KR102157742B1 - Method for preparing hydrogel for preventing surgical adhesions, hydrogel, film and porous material using the same - Google Patents

Abstract

The present invention relates to a method of preparing a hydrogel for preventing long-term adhesion, a hydrogel using the same, a film, and a porous material, and more particularly, a mixture of 0.5 wt% to 10 wt% of an animal cartilage-derived extracellular matrix powder and the remainder of the solvent A method of preparing an injection-type hydrogel for preventing long-term adhesion, comprising the step of preparing a radiation and irradiating a radiation of 1 to 50 kGy to the mixture, an injection-type hydrogel for preventing long-term adhesion prepared by this method, and a drying step. It relates to a method of manufacturing a dry hydrogel for preventing long-term adhesion, a film for preventing long-term adhesion obtained as a result, and a porous material for preventing long-term adhesion.

Description

Manufacturing method of hydrogel for preventing long-term adhesion, hydrogel, film, and porous material using the same.

It relates to a method of manufacturing a hydrogel for preventing long-term adhesion, a hydrogel, a film, and a porous material using the same.More specifically, an extracellular matrix derived from animal cartilage is used as the main material, and any additional material for crosslinking and controlling physical properties. It does not include a safe and efficient long-term adhesion prevention hydrogel composition having appropriate physical properties using ionization energy, and a film and a porous material using the same.

Cartilage, like other connective tissues, is composed of connective tissue cells and extracellular matrix, but unlike intrinsic connective tissues, cartilage is a special connective tissue that contains a hard and flexible matrix. The connective tissue cells of cartilage are mostly composed of one type of cartilage cell or chondrocyte, and they are located in the cartilage lacuna, an empty space in the cartilage matrix. The extracellular matrix is a lymphatic, avascular, and innervous tissue, which is divided into fibers and ground substances, and has tissue-specific differences compared to the extracellular matrix of other tissues. The fiber component is mostly composed of type II collagen (type II collagen), but some cartilage is rich in elastic fibers, and the intangible material is mainly sulfated glycosaminoglycan. Constituent protein sugar (proteoglycan) is the main component. In the cartilage matrix, a large number of protein sugar molecules are linked to hyaluronic acid, one of the glycosaminoglycans, by a link protein to form a macromolecule. This macromolecule is also bound to a collagen fiber. Protein sugar molecules are attached to the glial fibers by chondronectin, an intangible glycoprotein.

In general, glutaraldehyde, a chemical crosslinking agent, is used to improve the physical properties of the cartilage-derived extracellular matrix, but glutaraldehyde has a problem in that it is inconvenient to remove after washing and that it causes cytotoxicity and inflammation due to residuals. .

On the other hand, Korean Patent Publication No. 10-2017-0110882 discloses a hydrogel for preventing organ adhesion and a hydrogel film using the same, and the extracellular matrix component derived from animal cartilage is the main component that plays a role in preventing organ adhesion, but only these components are irradiated with radiation. Since gelation is not performed by the gel, the physical properties are not suitable for use to prevent long-term adhesion, and rather contains a large amount of sodium carboxymethylcellulose (CMC) as the main component excluding the solvent, and as a result, the result of film form when irradiated with radiation. Only is obtained.

In addition, Korea Laid-Open Publication No. 10-2017-0114692 relates to a composition for preventing long-term adhesion and an injection-type hydrogel using the same, and also contains a large amount of sodium carboxymethylcellulose (CMC) for gelation, and manufacture of an injection-type hydrogel. For this, a technology for controlling physical properties by including polyethylene glycol (PEG) as an additional component is disclosed.

However, in the case of the above-described prior art, since a large amount of components for controlling physical properties are included than the components for controlling physical properties than the components of the cartilage-derived extracellular matrix of animals that function to prevent adhesion, the extracellular matrix components derived from the cartilage of the animals are the main component, but the physical properties of the hydrogel are controlled. If technology is provided, it is expected to be widely applied in related fields.

Accordingly, one aspect of the present invention is to provide a hydrogel composition for preventing long-term adhesion, comprising an animal cartilage-derived extracellular matrix as a main component, and controlling physical properties.

Another aspect of the present invention is to provide a method of preparing a hydrogel composition for preventing long-term adhesion, comprising an animal cartilage-derived extracellular matrix as a main component as described above, and controlling physical properties.

Another aspect of the present invention is to provide a method of manufacturing a dry hydrogel for preventing long-term adhesion using the hydrogel of the present invention.

Another aspect of the present invention is to provide a film for preventing long-term adhesion prepared using the hydrogel composition for preventing long-term adhesion.

Another aspect of the present invention is to provide a porous material for preventing long-term adhesion prepared by using the hydrogel composition for preventing long-term adhesion.

Accordingly, according to one aspect of the present invention, preparing a mixture of 0.5 wt% to 10 wt% of animal cartilage-derived extracellular matrix powder and the remainder of the solvent; And there is provided a method for producing an injection-type hydrogel for preventing long-term adhesion, comprising the step of irradiating the mixture with radiation of 1 to 50 kGy.

According to another aspect of the present invention, there is provided an injection-type hydrogel for preventing long-term adhesion prepared by the method of the present invention.

According to another aspect of the present invention, the step of preparing the hydrogel of the present invention; And there is provided a method of manufacturing a dry hydrogel for preventing long-term adhesion, comprising the step of drying the hydrogel.

According to another aspect of the present invention, there is provided a film for preventing long-term adhesion obtained by naturally drying the injection-type hydrogel for preventing long-term adhesion prepared by the present invention at a temperature of 5 to 35°C.

According to another aspect of the present invention, there is provided a porous material for preventing long-term adhesion obtained by freeze-drying the injection-type hydrogel for preventing long-term adhesion prepared by the present invention.

According to the present invention, a technique for controlling the physical properties of the hydrogel by including the cartilage-derived extracellular matrix component of an animal that has an anti-adhesion function as a main component, and involves physical and chemical crosslinking, is provided to prepare a hydrogel with improved volume compatibility. I can.

1 schematically shows a process chart of manufacturing an irradiated cartilage-derived extracellular matrix hydrogel.
2 is a photograph of a radiation-crosslinked injection-type cartilage-derived extracellular matrix hydrogel.
3 is a photograph showing the change in radiation-crosslinked membrane-type cartilage-derived extracellular matrix hydrogel according to the radiation dose and the concentration of cartilage-derived extracellular matrix.
Figure 4 is a photograph showing the change in diameter according to the radiation dose of the radiation-crosslinked membrane-type cartilage-derived extracellular matrix hydrogel.
5(a) is a graph showing the gelation rate according to the concentration of cartilage-derived extracellular matrix upon irradiation, and FIG. 5(b) is a graph showing the gel strength according to the concentration of cartilage-derived extracellular matrix upon irradiation.
Figure 6 (a) is a graph showing the gelation rate according to the cartilage-derived extracellular matrix solvent during irradiation, and Figure 6 (b) is a graph showing the gelation rate of cartilage-derived extracellular matrix according to the solvent (PBS) concentration during irradiation 6(c) is a graph showing the gelation rate of cartilage-derived extracellular matrix according to the concentration of the solvent (SBF) upon irradiation.
7(a) is a graph showing the gel intensity of cartilage-derived extracellular matrix according to the type of solvent during irradiation, and FIG. 7(b) is a graph showing the gel intensity of cartilage-derived extracellular matrix according to the solvent (PBS) concentration during irradiation. It is a graph, and FIG. 7(c) is a graph showing the gel strength of cartilage-derived extracellular matrix according to the concentration of the solvent (SBF) upon irradiation with radiation.
8 is a photograph showing the morphological structure inside the lyophilized CAM hydrogel.
9 is a photograph showing a room temperature dried CAM hydrogel.
FIG. 10 is a photograph showing the procedure of a radio-crosslinked cartilage-derived extracellular matrix hydrogel implantation experiment in a mouse subcutaneous.
11 shows the results of observation of H&E and SO stained tissues of a cartilage-derived extracellular matrix hydrogel implanted for 7 days in a mouse subcutaneous tissue.
FIG. 12 shows the results of H&E and SO staining of a cartilage-derived extracellular matrix hydrogel implanted for 28 days in a mouse subcutaneous tissue.

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

According to the present invention, additional components such as sodium carboxymethylcellulose (CMC) and polyethylene glycol (PEG) are not required to control the gelation and physical properties of the hydrogel, and the extracellular matrix component derived from animal cartilage is the main component. There is provided a method of manufacturing a hydrogel in which physical properties of the hydrogel are variously controlled.

In more detail, the method of manufacturing the injection-type hydrogel for preventing long-term adhesion of the present invention comprises a mixture of 0.5 wt% to 10 wt% of cartilage-derived extracellular matrix (CAM) powder and the remainder of the solvent. Preparing; And irradiating the mixture with radiation of 1 to 50 kGy.

According to the manufacturing method of the hydrogel of the present invention, the gelation of the injection-type hydrogel for preventing adhesion and the degree of decomposition thereof can be controlled by the solvent and the radiation dose.

In the step of preparing the mixture, the mixture is a mixture of 0.5 wt% to 10 wt% of animal cartilage-derived extracellular matrix powder and the remainder of the solvent, preferably 2 wt% of cartilage-derived extracellular matrix powder To 10 wt%.

In the crosslinking reaction of hydrogels, H or OH radicals, which are secondary free radicals generated by radiolysis of water molecules, form radicals in the molecular chain of CAM, resulting in gelation with a three-dimensional network structure containing water. Therefore, when the cartilage-derived extracellular matrix powder is contained in an amount of less than 0.5 wt%, the gelation of the injection-type hydrogel for preventing adhesion tends to be insufficient, and when it exceeds 10 wt%, the viscosity of the animal cartilage-derived extracellular matrix is There is a problem that it increases and does not dissolve 100%, resulting in a non-uniform solution.

On the other hand, the solvent which can be used in the present invention is water or a ^{Na +, K +, Mg 2+} , Ca 2+, Cl - consisting, HPO ₄ ^2-, HPO ₄ ²⁺ and SO ₄ ^{2- -,} HCO ₃ It is preferably an aqueous solution containing at least one ion or salt selected from the group, but is not limited thereto.

Aqueous solution containing the ions or salts, for ^{example, 142.0 mM Na +, 5.0 mM} K +, 1.5 mM Mg 2+, 2.5 mM Ca 2+, 148.8 mM Cl -, 4.2 mM HCO 3 -, 1.0 mM HPO4 ^It may be a simulated body fluid (SBF) containing ^2- and 0.5 mM SO ₄ ^2- , phosphate buffered saline (PSB), or a combination thereof.

Further, the solvent is preferably a pH of 7.40±0.04, and if the pH is lower than this, there is a problem that the protein is transformed in the acidity of the CAM components, and in the case of high, the extracellular matrix components derived from animal cartilage have a basic molecular weight. There is a diminishing problem.

The manufacturing method of the injection-type hydrogel for preventing long-term adhesion of the present invention includes preparing a mixture of an animal cartilage-derived extracellular matrix powder and the remainder of the solvent, and subsequently irradiating the mixture with 1 to 50 kGy of radiation. For example, the irradiation of the radiation may be performed at a dose of 25 to 50 kGy.

In performing the step of irradiating the radiation, if the dose is less than 1 kGy, gelation of the hydrogel tends to be insufficient, and if it exceeds 50 kGy, the protein in the extracellular matrix derived from cartilage of the animal is modified through an oxidation reaction. There is a problem that the molecular weight of collagen is reduced.

In this case, the step of irradiating the radiation is performed by radiation selected from the group consisting of gamma rays, electron rays, ultraviolet rays, and X-rays, and is preferably performed by gamma rays. In addition, the gamma ray 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, the gamma ray may be cobalt (Co)-60, but is not limited thereto.

In the extracellular matrix of cartilage, there are lubrisine, biglycan, dicolin, fibromodulin, etc. that prevent cell adhesion specific to the tissue compared to the extracellular matrix of other tissues, preventing adhesion between organs and organs. The effect can be improved. On the other hand, the material for the extracellular matrix membrane derived from cartilage of an animal that can be used in the present invention can be obtained from animals other than humans, and for example, can be obtained from animals such as pigs, horses, cows, etc. It is not limited, it is more preferable that it is a porcine cartilage-derived extracellular matrix membrane.

According to the present invention, there is provided an injection-type hydrogel for preventing long-term adhesion, manufactured by the method of manufacturing an injection-type hydrogel for preventing long-term adhesion as described above.

The hydrogel of the present invention is physicochemically crosslinked by controlling the degree of ionization of the molecular chains of the bio-derived extracellular matrix in various solvents using radiation ionization energy, and is a material for preventing long-term adhesion with excellent biocompatibility having appropriate physical properties and biodegradability.

Further, there is provided a method of manufacturing a dry hydrogel for preventing long-term adhesion, further comprising a step of drying the hydrogel subsequent to the method for producing an injection-type hydrogel for preventing long-term adhesion as described above.

According to the method of manufacturing a dry hydrogel for preventing long-term adhesion of the present invention, various types of medical biomaterials such as a dry film and a porous material can be obtained.

Drying the hydrogel may be performed by freeze drying or natural drying at a temperature of 5 to 35°C.

In more detail, according to the present invention, a film for preventing long-term adhesion obtained by naturally drying the injection-type hydrogel for preventing long-term adhesion prepared by the method of manufacturing the injection-type hydrogel for preventing long-term adhesion of the present invention at a temperature of 5 to 25°C is provided. I can.

The injection-type hydrogel for preventing adhesion as described above may be suitably used, for example, for fixing the vocal cord's ringing plate during vocal cord nodules.

According to the present invention, there can be provided a porous material for preventing long-term adhesion obtained by freeze-drying an injection-type hydrogel for preventing long-term adhesion, which is produced by the method of manufacturing an injection-type hydrogel for preventing long-term adhesion of the present invention.

The porous material as described above has a shape similar to that of a sponge, for example, a cell support for tissue engineering, a dental shield, artificial skin, an anti-adhesion agent, an ophthalmic cornea and conjunctiva treatment material, a cornea treatment agent, a basic treatment for cartilage damage. Materials, bone marrow stimulation auxiliary materials for cartilage damage treatment (surgical area cover), insertion gel for vocal cord prosthesis, replenishment of spinal nucleus, and basic materials for therapeutic insertion gels, etc. can be suitably used.

As described above, according to the present invention, there is provided a technique for controlling the physical properties of the hydrogel by including an animal cartilage-derived extracellular matrix component as a main component, and physical and chemical crosslinking, thereby improving the volumetric compatibility. , It is possible to manufacture various types of biomaterials based on this.

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

Example

The present invention uses radiation ionization energy to dissolve the decellularized extracellular matrix of animal cartilage, which is a living body-derived extracellular matrix, with various solvents (distilled water (H ₂ O), PBS, SBF), and then dissolve the aqueous solution to the radiation ionization energy irradiation dose. It relates to the manufacture of a cartilage-derived extracellular matrix hydration gel that can control the gelation rate and gel strength by adjusting the degree of crosslinking accordingly, and it is effective because it can give a sterilization effect even in the sealed state during the irradiation process. It is possible to manufacture a safe cell therapy or a support for drug delivery and tissue engineering. The manufacturing process of the hydrogel composition for preventing long-term adhesion according to the present invention will be described in more detail below.

1. From cartilage Extracellular matrix Preparation of powder

In order to manufacture 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" For reference, a pig knee cartilage in a facility meeting the standards was purchased and used.

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

Cells and genetic materials present in the porcine cartilage powder were removed, and decellularization was performed as follows to obtain only pure extracellular matrix components.

To this end, the pork cartilage powder prepared above was treated with 500 ml of a stock solution (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, the cartilage powder was added to a 0.1% SDS (Sodium dodecyl sulfate, Bio-rad, USA) solution, followed by stirring at 200 rpm for 2 hours. After the SDS treatment was completed, the cartilage powder was repeatedly washed 5 times with 3rd distilled water, and the exchange of the washing water was performed under the above-described centrifugation conditions. 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, it was washed 5 times with 3rd distilled water as described above. The decellularized cartilage powder was cooled in a -80°C cryogenic freezer and freeze-dried for 3 days. The dried cartilage powder was crushed in the same manner as described above to finally obtain a cartilage powder powder having a size of about 10 μm and stored at -80°C until necessary.

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

2. Preparation of solvent

(1) Phosphate Buffered Saline aqueous solution preparation

The PBS buffer was prepared by dissolving 0.8 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO ₄ and 0.24 g KH ₂ PO ₄ in 800 ml of tertiary distilled water at room temperature, and then fixing the pH to 7.4 with 0.1 M HCl. 1L PBS aqueous solution was prepared by adding, and this is referred to as "1X PBS" buffer.

Further, "3X PBS" and "5X PBS" were additionally prepared, and these were concentrated to 3 times and 5 times the concentration of PBS in the "1X PBS" buffer, respectively.

(2) Preparation of an aqueous solution of simulated body fluid

SBF was prepared buffer ^{142.0 Na +, 5.0K +, 1.5} Mg 2+, 2.5 Ca 2+, 148.8 Cl in the distilled ^{water-ion _{concentration, 1.0 HPO 4 2-, 0.5 SO}} 4 2- (-, 4.2 HCO 3 mM) at the concentration of SBF (36.5 ^o C), NaCl, NaHCO ₃ , KCl, K ₂ HPO 3H ₂ O, MgCl ₂ 6H ₂ O, CaCl ₂ , Na ₂ SO ₄ After dissolving reagents in distilled water, finally As a result, tris-hydroxymethyl aminomethane (TRIS) and 0.1M HCl were used to prepare an SBF solution having a pH of 7.4 at 36.5° C., which is referred to as “1X SBF” buffer.

Further, "3X SBF" and "5X SBF" were additionally prepared, and these were concentrated to 3 times and 5 times the concentration of SBF in the 1X SBF" buffer, respectively.

3. Cartilage-derived Extracellular matrix (CAM) aqueous solution preparation

Each of the water-soluble porcine cartilage-derived extracellular matrix prepared in 1 above was used in 3rd distilled water (diH ₂ O, Comparative Preparation Example 1), and the phosphate buffered saline (PBS) prepared in 2 above, Preparation Example 1) , After treatment with the analog body fluid (Simulated body fluid (SBF), Comparative Preparation Example 2), the mixture was stirred at 500 rpm for 6 to 12 hours at 4°C. The concentration of the prepared cartilage-derived extracellular matrix aqueous solution was prepared at 0.5 to 10 wt%.

4. Cartilage-derived Extracellular matrix (CAM) irradiation of aqueous solution

(1) Preparation of injection-type hydrogel placed in a syringe

After putting the cartilage-derived extracellular matrix aqueous solution dissolved in various solvents into a 1 ml syringe and a 48-well cell culture plate, the radiation dose was irradiated at 0, 1, 5, 10, 25, and 50 kGy, respectively. At this time, the radiation was gamma ray ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Korea Atomic Energy Research Institute Jeongeup Radiation Research Institute), and was irradiated with 25 kGy (dose rate; 10 kGy/hr).

The results of irradiation with radiation are shown in Fig.2, and as shown in Fig.2(a), before irradiation, the cartilage-derived extracellular matrix aqueous solution was a transparent solution, but after irradiation, crystals crosslinked by crosslinking reaction of the molecular chains of the extracellular matrix It was confirmed that it changed to opaque white due to light reflection due to the castle structure. In addition, as shown in FIG. 2(b), it was confirmed that as the radiation dose and CAM concentration increased, the cartilage-derived extracellular matrix hydrogel was changed from a liquid (sol) to a solid (gel) state according to the degree of crosslinking.

(2) Manufacture of membrane hydrogel

After putting the cartilage-derived extracellular matrix aqueous solution dissolved in various solvents into a 48-well cell culture plate, radiation doses of 5, 7, 10 and 25 kGy were respectively irradiated. At this time, the radiation was gamma rays ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Korea Atomic Energy Research Institute Jeongeup Radiation Research Institute), and was irradiated with 25 kGy (dose rate; 10 kGy/hr).

The results of irradiation with radiation are shown in Fig. 3, and as shown in Fig. 3(a), before irradiation, the cartilage-derived extracellular matrix aqueous solution is a crosslinked crystal due to the crosslinking reaction of the molecular chains of the extracellular matrix after irradiation in a transparent solution. It was confirmed that the brightness of white increased as the degree of crosslinking increased due to the increase in the sexual structure. In addition, as shown in FIG. 3(b), as the concentration of cartilage-derived extracellular matrix increases, the crosslinking degree increases due to the increase in crosslinked crystalline structure due to crosslinking reaction of molecular chains of the extracellular matrix after irradiation. It was confirmed that the brightness increased.

As shown in FIG. 3(c), when the same volume of cartilage-derived extracellular matrix aqueous solution was added to the well of the 48-well cell culture plate, the thickness of the crosslinked hydrogel did not change even when the radiation dose and concentration were increased. Confirmed.

Meanwhile, as shown in FIG. 4, it was confirmed that the size of the cartilage-derived extracellular matrix hydrogel decreased as the gelation rate increased as the radiation dose and CAM concentration increased. The reason can be interpreted to be that the moisture content per unit area decreases because the crosslinking point of the three-dimensional network structure increases.

5. Cartilage-derived Extracellular matrix (CAM) According to concentration Hydrogel Gelation rate And Gel strength analysis

After putting the cartilage-derived extracellular matrix aqueous solution dissolved in various solvents into a 48-well cell culture plate, radiation doses of 5, 7, 10 and 25 kGy were respectively irradiated. At this time, the radiation was gamma rays ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Korea Atomic Energy Research Institute Jeongeup Radiation Research Institute), and was irradiated with 25 kGy (dose rate; 10 kGy/hr).

In order to remove the remaining CAM without participating in the crosslinking reaction by irradiation of radiation, it was washed with water at 30° C. for 72 hours using third distilled water. The crosslinked CAM hydrogel after washing with water was placed in a 70 ^o C oven and dried for 48 hours.

At this time, the gelation rate was expressed as a percentage by substituting the following equation (1) and dividing the weight ( W _d ) of the dried gel after irradiation with water by the weight ( W _i ) of the dried CAM before irradiation.

Gelation rate (%) = W _d / W _i × 100 (1)

In other words, when irradiated with gamma rays, the crosslinking reaction of the CAM hydrogel is a three-dimensional network containing water because H or OH radicals, which are secondary free radicals generated by radiolysis of water molecules, form radicals in the molecular chains of CAM. The structured gelation takes place. However, it was observed that the crosslinking reaction of CAM occurs less and the gelation rate of CAM increases at high gamma-ray doses because radicals are difficult to form in the molecular chains of CAM at low ionization energy doses.

Therefore, the radiation dose for preparing the hydrogel was tested using a dose of 5 to 50 kGy. When gamma rays were irradiated at a dose of 5 kGy, the gelation rate of 0.5 wt% CAM hydrogel using distilled water solvent was 67.4±2.6%, and it was confirmed that the gelation rate increased to 89.3±2.8% as the CAM concentration increased.

As described above, it was observed that the gelation rate of the crosslinked CAM hydrogel increased as the gamma ray dose increased. However, referring to FIG. 5(a), it was confirmed that the concentration of the extracellular matrix had a greater effect on the gelation rate of the cartilage-derived extracellular matrix than the radiation dose.

(2) gel strength

After putting the cartilage-derived extracellular matrix aqueous solution dissolved in various solvents into a 48-well cell culture plate, radiation doses of 5, 7, 10 and 25 kGy were respectively irradiated. At this time, the radiation was gamma rays ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Korea Atomic Energy Research Institute Jeongeup Radiation Research Institute), and was irradiated with 25 kGy (dose rate; 10 kGy/hr).

The mechanical properties of the hydrogel according to the radiation dose and the type of solvent of CAM crosslinked with radiation (gamma ray) were measured using a texture analyzer (TX-XT2i, stable microsystem Co. Ltd. Surrey England). In order to remove the remaining polymer without participating in the crosslinking reaction, the average thickness of the hydrogel immersed at 30° C. for 72 hours was 5 mm and the initial diameter was 20 mm, and 7 specimens were prepared and measured for each experimental group. When measuring the compressive strength, the cross head speed was 10 mm/min, and the value when the specimen was deformed by 50% was measured.

When irradiated with gamma rays, the crosslinking reaction of the CAM hydrogel is caused by the formation of a radical in the molecular chain of CAM, which is a secondary free radical generated by radiolysis of water molecules. Gelation is achieved with a mesh structure. However, it is difficult to form radicals in the molecular chain of CAM at a low ionization energy dose of gamma rays, so that the crosslinking reaction of CAM occurs less and the gel strength of CAM increases at high gamma radiation doses as shown in Fig. 5(b). Observed.

Therefore, in this study, an experiment was conducted by fixing a radiation dose of 5 to 50 kGy to prepare a hydrogel. When irradiated with a dose of 5 kGy of gamma rays, the gel strength of 0.5 wt% CAM hydrogel using distilled water solvent was 0.041±0.312%, and the gel strength increased to 0.520±0.044% as the CAM concentration increased.

As described above, it was observed that the gel strength of the crosslinked CAM hydrogel increased as the gamma ray dose increased. However, referring to FIG. 5(b), it was confirmed that the concentration of the extracellular matrix had a greater effect on the gelation rate of the cartilage-derived extracellular matrix than the radiation dose.

6. Depending on the solvent during irradiation Gelation rate and Gel strength

After putting the cartilage-derived extracellular matrix aqueous solution dissolved in various solvents into a 48-well cell culture plate, radiation doses were irradiated at 1, 25, and 50 kGy, respectively. At this time, the radiation was gamma rays ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Korea Atomic Energy Research Institute Jeongeup Radiation Research Institute), and was irradiated with 25 kGy (dose rate; 10 kGy/hr).

In order to check the gelation rate and gel strength of the crosslinked CAM hydrogel according to the type and concentration of the solvent during irradiation with radiation (gamma ray), the weight and strength of the hydrogel from which the remaining CAM was removed without participating in the crosslinking reaction was measured. Specific gelation rate and gel strength were calculated in the same manner as in 6.

As a result, as can be seen in FIGS. 6 and 7, at a gamma-ray dose with low ionization energy, it is difficult to form a cross-link between radicals and ionic substances in the molecular chain of CAM, so that the cross-linking reaction of CAM occurs less, and at high gamma-ray doses. It was observed that the double physicochemical crosslinking reaction was activated by the participation of ionized CAM, PBS, and SBF, thereby increasing the gelation rate and gel strength.

In particular, it was confirmed that as the concentration of ionic substances in PBS and SBF increased, the gelation rate and gel strength of CAM increased. In more detail, as can be seen in Figs. 6(a) and 7(a), the gelation rate of 0.5 wt% CAM dissolved in distilled water is 57.4±2.6% when gamma rays are irradiated at a dose of 1 kGy, and PBS and SBF It was increased to 80.5±3.2% by ionic substances in aqueous solution. In addition, as can be seen in Figs. 6(b) and 6(c), and Figs. 7(b) and 7(c), as the gamma ray dose and the concentration of ions increase, chemical crosslinking by ionic substances increases. As a result, it was observed that the gelation rate and gel strength of the CAM hydrogel increased.

7. Cartilage-derived Extracellular matrix (CAM) Hydrogel Morphological analysis according to drying method

(1) freeze drying

It was confirmed using a scanning electron microscope (FE-SEM, Hitachi S-4800, Japan) in order to observe the morphological characteristics of the radiation-crosslinked CAM hydrogel.

Cartilage-derived extracellular matrix aqueous solution dissolved in a concentration of 2wt% and 4wt% using a distilled water solvent was added to a 48-well cell culture plate, and then the radiation dose was irradiated at 5, 10, 25 and 50 kGy, respectively. At this time, the radiation was gamma rays ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Korea Atomic Energy Research Institute Jeongeup Radiation Research Institute), and was irradiated with 25 kGy (dose rate; 10 kGy/hr).

To remove the remaining polymer without participating in the crosslinking reaction, wash the hydrogel immersed for 72 hours at 30°C for more than 3 times with distilled water and store it in a -80 ^° C quick freezer for 2 days and then use a freeze dryer. It was dry. The dried sample was coated with platinum for 60 seconds using a sputter coater in order to obtain a high-resolution image, and confirmed by using a 15 kV electron beam and a distance of 8.3 mm.

As a result, the morphological structure inside the freeze-dried CAM hydrogel as shown in FIG. 8 is a three-dimensional porous structure created by sublimation of frozen water crystals when the hydrogel containing water in the three-dimensional network of CMC crosslinked by gamma rays is freeze-dried. Was observed to form.

On the other hand, as can be seen in FIG. 8, even if the radiation dose is increased, the pore sizes of the hydrogel having a porous structure are almost similar to each other, but in the 4 wt% CAM hydrogel rather than 2wt% CAM, the pore size decreases and the number of pores increases. Was observed. The reason for this is that the CMC hydrogel crosslinked by gamma rays is interpreted to be because the volume that can contain water decreases due to the increase in the mass of the CAM having a three-dimensional network structure inside the hydrogel as the CAM content increases.

(2) room temperature drying

It was confirmed using a scanning electron microscope (FE-SEM, Hitachi S-4800, Japan) in order to observe the morphological characteristics of the radiation-crosslinked CAM hydrogel.

Cartilage-derived extracellular matrix aqueous solution dissolved in a concentration of 2wt% and 4wt% using a distilled water solvent was added to a 48-well cell culture plate, and then the radiation dose was irradiated at 5, 10, 25 and 50 kGy, respectively. At this time, the radiation was gamma rays ( ⁶⁰ CO, MDS Nordion, Canada, IR 221 n wet storage type C-188, Korea Atomic Energy Research Institute Jeongeup Radiation Research Institute), and was irradiated with 25 kGy (dose rate; 10 kGy/hr).

In order to remove the remaining polymer without participating in the crosslinking reaction, the hydrogel immersed at 30° C. for 72 hours was washed three or more times with distilled water and then dried by a drying method at a temperature of 20° C. for 2 days.

After that, the structure was confirmed using a scanning electron microscope (FE-SEM, Hitachi S-4800, Japan) by the same process as in 7.(1). As a result, it was confirmed that the surface of the film was uniform as shown in FIG. 9.

8. In vivo Cartilage-derived Extracellular matrix (CAM) hydrogel Biodegradability evaluation

A dry hydration gel using a cartilage-derived extracellular matrix aqueous solution dissolved in a distilled water solvent at a concentration of 2 wt% CAM among the dried hydration gels prepared in 7. (1) was prepared as shown in Fig. 10 in rats (Rat, 7-8 weeks old). It was inserted into the subcutaneous tissue between the skin and the muscle and observed for 30 days.

Tissues were collected on the 7th and 28th days after transplantation, and the biodegradability of the cartilage-derived extracellular matrix hydrogel was observed in vivo through histological staining.

As can be seen in FIG. 11, which is the result of 7 days after transplantation, the CAM hydrogel was found to have low degradability in vivo because the crosslinking rate increased as the radiation dose increased. The crosslinked CAM at 50 kGy of radiation dose did not change morphologically, but as the dose decreased, the degree of crosslinking was lower, so it was confirmed that enzymatic degradation and hydrolysis in vivo were accelerated as the water content that could contain water increased. . On the other hand, no inflammatory cells were observed in the H&E and S-O stained tissues, and no growth of blood vessels penetrating from the outside to the inside was also observed.

On the other hand, as can be seen in Figure 12, which is the result of 28 days after transplantation, it was confirmed that all CAM hydrogels were absorbed into the body through enzymatic digestion and hydrolysis.

Therefore, it was confirmed that the radiation-crosslinked cartilage-derived extracellular matrix hydrogel according to the present invention decomposes the physically crosslinked chains of radiation through an in vivo decomposition mechanism within 30 days.

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 of ordinary skill in the field.

Claims (13)

As a solvent of the cartilage-derived extracellular matrix powder 0.5 wt% to 10 wt% and the balance animal ^{Na +, K +, Mg 2+} , Ca 2+, Cl -, HCO 3 -, HPO 4 2-, HPO 4 2+ And preparing a mixture of an aqueous solution containing at least one ion or salt selected from the group consisting of SO ₄ ^2- ; And
Irradiating the mixture with radiation of 1 to 50 kGy
Containing, a method of manufacturing an injection-type hydrogel for preventing long-term adhesion.
The method of claim 1, wherein the mixture comprises 2 wt% to 10 wt% of an animal cartilage-derived extracellular matrix powder.
delete The method of claim 1 wherein the solvent is ^{142.0 mM Na +, 5.0 mM K} +, 1.5 mM Mg 2+, 2.5 mM Ca 2+, 148.8 mM Cl -, 4.2 mM HCO 3 -, 1.0 mM HPO4 2- and 0.5 mM A method for producing an injection-type hydrogel for preventing long-term adhesion, which is a simulated body fluid (SBF), phosphate buffered saline (PSB), or a combination thereof containing SO ₄ ^2- .
The method of claim 1, wherein the solvent is pH 7.40±0.04, for preventing long-term adhesion.
The method of claim 1, wherein the irradiating the radiation is performed at a dose of 25 to 50 kGy.
The method of claim 1, wherein the irradiating the radiation is performed by radiation selected from the group consisting of gamma rays, electron rays, ultraviolet rays, and X-rays.
The method of claim 1, wherein the animal cartilage-derived extracellular matrix membrane is a porcine cartilage-derived extracellular matrix membrane.
An injection-type hydrogel for preventing long-term adhesion prepared according to any one of claims 1, 2, and 4 to 8.
Preparing a hydrogel obtained according to any one of claims 1, 2, and 4 to 8; And
Drying the hydrogel
Containing, a method for producing a dry hydrogel for preventing long-term adhesion.
The method of claim 10, wherein the drying of the hydrogel is performed by freeze drying or natural drying at a temperature of 5 to 35°C.
A film for preventing long-term adhesion obtained by naturally drying the injection-type hydrogel for preventing long-term adhesion prepared according to any one of claims 1, 2 and 4 to 8 at a temperature of 5 to 35°C.
A porous material for preventing long-term adhesion obtained by freeze-drying the injection-type hydrogel for preventing long-term adhesion prepared according to any one of claims 1, 2, and 4 to 8.

Images