KR20200113689A - Gellan gum hydrogel composition comprising agar or agarose, method of manufacuring the same and use of the same - Google Patents

Abstract

The present invention relates to an agar-gellan gum hydrogel composition, and a composition for regenerating cartilage and treating osteoarthritis using the same. More specifically, the present invention relates to: a hydrogel composition which is usable as a cartilage cell carrier superior than conventional hydrogel for regeneration of cartilage and treatment of osteoarthritis, and can be used by being directly injected to joints of the human body for fillers or treatment; and a composition for regenerating cartilage and treating and preventing osteoarthritis using the hydrogel composition.

Description

Gellan gum hydrogel composition comprising agar or agarose, method of manufacuring the same and use of the same}

The present invention relates to a gellan gum hydrogel composition containing agar or agarose, a method for preparing the same, and a use thereof, and more particularly, suitable for enduring cartilage load in damaged cartilage areas such as osteoarthritis, and as a cartilage cell delivery system It relates to a composition for cartilage regeneration and osteoarthritis treatment and prevention comprising the biocompatible hydrogel composition, a method of preparing the biocompatible hydrogel composition, and the biocompatible hydrogel composition serving as a role.

In case of cartilage inflammation or osteomalacia with relatively little cartilage damage, oral preparations are used, and for severe cartilage damage, various cartilage treatment methods such as artificial cartilage insertion, autologous osteocartilage insertion, autologous cartilage cell transplantation, and stem cell transplantation are used.

However, when only cells are injected directly into the affected area, the effect is insignificant and there is a limitation in adhesion, migration, and proliferation of the injected chondrocytes, so a method of injecting the scaffold and cells together is mainly used. In particular, as a transporter for cartilage cells and cartilage stem cells, hydrogels enable continuous treatment, and are also used as fillers for physically damaged areas.

Therefore, in order for the hydrogel to be used as a cartilage treatment, it is biocompatible, and strength and fatigue sufficient to withstand the load of cartilage are very important (Non-Patent Document 1).

Gellan gum is widely used in the biomedical field (Patent Document 1), and is transparent and has good stability by heat and acid. In addition, it has excellent mechanical properties and biocompatibility (Non-Patent Document 2).

However, when crosslinking gellan gum, it is difficult to pass through the injection needle and the compressive strength is low, making it unsuitable to withstand cartilage load. In other words, if the characteristics related to the convenience of use are not improved, it will inevitably suffer from generalization. In addition, it has lower properties compared to other hydrogels in proliferation of chondrocytes.

Therefore, there is an urgent need to find a way to improve the availability of gellan gum as a cartilage cell carrier as described above, thereby improving the proliferation rate of chondrocytes and improving mechanical properties.

Korean Patent Application Publication No. 10-2012-0091717

Osteoarthritis and Cartilage, 14, 13-29 (2006) Acta Biomater, 10(8):3650-63 (2014)

An object of the present invention is to prepare a hydrogel without physical properties, cell affinity, and crosslinking agent using agar or agarose, thereby providing a gellan gum hydrogel composition comprising agar or agarose capable of improving its function as a chondrocyte carrier. To provide.

Another object of the present invention is a gellan gum hydrogel composition comprising agar or agarose capable of improving the function as a chondrocyte carrier by preparing a hydrogel without physical properties, cell affinity, and crosslinking agent using agar or agarose. It is to provide a method of manufacturing.

Another object of the present invention is to provide a use of a gellan gum hydrogel composition comprising agar or agarose.

In order to achieve the above object, the present invention provides a gellan gum hydrogel composition comprising a gellan gum hydrogel as a main component and agar or agarose including agar or agarose as an auxiliary component.

In addition, the present invention comprises the steps of preparing agar or agarose solution by dissolving agar or agarose in distilled water; Preparing and heating an aqueous gellan gum solution; And cooling the heated gellan gum aqueous solution, and then adding and mixing the agar or agarose solution thereto. It provides a method for preparing a gellan gum hydrogel composition comprising agar or agarose.

In addition, the present invention provides a composition for cartilage regeneration and osteoarthritis treatment and prevention comprising a gellan gum hydrogel composition containing the agar or agarose and a pharmaceutically acceptable excipient.

In addition, the present invention provides a medical kit for cartilage regeneration and osteoarthritis treatment and prevention, in which the gellan gum hydrogel composition containing the agar or agarose is filled.

The present invention constitutes a new biocompatible agar-gellan gum hydrogel composition as described above, and effectively delivers cartilage cells to the treatment site, thereby expressing a regeneration effect against cartilage damage in the corresponding site, as well as providing the user's hydrogel delivery system. There is an effect of providing a better delivery system by improving the injectability for product development to be used.

In particular, the agar-gellan gum hydrogel composition of the present invention is a natural substance, which is suitable as a cartilage cell delivery system because it has low cytotoxicity, inhibits inflammatory reactions, protects cells from cartilage load, and enables continuous cell release.

The method of manufacturing the chondrocyte delivery system has an effect of reducing the inflammatory reaction of the crosslinking agent in the human body by suggesting a method of increasing the injectability of the hydrogel, and also suggesting a method of preparing gellan gum in a hydrogel state without a crosslinking agent. .

1 is a picture of a hydrogel after preparing by adding agar in each content (0 wt%, 0.2 wt%, 0.4 wt%, 0.6 wt%, 0.8 wt%) according to the manufacturing method of Example 1 of the present invention. .
2 is a graph showing the compressive strength of agar-gellan gum hydrogel prepared according to the present invention.
3 is a graph showing the extrusion force of agar-gellan gum hydrogel prepared according to the present invention.
4 is a graph showing the moisture content of the agar-gellan gum hydrogel prepared according to the present invention.
5 is a graph showing the cytotoxicity of the agar-gellan gum hydrogel prepared according to the present invention.
Figure 6a shows the results of measuring the pore size of the agar-gellan gum hydrogel prepared according to the present invention, Figure 6b is a photograph taken by SEM of the morphology and the morphology of the cells cultured by seeding chondrocytes.
7 shows the results of measuring MTT by seeding chondrocytes on the agar-gellan gum hydrogel prepared according to the present invention and culturing for 1, 7, 14, and 21 days.
Figure 8 shows the results of confocal microscopy photographing by seeding chondrocytes on the agar-gellan gum hydrogel prepared according to the present invention and culturing for 1, 7, 14 days.

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

One embodiment of the present invention relates to a gellan gum hydrogel composition comprising agar or agarose, comprising a gellan gum hydrogel as a main component and agar or agarose as an auxiliary component.

The gellan gum hydrogel composition of the present invention is an invention calculated as a result of an effort to develop a method of minimizing additional surgery and side effects, unlike conventional cartilage treatment surgery, and essentially maintains the proliferation and function of cartilage cells. Provides an injectable hydrogel that can effectively regenerate damaged areas of cartilage using biocompatible materials for tissue engineering.

The tissue-engineered biocompatible hydrogel according to the present invention is prepared in a composition containing agar or agarose in gellan gum, and provides cartilage regeneration and arthritis treatment effects on the cartilage area of the joint area as well as cartilage proliferation function. Since it can function as a filler, it exhibits excellent effects in cartilage regeneration and osteoarthritis treatment and prevention.

The present invention is a preferred composition, and basically, gellan gum as a main component, and agar or agarose may be mixed as an auxiliary component.

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 modulus of elasticity and stiffness that can be adjusted during manufacture. When the temperature of the gellan gum hydrogel falls below a certain temperature, it forms a secondary coil helix structure to gel.

Because gellan gum has a low toxicity rate and is easy to handle, if a hydrogel is prepared by mixing it with curcumin, it can be used as an infusion treatment without surgical intervention.

Agar is a natural substance with good biocompatibility that has been approved by the FDA, and is widely applied in various fields such as food and medicine. In addition, since it has relatively high strength and has rheological properties of shear-thinning in particular, it serves as an important additive for making gellan gum injectable. In addition, it has good interaction with cells and is good for cell adhesion and proliferation.

In the present invention, by using an agar having excellent biocompatibility and mechanical properties and, in particular, shear-thinning properties, problems of existing gellan gum can be solved. In addition, since agar has a good interaction with cells and aids in cell adhesion and proliferation, it is also effective in proliferation of chondrocytes.

On the other hand, in the gellan gum hydrogel composition of the present invention, agarose, which may be included instead of agar, may be purely separated from agar consisting of agaropectin and agarose.

The agarose is a straight chain consisting of a β-D-galactopyranose residue bonded to the 1,3 position and a 3,6-anhydro-α-L-galactopyranose residue bonded to the 1,4 position As a polysaccharide of the shape, it can be obtained, for example, by adding an ammonium salt to an aqueous agar solution to precipitate and remove agarofectin.

The agarose is dissolved in warm water, and a solution of about 1% or more becomes a gel when cooled. Since this gel has a net-like structure and can freely diffuse polymeric substances, it is used as a reagent for biochemical research, such as an electrophoretic support or a gel filtration filler.

In the gellan gum hydrogel composition comprising the agar or agarose of the present invention, the gellan gum is based on 0.5 to 2% by weight, preferably 0.7 to 1.5% by weight, more preferably 50 ml based on the total weight of the composition It may be included as 1% by weight.

In the gellan gum hydrogel composition comprising the agar or agarose of the present invention, the agar is 0.2 to 2% by weight, preferably 0.5 to 2% by weight, based on the total weight of the composition, when considering the solubility of the agar, More preferably, it may be included in 0.5 to 0.8% by weight based on 50ml.

When the agar or agarose is contained in an amount of 0.5% by weight or more based on the weight of the total composition, it is possible to maintain the hydrogel state without a crosslinking agent, so if an appropriate composition ratio of agar and gellan gum is used, it can be prepared without a crosslinking agent.

In the gellan gum hydrogel composition comprising the agar or agarose of the present invention, the agarose is 0.2 to 3% by weight, preferably 0.5 to 2% by weight, more preferably 50 It may be included in 0.5 to 0.8% by weight based on ml.

In the gellan gum hydrogel composition containing the agar or agarose of the present invention, the agar or agarose is contained in 0.2 to 0.5% by weight relative to the total weight of the composition, and calcium chloride as a crosslinking agent is contained in the total weight of the composition 0.02 to 0.05% by weight, preferably 0.03% by weight based on 50ml may be included.

When the calcium chloride is included in less than 0.02% by weight, crosslinking is not properly performed, and when it is included in 0.05% by weight or more, there is a problem that the injection needle does not pass well.

The gellan gum hydrogel composition comprising agar or agarose of the present invention is a silver additive, such as a growth factor of TGF-β1, BMPs, etc. of one or more components in the natural hydrogel composition in an amount of 0 to 5% by weight It may be added as, but is not limited thereto. When these additives are used, it can be expected to improve the recovery rate of cartilage regeneration.

The gellan gum hydrogel composition containing the agar or agarose of the present invention may be injectable.

Another embodiment of the present invention is to prepare an agar or agarose solution by dissolving agar or agarose in distilled water; Preparing and heating an aqueous gellan gum solution; And cooling the heated gellan gum aqueous solution, and then adding and mixing the agar or agarose solution thereto. It relates to a method for preparing a gellan gum hydrogel composition comprising agar or agarose.

First, agar or agarose and gellan gum are each prepared in a solution phase and used.

According to a preferred embodiment of the present invention, in the step of preparing an agar or agarose solution, since agar or agarose is a water-soluble material, it may be preferably dissolved in distilled water to prepare a solution.

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 80 to 100°C, and heated for 10 to 40 minutes under stirring to prepare an aqueous gellan gum solution.

According to the present invention, after cooling the heated gellan gum aqueous solution, the agar or agarose solution is added thereto, followed by mixing.

According to a preferred embodiment of the present invention, in this process, the gellan gum aqueous solution is, for example, cooled to 40 to 70°C in a heated state to generate a gellan gum hydrogel. After cooling, the prepared agar or The agarose solution can be added and mixed. Preferably, the concentration of the gellan gum hydrogel produced at this time may be prepared at 0.7 to 2%.

According to a preferred embodiment of the present invention, in the mixing of the aqueous gellan gum solution and the curcumin solution, the amount after mixing is 0.5 to 2% by weight, preferably 0.7 to 1.5% by weight, based on the total weight of the composition. More preferably, it may be mixed to be 1% by weight based on 50 ml.

In addition, the agar is 0.2 to 2% by weight, preferably 0.5 to 2% by weight, more preferably 0.5 to 0.8% by weight based on 50 ml of the total weight of the composition, and the agarose is It may be mixed so as to be 0.2 to 3% by weight, preferably 0.5 to 2% by weight, and more preferably 0.5 to 0.8% by weight based on 50 ml of the total weight.

According to the present invention, through the above process, a gellan gum hydrogel containing agar or agarose as desired in the present invention can be prepared.

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

Meanwhile, according to the present invention, the hydrogel composition containing agar or agarose as described above can be used as a composition for cartilage regeneration and osteoarthritis treatment and prevention.

According to a preferred embodiment of the present invention, when used as such a composition for treatment and prevention, a pharmaceutically acceptable excipient may be used as an additive.

As an example of the excipient, at least one selected from collagen, alginate, demineralized bone meal, gellan gum, silk, hyaluronic acid, and PLGA may be used to impart suitable pharmaceutical properties and formulation characteristics, but is not limited thereto.

In addition, in the present invention, since the hydrogel composition containing agar or agarose as described above can be applied as a filler for the purpose of regeneration and treatment of cartilage, the present invention also includes the application of these uses. These fillers can be used for the treatment of cartilage damaged by abrasion, rupture, and 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 as described above may be prepared in the form of a kit filled in, for example, a disposable syringe.

The kit may be vacuum-packed by an autoclave method, radiation sterilization, etc. to maintain hygiene, and the amount of the composition per kit may be composed of disposable charging units such as 0.1 to 1 ml.

According to a preferred embodiment of the present invention, the composition for cartilage regeneration and osteoarthritis treatment and prevention may be used by direct injection into the human body, and in this case, for example, the above kit may be preferably used.

According to a preferred embodiment of the present invention, the composition for cartilage regeneration and osteoarthritis treatment and prevention may be administered by injecting directly into the cartilage or joint area with a syringe, and the site is abnormal, before or after surgery or immediately after surgery in the course of surgery. It can be preferably injected and used.

According to a preferred embodiment of the present invention, the composition for the treatment and prevention of cartilage regeneration and osteoarthritis may be administered by direct injection in a dosage of 0.1 to 1 ml based on an adult in the case of a general patient.

In the case of a patient after surgery, it can be administered by injecting at a dosage of 0.1 to 1 ml based on an adult. This dosage can be adjusted according to the patient's condition, and it is necessary to inject at a maximum of 1 ml and at least 0.1 ml, and it can be administered repeatedly every one week or one month depending on the degree of cartilage damage or regeneration. .

According to the present invention, when using the agar according to the present invention or gellan gum hydrogel containing agarose according to the present invention, various problems such as side effects or delayed treatment after surgery, which have been a problem in the existing cartilage treatment surgery method, are solved at once. It can be solved, and in particular, it can be applied as a method of minimizing additional surgery and side effects, and since it is a tissue engineering biomaterial that is effective in proliferation and function maintenance of cartilage cells, it is possible to effectively regenerate damaged parts of cartilage.

As described above, the gellan gum hydrogel containing agar or agarose according to the present invention is simpler than other conventional methods for preparing hydrogels, maintains a gel form, and is easy to store. In addition, after cell growth is completed, it is naturally biodegraded and absorbed by the body, so it has the advantage of being biocompatible over other materials.

In addition, according to a preferred embodiment of the present invention, in order to reduce the extrusion force of the hydrogel, the hydrogel solution is hardened by putting it in a syringe, and when the particle size is reduced using a needle of each size, a composition having a desired extrusion force can be prepared. I can.

Hereinafter, the present invention will be described in detail by examples, and the following examples are for illustrative purposes only, and the contents of the present invention are not limited by the following examples.

Example 1: agar- Gellan gum Hydrogel Manufacturing method

0.2 wt (%), 0.4 wt (%), 0.6 wt (%), 0.8 wt (%) agar was added to 90° C. of distilled water and stirred for 30 minutes. Thereafter, when the agar was completely dissolved, the agar solution was lowered to 80° C., gellan gum 1% by weight was added and stirred for 30 minutes.

0.03 wt (%) of CaCl ₂ was added and stirred for 10 minutes, and then 0.1 g, 0.2 g, 0.3 g, and 0.4 g of agar were dissolved in tertiary distilled water at 90° C. to prepare a 50 mL solution.

0.5 g of gellan gum was added to each agar solution, and 0.015 g of CaCl ₂ was added to each solution. After crosslinking, the resulting solution was poured into a Petri dish at room temperature (25°C) to prepare a hydrogel. The hydrogel prepared above was manufactured in various sizes by punching according to use.

1 is a photograph showing a hydrogel prepared by adding agar in each content (0, 0.2, 0.4, 0.6, 0.8% by weight) according to the manufacturing method of Example 1 above.

Example 2: Collection and culture of knee cartilage cells

Chondrocytes were isolated from knee cartilage collected from a 6-week-old White New Zealand rabbit. After collecting the cartilage, it was put in PBS and washed 2-3 times.

After washing, chop the cartilage into 1 mm ² or less, 10 mg/ml Collagenase A (Roche Diagnostics GmbH), 1 mg/ml Hyaluronidase (Roche Diagnostics GmbH), 0.7 mg/ml DNase (Roche Diagnostics GmbH). ) Into the mixed solution and overnight at 37°C and 5% CO ₂ .

The digested solution was centrifuged under conditions of 1200 rpm for 3 minutes, and cultured in D-MEM/F12 medium containing 10% fetal bovine serum (Hyclone technologies) and 1% penicillin/ascorbic acid in a cell culture dish.

Example 3: Hydrogel Seeding my chondrocytes

Chondrocytes were cultured in hydrogels for cell encapsulation. Mix well at a cell density of 2×10 ⁶ cells/mL in agar-gellan gum hydrogel solution at 40°C. 100 μL hydrogel was dispensed into a silicone mold having a diameter of 6 mm and a height of 3 mm, and then hardened at room temperature.

After that, it was put in a 24-well plate and incubated under conditions of 5% CO ₂ , 37°C, and 1 mL of culture medium added (10% FBS-added DMEM/F12 medium (Lonza, USA)).

Experimental example 1: agar- Gellan gum Hydrogel characteristic

In order to evaluate the physical properties of the agar-gellan gum hydrogel, the results of measuring the compressive strength by applying a force of 1N to the prepared support to the surface of the support at a speed of 10 mm/sec are shown in FIG. 2. As shown in Figure 2, it was confirmed that the compressive strength increased as the agar increased, and increased up to about 2.5 times.

Fig. 3 shows the results of the extrusion force test to confirm the possibility of injection of the hydrogel. At this time, the needle size was measured as 23G. As shown in FIG. 3, the extrusion force increased as the agar was added.

In addition, the result of measuring the water absorption of the hydrogel is shown in FIG. 4. As shown in FIG. 4, it slightly increased at 0.2 weight (%) than the control group, and then decreased as agar was added.

Experimental example 2 : Hydrogel Cytotoxicity assessment

In order to investigate the toxicity of the hydrogel, the results of the MTT cytotoxicity evaluation are shown in FIG. 5. As shown in FIG. 5, the control group showed high cytotoxicity, and as the agar was added, the cell survival rate was at least 70%, and the toxicity decreased.

Experimental example 3: cell survival, cell death, proliferation and Morphology Confirm

The agar-gellan gum hydrogel seeded with chondrocytes was observed using Scanning Electron Microscopy (SEM), MTT assays, and Confocal Laser Scanning Microscope (CLSM) to observe the survival rate, morphology, and proliferation of cells.

Rabbit chondrocytes were collected, seeded on hydrogel, cultured for a certain time, and treated according to the experiment.

In order to measure cell proliferation and morphology, the seeded hydrogel was fixed with 2.5% glutaraldehyde, and the results confirmed using SEM are shown in FIGS. 6A and 6B.

As shown in FIGS. 6A and 6B, as the amount of agar increased, cell proliferation was better, and cell morphology was well attached to the hydrogel. ECM can also be seen to increase further. In addition, the pore size of each hydrogel also increased.

The results of MTT assays for confirming the proliferation rate of cells are shown in FIG. 7. As shown in FIG. 7, it can be seen that the group containing 0.8% by weight of agar has the best proliferation rate.

In addition, after staining the cells through live and dead fluorescence staining, the result of observing the survival rate of the cells through CLSM is shown in FIG. 8. As shown in FIG. 8, the survival rate of chondrocytes increases as agar is added. It could be confirmed that it increases.

As described above, the present invention has been described with reference to the preferred embodiments of the present invention, but those skilled in the art of the present invention have the present invention within the scope not departing from the spirit and scope of the present invention described in the following claims. It will be appreciated that various modifications and changes can be made to the invention.

Claims (11)

A gellan gum hydrogel composition comprising agar or agarose, comprising a gellan gum hydrogel as a main component and agar or agarose as an auxiliary component. The gellan gum hydrogel composition comprising agar or agarose according to claim 1, wherein the gellan gum is contained in an amount of 0.5 to 2% by weight based on the total weight of the composition. The gellan gum hydrogel composition according to claim 1, wherein the agar contains 0.2 to 2% by weight based on the total weight of the composition. The gellan gum hydrogel composition according to claim 1, wherein the agarose is contained in an amount of 0.2 to 3% by weight based on the total weight of the composition. The agar according to claim 1, wherein the agar or agarose is contained in an amount of 0.2 to 0.5% by weight based on the total weight of the composition, and calcium chloride as a crosslinking agent is contained in an amount of 0.02 to 0.05% by weight based on the total weight of the composition. Or gellan gum hydrogel composition comprising agarose. The gellan gum hydrogel composition according to claim 1, wherein the composition is injectable. Dissolving agar or agarose in distilled water to prepare an agar or agarose solution;
Preparing and heating an aqueous gellan gum solution; And
After cooling the heated gellan gum aqueous solution, adding and mixing the agar or agarose solution thereto; a method for producing a gellan gum hydrogel composition comprising agar or agarose. The method of claim 7, wherein the step of preparing and heating the gellan gum aqueous solution is characterized in that the gellan gum powder is added to distilled water heated to a temperature of 80-100°C and heated for 10-40 minutes under stirring to prepare an aqueous gellan gum solution Method for producing a gellan gum hydrogel composition comprising agar or agarose. The method of claim 7, after cooling the heated gellan gum aqueous solution. In the step of adding and mixing the agar or agarose solution, the agar or agarose solution is mixed after cooling the heated gellan gum aqueous solution to 40 to 70°C,
The gellan gum is 0.5 to 2% by weight based on the total weight of the composition, the agar is 0.2 to 2% by weight based on the total weight of the composition, and the agarose is 0.2 to 3% by weight based on the total weight of the composition Method for producing a gellan gum hydrogel composition comprising agar or agarose, characterized in that mixing so as to be. A composition for cartilage regeneration and osteoarthritis treatment and prevention comprising a gellan gum hydrogel composition comprising agar or agarose according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient. A medical kit for cartilage regeneration and osteoarthritis treatment and prevention, in which a gellan gum hydrogel composition comprising agar or agarose according to any one of claims 1 to 6 is filled.

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