KR101902194B1 - Injectable Curcumin/Gellan Gum Hydrogels for Cartilage Regeneration - Google Patents

Abstract

The present invention relates to a gellan gum hydrogel composition containing curcumin and a composition for treating cartilage regeneration and osteoarthritis using the same, and more particularly, to a composition for treating cartilage regeneration and osteoarthritis, The present invention relates to a composition of a gellan gum hydrogel composition which is usable and biologically compatible with curcumin which is usable and tissue engineering, and relates to a composition for treating cartilage regeneration and osteoarthritis using such a composition.

Description

Technical Field [0001] The present invention relates to a gellan gum hydrogel composition containing curcumin and a composition for treating cartilage regeneration and osteoarthritis using the same. [0002] Injectable Curcumin / Gellan Gum Hydrogels for Cartilage Regeneration [

The present invention relates to a gellan gum hydrogel composition containing curcumin and a composition for treating cartilage regeneration and osteoarthritis using the same, and more particularly, to a composition for treating cartilage regeneration and osteoarthritis, The present invention relates to a composition of a gellan gum hydrogel composition which is usable and biologically compatible with curcumin which is usable and tissue engineering, and relates to a composition for treating cartilage regeneration and osteoarthritis using such a composition.

If articular cartilage is damaged due to various causes, orthopedic research is needed because it does not heal naturally but shifts to arthritis. In other words, if the cartilage damage area is 1 ~ 4 ㎟, initial therapy is possible, but medication, physical therapy and pain control are performed. If the damage is more than 20 ㎟, And the cartilage regeneration method is needed. Surgical treatments include laparotomy and marginal resection, micro-perforation, and autologous osteochondral graft. [Korean Registered Patent No. 10-1351333 (Kang Gil Sun) et al.

Chondrocytes may be damaged by the temperature of the fluid, and the marginal resection may cause additional cartilage necrosis outside the cartilage to be removed. Micro-perforation is a treatment that should be avoided in the case of severe cartilage damage. The healing tissue is terminated with fibrocartilage and is worn without perfect regeneration. In particular, autologous osteochondral grafting has the disadvantage of requiring at least two operations to secure the chondrocytes for transplantation.

Therefore, there is a need for a tissue engineering method for cartilage regeneration which improves the disadvantages of these surgical methods.

Gellan Gum, a tissue engineering biomaterial, is a polymeric polysaccharide obtained by pure culture fermentation using Pseudomonas elodea isolated from Eelodia spp ., And is composed of 1: 1: 2 gluconic acid, rhamnose and glucose. Gellan gum hydrogel is colorless and transparent, has excellent acid and heat, has physical properties that can control physical properties through pH and ion addition, and shows high biocompatibility. In particular, glucan, a component of gellan gum, is known to produce an environment similar to that of the actual cartilage glycosaminoglycan, making it suitable for cartilage regeneration studies (Lucilia P. da Silva, Mariana T. Cerqueira, Rui A. Sousa, Rui L. Reis, Vitor M. Correlo, Alexandra P. Marques, Engineering cell-adhesive gellan gum spongy-like hydrogels for regenerative medicine purposes, Acta Biomaterialia , 10, 4787-4797 (2014)

In addition, curcumin is a spice of ginger and polyphenol extracted from Curcumalonga Linn (Zingiberaceae) roots and has been reported to have antioxidant and anti-inflammatory effects.

With respect to the use of curcumin, Korean Patent Laid-Open Publication No. 2016-0014707 has proposed a technique using curcumin or the like as a composition for joint health to control the rigidity of joints, the deterioration of cartilage and the function thereof, 2016-524614 and 2016-520609 propose a technique of using olefuran and hydrocortisol and using curcumin in combination for the purpose of preventing and treating cartilage destruction.

However, the description of the components applied to such cartilage tissue is merely an application example for only minor improvement through oral administration or application to health food, and it is difficult to expect an active therapeutic effect on cartilage damage.

Thus, despite the necessity of the above-mentioned injection-type hydrogel using the tissue engineering biomaterial, there has been no research on this.

In order to solve the problem that has not been solved by the conventional art, it is a problem to develop a method for cartilage regeneration and osteoarthritis treatment through a noninvasive tissue engineering method capable of minimizing a surgical site . The present inventors have long studied to solve the above problems, and as a result, they have developed cartilage-containing gellan gum hydrogel-injected arthritis therapeutic agent for cartilage treatment, and confirmed cartilage regeneration.

Accordingly, it is an object of the present invention to provide a gellan gum hydrogel composition for injecting cartilage therapy in which curcumin is combined with anti-inflammatory, antioxidant and cartilage regenerating effects for treating cartilage defects and arthritis.

Another object of the present invention is to provide a composition for treating and preventing osteoarthritis and cartilage regeneration using a gellan gum hydrogel composition containing curcumin which can be directly applied to a joint region of a human body.

It is still another object of the present invention to provide a filler for cartilage regeneration using a gellan gum hydrogel composition containing curcumin.

Another object of the present invention is to provide a medical kit containing a composition for treating and preventing osteoarthritis and osteoarthritis using gellan gum hydrogel composition containing curcumin.

In order to solve the above problems, the present invention provides a gellan gum hydrogel composition comprising curcumin, wherein gellan gum hydrogel is a main component and curcumin is contained as an auxiliary component.

The present invention also relates to a method for producing a curcumin solution, Preparing and heating an aqueous solution of gellan gum; Cooling the heated gellan gum aqueous solution, and then adding and mixing the curcumin solution. The present invention also provides a method for producing a gellan gum hydrogel composition comprising curcumin.

The present invention also provides a composition for the treatment and prevention of cartilage regeneration and osteoarthritis, which comprises a pharmaceutically acceptable excipient in a gellan gum hydrogel composition containing curcumin of the above composition.

In addition, the present invention provides a medical kit for cartilage regeneration and treatment and prevention of osteoarthritis in which a gel injector for injecting human body is filled with a gellan gum hydrogel composition containing curcumin.

When a new biocompatible hydrogel composition as described above is directly injected into a joint region of a human body, the present invention not only manifests a regeneration effect on the cartilage of the region, but also can suppress and treat osteoarthritis, There is an excellent effect in the treatment and prevention of osteoarthritis.

Particularly, the hydrogel composition of the present invention is very useful as a tissue engineering method, so that there is a remarkable effect that the side effects due to cartilage surgery can be greatly reduced and the operation time can be greatly shortened.

In addition, the present invention has an excellent effect of improving the tissue engineering characteristics of a surgical site by using it as a filler in a surgical procedure of a cartilage site, and enabling prompt treatment after surgery.

Figure 1 is a photograph of a suppository containing curcumin added to gellan gum hydrogel according to the preparation method of Example 1 of the present invention (0 wt.%, 5 wt.%, 10 wt.%, 20 wt.%) to be.
FIG. 2 is a graph showing the proliferation rate of rabbit chondrocytes sown on a curcumin-gellan gum hydrogel support prepared according to the present invention.
FIG. 3 is a photograph of the supernatant of curcumin-gellan gum hydrogel support prepared according to the present invention after lyophilization, and showing the surface of the supporter and the rabbit chondrocytes sown thereon by SEM after 1 week and 3 weeks after sowing, respectively.
4 is a graph showing the compressive strength of a curcumin-gellan gum hydrogel support prepared according to the present invention
FIG. 5 is a photomicrograph showing electrophoresis of cartilage-related marker gene expression in rabbit cartilage cells seeded on each hydrogel support prepared according to the present invention at the ratio of curcumin-gellan gum.

Hereinafter, the present invention will be described in more detail with reference to an embodiment.

The present invention is based on the result of an attempt to develop a treatment method of minimizing additional surgery and side effects unlike the conventional cartilage treatment method, which is essentially a tissue-engineered biomaterial suitable for proliferation and function maintenance of cartilage cells Is used to provide an injectable hydrogel capable of effectively regenerating cartilage damaged portions.

The tissue engineering biocompatible hydrogel according to the present invention is made of gellan gum containing curcum, which is a polymer, and provides cartilage regeneration and arthritis treatment effect on the cartilage area of the joint region as well as cartilage proliferation function, It can also function as an effective agent for cartilage regeneration and osteoarthritis treatment and prevention.

The present invention can be basically composed of gellan gum as a main component and curcumin as an auxiliary component as a preferable composition.

According to a preferred embodiment of the present invention, the gellan gum hydrogel used as a main component in the present invention 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. Since gellan is low in toxicity and easy to handle, hydrogels can be mixed with curcumin to be used as an injectable therapy without surgery.

According to a preferred embodiment of the present invention, Curcumin is known to have a high antiinflammatory effect due to its stable molecular structure, has excellent efficacy in rheumatoid arthritis, and has a positive effect on chronic diseases caused by aging .

According to a preferred embodiment of the present invention, the gellan gum and curcumin may be contained in a proportion of preferably 99-70% by weight to 1-30% by weight. More preferably, gellan gum and curcumin may be contained in an amount of 95 to 80% by weight: 5 to 20% by weight, and most preferably 90 to 80% by weight: 10 to 20% by weight.

According to the present invention, for example, TGF-β1, BMPs, etc., which are growth factors, can be added as an additive to the gellan gum hydrogel composition containing curcumin.

According to a preferred embodiment of the present invention, the curcumin-containing gellan gum hydrogel can be prepared by the following method.

First, curcumin and gellan gum are each prepared in solution.

According to a preferred embodiment of the present invention, in the step of preparing the curcumin solution, since curcumin is a lipid-soluble substance, it can be dissolved in an alcohol to prepare a solution. At this time, lower alcohols having 1 to 4 carbon atoms can be used as alcohol, and ethanol is most preferably used.

According to a preferred embodiment of the present invention, an aqueous gellan gum solution is prepared and heated. Preferably, the gellan gum powder is placed in distilled water heated to a temperature of 80-100 ° C and heated for 10-40 minutes under stirring to prepare an aqueous solution of gellan gum.

According to the present invention, the heated gellan gum solution is cooled, and then the curcumin solution is added thereto and mixed.

According to a preferred embodiment of the present invention, when the gellan gum aqueous solution is cooled, for example, at 40-70 ° C in a heated state, gellan gum hydrogel is produced. After cooling, the prepared gellan gum hydrogel is mixed with the prepared curcumin solution Can be added and mixed. Preferably, the concentration of the resulting gellan gum hydrogel can be made 0.7-2%.

According to a preferred embodiment of the present invention, the gellan gum aqueous solution and the curcumin solution are mixed so that the mixing ratio after mixing is preferably in the range of 99-70% by weight of gellan gum and 1-30% by weight of curcumin.

According to the present invention, gellan gum hydrogel containing curcumin can be prepared according to the present invention.

According to a preferred embodiment of the present invention, such a hydrogel composition has biocompatibility with tissue engineering properties suitable for the human body.

According to a preferred embodiment of the present invention, in the new hydrogel composition of the present invention, one or more components such as TGF-β1 and BMPs, which are growth factors, for example, are added as additives in an amount of 0-5 wt% can do. When these additives are used, an improvement in the recovery rate of cartilage regeneration can be expected.

Meanwhile, according to the present invention, the hydrogel composition containing curcumin as described above can be used as a composition for treating and preventing osteoarthritis and cartilage regeneration.

According to a preferred embodiment of the present invention, when used as a composition for such treatment and prevention, pharmaceutically acceptable excipients may be used as additives. Examples of such excipients include collagen, alginate, demineralized bone meal, gellan gum, silk, hyaluronic acid, and PLGA. One or more of these excipients may be used to impart proper pharmacological properties and characterize the formulation.

In addition, since the hydrogel composition containing curcumin as described above can be applied as a filler for regeneration and treatment of a cartilage region, the present invention also includes the application of such a use. Such fillers can be used for the treatment of cartilage damaged by abrasion, rupture or degenerative arthritis.

According to a preferred embodiment of the present invention, the composition for treating and preventing cartilage regeneration and osteoarthritis of the present invention may be manufactured in the form of a kit packed in a disposable syringe, for example.

The kit used herein may be packed in a vacuum state by a high pressure steam sterilization method, a radiation sterilization method, or the like to maintain a hygienic state. The amount of the composition per kit may be 0.1 to 1 ml.

According to a preferred embodiment of the present invention, the composition for treating and preventing cartilage regeneration and osteoarthritis can be directly injected into a human body. In this case, for example, the kit as described above can be preferably used.

According to a preferred embodiment of the present invention, the composition for treating and preventing cartilage regeneration and osteoarthritis can be injected directly into a cartilage or a joint region with a syringe, and the site can be administered after an abnormal patient, And can be preferably used by injection.

According to a preferred embodiment of the present invention, the composition for treating and preventing cartilage regeneration and osteoarthritis can be administered by injection by direct injection at a dose of 0.1 to 1 ml on an adult basis. In the case of postoperative patients, doses of 0.1 to 1 ml may be administered on an adult basis. These doses can be adjusted according to the condition of the patient, and it is necessary to inject them at a dose of max. 1 ml and a minimum of 0.1 ml, and it is possible to administer them repeatedly for 1 week or 1 month depending on degree of cartilage damage or degree of regeneration .

According to the present invention, when the gellan gum hydrogel containing the curcumin according to the present invention is used, it is possible to solve various problems such as postoperative side effects and delay in treatment, which were problems in the conventional cartilage treatment method, In particular, it can be applied as a method of minimizing additional surgery and side effects, and it is a tissue engineering biomaterial that is effective for the proliferation and function maintenance of cartilage cells, thereby effectively regenerating cartilage damaged parts.

As described above, the gellan gum hydrogel containing curcumin according to the present invention is simpler than the conventional method for producing hydrogels, can maintain the gel shape, and is easy to store. In addition, after cell growth has been completed, it is naturally biodegraded and absorbed into the body, so it has a biocompatible advantage over other materials.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following Examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  One: Curcumin - Gellan gum Hydrogel  Produce

The dissolution of the lipophilic substance curcumin is carried out using EtOH. 0.7% gellan gum (Sigma) (0.7 g) was added to distilled water (100 ml) heated to 90 ° C and stirred for 20 minutes. When the gellan gum powder is completely dissolved, add 0.03% calcium chloride (0.03 g) and dissolve for 10 minutes. The curcumin aqueous solution is prepared by dissolving in 200 μL EtOH. The aqueous solution of gellan gum is immersed in an aqueous solution of gellan gum whose temperature is lowered to 60 ° C in an amount of 0 wt.%, 5 wt.%, 10 wt.%, 20 wt.%. After mixing the gellan gum aqueous solution and the curcumin aqueous solution, pour into the Petri dish and harden at room temperature. Cut the hard gel to 8 mm in diameter and 4 mm in thickness using a bioxifon.

Figure 1 is a photograph of a suppository containing curcumin added to gellan gum hydrogel according to the preparation method of Example 1 of the present invention (0 wt.%, 5 wt.%, 10 wt.%, 20 wt.%) to be.

Example  2: Collection and culture of knee cartilage cells

The knee cartilage cells were isolated from the knee cartilage collected from 6 - week - old White New Zealand rabbits. Knee cartilage is harvested, chopped to less than 1 mm2, placed in PBS, and centrifuged at 12000 rpm for 3 minutes. Finely Sun cartilage 10 mg / ml collagenase A (Roche Diagnostics GmbH), 1 mg / ml hyaluronidase (Roche Diagnostics GmbH), 0.7 mg / ml DNase (Roche Diagnostics GmbH) to put in a mixed solution of 37 ℃, in 5% CO ₂ Disassemble for 3 hours.

The dissociated solution was centrifuged again and cultured in D-MEM F12 media containing 10% fetal bovine serum (Hyclone technologies) and 1% penicillin / ascorbic acid in a cell culture dish.

Experimental Example  1: Evaluation of cell proliferation rate

As a result of MTT (dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay, cell proliferation rate was found to increase with time in the hydrogel support (10 wt% I could confirm.

FIG. 2 is a graph showing the proliferation rate of rabbit chondrocytes sown on a curcumin-gellan gum hydrogel support prepared according to the present invention.

Experimental Example  2: cell Morphology  Confirm

The prepared hydrogel support (10 wt%) was lyophilized and the surface porosity of the support was observed by SEM. After rabbit knee cartilage cells were harvested, they were cultured in supporters for 1 week and 3 weeks, fixed with 2.5% glutaraldehyde, and morphology of chondrocytes was confirmed. Cell proliferation was the highest in gellan gum hydrogel containing 10 wt.% Curcumin, and extracellular matrix was also formed.

FIG. 3 is a photograph of the supernatant of curcumin-gellan gum hydrogel support prepared according to the present invention after lyophilization, and showing the surface of the supporter and the rabbit chondrocytes sown thereon by SEM after 1 week and 3 weeks respectively after sowing.

Experimental Example  3: Analysis of the compressive strength of the support

To evaluate the physical properties of the support, compressive strength was measured by applying a force of 1 N to the support at a speed of 10 mm / sec. The compressive strength increased with the addition of curcumin.

4 is a graph showing the compressive strength of a curcumin / gellan gum hydrogel support prepared according to the present invention

Experimental Example  4: Analysis of gene expression

When the gene phenotypes of the hydrogels containing gellan gum hydrogels and curcumin were compared, there was no significant difference and all of them were expressed well. ECM-associated gene expression results, such as Aggren and type II collagen, suggest that curcumin-incorporated gellan gum hydrogels may provide a more suitable microenvironment that enhances the synthesis of the desired ECM.

FIG. 5 is a photomicrograph showing electrophoresis of cartilage-related marker gene expression in rabbit cartilage cells seeded on each hydrogel support prepared according to the present invention at the ratio of curcumin-gellan gum.

Claims (8)

A gellan gum hydrogel comprising a gellan gum aqueous solution, wherein the hydrogel composition comprises a gellan gum hydrogel having a gellan gum concentration of 0.7-2 wt% in the hydrogel and a curcumin solution dissolved in ethanol, And curcumin in a proportion of 99-70% by weight to 1-30% by weight, based on the total weight of the gel.
delete Dissolving curcumin in ethanol to prepare a curcumin solution;
Adding gellan gum powder into distilled water heated to 80-100 ° C and heating for 10-40 minutes to prepare an aqueous solution of gellan gum;
The heated gellan gum aqueous solution was cooled to 40-70 ° C to prepare a gellan gum hydrogel, a gellan gum hydrogel was prepared so that the concentration of gellan gum contained in the hydrogel was 0.7-2 wt% The mixture is mixed with the gellan gum hydrogel and the curcumin solution so that the mixing ratio of gellan gum and curcumin is in the range of 99-70% by weight of gellan gum and 1-30% by weight of curcumin
≪ / RTI > wherein the composition comprises curcumin.
delete delete delete A composition for treating and preventing cartilage regeneration and osteoarthritis, comprising a pharmaceutically acceptable excipient in a gellan gum hydrogel composition comprising curcumin according to claim 1.
A medical kit for cartilage regeneration and treatment and prevention of osteoarthritis in which a gel injecting syringe is filled with a gellan gum hydrogel composition containing curcumin according to claim 1.

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