrogel is preferably 0.5 to 1% by weight, more preferably 0.7 to 1% by weight. That is, the gellan gum hydrogel used in the present invention is composed of pure gellan gum and a hydrogel in which hesperidin is blended and blended. When a bi-os punch is used after cross-linking after adding hesperidin, for example, it is composed of a cylindrical hydrogel composition .

According to a preferred embodiment of the present invention, the concentration of hesperidin is preferably 5 to 20% by weight based on gellan gum. If the content is too low, there is no addition effect, and if it is too high, hypertrophy and cell death due to cell growth may occur, which may cause undesirable problems.

According to a preferred embodiment of the present invention, the hydrogel composition of the present invention may be a ready-to-inject dosage form, for example, when injected into a knee cartilage damage site. Therefore, the composition for treating cartilage damage in the hydrogel form of the present invention has remarkably convenient effect in use compared to the conventional one.

Therefore, the present invention provides a composition for treating cartilage damage comprising the above-described gelatin gum hydrogel composition containing hesperidin as an active ingredient.

The composition for treating cartilage damage according to the present invention is injected into the body to be effective as an injection for cartilage treatment because of its high elasticity and durability unique to gellan gum as well as the effect of hesperidin which helps collagen production and anti-inflammatory action to relieve inflammation.

In addition, the composition according to the present invention has no cytotoxicity and has an advantage of being insoluble due to inflammatory reaction or toxic substances.

According to the present invention, when the composition for treating cartilage damage according to the present invention is used, it is possible to improve the cartilage regeneration effect, for example, by collecting the autologous knee cartilage and treating various methods The disadvantages can be minimized.

In addition, according to the present invention, at least one selected from hyaluronic acid and TGF-? 1 may be added to the composition for treating cartilage damage as described above in a conventional manner.

In addition, the composition for treating cartilage damage as described above can be provided as an injectable therapeutic agent for injectable arthritis by having an appropriate mechanical strength for injection. In other words, the hydrogel composition according to the present invention can be applied as an injectable drug with a concentration of 1 mg of hesperidin / 1% gellan gum when injected into a cartilage damaged area. In this case, the cartilage regenerating effect is generally observed after 14 to 21 days Since the therapeutic effect on the cartilage damage site is made, the cartilage can be easily damaged and damaged.

The present invention includes a therapeutic agent for injectable arthritis comprising the composition for treating cartilage damage as described above.

According to a preferred embodiment of the present invention, the composition for treating cartilage damage according to the present invention can control its dose within a range of 3-5 ml according to a cartilage damage site when injected in an injection form. Preferably, it is administered within the range of 3-5 ml once per osteoarthritis patient on an adult basis, preferably 60 kg body weight.

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

When the composition for treating cartilage damage of the present invention is injected into the body, it can be effectively used as a therapeutic agent for cartilage damage, because it protects the joint tissue, inhibits antioxidative effect and bacterial infection, and is used as an injection type because of high elasticity and durability.

In particular, in order to improve the cartilage regeneration effect, the self-knee cartilage can be collected to minimize the disadvantages of existing autologous chondrocyte implantation.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples serve to illustrate the present invention and are not intended to limit the scope of the present invention.

Comparative Example and Example 1-3: Preparation of a Hesperidin / Gellan gum Hydrogel Composition

The dissolution of the lipid-soluble material hesperidin is carried out using EtOH. Gellan gum (0.7 g) (Sigma) was dissolved in distilled water (100 ml) of tertiary water at 90 ° C, and the aqueous 0.7% gellan gum solution was stirred and heated for 20 minutes. When the gellan gum powder is completely dissolved, add 0.03% calcium chloride (0.03 g) and dissolve for 10 minutes.

. (Comparative Example), 1 mg (Example 1), 2 mg (Example 2) and 4 mg (Example 3) of aqueous solution of hesperidin are added to the gellan gum aqueous solution whose temperature is lowered to 60 캜. The gellan gum aqueous solution and the hesperidin aqueous solution were mixed, poured into Petri dishes, and hardened at room temperature to obtain a hydrogel composition.

The hydrogel thus obtained is in the form of a hard gel, and is cut into a diameter of 6 mm and a height of 5 mm using a bioxy punch, and a photograph of the product is shown in FIG.

FIG. 1 is a photograph showing the preparation of a composition for cartilage regeneration containing hesperidin and gellan gum of the present invention in which gelatin gum hydrogel was added with the amounts of hesperidin (0 mg, 1 mg, 2 mg, and 4 mg) to be.

Experimental Example:

(1) Collection and culture of knee cartilage cells

To obtain chondrocytes, 2-week-old female New Zealand white rabbits (Hanil laboratory animal center, Wanju, Korea) were used. The cartilage was cut between the knee joints of rabbits and then washed several times with PBS (pH 7.4, Gibco, NY, USA), and the chondrocytes were scraped off. The scraped chondrocytes were placed in a conical tube and centrifuged twice at 1200 rpm, 4 ° C for 3 minutes.

Dulbecco's modified eagle medium (DMEM / F-12, Gibco), 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic (100 units / mL penicillin and 100 μg / mL streptomycin) were added to the centrifuged chondrocytes 10 mL of the mixed culture and 200 μL of 0.2 wt% collagenase type A (Roche, Indianapolis, USA) were added and the collagen was degraded in an incubator at 36 ° C and 5% CO ₂ for 1 day. After 1 day, the cells were centrifuged at 1200 rpm at 4 ° C for 3 minutes, pipetted, and the cells were dispensed into a cell culture dish together with the culture medium, and the culture medium was changed every 3 days. Cells used for seeding were Passage 2 cells at 1 × 10 ⁵ cells / scaffold per support.

(2) Analysis of physical properties and efficacy

(2-1) Evaluation of cell proliferation rate

As a result of MTT (dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay, the cell proliferation rate was increased in all groups with increasing time Respectively. In addition, it showed the best cell growth rate at 1 mg H / G at all time points.

The results are shown in Fig. Here, the proliferation rate of rabbit chondrocytes sown on the hesperidin / gellan gum hydrogel support can be confirmed.

(2-2) Confirmation of cell morphology

The prepared hydrogel supports were lyophilized and the surface porosity of the support was observed by SEM. After rabbit knee cartilage cells were harvested, they were cultured on a scaffold for 2 weeks, fixed with 2.5% glutaraldehyde, and the morphology of chondrocytes was confirmed.

FIG. 3 is a photograph of the rabbit chondrocytes sown on the prepared hesperidin / gellan gum hydrogel support through BIo-LV SEM 2 weeks after sowing. In this case, the cell proliferation was the most prominent in gellan gum hydrogel containing 1 mg hesperidin, and extracellular matrix was also formed.

(2-3) Analysis of compressive strength of the support

A laboratory press (TMS-Pro, Food Technology Corporation, Sterling, USA) was used to measure the compressive strength of the hesperidin / gellan gum backing. At this time, the measurement speed was set to 1 mm / sec, and the measurement force was set to 0.5 N to measure the compressive strength. The load at the fracture of the support crack was divided by the cross-sectional area of the support.

4 is a graph showing the compressive strength of a hesperidin / gellan gum hydrogel support. As a result, the compressive strength of gellan gum hydrogel containing 0 mg hesperidin was more than 5 N and the gel strength of gelatin gel hydrogel containing 1, 2 and 4 mg hesperidin was decreased with increasing hesperidine content.

(2-4) Analysis of gene expression

 When the gene phenotypes of the hydrogels containing gellan gum hydrogel and hesperidin were compared, it was confirmed that the expression was better in gellan gum hydrogel containing hesperidin.

5a, 5b and 5c show that the expression of the cartilage-related marker gene of rabbit chondrocytes seeded on a gellan gum hydrogel support by the content of hesperidin in the hesperidin / gellan gum hydrogel support, (Housekeeping gene) RT-PCR using Beta-actin.

Here, ECM-related gene expression results, such as aggrecan and type I and type II collagen, suggest that hesperidin-incorporated gellan gum hydrogels may provide a more suitable microenvironment that enhances the synthesis of the desired ECM .

(2-5) Experimental results

From the above experimental results, the hesperidin / gellan gum hydrogel according to the present invention is simpler than the conventional preparation method, can maintain the gel shape, and is easy to store. In addition, after cell growth is completed, it is naturally biodegraded and absorbed into the body.

In addition, in the case of the example, it was confirmed that it is remarkably effective in the treatment of cartilage damage due to its excellent various properties and growth effect of cells compared to the comparative example in which hesperidin was not used.

Claims (8)

Wherein the hesperidin is combined with gellan gum in the form of a hydrogel as an effective ingredient for alleviating cartilage regeneration and bone inflammation.
The gellan gum hydrogel composition according to claim 1, wherein the concentration of the gellan gum hydrogel is 0.5 to 1 wt%.
The hesperidin-containing gellan gum hydrogel composition according to claim 1, wherein the concentration of hesperidin is 5 to 20% by weight based on the gellan gum.
A composition for treating cartilage damage comprising a gellan gum hydrogel composition containing hesperidin according to any one of claims 1 to 3.
The composition for treating cartilage damage according to claim 4, which is used in the form of an injection.
A therapeutic agent for injectable arthritis comprising a composition for treating cartilage damage according to claim 4.
[Claim 7] The therapeutic agent for arthritis according to claim 6, wherein the dosage can be adjusted in the range of 3-5 ml depending on the cartilage damage site.
[Claim 7] The therapeutic agent for arthritis for injectable use according to claim 7, which is administered repeatedly on a weekly or monthly basis depending on the degree of cartilage damage and degree of regeneration.

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