KR20130123080A - Crosslinked hyaluronic acid epoxide and manufacturing method thereof - Google Patents

Abstract

The present invention relates to crosslinked hyaluronic acid epoxide and, more specifically, to a crosslinked hyaluronic acid epoxide containing natural hyaluronic acids by mixing crosslinked hyaluronic acid epoxide and the natural hyaluronic acids. The crosslinked hyaluronic acid epoxide is able to easily control rheological properties such as viscoelasticity and degradation period according to the composition and improve adhesion to biological tissues. The crosslinked hyaluronic acid epoxide containing the natural hyaluronic acid has superior biocompatibility, biodegradation, and physical stability, thereby being used as a dental filler, an adjuvant for orthopedics, a filler for plastic surgery, an anti-adhesion agent, a cell support, or a drug delivery system. [Reference numerals] (AA) Complex viscosity (cP);(BB) Control group 1;(CC) Test group 1;(DD) Test group 2;(EE) Test group 3;(FF) Test group 4

Description

Crosslinked hyaluronic acid epoxide and manufacturing method

The present invention relates to a hyaluronic acid epoxide crosslinked product and a method for preparing the same, and more particularly, by mixing natural hyaluronic acid with a hyaluronic acid epoxide crosslinked product, the rheological properties such as viscoelasticity and decomposition period can be easily changed depending on the composition. The present invention relates to a hyaluronic acid epoxide crosslinked product including natural hyaluronic acid capable of regulating and improving adhesion to biological tissues, and a method for preparing the same.

Hyaluronic acid is a type of polysaccharide that is linearly linked with repeating units composed of N-acetyl-glucosamine and D-glucuronic acid. It is a biopolymer substance and is first separated from the liquid filling the animal's eye [Meyer K. et (synovial fluid, pleural fluid, skin, rooster, and the like) of animal placenta, joints since it is known to be present in many cases, Streptococcus microorganisms Streptococcus equi, Streptococcus zooepidemecus, etc. are also produced.

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, many efforts have been made to develop structurally stable hyaluronic acid crosslinks (Laurent, T.C., et al., Portland Press Ltd, London, 1998).

Such hyaluronic acid crosslinked products have been developed for various uses such as molding aids, anti-adhesion agents after surgery, joint function improvers, drug carriers, cell culture supports, etc. As one of the methods for preparing hyaluronic acid crosslinked products, a hyaluronic acid is added by adding a crosslinking agent. The hyaluronic acid crosslinked with each other has excellent biocompatibility, physical stability and biodegradability, and methods for preparing the same are disclosed in US Pat. Nos. 4,716,154, 4,716,224, 4,963,666, 5,827,937, and the like.

However, in order to control the rheological properties such as the viscoelasticity of the hyaluronic acid crosslinked body produced by the method of these patents or the decomposition period, the concentration of the hyaluronic acid reacted in the preparation of the crosslinked body and the ratio of the crosslinking agent added must be controlled from the beginning. . In general, in order to improve the rheological properties or the decomposition period, it is necessary to increase the reaction concentration or to add a large amount of crosslinking agent. There is a problem.

Accordingly, an object of the present invention is to solve the problems of the prior art to easily control the rheological properties such as viscoelasticity and the decomposition period according to the composition, hyaluronic acid epoxide crosslinking which can improve the adhesion to biological tissues And to provide a method for producing the same.

The present invention provides a hyaluronic acid epoxide crosslinked product containing natural hyaluronic acid by using a mixture of natural hyaluronic acid in such a manner that natural hyaluronic acid is added to the hyaluronic acid epoxide crosslinked product, and a method of manufacturing the same.

Through the present invention, it is possible to easily control the rheological properties such as viscoelasticity of the hyaluronic acid epoxide crosslinked body, stability in the body, etc., and improve adhesion and biocompatibility to biological tissues.

In addition, the hyaluronic acid epoxide crosslinked product including the natural hyaluronic acid of the present invention has excellent biocompatibility, biodegradability, physical stability and the like, so that it can be used as a dental filler, orthopedic adjuvant, plastic surgery filler, cell support, drug carrier. Etc. can be used for various purposes.

1 is a graph showing a comparison of complex viscosity and tan δ with or without natural hyaluronic acid of a hyaluronic acid epoxide crosslinked product according to Test Example 1 of the present invention.
Figure 2 is a graph showing the adhesion (tack) of the hyaluronic acid epoxide crosslinked by the presence or absence of natural hyaluronic acid in accordance with Test Example 2 of the present invention.
Figure 3 is a graph showing a comparison of the composite viscosity according to the composition of the natural hyaluronic acid of the hyaluronic acid epoxide crosslinked body according to Test Example 3 of the present invention.
4 is in In the animal experiment of Test Example 4 evaluating the degradation performance in vivo , it is a graph showing the degree of degradation of the experimental group (3 types) including the natural hyaluronic acid of the present invention and the crosslinked product (comparative group 1) containing no natural hyaluronic acid. .
5 is an animal experiment of Test Example 5 evaluating the degree of bone regeneration, the experimental group 6 using the hyaluronic acid epoxide crosslinked product containing the natural hyaluronic acid of the present invention and the crosslinked body not containing natural hyaluronic acid (comparative group) 1) and bone regeneration in the control group that did not receive any treatment at the bone defect.
6 is a microorganism test of Test Example 6 to confirm the antibacterial performance, Bacillus subtillis , Escherichia coli , Candida It is a photograph showing the antimicrobial activity against albicans strain.

The present invention provides a hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid. Hereinafter, the present invention will be described in detail.

Specifically, natural hyaluronic acid is added to a crosslinked product obtained by crosslinking hyaluronic acid with hyaluronic acid using an epoxide crosslinking agent having two or more epoxy reactors.

The term "hyaluronic acid" as used herein is meant to include both hyaluronic acid, hyaluronic acid salts, or mixtures of hyaluronic acid and hyaluronic acid salts. 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 is not limited thereto.

In addition, the present invention comprises the steps of (i) dissolving the hyaluronic acid or hyaluronic acid salt in an aqueous alkali solution; (ii) crosslinking by adding an epoxide crosslinking agent to the hyaluronic acid or hyaluronic acid salt solution; (iii) washing the crosslinking reactant several times to remove unreacted reactant; (iv) grinding the washed product to adjust particle size; It provides a method for producing a hyaluronic acid epoxide crosslinking comprising natural hyaluronic acid, and (v) adding and mixing the natural hyaluronic acid.

The epoxide crosslinking agent used in the present invention is 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether Hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and the like. Poly (tetramethylene glycol diglycidyl ether), neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, Diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, and the like. (1,2- (bis (2,3-epoxypropoxy) ethylene), pentaerythritol polyglycidyl ether, or sorbitol And sorbitol polyglycidyl ether. More preferably, 1,4-butanediol diglycidyl ether is used, but the present invention is not limited thereto.

The crosslinking agent containing two or more epoxy functional groups reacts with an alcohol group of hyaluronic acid to form an ether bond, thereby crosslinking.

The crosslinking density of the hyaluronic acid epoxide crosslinked product may be 1 mol% to 100 mol%, and more preferably 5 mol% to 50 mol%. When the crosslinking density is less than 1 mol%, the physical properties are lowered and the water absorption capacity is increased, which makes purification difficult. When the crosslinking density is more than 100 mol%, the possibility of a large amount of crosslinking agent remaining is increased.

The final reaction concentration of the hyaluronic acid epoxide crosslinked product is preferably 0.05 mg / ml to 50 mg / ml. That is, when the final reaction concentration is less than 0.05 mg / ml cross-linking reaction is difficult to achieve a satisfactory cross-linking body, when the concentration exceeds 50 mg / ml is too high concentration occurs a problem that the manufacturing process is not smooth .

In addition, the concentration of the natural hyaluronic acid to be added is not particularly limited, but if it is added more than necessary, there is a problem that the physical viscoelasticity is very low, 0.01% to 80% of the final reaction concentration of the hyaluronic acid epoxide crosslinked product, more It is preferably characterized in that 0.1% to 40%.

In the step (ii) of the method for preparing a hyaluronic acid epoxide crosslinked product comprising a natural hyaluronic acid of the present invention, hyaluronic acid or a hyaluronic acid salt alone, or a hyaluronic acid or an ether covalent bond to a hyaluronic acid or a hyaluronic acid salt solution A hyaluronic acid epoxide crosslinked product can also be obtained by adding the polymer which can be formed and reacting with an epoxide crosslinking agent.

The polymer capable of forming an ether covalent bond with hyaluronic acid is a natural polymer of collagen, alginic acid, heparin, gelatin, elastin, fibrin, laminin, fibronectin, proteoglycan, heparan sulfate, chondroitin sulfate, dermatansulfate and keratan sulfate. Characterized in that the one or more selected from the group consisting of.

The polymer capable of forming an ether covalent bond with hyaluronic acid may be a synthetic high molecular weight polyethylene oxide, polyvinyl alcohol, polypropylene oxide, polyethylene oxide-polypropylene oxide copolymer, polyethylene oxide-poly Polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, polyoxyethylene alkylphenols, Fatty acid esters, polyoxyethylene stearates, and the like.

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

(Scheme 1)

The weight ratio of the hyaluronic acid epoxide crosslinking agent 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 of the hyaluronic acid epoxide crosslinking agent is less than 1 part by weight, the physical properties are lowered and the water absorption capacity is increased, which makes purification difficult. When the weight ratio exceeds 100 parts by weight, the possibility that a large amount of crosslinking agent remains is increased.

Hyaluronic epoxide crosslinked product according to the present invention is characterized in that it can be produced in various forms such as bead gel, sponge, powder, membrane or sheet according to the method of obtaining the hydrate, preferably the following Examples and As prepared in the form of bead gel, but is not limited thereto.

In the present invention, it is possible to improve viscoelasticity and decomposition period by adding natural hyaluronic acid to the obtained hyaluronic acid epoxide crosslinked product. In addition, the hyaluronic acid epoxide crosslinked product containing natural hyaluronic acid has a high adhesion and binds well to biological tissues, thereby having excellent biocompatibility. In addition, since it has stability against degrading enzymes, it maintains the volume in the body for a certain time, and then completely decomposes and is absorbed into the living body.

Due to these properties, the hyaluronic acid epoxide crosslinked product including the natural hyaluronic acid prepared in the present invention may be used for dental fillers, orthopedic aids, plastic surgery fillers, anti-adhesion agents, cell supports or drug carriers. Can be.

In addition, the hyaluronic acid epoxide cross-linked body including the natural hyaluronic acid of the present invention may further include an antibacterial or anti-inflammatory natural substance, the antimicrobial or anti-inflammatory natural substance is rosemary, cloves, white porcelain, turmeric, green tea, black beans Seedlings, rose leaves, peony root, bellflower, bean sprouts, colored barley seedlings, camellia petals, buckwheat, grapefruit, licorice, rhubarb, astragalus, yellow white, golden, cinnamon, herb, bokbunja, gallula, juniper, forsythia, red pepper, mint It may be a snakeberry, mulberry, triticale, pine, wormwood, eosungcho, Yoshino cherry tree, stalk or double stem extract. Hereinafter, embodiments of the present invention will be described in detail.

(Example)

Example  1: hyaluronic acid including natural hyaluronic acid Epoxide Crosslinked  Produce

Hyaluronic acid solution in which 10% by weight of hyaluronic acid was dissolved in 0.25N NaOH solution was prepared in each of four reactors. 1,4-butanediol diglycidyl ether (BDDE), a crosslinking agent, was added at a rate of 20 parts by weight based on 100 parts by weight of hyaluronic acid of the solution, and reacted at room temperature for 30 hours. The reaction product was washed several times with phosphate buffer solution to remove unreacted material, and the washed product was pulverized to adjust the particle size to 100 μm to 500 μm. 0.5%, 1.25%, 2%, 2.75% by weight of natural hyaluronic acid was further added to the hyaluronic acid included in the reaction. All prepared samples were sterilized at 121 ° C. for 20 minutes, and depending on the amount of natural hyaluronic acid added, Experimental Group 1 (0.5% by weight), Experimental Group 2 (1.25% by weight), Experimental Group 3 (2% by weight), and Experimental Group 4 (2.75% by weight) ).

Comparative Example  1: hyaluronic acid without natural hyaluronic acid Epoxide Crosslinked material Manufacturing

A hyaluronic acid solution was prepared by dissolving hyaluronic acid in a 0.25 N NaOH solution at 10% by weight. 1,4-butanediol diglycidyl ether (BDDE), a crosslinking agent, was added at a rate of 20 parts by weight based on 100 parts by weight of hyaluronic acid of the solution, and reacted at room temperature for 30 hours. The reaction product was washed several times with phosphate buffer solution to remove unreacted materials, and the washed product was pulverized to adjust the particle size to 100 μm to 500 μm and then sterilized at 121 ° C. for 20 minutes. The crosslinked product prepared in Comparative Example 1 was named Comparative Group 1.

Example  2: hyaluronic acid according to the composition of natural hyaluronic acid Epoxide Crosslinked  Produce

Hyaluronic acid solution in which 10% by weight of hyaluronic acid was dissolved in 0.25N NaOH solution was prepared in each of five reactors. 1,4-butanediol diglycidyl ether (BDDE), a crosslinking agent, was added at a rate of 20 parts by weight based on 100 parts by weight of hyaluronic acid of the solution, and reacted at room temperature for 30 hours. The reaction product was washed several times with phosphate buffer solution to remove unreacted material, and the washed product was pulverized to adjust the particle size to 100 μm to 500 μm. Natural hyaluronic acid composition ratio to the hyaluronic acid contained in the reaction was adjusted to 15%, 20%, 40%, 60%, 80%, and then sterilized for 20 minutes at 121 ℃. The reactants were named Experimental Group 5 (15%), Experimental Group 6 (20%), Experimental Group 7 (40%), Experimental Group 8 (60%), and Experimental Group 9 (80%), depending on the composition.

(Test example)

Test Example  1: depending on the amount of natural hyaluronic acid added Rheological  characteristic

In order to compare the rheological properties of the hyaluronic acid epoxide crosslinked body prepared in Example 1 and Comparative Example 1 according to the concentration of natural hyaluronic acid, the rheometer was used in a frequency range of 0.02 Hz to 1 Hz. Complex viscosity and tan δ were measured.

1 is a comparison of the physical properties according to the amount of natural hyaluronic acid addition of the hyaluronic acid epoxide cross-linked product containing natural hyaluronic acid prepared in the present invention, hyaluronic acid epoxide cross-linking including the natural hyaluronic acid prepared in Example 1 The composite viscosity of the sieve was about 1.4 to 2.7 times higher than that of the hyaluronic acid epoxide crosslinked product containing no natural hyaluronic acid prepared in Comparative Example 1, and the hyaluronic acid epoxide crosslinked product containing natural hyaluronic acid had excellent composite viscosity. It was confirmed to have a value (see FIG. 1A). In addition, the hyaluronic acid epoxide crosslinked product including the natural hyaluronic acid prepared in Example 1 in tanδ value was 2.5 times higher than the hyaluronic acid epoxide crosslinked product containing no natural hyaluronic acid prepared in Comparative Example 1. It can be seen that the hyaluronic acid epoxide crosslinked product including natural hyaluronic acid is formed into a structure having high stability (see FIG. 1B).

Test Example  2: according to the addition amount of natural hyaluronic acid Adhesion

In order to measure the tack value according to the concentration of natural hyaluronic acid of the hyaluronic acid epoxide crosslinked body prepared in Example 1 and Comparative Example 1, after loading on the plate-plate of the rheometer, 0.1 second Rotation was performed at a shear rate of 10 ⁻¹ , and the value of normal force applied when the upper plate was dropped at a speed of 0.1 mm / s was measured.

Figure 2 is a comparison of the talc value according to the concentration of the hyaluronic acid epoxide cross-linked product containing natural hyaluronic acid prepared in Example 1, the higher the addition amount of the natural hyaluronic acid is the tag value of the hyaluronic acid epoxide cross-linked It confirmed that it became high. That is, it was confirmed that the hyaluronic acid epoxide crosslinked product including the natural hyaluronic acid of the present invention has higher tissue adhesion than the comparative group.

Test Example  3: hyaluronic acid according to the composition of natural hyaluronic acid Epoxide Rheological of the Crosslinked Body  characteristic

In order to compare the rheological properties of the hyaluronic acid epoxide crosslinked body prepared in Example 2 and Comparative Example 1 according to the composition of the natural hyaluronic acid, the rheometer was used in a frequency range of 0.02 Hz to 1 Hz. Complex viscosity was measured.

Figure 3 is a comparison of the physical properties according to the composition of the natural hyaluronic acid composition of the hyaluronic acid epoxide crosslinked product including the natural hyaluronic acid prepared in Example 2, showing that the physical properties can be adjusted according to the composition of the natural hyaluronic acid.

Test Example  4: hyaluronic acid according to the composition of natural hyaluronic acid Epoxide Crosslinked in vivo  Degradation Characteristic Evaluation

Animal model (SD rat) was used to evaluate the degradation characteristics according to the composition of the natural hyaluronic acid of the hyaluronic acid epoxide crosslinked product including the natural hyaluronic acid prepared in Example 2. Anesthesia was injected into the lower abdomen of the rat, anesthetized, 0.5 ml of the sample was injected into the back of the anesthetized rat, and the remaining amount after 4 weeks and 12 weeks was measured. Comparative Group 1 prepared in Comparative Example 1 and Experimental Groups 6, 7, and 8 prepared in Example 2 were compared and observed.

It was confirmed from FIG. 4 that the decomposition period of the hyaluronic acid epoxide crosslinked product including the natural hyaluronic acid can be easily adjusted according to the amount of the natural hyaluronic acid added.

Test Example  5: hyaluronic acid Epoxide Crosslinked Bone regeneration  Degree evaluation

In order to confirm and compare the bone regeneration effect of Experimental Group 6 prepared in Example 2 and Comparative Group 1 prepared in Comparative Example 1, 8-week-old SD rats were used as model animals. After anesthetizing the experimental animal, cut the skin at the center of the forehead by about 4cm, cut the periosteum and put it aside, and use an 8mm diameter dental drill while being careful not to damage the meninges and blood vessels passing through the two medians. Size 8mm diameter with critical size defect [J. P Schmitz et al., Clin. Orthop. Relat. Res., 205, 299 (1986)], the experimental group 6 and the comparative group 1 was formed into a shape of Φ8mm * 2mm to fill the defect site, and the periosteum and skin were sutured, respectively. In addition, the control group sutured without any treatment to the bone defect was also tested. After breeding for 12 weeks, animals were sacrificed and bone defects were collected. The bone regeneration effect was evaluated by micro-CT and soft X-ray.

As shown in FIG. 5, it was observed that the comparative group 1 and the experimental group 6 had superior bone regeneration compared to the control group, and the bone superior in the experimental group 6 containing natural hyaluronic acid compared to the comparative group 1 containing no natural hyaluronic acid. Regeneration effect was shown. Based on these results, it was confirmed that the hyaluronic acid crosslinked product including natural hyaluronic acid has excellent bone regeneration ability.

Test Example  6: hyaluronic acid including natural hyaluronic acid Epoxide Crosslinked  Antimicrobial Performance Evaluation

Experimental group 6 prepared in Example 2 was mixed with a natural antimicrobial clove extract and white porcelain extract at a concentration of 1 mg / ml and lyophilized to prepare a sheet form, the antimicrobial performance was evaluated by the paper disc method. Cut hyaluronic acid epoxide crosslinked sheet containing antimicrobial substance into 8 mm diameter and each 1 x 10 ⁶ ~ 10 ⁷ cfu / ml strain (bacteria- Bacillus subtillis , Escherichia coli ; Fungus- Candida albicans ) was placed on the culture plate, and the antimicrobial zone was formed after 24 hours for bacterial culture and 48 hours for fungal culture.

As shown in Figure 6, the hyaluronic acid epoxide cross-linked sheet comprising a natural hyaluronic acid containing a natural antibacterial material Bacillus subtillis , Escherichia coli , Candida It was confirmed that the strain of albicans has antibacterial activity.

Claims (16)

Hyaluronic acid epoxide crosslinked product containing natural hyaluronic acid. The hyaluronic acid epoxide crosslinked product according to claim 1, wherein the hyaluronic acid is a hyaluronic acid, a hyaluronic acid salt, or a mixture of hyaluronic acid and a hyaluronic acid salt. The hyaluronate salt according to claim 2, wherein the hyaluronate salt is at least one selected from the group consisting of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate and tetrabutyl ammonium hyaluronate. A hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid, characterized in that. The hyaluronic acid epoxide crosslinked product according to claim 1, further comprising natural hyaluronic acid, characterized in that it further comprises an antibacterial or inflammatory natural substance. The method of claim 4, wherein the antimicrobial or inflammatory natural substance is rosemary, cloves, white porcelain, turmeric, green tea, black bean seedling, rose leaf, peony root, bellflower, bean sprouts, colored barley seedling, camellia leaf, buckwheat, grapefruit, licorice, yellow lotus Selected from the group consisting of Astragalus, Yellow white, Golden, Cinnamon, Herb, Bokbunja, Ginseng, Juniper, Forsythia, Red pepper leaf, Peppermint, Snakeberry, Mulberry, Triticale, Pine, Rootworm, Estuary, Yoshino cherry, Sprout and Sprout stem extract A hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid, characterized in that at least one. A dental filler, an orthopedic adjuvant, a plastic surgery filler, an anti-adhesion agent, a cell support or a drug carrier, comprising the hyaluronic acid epoxide crosslinked product comprising the natural hyaluronic acid of claim 1. (i) dissolving hyaluronic acid or a hyaluronic acid salt in an aqueous alkali solution;
(ii) crosslinking by adding an epoxide crosslinking agent to the hyaluronic acid or hyaluronic acid salt solution;
(iii) washing the crosslinking reactant several times to remove unreacted reactant;
(iv) grinding the washed product to adjust particle size; And
(v) adding and mixing natural hyaluronic acid; a method of producing a hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid comprising a. The method of claim 7, wherein the hyaluronic acid epoxide crosslinked product is prepared in the form of a bead gel, a sponge, a powder, a membrane, or a sheet. The method of claim 7, wherein the epoxide crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1 , 6-hexanediol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, poly (propylene glycol) diglycidyl ether ), Polytetramethylglycol diglycidyl ether (poly (tetramethylene glycol) diglycidyl ether), neopentyl glycol diglycidyl ether (neopentyl glycol diglycidyl ether), polyglycerol polyglycidyl ether (diglycerol polyglycidyl ether) Glycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl et her), 1,2- (bis (2,3-epoxypropoxy) ethylene (1,2- (bis (2,3-epoxypropoxy) ethylene), pentaerythritol polyglycidyl ether) or sorbitol A method for producing a hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid, characterized in that at least one selected from sorbitol polyglycidyl ether. 8. The method of claim 7, wherein the hyaluronic acid epoxide crosslinked product has a crosslinking density of 1 mol% to 100 mol%. 9. The method of claim 7, wherein the final reaction concentration of the hyaluronic acid epoxide crosslinked product is 0.05 mg / ml to 50 mg / ml. The method of claim 7, wherein the concentration of the natural hyaluronic acid is 0.01% to 80% of the final reaction concentration of the hyaluronic acid epoxide crosslinked product. 8. The hyaluronic acid epoxide derivative is obtained by adding a polymer capable of forming an ether covalent bond with hyaluronic acid to a solution of hyaluronic acid or a hyaluronic acid salt in step (ii) and reacting with an epoxide crosslinking agent. A method for producing a hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid. The method of claim 13, wherein the polymer capable of forming an ether covalent bond with hyaluronic acid is collagen, alginic acid, heparin, gelatin, elastin, fibrin, laminin, fibronectin, proteoglycan, heparan sulfate, chondroitin sulfate, dermatansulfate, and keratin. A method for producing a hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid, characterized in that at least one member selected from the group consisting of rattan sulfate. The polymer according to claim 13, wherein the polymer capable of forming an ether covalent bond with hyaluronic acid is polyethylene oxide, polypropylene oxide, polyvinyl alcohol, polyethylene oxide-polypropylene oxide copolymer, polyethylene oxide-polylactic acid copolymer, polyethylene Oxide-polylactic glycolic acid copolymer, polyethylene oxide-polycaprolactone copolymer, polybutylene oxide, polyoxyethylene alkyl ethers, polyoxy-ethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters and polyoxy A method for producing a hyaluronic acid epoxide crosslinked product comprising natural hyaluronic acid, characterized in that at least one member selected from the group consisting of ethylene stearate. 8. The method of claim 7, wherein the weight ratio of the epoxide crosslinking agent is 1 to 100 parts by weight based on 100 parts by weight of the repeating unit of hyaluronic acid.

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