KR20100111383A - The method of producing an anti-adhesion using non-chemical cross-linking of the esterified atelocollagen and hyaluronic acid - Google Patents

Abstract

PURPOSE: A method for manufacturing an anti-adhesion agent of porous film, film, gel, and microsome using esterification collagen is provided to ensure safety and biocompatibility. CONSTITUTION: A method for manufacturing an anti-adhesion agent using esterification collagen and hyaluronic acid comprises: a step of treating the natural collagen with pepsin to obtain atelocolagen; a step of adding 0.5-3 wt% of atelocollagen to 65-85 wt% of ethanol solution and stirring; a step of adjusting by pH 7.4 using 0.5M of acetic acid or hydrochloric acid; a step of reacting at 4°C for a week to obtain esterification collagen; a step of freeze-drying the esterification collagen; and a step of preparing a complex of esterification collagen and hyaluronic acid.

Description

The method of producing an anti-adhesion using non-chemical cross-linking of the esterified atelocollagen and hyaluronic acid}

The present invention relates to a method for producing an esterified collagen using pig-derived natural collagen and a non-adhesion agent of a porous membrane, a film, a gel, and a microsomal complex through non-chemical crosslinking of esterified collagen and hyaluronic acid. In particular, the present invention relates to biocompatibility using esterified collagen and hyaluronic acid complexes, and physical separation of tissues and wounds, as well as a method for preparing anti-adhesion agents that help regeneration of wounds.

After surgery, adhesions occur between the wound and tissues or organs. Tissue adhesion occurs in almost every part of the human body, especially after surgery or obstetrics surgery. This phenomenon is due to the fact that the dissolution and absorption of the exudate from the damaged tissues are not achieved, so that fibrous tissues are formed and the collagen deposition occurs through the binding to fibroblasts. Adhesions in the tissues of the surgical site have a great effect on the recovery of the patient after surgery, and the risk of reoperation due to adhesions is also related to mortality.

Various researches and developments are underway to prevent adhesions where prevention is essential. In the United States, research is being conducted on biodegradable polymers such as cellulose to prevent tissue adhesion, and commercialized products include Interceed ™ (Jonhson & Johnson Medical Inc.), Seprafilm ™ (Genezyme Corp.), and Integel ™ (LifeCore Biomedical). Etc. However, they are simply focused on the function of separating tissue from the surgical site and thus have not been proven effective as anti-adhesion agents. Domestic patients who rely on income have to spend a lot of money to prevent adhesion.

Conventional techniques include "Methods and compositions based on inhibition of cell invasion and fibrosis by anionic polymers, US Patent 6756362, 2004", "Water Insoluble Derivatives of Hyaluronic Acid, US Patent 5017229,1991", "Lee WM et al., A new anti-adhesion film synthesized from polygalacturonic acid with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide crosslinker, Biomaterials, Vol. 26 No. 18, Jun 2005 "," Sawada T et al., Cross-linked hyaluronate hydrogel prevents adhesion formation and reformation in mouse uterine horn model, Human Reproduction, Vol. 16, No. 2, Feb 2001 "," Shunquing T et al., Agarose / collagen composite scaffold as an anti-adhesive sheet, Biomedical Materials, Vol. 2, No. 3, Jul 2007 ", but the focus is only on physical separation of wounds and tissues, and short residence times in the body prevent them from staying at the site of injury for enough time to achieve antiadhesion effects.

Expanded poly tetrafluoroethlene (ePTFE), chitosan, polyglycol acid, polyvinly alcohol (PVA), agarose and collagen are used as anti-adhesion materials. However, synthetic materials such as ePTFE are not biodegradable and can cause inflammation between the surgical site and the tissue. Even with biodegradable materials, the physical membranes on the wounds may not be able to supply nutrients smoothly, which may slow recovery of the wounds. Anti-adhesion agents should prevent the penetration of extracellular matrix into the wound area, the wound area within the body should be decomposed over several weeks during healing, and the supply of nutrients for regeneration to the wound area should be made smoothly. It must be completely decomposed and absorbed within. Therefore, the development of new anti-adhesion agents that help to protect and treat wounds from tissues as well as the absorption of exudate, which is the cause of adhesion to tissues after surgery, has a considerable replacement effect not only for the pain of patients suffering from postoperative tissue adhesion but also for the economic part. You can reap.

The present invention relates to a method for preparing an anti-adhesion agent using a non-chemical crosslinking method using a human-derived polymer suitable for a living body as well as providing an environment that helps to heal damaged tissue as well as preventing tissue adhesion after surgery. The anti-adhesion agent is prepared from a human-derived polymer compound such as collagen or hyaluronic acid so that the anti-adhesion agent may play a role in helping to restore the wound in addition to separating the damaged tissue from other tissue surfaces.

Collagen, used as an anti-adhesion material, is extracted from the dermal layer of pigs. Non-immunized collagen is obtained by removing the ateloptide of the extracted collagen. Non-immunized collagen is esterified to the carboxyl group (CHOOH) of collagen using ethanol to produce esterified collagen having the properties of a cation. Hyaluronic acid, which has hydrophilic properties, is a human-derived polymer with anionic properties and has biocompatibility, but has a short residence time in vivo. To compensate for this drawback, complexes are made using non-chemical crosslinking of hyaluronic acid and esterified collagen. Hyaluronic acid, a hydrophilic polymer of this complex, prevents adhesion to tissues and stays in the body until tissues are regenerated in vivo through non-chemical crosslinking with collagen, and prevents adhesions. To help. It is safely degraded and absorbed in vivo.

The present invention is to provide a method for preparing an anti-adhesion agent that provides an effect of preventing tissue adhesion after surgery and regeneration of a wound site using esterified collagen and hyaluronic acid. It removes the telopeptide of collagen to prevent the induction of immune response, and through the esterification reaction of collagen to make esterified collagen having the property of cation, to obtain a complex through non-chemical crosslinking with hyaluronic acid, which is an anion property. This can maximize the effect of the anti-adhesion by increasing the residence time in the living body and provides an excellent anti-adhesion agent that has the effect of helping to absorb exudates and regeneration of the wound site by securing the disadvantages of the existing anti-adhesion agent. Is in.

Collagen extracted from pigs is used to remove collagen telopeptides to create collagen that does not induce an immune response. Collagen without telopeptides does not induce an immune response unlike conventional collagen. It also cationizes to the surface of collagen through reaction with ethanol. Esterized collagen with cationic properties creates a complex without chemical crosslinking with hyaluronic acid with anionic properties by non-chemical bonding. The reaction complex of esterified collagen and hyaluronic acid is a biocompatible anti-adhesion agent that prevents tissue adhesion after surgery. Existing anti-adhesion agents are mainly focused on the role of physical separation of tissues and wounds or made of materials that are not suitable for living organisms, causing inflammatory reactions. Complementing this drawback, the present invention can absorb the exudate and help the tissue wound as well as the long retention time in the body through the esterified collagen and hyaluronic acid complex, which is a biocompatible material, and prevents adhesion between damaged and surrounding tissues. An anti-adhesion agent has been invented that provides an environmental environment.

The present invention relates to an anti-adhesion agent using a biocompatible material of esterified collagen and hyaluronic acid complex, which is an anti-adhesion agent that helps regeneration of a wound site rather than a simple physical separation between the tissue and the wound site of a conventional anti-adhesion film. It is a way. Non-immunized natural collagen extracted from pigs can prevent immune reactions and convert to esterified collagen to have cationic properties, forming complexes with hyaluronic acid with anionic properties easily by non-chemical crosslinking. In addition, the esterified collagen and hyaluronic acid complex is longer than the in vivo residence time of each of the collagen and hyaluronic acid, and retains the characteristics of collagen and hyaluronic acid, thereby helping to prevent the adhesion and to recover the exudation and wound healing. Unlike conventional anti-adhesion membranes made by chemical crosslinking with chemical or synthetic materials, it is a material suitable for living organisms and manufactured by non-chemical methods to produce safer anti-adhesion agents that can prevent inflammation of wounds. to provide. In addition, the patients who suffer from prevention of post-adhesion adhesion can have the effect of preventing the adhesion and the substitution effect of the anti-adhesion agent, which mostly depends on the import.

Unlike the anti-adhesion agent made by chemical crosslinking of the conventional synthetic material, the present invention removes telopeptide from natural collagen extracted from pigs to obtain collagen that does not cause an immune reaction, and adds ethanol to esterified collagen through esterification. It is a biocompatible polymer that has anionic properties of hyaluronic acid, and it is characterized in that it is used as a bioadhesion anti-adhesion agent by making a complex through non-chemical crosslinking with esterified collagen of cationic properties obtained by reaction with ethanol. have.

In the present invention, the preparation of the anti-adhesion agent is in the preparation method of making a composite in the form of a porous membrane, a film, a gel, and a microbody using esterified collagen and hyaluronic acid. The esterified collagen and hyaluronic acid complexes can be used not only as anti-adhesion agents but also as cartilage regeneration membranes, cell carriers, and artificial skins made of collagen and hyaluronic acid. Referring to the production method of the anti-adhesion agent of the present invention in more detail,

Step 1 to obtain atelo collagen from which telopeptides were removed by treating natural collagen extracted from pigs with pepsin enzyme,

Add 0.5 ~ 3wt% of the atelocollagen to 65 ~ 85wt% ethanol solution to make it homogeneous, adjust to pH7.4 with 0.5M acetic acid or hydrochloric acid, and then react for 1 week at 4 ℃ for collagen esterification Step 2 to obtain a solution,

Lyophilizing the esterified collagen solution to prepare esterified collagen,

It is a method for preparing an anti-adhesion agent using esterified collagen and hyaluronic acid comprising four steps of obtaining an esterified collagen and a hyaluronic acid complex in one form selected from a porous membrane, a film, a gel, and a microsomal form.

In the above, the esterified collagen and the hyaluronic acid complex of the porous membrane, the film, the gel, and the microsomal form can be obtained as follows.

(1) esterified collagen and hyaluronic acid complexes in the form of porous membranes,

While maintaining the 0.5 ~ 3wt% esterified collagen purified solution obtained in step 3 at 4 ° C to ensure homogeneity for 24 hours, the stirred solution was sprayed onto a thin plate to form a predetermined thickness and then lyophilized. To prepare a porous membrane of the esterified collagen on the spongy body, and the porous membrane was immersed in an aqueous solution of 0.5-5 wt% of hyaluronic acid at 4 ° C. for 24 hours, reacted, and then freeze-dried to form a porous membrane composite of esterified collagen and hyaluronic acid. You get This porous membrane composite can be used as an anti-adhesion agent and the porous membrane composite is crosslinked by a non-chemical method, DHT (dehydrothermal) method. Here, the DHT treatment is a non-chemical crosslinking method in which the porous membrane composite is placed in a vacuum oven and reacted at 100 to 120 ° C. for 24 hours, thereby improving physical properties of the porous membrane composite. Also, EDC [1-ethyl-3- (3- dimethylaminopropyl) carbodiimide] can be used to improve physical properties.

(2) esterified collagen and hyaluronic acid complexes in film form;

Agitated for 24 hours while maintaining 0.5 ~ 3wt% of the esterified collagen obtained in the step 3 at 4 ° C and spraying the stirred solution on a thin plate to form a constant thickness, followed by aerosol at 4 ° C. While drying, a film of esterified collagen was prepared, and the film was immersed in an aqueous solution of 0.5-5 wt% of hyaluronic acid, maintained at 4 ° C. for 24 hours, and then lyophilized to obtain an esterified collagen and hyaluronic acid film complex. do. The film can be used as an anti-adhesion agent, and can be crosslinked by DHT (dehydrothermal) method, which is similar to the porous membrane, or by crosslinking by EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide]. An improved esterified collagen and hyaluronic acid film composite is obtained.

(3) esterified collagen and hyaluronic acid complexes in gel form,

While maintaining a predetermined amount of 0.5 ~ 3wt% purified water and 0.5 ~ 5wt% hyaluronic acid aqueous solution of the esterified collagen obtained in the three step process to each of the syringe to inject each solution at the same time by mixing and homogenizing Esterified collagen and hyaluronic acid complexes in gel form are obtained.

(4) esterified collagen and hyaluronic acid complexes in microsomal form,

While stirring and maintaining a 0.5-5 wt% aqueous solution of hyaluronic acid at a speed of 50 rpm to 1000 rpm, an aqueous solution of 0.5-3 wt% of the esterified collagen obtained in the step 3 was added dropwise at a rate of 1 ml / min to 50 ml / min and hyaluronic acid. The microsomal complex of esterified collagen and hyaluronic acid, which was reacted at 4 ° C. for 24 hours to facilitate complex formation of lonic acid and esterified collagen, and then removed excess hyaluronic acid with purified water to freeze-dry only the complex of microsomal form. You get

The formation of atelo collagen porous membrane or film does not have a significant difference in the process, and the porous membrane can be formed by lyophilizing the porous membrane, and the film is dried with air, so that the tissue is dense and denser than the porous membrane.

In the method of crosslinking the porous membrane composite, the film composite, and the microsomal composite, the method of crosslinking with EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide] is EDC in a high concentration (94-96wt%) ethanol solution. The complexes were immersed in a solution in which 10 to 100 mmol was dissolved and maintained at 4 ° C. for 24 hours to be reacted, followed by washing with purified water to remove the excess EDC and freeze-drying to obtain each crosslinked complex.

Preparation of the anti-adhesion using esterified collagen and hyaluronic acid in the same manner as described above to determine the collagen content test, solubility test, contact angle measurement test of the composite.

Example (1)

Esterized Collagen Manufacturing

(1) Add 75 wt% ethanol solution to 2 wt% atelo collagen to add homogeneity, adjust to maintain pH 7.4 with 0.5 M acetic acid and maintain at 4 ° C. for 1 week to react the esterified collagen solution. Obtained and lyophilized to prepare esterified collagen.

Example (2)

Preparation of complexes of esterified collagen and hyaluronic acid in the form of porous membranes

(1) The aqueous solution of 1.5 wt% of esterified collagen in Example (1) was stirred for 24 hours while maintaining at 4 ° C. and then lyophilized to obtain an esterified collagen porous membrane.

1 is a state diagram of the esterified collagen porous membrane after lyophilization.

(2) The porous membrane was immersed in an aqueous solution of 2% by weight of hyaluronic acid purified water, reacted at 24 ° C. for 24 hours, and then lyophilized to obtain a porous membrane composite of esterified collagen and hyaluronic acid.

(3) The porous membrane composite is crosslinked by a nonhydrochemical DHT (dehydrothermal) method.

Specifically, the porous membrane composite is placed in a vacuum oven and reacted at 100 to 200 ° C. for 24 hours for crosslinking or crosslinking with EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide].

Here, the EDC crosslinking method is by immersing the porous membrane in a solution of 10 ~ 100 mmol molten EDC in 95% ethanol solution and kept at 4 ℃ for 24 hours to react, then washed excess EDC with purified water and freeze-dried porous membrane complex Get

Example (3)

Preparation of complex of esterified collagen and hyaluronic acid in film form

(1) While stirring the aqueous solution of purified water of 2 wt% of esterified collagen in Example (1) at 4 ° C. for 24 hours, the stirring solution was sprayed onto a plate of plastic or metal to form a constant thickness, and then 4 ° C. It is kept in air and dried by air to obtain an esterified collagen film.

2 is a state diagram of an esterified collagen film.

(2) The film was immersed in a 2 wt% aqueous solution of hyaluronic acid purified water, reacted at 4 ° C. for 24 hours, and then lyophilized to obtain a film complex of esterified collagen and hyaluronic acid.

(3) A film composite is obtained by treating in the same manner as in (2) and (3) of Example (2).

Figure 3 is a state diagram of the reaction film complex of esterified collagen and hyaluronic acid.

Example (4)

Preparation of complexes of esterified collagen and hyaluronic acid in gel form

(1) A gel complex is obtained by injecting a 2 wt% aqueous solution of purified water of esterified collagen and a 2 wt% aqueous solution of hyaluronic acid purified water into different syringes to simultaneously inject and mix the respective injection solutions.

Figure 4 is a gel complex state of esterified collagen and hyaluronic acid.

Example (5)

Preparation of complexes of esterified collagen and hyaluronic acid in microsomal form

(1) Using a syringe or a separatory funnel on the stirring solution while maintaining a 2 wt% aqueous solution of hyaluronic acid purified water at a speed of 50 rpm to 1000 rpm, 1 ml / min to 50% aqueous solution of 2 wt% esterified collagen purified water of Example (1) After dropping at a rate of ㎖ / min at 4 ℃ to react for 24 hours and then washed with purified water to obtain only the microsomal complex from which excess hyaluronic acid was removed to obtain a microsomal complex by lyophilization.

(2) The microsomal complex obtained by crosslinking the microsomal complex by the DHT method or crosslinking by EDC as in the method of (3) of Example (2) is obtained.

Example (6)

Collagen content test of esterified collagen porous membrane and porous membrane composite

1. An esterified collagen porous membrane is made according to (1) of Example (2) and a porous membrane composite is prepared according to (2) of Example (2).

2. Dissolve the complex using 0.5 M acetic acid.

3. Add Sircol dye to the solution of the complex dissolved in acetic acid and react for 30 minutes at room temperature.

4. Centrifuge at least 1000g for 10 minutes.

5. Discard the supernatant and take only pellets.

6. Dissolve the pellet by adding 0.5 M NaOH to the pellet.

7. Take a portion of the solution and measure it at 550 nm using a spectrophotometer.

8. Calculate the collagen content in the composite from the measured values and the standard curve.

As a result, the higher the concentration of hyaluronic acid, the lower the collagen content. It can be seen that the higher the hyaluronic acid is combined with the esterified collagen to form a complex.

Fig. 5 is a table measuring the content of collagen contained in the esterified collagen membrane and the hyaluronic acid complex.

Example (7)

Solubility test of esterified collagen porous membrane and porous membrane composite

1. An esterified collagen porous membrane is made according to (1) of Example (2) and a porous membrane composite is prepared according to (2) of Example (2).

2. The weight of the prepared porous membrane composite is measured.

3. Add 0.006 wt% of propynparaben and 0.054 wt% of methylparaben to the purified water and add the porous membrane composite to the purified water. The complex placed in purified water is reacted at 37 ° C.

4. Take out the reacted complex for a certain time and measure the weight after lyophilization.

5. Solubility of the complex is calculated by measuring the weight of the purified water before and after the reaction.

As a result, esterified collagen was dissolved when reacted with purified water, but the porous membrane composite was not dissolved. In addition, the higher the concentration of hyaluronic acid was shown to increase the solubility. This indicates that the complexed with hyaluronic acid rather than the esterified collagen alone can increase the physical properties and increase the residence time in vivo.

6 is a shape in which only the esterified collagen membrane is reacted with purified water. 7 is a state comparing the solubility of the esterified collagen membrane and the esterified collagen and hyaluronic acid complex that does not form a complex. Observed after 18 hours in purified water. 8 is a result table of the solubility test of the membrane of the esterified collagen and the hyaluronic acid complex.

Example (8)

Measurement of contact angle between esterified collagen film and film composite

1. An esterified collagen film is made according to (1) of Example (3), and a film composite is made according to (2) of Example (3).

2. Measure the contact angle by dropping purified water on the surface of the composite (contact angle instrument: First ten Angstroms 100).

As a result, the higher the concentration of hyaluronic acid was confirmed to increase the hydrophilicity.

9 is a result of measuring the contact angle of the esterified collagen film and hyaluronic acid complex.

1 is a preparation of esterified collagen porous membrane

2 is a preparation of esterified collagen film

Fig. 3 shows esterified collagen porous membrane and hyaluronic acid complex (left) and esterified collagen film and hyaluronic acid complex (right).

4 is a gel-form complex of esterified collagen and hyaluronic acid

5 is a collagen content of the esterified collagen porous membrane and hyaluronic acid complex

6 is the solubility of esterified collagen porous membranes over time.

7 is a (right) solubility comparison of esterified collagen porous membrane (left) and esterified collagen porous membrane and hyaluronic acid complex (right) after 18 hours in purified water.

8 is a solubility test table (top) and graph (bottom) of the esterified collagen porous membrane and hyaluronic acid complex

9 is a contact angle measurement of the esterified collagen film and hyaluronic acid complex

Claims (10)

Step 1 to obtain atelo collagen from which telopeptides were removed by treating natural collagen extracted from pigs with pepsin enzyme, Add 0.5 ~ 3wt% of the atelocollagen to 65 ~ 85wt% ethanol solution to make it homogeneous, adjust to pH7.4 with 0.5M acetic acid or hydrochloric acid, and then react for 1 week at 4 ℃ for collagen esterification Step 2 to obtain a solution, Lyophilizing the esterified collagen solution to prepare esterified collagen, A method for preparing an anti-adhesion agent using an esterified collagen and hyaluronic acid comprising four steps of obtaining a complex of esterified collagen and hyaluronic acid in one form selected from a porous membrane, a film, a gel, and a microsomal form. The method of claim 1, wherein the esterified collagen and hyaluronic acid porous membrane composite is stirred to ensure homogeneity for 24 hours while maintaining the purified aqueous solution of 0.5 ~ 3wt% of the esterified collagen obtained in step 3 at 4 ℃ and stirred The solution was sprayed onto a thin plate to form a certain thickness, and then lyophilized to prepare a porous membrane of esterified collagen on the spongy body, and the porous membrane was immersed in an aqueous solution of 0.5-5 wt% of hyaluronic acid at 4 ° C. for 24 hours. A method of preparing an anti-adhesion agent using esterified collagen and hyaluronic acid, characterized in that the porous membrane complex of esterified collagen and hyaluronic acid reacted and lyophilized. The ester of claim 1, wherein the porous membrane composite is a porous membrane composite obtained by crosslinking the porous membrane composite of claim 2 with a dehydrothermal (DHT) method or EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide]. Method for preparing anti-adhesion agent using hydrogenated collagen and hyaluronic acid. The method of claim 1, wherein the esterified collagen and hyaluronic acid film composite is stirred for 24 hours while maintaining the aqueous solution of 0.5 ~ 3wt% of the esterified collagen obtained in the three step process at 4 ℃ spraying the stirring solution on a thin plate To form a constant thickness, and then dried with air while maintaining 4 ℃ to prepare an esterified collagen film, the film was immersed in an aqueous solution of 0.5-5 wt% of hyaluronic acid at 4 ℃ for 24 hours to react and then lyophilized A method for producing an anti-adhesion agent using esterified collagen and hyaluronic acid, characterized in that the film complex of the esterified collagen and hyaluronic acid. The method of claim 1, wherein the film complex is esterified collagen, characterized in that the film complex obtained by crosslinking the film complex of claim 4 by DHT (dehydrothermal) method or EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide] Method for preparing an anti-adhesion agent using hyaluronic acid. The method of claim 1, wherein the esterified collagen and hyaluronic acid gel complex is injected into the syringe by injecting a predetermined amount of 0.5-3wt% aqueous solution of collagen ester and 0.5-5wt% aqueous solution of hyaluronic acid obtained in the three-step process, respectively. A method for preparing an anti-adhesion agent using esterified collagen and hyaluronic acid, characterized in that the gel complex is mixed and homogenized by simultaneously injecting together. The method according to claim 1, wherein the esterified collagen and hyaluronic acid microsomal complex is a 0.5 ~ 5wt% aqueous solution of hyaluronic acid while stirring and maintaining at a rate of 50rpm to 1000rpm, the aqueous solution of 0.5-3wt% esterified collagen thereon 1ml / min Esterified collagen and hyaluronic acid, characterized in that the microsomal complexes were removed by hydration of only the microsomal complexes by removing the excess hyaluronic acid with purified water and reacting at 4 ° C. for 24 hours at a rate of ˜50 ml / min. Method for preparing the anti-adhesion using. The microsomal complex according to claim 1, wherein the microsomal complex is an microsomal complex obtained by crosslinking the microsomal complex of claim 6 with a DHT (dehydrothermal) method or EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide]. Method for producing coalescing agent using collagen and hyaluronic acid. The method of claim 3 and 5, wherein the crosslinking treatment method of dehydrothermal (DHT) is a non-chemical crosslinking method of putting a porous composite or a film composite into a vacuum oven and reacting at 100 to 120 ° C for 24 hours. Method for producing coalescing agent using hyaluronic acid. The method according to claim 3 and 5, the crosslinking method of EDC treatment is a porous membrane or film composite to react 10 ~ 100ml of EDC in 94 ~ 96wt% ethanol solution and then washed with purified water to remove excess EDC and freeze A method for producing coalescing agents using esterified collagen and hyaluronic acid, characterized in that the drying method.

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