KR101413510B1 - Method of preparing raw materials for transplantation using biocompatible polymers - Google Patents

Abstract

A hyaluronic acid derivative film containing a polymer containing a hydroxy (-OH) end group produced by reacting an epoxy crosslinking agent with a solution of a polymer containing a hydroxy (-OH) end group and hyaluronic acid has physical strength , Stability in the body, flexibility, adhesion to living tissues and biocompatibility.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a biocompatible polymer (biocompatible polymer)

The present invention relates to a method for producing a hyaluronic acid epoxide derivative film, and more particularly, to a method for producing a hyaluronic acid epoxide derivative film by reacting an epoxide crosslinking agent with a solution of a polymer containing a hydroxy (-OH) end group and hyaluronic acid, The present invention relates to a method for producing a hyaluronic acid derivative containing a polymer containing a hydroxy-terminated group in the form of a film having improved stability, flexibility, adhesion to biotissue and biocompatibility.

Hyaluronic acid, first isolated from the vitreous humor by Meyer and Palmer in 1934, is a biopolymer that is widely found in nature as a polyanionic mucopolysaccharide [Meyer K. et al ., Journal of Biology and Chemistry 107 629-34 (1934)]. Hyaluronic acid is present in abundance in the connective tissues of the placenta, eyes, and joints of animals, and is also produced in Streptococcus microorganisms such as Streptococcus equi, Streptococcus zooepidem i cus, and the like. A repeating unit in which glucuronic acid and N-acetylglucosamine are connected in a β (1,3) glycosidic bond is β (1) , 4) a long chain structure continuously connected by glycosidic bonds [Balazs EA et al ., Biochemical Journal , 235, 903, 1986; Toole BP et al ., Journal of Internal Medicine , 242, 35-40 (1997)).

Hyaluronic acid is excellent in biocompatibility and has high viscoelastic properties in a solution state, and is widely used not only for cosmetic applications such as cosmetic additives but also for a variety of medicines such as eye surgery aids, joint function improvers, drug delivery materials and eyedrops. However, hyaluronic acid itself is easily decomposed in vivo or in acid or alkali conditions and its use is limited. Therefore, much effort has been made to develop structurally stable hyaluronic acid derivatives [Laurent TC et al ., Portland Press Ltd, London, 1998).

Since hyaluronic acid derivatives have excellent biocompatibility, physical stability and biodegradability, they are being developed for various purposes such as molding aids, joint function improvers, drug delivery vehicles, cell culture scaffolds, and postoperative adhesion inhibitors.

Among the methods of derivatizing hyaluronic acid, the techniques of derivatization using an epoxide crosslinking agent have been developed in various forms such as solutions, gels, fibers, sponges, films, and the like. US Patent Nos. 4,500,676, 4,713,448, 4,716,224 , 4,716,154, 4,886,787, 4,963,666, 5,827,937, etc. disclose their preparation methods.

However, the hyaluronic acid epoxide derivative film has a problem of shrinkage during the drying process, which limits the production of a uniform film and weakens physical strength during purification.

In order to overcome the above problems of the production of the hyaluronic acid epoxide derivative film, Korean Patent Laid-Open Publication No. 2009-0012439 has developed a film having improved flexibility and physical strength by surface cross-linking of HA and CMC. However, When the physical strength is weak and the CMC composition rises due to the wet state, the rate of the biodegradation is slowed, and the CMC is left longer than necessary, causing a foreign matter reaction.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to solve the problems encountered in drying and refining hyaluronic acid derivatives without using the CMC derivative part, And to provide a method for producing a hyaluronic acid epoxide derivative film having high strength.

The hyaluronic acid epoxide derivative film produced by the present invention is characterized by excellent physical strength, stability in the body, flexibility, adhesion to living tissue and biocompatibility.

The above and other objects of the present invention can be achieved by the present invention described below.

Disclosure of the Invention The present invention has been conceived to solve the problems occurring in the prior art, and it is an object of the present invention to provide a hyaluronic acid epoxide derivative using a polymer containing a hydroxy end group to prevent shrinkage and physical strength deterioration The present invention provides a method for producing a hyaluronic acid epoxide derivative film, which has improved uniformity and excellent physical strength, stability in the body, flexibility, adhesion to living tissue, and biocompatibility.

Through the present invention, it is possible to improve uniformity of hyaluronic acid epoxide derivative film, physical strength, stability in body, flexibility, adhesion to living tissue and biocompatibility.

In addition, the hyaluronic acid epoxide derivative film of the present invention has excellent biocompatibility, biodegradability, and physical stability, and can be used for various purposes such as wound dressings, cell supports, drug delivery systems, antioxidant coating films, and tissue adhesion inhibitors.

FIG. 1 shows a structure in which a polymer having a hydroxy end group is physically bonded to a structure in which hyaluronic acid is crosslinked according to an embodiment of the present invention. After the purification, most of the polymer including a hydroxy end group is removed Is a schematic view showing a structure in a state in which
2 is a schematic view showing a process for producing a hyaluronic acid epoxide derivative film containing a polymer containing a hydroxy end group of the present invention.
Fig. 3 is a photograph showing a comparison of the film form with or without a polymer containing a hydroxy end group according to Test Example 1 of the present invention.
4 is a graph showing an IR spectrum of a hyaluronic acid epoxide derivative film containing a polymer containing a hydroxy end group according to Test Example 2 of the present invention.
FIG. 5 is a graph showing the results of a comparison between a hyaluronic acid epoxide derivative film (experimental groups 1, 2 and 3) and commercialized adhesion inhibitor Interceed (comparison group 1) and a bone regeneration membrane Bio-Gide (comparison group 2) Fig.
6 is a chart comparing the tensile strength of the hyaluronic acid epoxide derivative film according to Test Example 4 of the present invention with or without an organic solvent treatment.
FIG. 7 is a photograph showing a comparison of the remaining duration of the hyaluronic acid epoxide derivative film in vivo according to Test Example 5 of the present invention through tissue staining.
Fig. 8 shows the results of evaluation of the anti-adhesion performance in Experimental Example 6 in which the experimental group (three kinds) using the hyaluronic acid epoxide derivative film of the present invention, Interceed (comparative group 1), commercialized anti- A graph showing the degree of adhesion and the adhesion strength in the unused control group.
Fig. 9 shows the results of the animal experiment of Test Example 7 in which the degree of bone regeneration was evaluated. The experimental group 3 using the hyaluronic acid epoxide derivative film of the present invention, the Bio-Gide (comparative group 2) This is a photograph showing the degree of bone regeneration in the control group in which no treatment was performed.
10 is a photograph showing antimicrobial activity against strains of Bacillus substilis, Escherichia coli and Candida albicans in the microorganism test of Test Example 8 for confirming the antibacterial performance.

The present invention provides a process for producing a hyaluronic acid epoxide derivative film containing a polymer containing a hydroxy (-OH) end group.

Hereinafter, the present invention will be described in detail.

Specifically, the present invention is a crosslinked hyaluronic acid using an epoxide crosslinking agent having two or more epoxide groups by mixing a polymer containing hyaluronic acid and a hydroxy end group. As shown in FIG. 1, the polymer containing a hydroxy end group prevents the shrinkage phenomenon in the process of solidifying into a film and gradually removes it in the purification process to prevent the shape of the hyaluronic acid derivative film from being deformed.

In addition, the hyaluronic acid epoxide derivative can be purified and then precipitated in an organic solvent to improve the physical strength weakened during the purification (Fig. 6).

The hyaluronic acid epoxide derivative film has a hyaluronic acid content of 50 to 90% by weight and a polymer containing the hydroxy end group of 10 to 50% by weight.

The crosslinking density of the hyaluronic acid epoxide derivative film may be from 1 mol% to 100 mol%, and more preferably from 5 mol% to 50 mol%. When the crosslinking density is less than 1 mol%, the water absorbing ability is high and the shape deformation occurs and the refining is difficult. When the crosslinking density is more than 100 mol%, the possibility of remaining a large amount of crosslinking agent is increased.

The hyaluronic acid epoxide derivative film comprises the steps of: (a) mixing a basic aqueous solution with a polymer containing hyaluronic acid and a hydroxy end group and reacting the polymer with an epoxide crosslinking agent to prepare a derivative solution; (b) drying the derivative solution with a film; (c) purifying the film and precipitating it in an organic solvent; And (d) drying the purified derivative.

More specifically, the hyaluronic acid epoxide derivative film of the present invention can be produced by (i) reacting a basic aqueous solution with a polymer containing hyaluronic acid, a hydroxy end group and an epoxide crosslinking agent to produce an epoxide derivative solution ; (ii) drying the epoxide derivative solution to prepare a primary film; (iii) purifying the primary film to remove an unreacted polymer containing a hydroxyl end group and a crosslinking agent; (iv) precipitating the purified primary film in an organic solvent to prepare a secondary film; And (v) re-swelling and drying the secondary film.

In the above preparation method, 50 to 500 parts by weight of the polymer containing the hydroxy end group is used relative to 100 parts by weight of hyaluronic acid. When the polymer containing hydroxy end groups is less than 50 parts by weight, the solution of the hyaluronic acid epoxide derivative does not prevent shrinkage due to drying of the film, and when it exceeds 500 parts by weight, the polymer containing hydroxy end groups is removed. There arises a problem that a long time is taken and a polymer containing a large amount of hydroxy end groups remains on the film after the purification.

In the present invention, the hyaluronic acid epoxide derivative can be prepared by reacting hyaluronic acid or hyaluronic acid salt alone or an hyaluronic acid or hyaluronic acid salt and a polymer capable of forming an ether covalent bond with hyaluronic acid by an epoxide crosslinking agent .

The hyaluronic acid salt is characterized in that it is at least one selected from the group consisting of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate and tetrabutylammonium hyaluronate, Sodium hyaluronate is used, but it is not limited thereto.

In the present invention, the polymer capable of forming an ether covalent bond with hyaluronic acid is selected from the group consisting of hyaluronic acid, collagen, alginic acid, heparin, gelatin, elastin, fibrin, laminin, fibronectin, proteoglycan, heparan sulfate, chondroitin sulfate, Sulfates, and sulfates.

In the present invention, the polymer containing a hydroxyl end group may be selected from the group consisting of polyethylene oxide, polyvinyl alcohol, polypropylene oxide, polyethylene oxide-polypropylene oxide copolymer, polyethylene oxide-polylactic acid copolymer, polyethylene oxide-polylactic glycolic acid copolymer Polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters and polyoxyethylene stearates. And the like.

The process of derivatizing hyaluronic acid of the present invention with an epoxide crosslinking agent is shown in Scheme 1 below.

[Reaction Scheme 1]

The basic aqueous solution used in step (a) of the process of the present invention may be NaOH aqueous solution, and the reaction temperature is preferably 10 ° C to 50 ° C, the reaction time is preferably 6 hours to 36 hours, and the reaction pressure is preferably atmospheric pressure.

The epoxide crosslinking agent of the step (a) of the present invention may be selected from the group consisting of polyethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, Ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, Poly (propylene glycol) diglycidyl ether, poly (tetramethylene glycol) diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol poly Polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylol glycidyl ether, (Trimethylpropane polyglycidyl ether), 1,2- (bis (2,3-epoxypropoxy) ethylene), pentaerythritol polyglycidyl And at least one selected from the group consisting of pentaerythritol polyglycidyl ether and sorbitol polyglycidyl ether.

The cross-linking agent comprising two or more epoxy functional groups reacts with the hydroxy (-OH) end group of hyaluronic acid to form an ether bond, thereby crosslinking.

The weight ratio of the hyaluronic acid epoxide crosslinking agent in the step (a) is 1 to 100 parts by weight, more preferably 5 to 50 parts by weight based on 100 parts by weight of the repeating unit of the hyaluronic acid. When the weight ratio is less than 5 parts by weight, the water absorbing ability is high and shape deformation occurs and it is difficult to refine. When the weight ratio is more than 50 parts by weight, it is likely to be fragile and a large amount of crosslinking agent remains.

The temperature for drying the solution in step (b) of the present invention is 20 to 80 캜, preferably 30 to 50 캜, and the time is 6 to 24 hours. The organic solvent precipitation time and re-drying time of the present invention may be varied depending on the capacity of the organic solvent and the capacity of the derivative.

The organic solvent used to improve the physical strength of the hyaluronic acid epoxide derivative film of the present invention may be selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, tetrahydrofuran , THF), acetone, aqueous acetone solution, C ₁ to C ₆ alcohol or alcohol aqueous solution such as methanol or ethanol.

The hyaluronic acid epoxide derivative film according to the present invention is characterized in that it can be produced in the form of membrane, sponge and powder by re-swelling in water and pulverizing and drying.

Since the hyaluronic acid epoxide derivative film according to the present invention has a high water absorption capacity, it can rapidly absorb exudates and blood from wounds and help the hemostatic effect and wound healing.

In addition, the hyaluronic acid epoxide derivative film has high physical strength (Fig. 4) and is easy to handle and can protect the affected part from surrounding tissues. In addition, since it has stability against decomposing enzymes, it forms a physical barrier for a certain period of time, and then is completely decomposed and absorbed into the living body (FIG. 6).

The hyaluronic acid epoxide derivative film may be used as a material for tissue repair, a bone regeneration inducing film or an anti-adhesion agent, but is not limited thereto.

In addition, the bone regeneration-inducing membrane or the anti-adhesion agent according to the present invention may further include antibacterial and anti-inflammatory natural substances. The antibacterial and anti-inflammatory natural substances include green tea, turmeric, black soybean seed, rose petal, Rosemary, pepper leaves, rosemary, peppermint, bamboo, mulberry, mulberry, mulberry, golden, cinnamon, cinnamon, grass, Pine, cauliflower, white porcelain, oriental herb, Yoshino cherry tree, sasae, or two stem extracts . Specific examples thereof include cloves or white porcelain .

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Changes and modifications may fall within the scope of the appended claims.

[Example]

Example  1 to 4: Crosslinking of usable polymers mixed with hyaluronic acid Presence or absence of formation

In this experiment, 1% by weight of hyaluronic acid, alginic acid, gelatin, and collagen were mixed with a 1% by weight hyaluronic acid solution at a ratio of 50/50 in order to determine whether the polymers capable of being mixed with hyaluronic acid were crosslinked to form an ether covalent bond After 5 wt% of polyethylene oxide was added, each of the epoxide crosslinking agents (1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene diglycidyl ether, neopentyl diglycidyl ether, Diallyl ether) was added in a proportion of 5 parts by weight based on 100 parts by weight of the repeating units of the mixed polymers. The crosslinking reaction was carried out at room temperature for 12 hours, and then the solid matter was taken out and stored in DI water for 1 hour. The solids were checked for dissolution in deionized water and the presence or absence of crosslinking was determined.

division matter Cross-linking agent Dissolution Example 1 Hyaluronic acid 1,4-butanediol diglycidyl ether Do not cost Polyethylene glycol diglycidyl ether Do not cost Polypropylene glycol diglycidyl ether Do not cost Neopentyl diglycidyl ether Do not cost Example 2 Alginic acid 1,4-butanediol diglycidyl ether Do not cost Polyethylene glycol diglycidyl ether Do not cost Polypropylene glycol diglycidyl ether Do not cost Neopentyl diglycidyl ether Do not cost Example 3 gelatin 1,4-butanediol diglycidyl ether Do not cost Polyethylene glycol diglycidyl ether Do not cost Polypropylene glycol diglycidyl ether Do not cost Neopentyl diglycidyl ether Do not cost Example 4 Collagen 1,4-butanediol diglycidyl ether Do not cost Polyethylene glycol diglycidyl ether Do not cost Polypropylene glycol diglycidyl ether Do not cost Neopentyl diglycidyl ether Do not cost

As shown in Table 1, solids prepared by mixing hyaluronic acid, alginic acid, gelatin, and collagen with respective epoxide crosslinking agents were confirmed to be swollen without being dissolved in deionized water. From the above results, it has been confirmed that ether covalent bonds are formed when hyaluronic acid, alginic acid, gelatin, and collagen are mixed with an epoxide crosslinking agent, and are stably present in deionized water.

Example  5: Hydroxy Horse short  Containing hyaluronic acid Epoxide  Preparation of derivative films

In this experiment, polypropylene oxide was used as a polymer containing a hydroxy end group. A solution of 10 wt% of hyaluronic acid and 30 wt% of polypropylene oxide in 0.25N NaOH was prepared in each of three reactors. 5 parts by weight, 10 parts by weight and 25 parts by weight of polyethylene glycol diglycidyl ether were added to 100 parts by weight of hyaluronic acid in the solution. After reacting at room temperature for 24 hours, it was spread in a square dish, spread evenly, and dried at room temperature for 12 hours to prepare a primary film. The primary film was washed with purified water to remove non-reactant and polypropylene oxide, and then precipitated in ethanol and shrunk to prepare a secondary film. The secondary film was re-swollen with purified water and dried to produce a final derivative film. The derivative films prepared for carrying out the experiments of the present invention were named Experimental group 1 (5 parts by weight), Experimental group 2 (10 parts by weight) and Experimental group 3 (25 parts by weight) according to the ratio of crosslinking agent.

Comparative Example  One: Hydroxy Horse short  Hyaluronic acid containing no polymer Epoxide  Preparation of derivative films

10% by weight of hyaluronic acid was dissolved in 0.25N NaOH to prepare a hyaluronic acid solution. 25 parts by weight of polyethylene glycol diglycidyl ether was added to 100 parts by weight of hyaluronic acid in the solution, followed by reaction at room temperature for 24 hours. The solution was spread in a square dish, spread out, and dried at room temperature for 12 hours to prepare a primary film, which was then washed with purified water to remove non-reactants. The hydrated derivative was precipitated in ethanol and shrunk to prepare a secondary film, followed by re-swelling with purified water and drying to prepare a final derivative film. The film of the derivative prepared in Comparative Example 1 was named experimental group 4.

[Test Example]

Test Example  One: Hydroxy Horse short  Comparison of film shape by presence or absence of polymer

In order to confirm the uniformity of the polymer film containing the hydroxy end groups, the primary film shapes prepared in Example 5 and Comparative Example 1 were compared with each other by photographs.

As shown in FIG. 3, the film containing no hydroxy-terminated polymer was shrunk to a great extent after drying, whereas a film containing a polymer containing a hydroxy end group had some shrinkage at the edge portion Curling was not seen. From the above results, it was confirmed that a polymer containing a hydroxy end group plays a role in preventing shrinkage in a process of solidifying into a film in a solution.

Test Example  2: hyaluronic acid Epoxide  Structural analysis of derivative films

1% by weight of hyaluronic acid was dissolved in deionized water (D-I water) to prepare a hyaluronic acid solution, and a polymer containing 10% by weight of a hydroxy end group was dissolved in deionized water to prepare a solution. The prepared solution was placed in a square dish and dried at room temperature for 12 hours to prepare a polymer film containing a hyaluronic acid film and a hydroxy end group. The structures of the hyaluronic acid film, the polymer film including the hydroxy end group, the experimental group 3 and the experimental group 4 were analyzed using ATR-IR.

FIG. 4 compares the IR spectra. The polymer film containing a hydroxyl end group showed a strong peak at 2885 cm ^-1 (CH, methylene), and similarly, a hydroxy end group Was observed at 2925 cm ^-1 , indicating that a mixture of hyaluronic acid and a polymer containing a hydroxyl end group was mixed. As a result, it was confirmed that the hyaluronic acid derivative containing the polymer containing the hydroxy end group remained a certain amount of the polymer including the hydroxy end group even after the purification.

Test Example  3: Hyaluronic acid derivative Epoxide  Physical strength measurement of derivatives

Interceed (J & J, USA) and Bio-Gide (Geistlich Pharma AG, Switzerland), which are commercialized anti-adhesion agents, commercially available products 1, 2 and 3 prepared in Example 5, The intensity values were compared and analyzed. For the measurement, the sample was cut to 3 × 1 cm, placed on the grip of a universal material testing machine (Instron, USA), and the force applied to the material was measured while pulling at a speed of 10 mm / min.

FIG. 5 shows the tensile strength (N) values of the experimental group and the comparative group. As the amount of cross-linking agent increased, the physical strength was increased. However, in the experimental group 1, the value was slightly lower than that of the comparative group 2 (Bio-Gide), but the difference was not statistically significant.

Based on these results, it was confirmed that the hyaluronic acid derivative film had a physical strength suitable for use as an adhesion inhibitor or a bone regeneration inducing membrane.

Test Example  4: Physical strength comparison with organic solvent treatment

In order to compare the physical strength of the HA treated and untreated HA treated HA in the process of Example 5, the HA HA hydrated in the preparation of Experiments 1, 2, 3 and Example 5 was dissolved in ethanol Samples were prepared by drying without sedimentation. Each sample was prepared as in Example 5 and the physical strength was measured.

As shown in FIG. 6, the samples prepared by precipitation in ethanol exhibited higher physical strength than the samples prepared without sedimentation. From the above results, it was confirmed that the process of treating an organic solvent such as ethanol increases the physical strength of the hyaluronic acid derivative.

Test Example  5: hyaluronic acid Epoxide  Of the derivative film In vivo  Evaluation of the remaining period

Animal experiments were conducted to evaluate the in vivo survival time of the sample prepared in Example 5. An 8 - week - old SD rat was used as an experimental animal. Anesthesia was injected into the lower abdomen of rats and anesthesia was performed. The specimens of 5 × 10 mm size were implanted at the back, and histological analysis (H & E staining) was performed at 2, 4, and 12 weeks.

The results of the experiment are shown in Fig. As shown in FIG. 7, experimental group 1 was observed in vivo until 2 weeks, but not at 4 weeks and 12 weeks. Experimental groups 2 and 3 were found to remain in vivo until 12 weeks. Based on the above results, it was confirmed that the hyaluronic acid epoxide derivative film shows a difference in the remaining period of time in vivo according to the content of the crosslinking agent, but the hyaluronic acid epoxide derivative was not easily decomposed in vivo and stably maintained for a certain period of time.

Test Example  6: hyaluronic acid Epoxide  Performance evaluation of anti-tissue adhesion of derivative films

An animal model (SD rat) was used to evaluate the tissue adhesion prevention performance of the sample prepared in Example 5. Anesthetics were injected into the lower abdomen of rats and anesthesia was performed. The abdomen of the anesthetized rats was dissected and a 1x2 cm wound was formed on the epidermis of the abdominal wall (peritoneum) using a bone burr, and the epidermis was slightly peeled off from the cecum in contact with the wound I wounded him. Tissue adhesion of the control group, Interceed, and experimental groups 1, 2 and 3 prepared in Example 5, which did not use any adhesion inhibitor between wound and wound, was compared and observed. Each sample was cut into 2 × 3 cm size. (AA Luciano, et al., 2000). The grading system used in this study was a total of 4 grades (0, 1, 2, 3, &Quot; Evaluation of commonly used adjuvants in the postoperative adhesions ", Am . J. Obstet . Gynecol ., 146, 88-92 (1983)).

In addition, the adhesion strength was measured in three steps (1, 2, 3, stronger adhesion strength), and the results were summed and averaged.

The degree of tissue adhesion and the degree of adhesion by the animal experiment are shown in Fig. As shown in FIG. 8, the degrees of adhesion of the experimental group 1, the experimental group 2 and the experimental group 3 of the present invention were 0.67 ± 0.82, 1.25 ± 0.96 and 0, respectively, as well as the negative control group of 2.6 ± 0.54 and the comparative group 1 of 2.5 ± 0.58 Which is lower than that of The values of the adhesion strength of the experimental group 1, the experimental group 2 and the experimental group 3 were 0.5 ± 0.54, 1 ± 0.8, 0, respectively, which was lower than that of the control group 1, which is 1.6 ± 0.54 and 1.75 ± 0.5.

As a result of the anti-adhesion performance test, it was confirmed that the hyaluronic acid epoxide derivative film of the present invention showed a slight difference according to the cross-linking ratio, but the value was lower than that of the control group as well as the comparative group 1, so that it was effective in preventing tissue adhesion.

Test Example  7: Hyaluronic acid Epoxide  Of the derivative film Bone regeneration  Degree evaluation

In order to confirm the bone regeneration effect of the experimental group 3 prepared in Example 5, 8-week-old SD rats were used as model animals. After anesthetizing the animal, the skin in the middle of the forehead was incised about 4 cm, and the periosteum was incised. While being placed aside, the dental drill with a diameter of 8 mm was cautiously taken care of the damage of the blood vessels passing through the dura mater and two mid- A critical size defect of 8 mm in diameter [J. P Schmitz et al ., Clin . Orthop . Relat . Res ., 205, 299 (1986)], and the experimental group 3 and the comparative group 2 were cut into a Φ 12 mm circular shape to cover the defect and seal the periosteum and skin, respectively. In addition, a control group in which the bone defects were sutured without any treatment was also tested. After 12 weeks of feeding, animals were sacrificed and bone defect was collected and evaluated for bone regeneration effect by Micro-CT and Soft X-ray.

As shown in FIG. 9, the experimental group 1 showed better bone regeneration than the control group, and showed the same bone regeneration effect as that of the comparative group 2 (Bio-Gide), which is a commercialized bone-induced regeneration membrane. Based on these results, it was confirmed that the hyaluronic acid derivative film has excellent bone regeneration ability.

Test Example  8: Hyaluronic acid Epoxide  Evaluation of antibacterial activity of derivative film

To evaluate the antimicrobial activity of the experimental group 3 prepared in Example 5, the natural antimicrobial clove extract and the white porcelain extract were mixed at a concentration of 1 mg / ml, respectively, and then the agar diffusion method (paper disc method) was used. The antimicrobial-added film was cut into 8 mm in diameter and placed on a culture dish in which 1 × 10 ⁶ to 10 ⁷ cfu / ml of each strain (Bacillus substilis, Escherichia coli; fungal-Candida albicans) was cultured. , And an antimicrobial zone formed after 48 hours of fungal culture.

As shown in FIG. 10, it was confirmed that a hyaluronic acid epoxide derivative film containing a natural antimicrobial substance had antibacterial activity in strains of Bacillus substilis, Escherichia coli and Candida albicans.

Claims (15)

(a) mixing a basic aqueous solution with a polymer containing hyaluronic acid and a hydroxy (-OH) end group and reacting the mixture with an epoxide crosslinking agent to prepare a derivative solution;
(b) a hyaluronic acid epoxide derivative film in which a polymer containing a hydroxy (-OH) terminal group is physically bonded to a structure in which hyaluronic acid is crosslinked by an ether bond upon film drying of the derivative solution, Lt; / RTI >
(c) The film is purified with water to obtain a film containing a polymer containing a residual hydroxy (-OH) end group and partially removing the physical bond of the polymer including the hydroxy (-OH) end group. ; And
(d) drying the film. < RTI ID = 0.0 >
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
(i) reacting a basic aqueous solution with a polymer containing hyaluronic acid, a hydroxy (-OH) end group and an epoxide crosslinking agent to prepare an epoxy derivative solution;
(ii) drying the epoxide derivative solution to prepare a primary film;
(iii) purifying the primary film to remove an unreacted hydroxy (-OH) end group-containing polymer and a crosslinking agent;
(iv) precipitating the purified primary film in an organic solvent to prepare a secondary film; And
(v) re-swelling and drying the secondary film.
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
And the polymer containing the hydroxy (-OH) end group is contained in an amount of 50 to 500 parts by weight based on 100 parts by weight of the hyaluronic acid
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
The polymer containing the hydroxy (-OH) terminal group may be at least one selected from the group consisting of polyethylene oxide, polyvinyl alcohol, polypropylene oxide, polyethylene oxide-polypropylene oxide copolymer, polyethylene oxide- polylactic acid copolymer, polyethylene oxide- Polyolefins such as polyethylene oxide-polycaprolactone copolymer, polybutylene oxide, polyoxyethylene alkyl ethers, polyoxy-ethylene-co-ester oil derivatives, polyoxyethylene sorbitan fatty acid esters and polyoxyethylene stearates And at least one group selected from the group consisting of
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
The hyaluronic acid epoxide derivative may be at least one selected from the group consisting of green tea, turmeric, black bean seed, rosacea, peony root, Further comprising antimicrobial and antiinflammatory natural substances selected from the group consisting of bokbunja, rhubarb, juniper, forsythia, red pepper leaf, rosemary, mint, bamboo, mulberry, saururus, pine, wormwood, white porcelain, Characterized in that
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
The hyaluronic acid epoxide derivative is characterized in that the hyaluronic acid epoxide derivative is produced by reacting with hyaluronic acid or hyaluronic acid alone or with an epoxide crosslinking agent together with a hyaluronic acid or a hyaluronic acid and a polymer capable of forming an ether covalent bond with hyaluronic acid To
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 6,
Wherein the hyaluronic acid salt is at least one selected from the group consisting of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate and tetrabutylammonium hyaluronate
Method for the production of hyaluronic acid epoxide derivative fills. The method according to claim 6,
The polymer capable of forming an ether covalent bond with the hyaluronic acid is selected from the group consisting of collagen, alginic acid, heparin, gelatin, elastin, fibrin, laminin, fibronectin, proteoglycan, heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate One or more species selected from the group consisting of
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
The epoxide crosslinking agent is selected from the group consisting of polyethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, ether, 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, poly (propylene glycol) diglycidyl ether, glycol diglycidyl ether, poly (tetramethylene glycol) diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycol ether, Trimethylpropane polyglycidyl ether, 1,2- bis (2,3-epoxypropoxy) ethylene, pentaerythritol (pentaerythritol) polyglycidyl ether, and sorbitol polyglycidyl ether. In the present invention,
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
Wherein 1 to 100 parts by weight of the crosslinking agent is subjected to a crosslinking reaction with respect to 100 parts by weight of the repeating unit of the hyaluronic acid
A process for producing a hyaluronic acid epoxide derivative film. 3. The method of claim 2,
Wherein the organic solvent is at least one organic solvent selected from the group consisting of dimethylsulfoxide, dimethylformamide, acetonitrile, tetrahydrofuran, acetone, aqueous acetone, C1 to C6 alcohol or an aqueous solution thereof
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
The reaction temperature in step (a) is 10 to 50 ° C, and the reaction time is 6 to 36 hours.
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
Wherein the temperature of step (b) is 20 to 80 ° C and the time is 6 to 24 hours.
A process for producing a hyaluronic acid epoxide derivative film. The method according to claim 1,
The hyaluronic acid epoxide derivative film is characterized in that it is re-swollen into moisture and is produced in the form of a membrane, a sponge or a powder by a pulverizing and drying method
A process for producing a hyaluronic acid epoxide derivative film.
The method according to claim 1,
The hyaluronic acid epoxide derivative film has a crosslinking density of 1 to 100 mol%
A process for producing a hyaluronic acid epoxide derivative film .

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