KR102048914B1 - Gellan-gum Hydrogels Composition containing Chondroitin Sulfate - Google Patents

Abstract

The present invention is characterized in that it is used as a composition for treating cartilage damage using an infusion hydrogel composition based on gellan gum hydrogel, which is a biocompatible natural material, and which contains chondroitin sulfate. 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 high elasticity and durability peculiar to gellan gum as well as the effect of chondroitin sulfate that protects joint tissues, prevents antioxidant effects and inhibits bacterial infection. . In addition, there is no cytotoxicity, there is an advantage that does not melt by inflammatory reactions or toxic substances. In addition, in order to enhance the cartilage regeneration effect, by collecting the autologous knee cartilage, it is possible to minimize the disadvantages occurring in the existing autologous chondrocyte transplantation.

Description

Gellan gum hydrogels composition containing chondroitin sulfate {Gellan-gum Hydrogels Composition containing Chondroitin Sulfate}

The present invention relates to a gellan gum hydrogel composition containing chondroitin sulfate, and more particularly, to a gellan gum hydrogel composition containing chondroitin sulfate that helps the proliferation of chondrocytes in vivo for cartilage treatment and cartilage using the same. It relates to a composition for treating damage.

Once the cartilage is damaged or degraded, it is difficult to regenerate, and if it is not regenerated early, the damaged cartilage is easily broken and worn down by mechanical pressure, causing inflammation and gradually increasing the defect site. Therefore, it is very important to develop a treatment method for early regeneration of damaged cartilage.

If cartilage is damaged at present, there are various methods of treatment depending on the degree of damage. Representative treatment methods include autologous chondrocyte transplantation, bone marrow stimulation using stem cells of bone marrow, cartilage regeneration treatment using adult stem cells and embryonic stem cells, and draining bone marrow cells through fine holes in damaged areas to treat damaged areas. Fine fracture surgery is used. [Z. Lin, C. Willers, J. Xu, and M. H Zheng, Tissue Eng., 12, 1971 (2006)].

However, the above-mentioned cartilage damage treatments have a disadvantage in that a difficult surgical method or various problems occurring during the treatment process occur. Therefore, histological methods are proposed to improve the various disadvantages and to provide effective treatment.

Biomaterials used in tissue engineering include cartilage regeneration materials such as 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 is excellent in odorless, colorless, heat resistance, and enzyme resistance. . In addition, it is possible to control the mechanical properties of the hydrogel through the addition of ions and pH adjustment, and there is no cytotoxicity has been studied in various biomaterials (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)).

In addition, chondroitin sulfate (CS) is a viscous polysaccharide (mucopolysaccharide, mucopolysaccharide) combined with D-glucuronic acid, N-acetyl-D-galactosamine and sulfuric acid. CS is distributed in substrates, organs, and body fluids of animal connective tissues, and is known to have various effects such as antioxidant effects, blood coagulation, alliance hardening inhibition and prevention, joint tissue protection, and bacterial infection inhibition.

Korean Patent No. 10-1220158 relates to an effective system for molecular imaging of a target cell, using a method of chemically connecting a target cell and a contrast agent using a biocompatible mediator polymer material having different functional groups at both ends. In the technique of molecular imaging method and the technique for the molecular imaging complex of the chemical bonding structure, it is mentioned that the possibility of using various polymer components such as chondroitin and gellan gum among dozens of polymer compounds.

However, the study of injectable hydrogels for the treatment of cartilage regeneration using the above-mentioned biomaterials, although there is a clear need, no effective approach to these uses has yet been made and there is little research.

Korean Patent Registration No. 10-1220158

1.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)

In order to improve the problems in the field of cartilage treatment as described above, the present inventors to solve the development of a hydrogel for cell therapy that can be cartilage regeneration only by local injection of the damaged area without any incision of the surgical site.

In the present invention, as a result of a long study for solving the problem, it was confirmed whether the gellan gum hydrogel containing chondroitin sulfate is helpful for proliferation of chondrocytes, and completed the present invention by confirming the injectable properties.

An object of the present invention is to provide a gellan gum hydrogel composition containing chondroitin sulfate with antioxidant, joint tissue protection, cartilage regeneration effect for the treatment of damaged cartilage as well as arthritis.

Another object of the present invention to provide a composition for treating cartilage damage comprising a gellan gum hydrogel composition containing chondroitin sulfate as an active ingredient.

In order to solve the problems of the present invention as described above, the present invention is chondroitin sulfate containing chondroitin sulfate, characterized in that it is combined with gellan gum as a component to relieve osteoporosis and to help the proliferation of chondrocytes. It provides a gellan gum hydrogel composition.

In another aspect, the present invention provides a composition for treating cartilage damage comprising a gellan gum hydrogel composition containing chondroitin sulfate as an active ingredient.

In addition, the present invention can be provided as a therapeutic agent for injectable arthritis by injecting a composition for treating damage to the above-described damage to the injury by having a mechanical strength suitable for injection.

The advantages and effects of the gellan gum hydrogel composition containing chondroitin sulfate according to the present invention are summarized as follows.

(i) The hydrogel composition of the present invention is due to the high elasticity and durability by using a natural polymer material suitable for living body unique to gellan gum, along with the effect of chondroitin sulfate, which protects joint tissues, prevents antioxidant effects and inhibits bacterial infection. It is suitable as an injection for cartilage treatment. Therefore, it can be used as a therapeutic agent for cartilage regeneration and osteoarthritis expressing a safe and excellent effect.

(ii) The present invention provides a composition prepared by a simple preparation method of gellan-gum hydrogel composition for treating cartilage damage containing chondroitin sulfate, which can be conveniently used for infusion. In addition, there is no cytotoxicity, there is an advantage that does not melt by inflammatory reactions or toxic substances.

(iii) According to the present invention by producing a histological injection-type hydrogel, it has the side effect of surgery, minimizing the surgical site and shortening the operation time.

Figure 1 is chondroitin sulfate prepared for the chondroitin sulfate-containing gellan gum hydrogel composition prepared in accordance with the present invention by varying the amount added to the gellan gum hydrogel by varying the content (0 wt.%, 10 wt. %, 20 wt.%, 30 wt.%) Product picture.
Figure 2 is a graph showing the compressive strength according to the content of CS / gellan gum hydrogel in the hydrogel composition according to the present invention.
FIG. 3 is a graph showing the proliferation amount of chondrocytes by MTT assay on a daily basis (1-21 day) after seeding chondrocytes in CS / gellan gum hydrogel composition by ratio.
Figure 4 is a SEM photograph taken to confirm the roughness and porosity of the surface of the support after lyophilizing the gellan gum and CS / gellan gum hydrogel composition.
5 is a graph quantifying gene expression of chondrocytes by GAPDH on a day-by-day basis (1-21 day) after seeding chondrocytes in CS / gellan gum hydrogel composition by ratio, and FIG. 5A is aggrecan, and FIG. 5B is ColI, FIG. 5C is a graph showing the results for the phenotype of Col II.

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

The present invention relates to the preparation of a hydrogel composition by using a combination of gellan gum with excellent biocompatibility and chondroitin sulfate, which is good for repairing cartilage damage, for arthritis and cartilage regeneration, which is still poorly studied. Provide the invention.

The present invention tried to invent a treatment method of changing the paradigm for conventional cartilage treatment to minimize the additional re-surgery, side effects, the scope of surgery unlike conventional cartilage treatment. As a result, an injectable hydrogel capable of effectively regenerating damaged cartilage sites was prepared using biomaterials that are biocompatible and have excellent cartilage cell proliferation and effective function maintenance.

According to an embodiment of the present invention, chondroitin sulfate (chondroitin sulfate) is a major component of connective tissues, including protein collagen along with cartilage, mainly contained in cartilage 20 to 40% and widely distributed in various connective tissues such as skin and umbilical cord It is known to have a positive effect on arthritis.

In addition, the gellan gum used in the present invention can be adjusted to the mechanical properties according to the crosslinking method or content when prepared by the hydrogel. In addition, gellan gum is suitable in vivo, non-cytotoxic and biodegradable over time. Therefore, when the gellan gum is mixed with chondroitin sulfate and prepared as a hydrogel, it can be used as an injection-injectable arthritis treatment agent without the need for an incision.

According to the present invention, it is characterized by a hydrogel composition comprising chondroitin sulfate and gellan gum that relieve osteoporosis and help the cartilage cell proliferation. In addition, the hydrogel composition according to the present invention can be used as a composition for treating cartilage damage.

According to the present invention, a hydrogel composition is composed of gellan gum hydrogel, which is a biocompatible natural material, and chondroitin sulfate is added thereto.

According to a preferred embodiment of the present invention, the concentration of gellan gum hydrogel is preferably 1 to 3% by weight, more preferably 1-2% by weight. In other words, the gellan gum hydrogel used in the present invention is composed of a hydrogel cross-linked with CaCl ₂ with tertiary distilled water mixed with pure gellan gum, and when chondroitin sulfate is added and mixed uniformly, chondroitin sulfate is contained. Gellan gum hydrogel composition.

According to a preferred embodiment of the present invention, the concentration of chondroitin sulfate is preferably added to chondroitin sulfate in an aqueous gellan gum solution but is 5 to 30% by weight relative to the gellan gum. If the content is too low, there is no addition effect, if too high, the mechanical strength of the hydrogel is reduced, which may cause undesirable problems.

According to a preferred embodiment of the present invention, the hydrogel composition of the present invention may be a formulation that can be directly injected using an injection when applied to a knee cartilage injury, for example. Therefore, the composition for treating cartilage damage of the hydrogel form of the present invention has a remarkably convenient effect compared to the conventional use.

Therefore, the present invention provides a composition for treating cartilage damage comprising a gellan gum hydrogel composition containing chondroitin sulfate as an active ingredient.

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 high elasticity and durability peculiar to gellan gum as well as the effect of chondroitin sulfate that protects joint tissues, antioxidant effects and inhibits bacterial infection.

In addition, the composition according to the present invention is not cytotoxic, there is an advantage that does not dissolve by inflammatory reactions or toxic substances.

According to 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, various methods of collecting the autologous knee cartilage and treating by the method occurring in autologous chondrocyte transplantation This can be minimized.

In addition, according to the present invention, additives such as collagen may be mixed and used in a conventional manner in the composition for treating cartilage damage as described above.

In addition, the composition for treating cartilage damage as described above may be provided as an injectable arthritis therapeutic agent for injection by having a mechanical strength suitable for injection. That is, the hydrogel composition according to the present invention, for example, 20 wt% chondroitin sulfate / gellan gum hydrogel can be applied as an injection when injected into the cartilage damage site, in this case, the cartilage regeneration effect is generally after 14 days to 21 days. The cartilage is damaged and the healing effect is achieved because it is possible to treat damaged cartilage.

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, the composition for treating cartilage damage of the present invention can be adjusted in the range of 0.1 to 1ml depending on the cartilage damage site when injected into the injection form. Preferably it is administered in the range of 0.1 ~ 1ml for patients with knee arthritis on an adult basis of 60Kg body weight.

According to a preferred embodiment of the present invention, the composition for administering cartilage damage of the present invention can be repeatedly administered weekly or monthly, 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 is suitable as an injection for cartilage treatment due to the high elasticity and durability peculiar to gellan gum as well as the effect of chondroitin sulfate that protects joint tissues, antioxidant effects and inhibits bacterial infection.

In addition, there is no cytotoxicity, there is an advantage that does not melt by inflammatory reactions or toxic substances.

In particular, in order to enhance the cartilage regeneration effect, by collecting the autologous knee cartilage, it is possible to minimize the disadvantages occurring in the existing autologous chondrocyte transplantation.

Hereinafter, the present invention will be described in detail by way of examples. The following examples are intended to illustrate the present invention, but are not intended to limit the scope of the present invention.

Comparative Examples and Examples 1-3: Chondroitin Sulfate / Gellan Gum Hydrogel Production Method

The gellan gum powder (2 wt%, 0.8g) was further added to tertiary distilled water (40 ml), heated at a temperature of 90 ° C. or higher for 20 minutes, and stirred until the gellan gum powder was completely dissolved. Chondroitin sulfate (CS) was then 0 (comparative), 10 wt% (Example 1), 20 wt% (Example 2), and 30 wt%, respectively, on the basis of gellan gum in the aqueous solution in which the gellam gum powder was dissolved. (Example 3) After the concentration, the solution is stirred until the reagent is completely dissolved. After that, CaCl ₂ (0.03 wt%) was added as a crosslinking agent and heated and stirred for 20 minutes. Gellan gum in an aqueous solution is dispensed into a petri dish and hardened, thereby preparing a hydrogel gellan gum having a diameter of 8 mm and a height of 4 mm using a biopsy punch.

Chondroitin sulfate (CS) was added to gellan gum hydrogel for each of the chondroitin sulfate-containing gellan gum hydrogel compositions thus prepared (0 wt%, 10 wt%, 20 wt%, 30 wt%). A photograph of the product is shown in FIG. 1.

Experimental Example:

(1) Chondrocyte Separation and Culture

Chondrocytes were isolated from the knee joints of 4 week old female New Zealand white rabbits (Hanil laboratory animal center, Wanju, Korea). The extracted knee cartilage was immediately washed three or more times with phosphate buffer solution (PBS, pH 7.4, Gibco, NY, USA). Tissue was dissolved in an incubator (37 ° C. 5% CO ₂ ) for 24 hours with 0.25 wt% collagenase type A (Roche, Applied Science, Germany) solution.

The lysed tissues were filtered through a 70 μm nylon mesh and centrifuged to obtain Dulbecco's modified eagle medium (DMEM, Gibco) and F-12 nutrient medium (Ham's F-12, Gibco), 10% fetal bovine serum (FBS, Gibco). ) And 1% antibiotics (100 units / mL penicillin and 100 μg / mL straptomycin) with 200 mM L-glutamine (Gibco), 50 μg / mL ascorbic acid (Sigma), 15 mM HEPES buffer 1 M (Gibco) Cell culture flasks were aliquoted using the mixed culture solution and cultured under 37% 5% CO 2 condition.

(2) Physical property and efficacy analysis

(2-1) Compressive strength analysis of support

To measure the compressive strength of hydrogels according to the content of each chondroitin sulfate, a universal property measuring instrument (TMS-Pro, Food Technology Corporation, Sterilng, USA) was used. As a result of setting the force of 1 N, the speed of 20 mm / sec, and the measuring distance of 2 mm, the compressive strength decreased as the content of chondroitin sulfate increased. The results are shown in comparison with FIG.

(2-2) Evaluation of Cell Proliferation Rate

MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) assays were performed to determine cell proliferation capacity in hydrogel scaffolds where chondrocytes were seeded. , 7, 14, and 21 days, the results showed that the cells proliferated over time. The results can be seen in FIG.

(2-3) Confirmation of Morphology of Support

SEM observation was performed to confirm the surface roughness of the pores and the support of the prepared hydrogel. First, the hydrogel supports were lyophilized and the morphology was confirmed in a sponge state. As a result, the pore size of the support containing 0, 10, 20 and 30 wt% of chondroitin sulfate was 84.66 ± 23, 64.53 ± 17, 39.33 ± 20, As the drug content increased to 33.12 ± 22 μm, the pore size gradually decreased and the coarseness also increased. SEM observation pictures for each product are shown in FIG. 4.

(2-4) Gene Expression Analysis

When comparing the gene expression levels of chondrocytes in hydrogels containing gellan gum and chondroitin sulfate, the aggrecan, Col I and Col II phenotypes associated with cartilage genes were highest in gellan gum hydrogels containing 20 wt% chondroitin sulfate. . Accordingly, it was confirmed that chondroitin sulfate plays a positive role in chondrocyte proliferation, but when it exceeds a certain amount, chondrocyte proliferation is more inhibited than when optimal.

5A is aggrecan, FIG. 5B is ColI, and FIG. 5C is a graph showing results of the phenotype of Col II.

(2-5) Experiment Result

From the above experimental results, the chondroitin sulfate / gellan gum hydrogel according to the present invention not only maintains the gel form but is also easy to store, and is naturally biodegraded and absorbed into the body after the growth of cells is completed. It has been found to have biocompatible advantages over other materials.

In addition, in the case of the Example it was confirmed that the physical properties and the growth effect of the cells significantly superior to the comparative example without using the chondroitin sulfate is effective in treating cartilage damage.

Claims (8)

delete delete delete It is a component that helps to relieve bone inflammation and helps to proliferate chondrocytes. Chondroitin sulfate is combined with gellan gum to form hydrogel. The gellan gum has a concentration of 1 to 3% by weight and the concentration of chondroitin sulfate is gellan. A composition for treating cartilage damage in the form of an injection comprising a gellan gum hydrogel composition containing chondroitin sulfate at 20% by weight relative to the gum.
delete Injectable arthritis therapeutic agent comprising a composition for treating cartilage damage according to claim 4.
The method of treating arthritis for injection according to claim 6, wherein the dosage of 0.1-1 ml can be adjusted according to the site of cartilage damage.
According to claim 7, Arthritis therapeutics for injection, characterized in that repeated administration, weekly or monthly depending on the degree of cartilage damage and regeneration.

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