KR102549899B1 - Method for adjusting adhesive force of animal cartilage-derived extracellular matrix membrane and animal cartilage-derived extracellular matrix membrane having adjustable adhesive force by using the same - Google Patents

Abstract

The present invention relates to a method for controlling the adhesiveness of an animal cartilage-derived extracellular matrix membrane and an animal cartilage-derived extracellular matrix membrane whose adhesiveness is controlled using the same, and more particularly, to at least one of irradiated animal cartilage-derived extracellular matrix membranes. A method for controlling the adhesive strength of an animal cartilage-derived extracellular matrix membrane, comprising applying an aqueous hydrogen peroxide solution to a portion thereof; and animal cartilage-derived extracellular matrix membranes whose adhesiveness is controlled by the method of the present invention.

Description

Method for adjusting adhesive force of animal cartilage-derived extracellular matrix membrane and animal cartilage-derived extracellular matrix membrane having adjustable adhesive force by using the same}

The present invention relates to a method for controlling the adhesiveness of animal cartilage-derived extracellular matrix membranes and an animal cartilage-derived extracellular matrix membrane whose adhesiveness is controlled by using the same, and more particularly, can be used without limitation in the shape or location of an organ. , It relates to animal cartilage-derived extracellular matrix membranes with controlled adhesion.

An extracellular matrix is a material derived from animals and humans, and has excellent biocomposite and physiological functions. Therefore, membranes made of extracellular matrix are evaluated as ideal cell therapy agents or scaffold materials for drug delivery and tissue engineering because they can provide excellent biofunctionality and biodegradability as well as low inflammatory response after transplantation into the living body.

The cartilage-derived extracellular matrix is an avascular and non-nervous tissue, and has tissue-specific component differentiation compared to the extracellular matrix of other tissues. Typically, cartilage tissue is rich in chondromodulin, thrombospondin, and endostatin, which inhibit angiogenesis, as well as lubricin and bigley, which prevent cell adhesion. Biglycan, Decorin, and Fibromodulin are present. However, despite high biocompatibility and functionality as a natural material, most of the currently commercialized anti-adhesion agents are manufactured in the form of films or gels and are designed to be suitable for use against intestinal adhesion models. However, since adhesion may occur in various parts, there is a problem in that the application of the current anti-adhesion agent is limited.

In particular, the adhesion of the anti-adhesion film to the surface of various wounds is insufficient, so that the anti-adhesion film cannot be accurately fixed to the required area, or for this reason, the work of fixing the anti-adhesion film with a surgical suture is required. At this time, the film Lack of flexible wire may cause problems such as tearing of the film. Therefore, the anti-adhesion film must have sufficient flexibility to be applicable to, for example, laparoscopic surgery, and at the same time, have sufficient tensile strength to sufficiently protect the treatment site, and at the same time, sufficient adhesive strength is expressed with respect to the target site It is desired to possess properties that may not require fixation by additional sutures.

In this regard, Korean Patent No. 10-1772316 discloses a method for controlling the adhesive strength of an animal cartilage-derived extracellular matrix membrane using radiation, but when only radiation is applied to the animal cartilage-derived extracellular matrix membrane as in the above patent, low At high doses, the effect of regulating adhesion is hardly obtained, and at high doses, denaturation of the collagen component, which is the main component of animal cartilage-derived extracellular matrix membranes, is induced, resulting in deterioration of physical properties of the membranes.

Therefore, it is expected that if a technology capable of adjusting the adhesive force while maintaining the physical properties of animal cartilage-derived extracellular matrix membrane is developed, it will be widely applied in related fields.

Accordingly, one aspect of the present invention is to provide a method for controlling the adhesiveness of an animal cartilage-derived extracellular matrix membrane.

Another aspect of the present invention is to provide an animal cartilage-derived extracellular matrix membrane having controlled adhesiveness by using the adhesiveness control method of the present invention.

According to one aspect of the present invention, there is provided a method for regulating the adhesive strength of an animal cartilage-derived extracellular matrix membrane, comprising applying an aqueous hydrogen peroxide solution to at least a portion of the animal cartilage-derived extracellular matrix membrane irradiated with radiation.

According to another aspect of the present invention, an animal cartilage-derived extracellular matrix membrane, the adhesiveness of which is controlled by the method for controlling the adhesiveness of an animal cartilage-derived extracellular matrix membrane of the present invention, is provided.

According to the present invention, it is possible to control the adhesion while maintaining the physical properties of the animal cartilage-derived extracellular matrix membrane. , it is possible to obtain an anti-adhesion agent with an extended application range.

Figure 1 confirms the effect of the application of an aqueous hydrogen peroxide solution to the anti-adhesion films obtained in Examples and Comparative Examples.
Figure 2 shows the results of SEM image analysis to confirm the effect of the application of an aqueous hydrogen peroxide solution to the anti-adhesion films obtained in Examples and Comparative Examples.
Figure 3 shows the swelling (%, swelling ratio) of the anti-adhesion film obtained in Examples and Comparative Examples according to the concentration of aqueous hydrogen peroxide solution.
Figure 4 shows an FTIR graph for analyzing component changes of the anti-adhesion film.
Figure 5 shows the results of measuring the adhesive force of the anti-adhesion film obtained in Examples and Comparative Examples according to the concentration of aqueous hydrogen peroxide solution.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, there is provided a method for adjusting the adhesiveness of an animal cartilage-derived extracellular matrix membrane while maintaining its physical properties, and in particular, hydrogen peroxide and radiation are used.

In the present invention, "cartilage-derived extracellular matrix" may be used interchangeably with "cartilage-derived decellularized extracellular matrix membrane", and "membrane" may be used interchangeably with "film". Meanwhile, in the present invention, since the cartilage-derived extracellular matrix membrane or film may be an anti-adhesion film, it may also be referred to as an "anti-adhesion film".

Hydrogen peroxide, which has the ability to oxidize organic substances such as proteins, is a weak acid and can form highly reactive free radicals since it is decomposed into hydroxyl radicals (OH·). Direct application of hydrogen peroxide to an animal cartilage-derived extracellular matrix membrane may cause degradation of the membrane itself. It is possible to control the adhesiveness of cartilage-derived extracellular matrix membranes.

More specifically, the method for controlling the adhesiveness of an animal cartilage-derived extracellular matrix membrane of the present invention includes applying an aqueous hydrogen peroxide solution to at least a portion of the animal cartilage-derived extracellular matrix membrane irradiated with radiation.

The step of applying the hydrogen peroxide aqueous solution may be performed entirely or partially on both sides or one side of the cartilage-derived extracellular matrix membrane of the animal, and more specifically, it may be performed on the entire membrane, both sides or one side, and also It may be the entire area, both sides or one side, or a necessary part of it.

Therefore, the step of applying the hydrogen peroxide aqueous solution may be performed by at least one method of dipping, spraying, and coating, and may be appropriately employed depending on the target application area, for example, coating or spraying (spray Ying), it is possible to apply the hydrogen peroxide aqueous solution only to one side.

The animal cartilage-derived extracellular matrix membrane of the present invention is irradiated with radiation, and can be obtained through a step of irradiating the membrane with radiation, wherein the radiation is selected from the group consisting of gamma rays, electron rays, ultraviolet rays, and X-rays, Preferably, it is a gamma ray. In addition, the gamma rays may be emitted from any one radioactive isotope selected from the group consisting of cobalt (Co) -60, krypton (Kr) -85, strontium (Sr) -90, and cesium (Cs) -137, preferably More specifically, the gamma ray may be cobalt (Co)-60, but is not limited thereto.

The irradiated animal cartilage-derived extracellular matrix membrane may be obtained by irradiating the animal cartilage-derived extracellular matrix membrane with radiation of greater than 0 and less than 50 kGy, for example, a total dose of 1 to 25 kGy. In the step of irradiating the radiation, when the total dose exceeds 50 kGy, the collagen, which is the main component of the cartilage-derived extracellular matrix membrane of the animal, is modified by the radiation, thereby reducing the actual collagen content and molecular weight. As a result, the animal There is a problem that the physical properties and biochemical substances of the cartilage-derived extracellular matrix membrane are reduced.

Furthermore, the step of irradiating the radiation is preferably performed at a dose of greater than 0 and less than 10 kGy per hour, for example, may be performed at a dose of 100 Gy to 10 kGy per hour. When the irradiation dose per hour exceeds 10 kGy, collagen may be deformed and consequently a substantial decrease in collagen content may occur even when the total dose is 25 kGy or less.

The hydrogen peroxide aqueous solution used in the present invention may be a hydrogen peroxide aqueous solution having a concentration of 0.1 to 30 wt%, for example, greater than 3 wt% to 30 wt%, preferably 5 to 30 wt% or 10 to 30 wt%. can If the concentration of hydrogen peroxide in the aqueous hydrogen peroxide solution is less than 0.1 wt%, the effect of improving adhesion may be insufficient, and if it exceeds 30 wt%, protein denaturation and mechanical properties of animal cartilage-derived extracellular matrix may be deteriorated due to increased oxidation of proteins. There is a problem that comes off.

Compared to the extracellular matrix of other tissues, the extracellular matrix of cartilage contains lubricin, biglycan, digolin, fibromodulin, etc., which prevent cell adhesion in a tissue-specific manner, preventing adhesion between organs. effect can be improved. On the other hand, the material for the animal cartilage-derived extracellular matrix membrane that can be used in the present invention is preferably obtained from animals other than humans, for example, pigs, horses, cows, etc. It is not limited, and it is more preferable that it is a porcine cartilage-derived extracellular matrix membrane.

The animal cartilage-derived extracellular matrix membrane may be a chemically crosslinked animal cartilage-derived extracellular matrix membrane, and the crosslinking agent capable of performing the chemical crosslinking is not particularly limited, and glutaraldehyde, EDC (1-ethyl-3 -(3-dimethylaminopropyl)carbodiimide), genipin, etc., and in particular, it can be performed by glutaraldehyde.

According to the present invention, an animal cartilage-derived extracellular matrix membrane, the adhesiveness of which is controlled by the method for controlling the adhesiveness of an animal cartilage-derived extracellular matrix membrane, is provided.

Control of the adhesiveness of animal cartilage-derived extracellular matrix membranes should be premised on maintaining the components and desirable physical properties of animal cartilage-derived extracellular matrix membranes as much as possible. This is maintained without being deformed, and desirable physical properties can be maintained.

Since the main component of the animal cartilage-derived extracellular matrix membrane is collagen, when deformation or denaturation of collagen occurs, the adhesion of the animal cartilage-derived extracellular matrix membrane is controlled, but the physical properties are deteriorated.

On the other hand, the cartilage-derived extracellular matrix membrane of animals whose adhesiveness is controlled according to the present invention can minimize deformation and denaturation of collagen in the process for adjusting the adhesiveness, while having adhesiveness in the range of 1.0 to 2.5 N.

When an aqueous hydrogen peroxide solution is applied to one side of the animal cartilage-derived extracellular matrix membrane, the adhesive strength of the corresponding side is significantly improved, so that the animal cartilage-derived extracellular matrix membrane can be used as an anti-adhesion agent in the required area without performing additional suturing. .

For example, the animal cartilage-derived extracellular matrix membrane with controlled adhesiveness may be a membrane with controlled adhesiveness on one side, and in this case, after surgery on an internal organ, the side with improved adhesiveness faces the surgical site so that additional suturing is performed. Even though it is not required, the side with low adhesive strength faces the outside, so it can effectively perform the role of preventing adhesion.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. Preparation of cartilage-derived extracellular matrix powder

To prepare cartilage-derived extracellular matrix powder from pigs, EN 12442 "Animal tissues and their derivatives utilized in the manufacture of medical devices, part 1; Analysis and management of risk, part 2; controls on sourcing, collection and handling" was followed. For reference, porcine knee cartilage from a facility that meets the criteria was purchased and used.

Cartilage was cut from pig cartilage to make cartilage pieces (about 20 X 30 mm), washed three times for 10 minutes using physiological saline, frozen at -80 ° C, and then freeze-dried for 3 days. The dried cartilage pieces were freeze-pulverized into a size of about 10 μm using a freeze mill (JAI, JFC-300, JAPAN) and stored at -80°C.

In order to remove cells and genetic material present in the porcine cartilage powder and obtain only pure extracellular matrix components, a decellularization process was performed as follows.

To this end, the porcine cartilage powder prepared above was treated with 500 ml of hypotonic buffer per 10 g and stirred at 200 rpm at 4° C. for 4 hours. In order to precipitate and separate the cartilage powder, it was treated for 30 minutes at 10,000 rpm using a centrifuge (US-21SMT, Vision, Korea). After removing the supernatant, cartilage powder was added to a 0.1% SDS (sodium dodecyl sulfate, Bio-rad, USA) solution and stirred at 200 rpm at 4°C for 2 hours. After the SDS treatment, the cartilage powder was repeatedly washed 5 times with tertiary distilled water, and the washing water was exchanged under the same centrifugation conditions as described above. Next, 200 ml of 500 U/ml DNase (Sigma, USA) was treated and stirred at 200 rpm in a 37° C. incubator for 12 hours. After Dnase treatment, the mixture was washed 5 times with tertiary distilled water as described above. The decellularized cartilage powder was cooled in a cryogenic freezer at -80° C. and then freeze-dried for 3 days. The dried cartilage powder was crushed in the same manner as described above to finally obtain a cartilage powder having a size of about 10 μm and stored at -80°C until needed.

100 ml of an aqueous hydrochloric acid solution per 4 g of decellularized cartilage powder was treated with pepsin (Pepsin; sigma, USA) and stirred at 200 rpm at 4° C. for 24 hours. After pepsin treatment, it was neutralized to pH 7.4 using NaOH solution. The solubilized cartilage powder was placed in a dialysis membrane (MWCO 1000, Spectrolab, USA) and then stirred in tertiary distilled water at 200 rpm at 4°C for 24 hours. Thereafter, the water-soluble cartilage powder was placed in a container, cooled in a cryogenic freezer at -80 ° C, and then freeze-dried for 3 days. The water-soluble cartilage powder was pulverized into powder having a size of about 10 μm in the same manner as described above, and finally stored in a -80° C. freezer until needed. The cartilage-derived extracellular matrix powder thus obtained is water-soluble.

2. Preparation of porcine cartilage-derived extracellular matrix (PCAM) membrane

Example 1

1.3 g of the water-soluble porcine cartilage powder obtained in 1. above was treated with 100 ml of tertiary distilled water and stirred at 200 rpm at room temperature for 1 hour. After putting the aqueous solution of pork cartilage powder into a centrifuge container, it was treated at 3000 rpm for 10 minutes. The supernatant was dispensed into 100 X 100 mm square silicon molds by 35 ml each using a pipette, and then dried on a clean bench for 48 hours. 1 ml of 0.1% glutaraldehyde solution (Sigma, USA) per 6 mg of the dried membrane was treated and stirred at 100 rpm at room temperature for 1 hour. The crosslinked membrane was washed three times with 1 ml of PBS solution per 6 mg at 100 rpm for 30 minutes, and then washed three times with tertiary distilled water. The washed membrane was treated with 1 ml of 4M NaCl solution per 6 mg at 100 rpm at room temperature for 30 minutes. The washed membrane was spread on a Teflon film in a clean bench and dried to finally prepare a porcine cartilage extracellular matrix membrane having a thickness of about 30 μm.

Furthermore, additional crosslinking was performed by irradiating radiation to the porcine cartilage extracellular matrix membrane chemically crosslinked with glutaraldehyde obtained above.

At this time, the radiation was gamma rays ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Jeongeup Radiation Research Center, Korea Atomic Energy Research Institute), and irradiated at 25 kGy (dose rate; 10 kGy/hr).

Comparative Example 1

An anti-adhesion film was manufactured in the same manner as in Example 1, except that glutaraldehyde solution treatment was not performed and radiation was not irradiated during the manufacturing process of the anti-adhesion film described in 3. above.

Comparative Example 2

The hydrogen peroxide aqueous solution was treated in the same manner as in Example 1, except that radiation was not irradiated during the manufacturing process of the anti-adhesion film described in 3. above.

Comparative Example 3

An anti-adhesion film was prepared by irradiating radiation in the same manner as in Example 1, except that the glutaraldehyde solution treatment was not performed during the manufacturing process of the anti-adhesion film described in 3. above.

3. Preparation of hydrogen peroxide solution

Aqueous hydrogen peroxide solutions were prepared by combining distilled water and 50% hydrogen peroxide (H ₂ O ₂ ) in various weight ratios. In this experiment, 0.3 to 30% hydrogen peroxide (H ₂ O ₂ ) was used. Meanwhile, distilled water was used as a control.

4. Application of aqueous hydrogen peroxide solution to cross-linked porcine cartilage decellularized extracellular matrix (PCAM) membrane

The hydrogen peroxide aqueous solution of various concentrations prepared in 3. was placed in a 50 ml glass bottle, and the anti-adhesion film, which is the cross-linked porcine cartilage decellularized extracellular matrix (PCAM) film obtained in 2., was immersed at 37 ° C. for 2 hours, respectively. The results are shown in Figure 1.

Comparative Example 1 (Non-radiated CAM, GA-X), in which both crosslinking by irradiation and crosslinking with glutaraldehyde were not performed, was suspended in the form of particles while losing the film shape, and was not irradiated but crosslinked with glutaraldehyde. (Non-radiated CAM, GA-O) Comparative Example 2 was confirmed to maintain the film shape, and was irradiated with 25 kGy of radiation of Comparative Example 3 but not crosslinked with glutaraldehyde (Radiated CAM (25 kGy), GA-X ) It was confirmed that the film was physically crosslinked by radiation, as the shape was maintained, and the crosslinked glutaraldehyde (Non-radiated CAM, GA-O) irradiated with 25 kGy radiation of Example 1 was in the form of a film. It was confirmed that there was no change.

5. Morphological Analysis of Porcine Cartilage Extracellular Matrix (PCAM) Membrane

After vacuum coating the surface of the film treated with hydrogen peroxide solution by the same process as in 4. with gold, the surface was imaged and analyzed through a scanning electron microscope (SEM, Scanning Electron Microscopy), and the results are shown in FIG. was

As shown in FIG. 2, the film of Comparative Example 1 lost its film shape and was suspended in particle form, making it impossible to measure, while the film of Comparative Example 2 was crosslinked with glutaraldehyde without irradiation (Non-radiated CAM, GA- O) As the hydrogen peroxide concentration increased, the collagen in the CAM was oxidized and the collagen fiber structure was crushed.

On the other hand, in Comparative Example 3 (Radiated CAM (25 kGy), GA-X), the film shape was maintained, and in Example 1 (Non-radiated CAM, GA-O), it was confirmed that there was no change in the film shape. Double cross-linking by taraldehyde and radiation was confirmed.

6. Swellability analysis of porcine cartilage decellularized extracellular matrix film

For the film obtained in Example 1, after adjusting the concentration of the hydrogen peroxide aqueous solution in 3. to 0.3%, 3% and 30%, the film was immersed in each concentration of hydrogen peroxide aqueous solution at 37 ° C. for 2 hours, and then distilled water. It was washed three times and dried at room temperature for 2 days. As a result, the swelling properties of each sample are shown in FIG. 3 .

For swelling stone analysis, distilled water was poured so that each dried film (first weighed) in the Petri dish was immersed in distilled water, and then the distilled water was removed after 120 minutes. Then, the Petri dish weighed together with the swollen film was placed on an analytical balance and the closest weight value was recorded. The value of swelling property was calculated by the following equation.

Swellability (%)=[(W _s -W _d )/(W _d )]*100

(Where, W _s represents a swollen film, and W _d represents a dried film)

As a result, as shown in Figure 3 (a), the film of Example 1 confirmed a significant increase in the moisture content of the film as the hydrogen peroxide concentration increased compared to the film of Comparative Example 2, and as shown in Figure 3 (b), the comparison When the film irradiated with 25 kGy of Example 3 but not cross-linked with glutaraldehyde (Radiated CAM (25 kGy), GA-X) was immersed in 30% hydrogen peroxide, the collagen fiber structure of CAM was destroyed by high oxidation power, resulting in a film It was confirmed that there is a problem of destroying the shape of However, in the case of the film soaked in 0.3 to 3% hydrogen peroxide, it was confirmed that the swelling property increased because the radiation crosslinking reaction acted more than the oxidation reaction of hydrogen peroxide.

7. Analysis of chemical bonding of porcine cartilage decellularized extracellular matrix film

In order to measure the chemical change in the film according to irradiation and application of an aqueous hydrogen peroxide solution, the films obtained from Example 1 and Comparative Examples 2 and 3 were dried at room temperature to analyze components and FT-IR (Tensor 37, Bruker, Germany) was analyzed, and as a result, as can be seen in FIG. 4, component analysis and chemical structure were analyzed, and it was confirmed that there was no change in the surface of the film.

In particular, since the positions of 3700-3000 cm ^-1 and 1600-1700 cm ^-1 , which are amide peaks of proteins of porcine cartilage extracellular matrix, do not change, it can be interpreted that the original physical properties are maintained because there is no change in composition and chemical structure. there is. On the other hand, it was confirmed that the 3000 cm ^-1 peak increased due to the formation of OH radicals as the protein was oxidized by hydrogen peroxide. From the results of this experiment, it can be seen that there is no change in chemical structure and composition for radiation dose and oxidizing agent.

8. Measurement of adhesion of porcine cartilage decellularized extracellular matrix (PCAM) film

In order to measure the change in adhesive strength of the porcine cartilage decellularized extracellular matrix film according to the concentration of the hydrogen peroxide aqueous solution, the dried film was cut into 1 cm x 1 cm same size and then analyzed using a texture analyzer (TX-XT2i, sable microsystem Co. Ltd. Surrey England) It was attached to the bottom of the pressure rod and fixed to the rat skin, and the pressure applied when the rod went up was measured after 10 minutes.

As a result, as can be seen in FIG. 5, in the film of Example 1, as the concentration of hydrogen peroxide increases, the cross-linked structure is decomposed and the water content is increased. could be observed to increase.

On the other hand, in the film of Comparative Example 2, it was confirmed that the increase in adhesive strength according to the increase in the concentration of hydrogen peroxide was not significant.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention described in the claims. It will be obvious to those skilled in the art.

Claims (13)

A method for regulating the adhesiveness of an animal cartilage-derived extracellular matrix membrane comprising the step of applying an aqueous hydrogen peroxide solution having a concentration greater than 3 to 30 wt% to at least a portion of the animal cartilage-derived extracellular matrix membrane irradiated with radiation.
The method of claim 1, wherein the irradiated animal cartilage-derived extracellular matrix membrane is obtained by irradiating the animal cartilage-derived extracellular matrix membrane with radiation at a total dose of greater than 0 and less than 50 kGy. control method.
delete delete The method of claim 1, wherein the radiation is selected from the group consisting of gamma rays, electron rays, ultraviolet rays, and X-rays.
The method of claim 1, wherein the step of applying the aqueous hydrogen peroxide solution is performed by at least one of dipping, spraying, and coating.
The method of claim 1, wherein the step of applying the aqueous hydrogen peroxide solution is performed entirely or partially on both sides or one side of the animal cartilage-derived extracellular matrix membrane.
The method of claim 1, wherein the animal cartilage-derived extracellular matrix membrane is a porcine cartilage-derived extracellular matrix membrane.
The method of claim 1, wherein the animal cartilage-derived extracellular matrix membrane is a chemically cross-linked animal cartilage-derived extracellular matrix membrane.
10. The method of claim 9, wherein the chemical crosslinking is performed by glutaraldehyde.
An animal cartilage-derived extracellular matrix membrane, the adhesiveness of which is controlled by the method for controlling the adhesiveness of an animal cartilage-derived extracellular matrix membrane according to any one of claims 1, 2, and 5 to 10.
The animal cartilage-derived extracellular matrix membrane according to claim 11, wherein the adhesive force of the animal cartilage-derived extracellular matrix membrane with controlled adhesive force is in the range of 1.0 to 2.5 N.
The animal cartilage-derived extracellular matrix membrane according to claim 11, wherein one side of the animal cartilage-derived extracellular matrix membrane having controlled adhesiveness has a controlled adhesiveness.

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