KR20190023794A - A preparing method of tissue repair treatment composition using the two step cross-linking and the composition therefrom - Google Patents

Abstract

The present invention relates to a method for producing a bio-composition for tissue restoration, which has excellent shape retention and good injection force, and a bio-composition produced therefrom, which lowers injection pressure to facilitate injection by allowing a first low degree crosslinking reaction to occur in order to improve injection force and reduce side effects; and which has good injection force and excellent shape retention through double crosslinking by allowing an ionic second-step crosslinking of a first-step crosslinking agent and a cationic biocompatible polymer crosslinking agent to proceed in vivo.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of preparing a biocomponent for tissue repair using double crosslinking, and a biocomposite prepared from the same. BACKGROUND OF THE INVENTION 1. Field of the Invention [0002]

The present invention relates to a method for producing a biocomponent for tissue repair having excellent shape retentivity and good main input and a biocomposite prepared from the same, and more particularly, And the ionic two-step crosslinking of the cross-linking agent using the first-step cross-linking agent and the cationic biocompatible polymer is allowed to proceed in the body, whereby the main input is excellent and the shape retention A method for producing a biocidal composition for excellent tissue repair, and a biocidal composition produced therefrom.

The skin consists of epidermis and dermis. The epidermis protects the skin from external stimuli, and the dermis is the layer that determines the skin condition. The epidermis is composed of ceramide, the dermis consists of hyaluronic acid, collagen and elastin. The dermis is 0.1 to 0.3 mm below the skin and has a thickness of 0.6 to 4 mm. As the dermis progresses, the thickness of the dermis becomes thinner. On the other hand, hyaluronic acid, which is one of the components of the dermis, is filled between collagen and elastin fibers, and as hyaluronic acid progresses, hyaluronic acid disappears and causes wrinkles.

As the aging of the skin progresses, many wrinkles occur around the face due to gravity, exposure to sunlight, and the like. Recently, as the interest in beauty has increased, there has been an increasing demand for improving the wrinkles. Therefore, patients tend to solve the need for beauty such as wrinkle improvement by using biomaterials with low downtime and short downtime for patients. Accordingly, various kinds of biomaterials for tissue repair are being researched and developed, and their demand is increasing.

Hyaluronic acid (hereinafter abbreviated as "HA") is a compound having a linear structure in which the repeating unit composed of N-acetyl-D-glucosamine and D-glucuronic acid is linear , And it is used in various forms such as a film or gel for preventing post-operative adhesion, an insert for improving wrinkles, a molding aid, etc. in the form of a hyaluronic acid derivative.

Wherein n is an integer of 1 or more.

Conventional biomaterials for hyaluronic acid (HA) tissue repair have been developed to increase the viscosity and elasticity of products by crosslinking using a single component chemical crosslinking agent such as BDDE (1,4-Butanediol diglycidyl ether) Has been developed. However, when cross-linking with synthetic chemicals such as BDDE, three types of unreacted cross-linking agents are causing side effects.

On the other hand, biomaterials for tissue repair to improve facial wrinkles are composed of two types, monophasic and biphasic. Biological materials for single-phase tissue repair are homogeneous gel, which has the advantage of allowing a smooth and delicate injection during the procedure, but it has a disadvantage of low shape retention. On the other hand, biomaterials for biopsy-type tissue restoration have the advantage that the volume of the skin is increased and the shape retentiveness is high when the treatment is performed by mixing the hyaluronic acid solution with the particle type gel. However, it has a disadvantage in that the main input is higher than that of a single-body biomaterial for tissue repair.

Accordingly, there is a continuing need for a biocompatible composition for tissue repair that has a low injection pressure and is easily injected and has excellent shape retaining ability.

Korean Patent Application No. 2010-0055556 entitled "Method for producing cationic polymer / hyaluronic acid microbeads and cationic polymer / hyaluronic acid microbeads chelated with metal ions"

It is an object of the present invention to provide a method for manufacturing a biocidal composition for tissue repair, which has a low main input through double crosslinking in two steps and is excellent in shape retention ability.

It is still another object of the present invention to provide a biocidal composition for tissue repair which has low main input and excellent shape retention ability through double crosslinking in two steps.

The present invention provides a method for preparing a biological composition for hyaluronic acid-chitosan complex hydrogel structure recovery, which has a low main input through double-crosslinking in two steps and is excellent in shape retention.

Another object of the present invention is to provide a biocompatible composition for hyaluronic acid-chitosan complex hydrogel structure recovery, which has low main input and excellent shape retention ability through double crosslinking in two steps.

The biocompatible composition for hyaluronic acid-chitosan complex hydrogel structure according to the present invention has a two-step crosslinking, that is, a low-cost cross-linking composition in vivo, and has excellent main input and excellent shape retention ability.

The first photograph of FIG. 1 shows the state 1 to 4 and the purified water 5 in which hyaluronic acid is dissolved in a basic solution, and then chitosan is added. The second photograph shows the order of reaction at pH 3.5, pH 4.0, pH 5.0 and pH 6.5 FIG.
FIG. 2 is a photograph showing the state color change according to the chitosan concentration added to the hyaluronic acid solution at room temperature,
FIG. 3 is a photograph showing a state color change according to a chitosan concentration added to a hyaluronic acid solution at 40 ° C,
4 is a graph showing the rheological characteristics of Examples and Comparative Examples using a rheometer,
FIG. 5 is a graph of particle size analysis of hyaluronic acid-chitosan complex hydrogel and Comparative Examples 1 and 2 compositions of Examples 1 to 4,
FIG. 6 is a graph showing the enzyme resistance characteristics of Examples and Comparative Examples,
7 is a view showing a method of injecting the hyaluronic acid-chitosan complex composition of the present invention.

The present invention

Cross-linking homogenized hyaluronic acid gel and BDDE (1,4-butanediol diglycidyl ether) to obtain a crosslinked hyaluronic acid derivative;

Dialyzing the crosslinked hyaluronic acid derivative;

Lyophilizing the crosslinked hyaluronic acid derivative;

Pulverizing the lyophilized crosslinked hyaluronic acid derivative;

To the solution having a concentration of 2 to 10% (w / v) of the crushed crosslinked hyaluronic acid derivative is added 1 to 10% (w / v) of crosslinked hyaluronic acid having a molecular weight of about 600,000 daltons to 3,000,000 daltons in a phosphate buffer solution Thereby obtaining an anionic hyaluronic acid gel; And

Chitosan complex hydrogel obtained by mixing the anionic hyaluronic acid obtained by the one-step crosslinking with the cationic chitosan gel to obtain a hyaluronic acid-chitosan complex hydrogel. A method for preparing a composition is provided.

The present invention also provides a biocidal composition for tissue repair, which is produced by the above production method.

Hereinafter, the present invention will be described in more detail.

The biologic composition for tissue repair according to the present invention, that is, the hyaluronic acid-chitosan complex hydrogel is obtained by double cross-linking, and is characterized in that it is composed of a first step crosslinking by chemical cross-linking, an anionic hyaluronic acid hydrogel and a cationic chitosan gel electrostatically Characterized in that it is obtained through two-step crosslinking by gravity.

The method for producing a biocompatible composition for hyaluronic acid-chitosan complex hydrogel structure repair of the present invention will be described in detail as follows.

The one-step crosslinking is a step for preparing an anionic crosslinked hydrogel.

Preparation of crosslinked hyaluronic acid derivative (1 step crosslinking)

1 g of caustic soda is dissolved in 100 ml of distilled water to prepare a 1% (w / v) caustic soda aqueous solution. Hyaluronic acid 10% (w / v) having a molecular weight of about 110 to 1.2 million Daltons was added thereto, and homogenized at 900 to 1,000 rpm for 180 minutes at room temperature using a homogenizer to obtain hyaluronic acid hydrogel .

After 2.5% (v / v) of a crosslinking agent BDDE (1,4-butanediol diglycidyl ether) was added to the hyaluronic acid hydrogel thus prepared, the mixture was stirred at room temperature for 30 minutes at 900 to 1,000 rpm using a homogenizer, For 30 to 40 hours. When the reaction is carried out at the above temperature and time, it is preferable to obtain 1 to 8% of the desired low cost pellets in the present invention.

In the case of a biocomponent for tissue repair, it is common to increase the cross-linking to about 20% in order to improve the shape retention ability. When the cross-linking degree is high, the shape retention is good when injected into the body, but injection is difficult and side effects cause necrosis, heterogenization or dizziness There is a possibility. In the case of the present invention, it is possible to minimize side effects due to high crosslinking by lowering the degree of crosslinking.

The prepared basic crosslinked hyaluronic acid derivative is adjusted to pH 6.5 to 7.5 with a 1N hydrochloric acid solution. As shown in Fig. 1, the pH is maintained at 6.5 to 7.5, and more preferably at pH 6.5 or more because the transparency is the most excellent and the product is excellent.

After the cross-linking reaction, dialyzed with PBS (phosphate buffer saline) and distilled water for 2 days to remove the remaining cross-linking agent BBDE to remove the residual cross-linking agent. The crosslinked hyaluronic acid derivative dialysis solution from which the crosslinking agent has been removed is lyophilized for 2 days to obtain a white crosslinked hyaluronic acid derivative, followed by pulverization.

The particles of the pulverized cross-linked hyaluronic acid derivative is preferably from ₅₀ 200 D to 300㎛.

Anionic  Bridged hyaluronic acid Hydrogel  Produce

The prepared crushed crosslinked hyaluronic acid derivative 4% (w / v) and unhybridized hyaluronic acid (molecular weight about 250 to 260 million daltons) 2% (w / v) were mixed in a buffer solution, Homogenized at 1,000 rpm for 20 minutes and aged for 12 hours in an incubator set at 30 DEG C to prepare an anionic biphasic hyaluronic acid hydrogel. FIG. 2 and FIG. 3 show the state of the hyaluronic acid hydrogel at room temperature and 40.degree. C., confirming that the product has the best quality and appearance at room temperature.

Hyaluronic acid-chitosan complex Hydrogel  Manufacturing (two-step bridge)

A mixture of 4% (w / v) of ground crosslinked hyaluronic acid derivative prepared in the first step crosslinking and 2% (w / v) of uncrosslinked hyaluronic acid (molecular weight about 250 to 260 million daltons) Biphasic anionic hyaluronic acid hydrogel was prepared by homogenization at 900 to 1,000 rpm for 20 minutes using a centrifuge.

A buffer solution containing 0.01 to 0.5% (w / v) of chitosan prepared in advance was mixed and agitated at 900 to 1,000 rpm for 30 minutes using a homogenizer, followed by aging for 12 hours in an incubator set at 30 캜 to obtain an anionic hyaluronic acid A hyaluronic acid-chitosan complex hydrogel in which cationic chitosan is bound by electrostatic attraction is prepared.

FIG. 2 and FIG. 3 show the state of the hyaluronic acid-chitosan complex hydrogel at room temperature and 40.degree. C. It can be confirmed that the product of 0.001 to 1.0% (w / v) in chitosan has the best quality and appearance.

A method of injecting the hyaluronic acid-chitosan complex hydrogel produced by the present invention into the body will be described below.

The anionic crosslinked hyaluronic acid hydrogel prepared by the one-step crosslinking was injected into the first inlet 100 using the double filler injector shown in FIG. 7, and the cationic chitosan gel was injected into the second inlet 200 When the rear piston 300 is pushed and injected, the composition in the first inlet and the second inlet is collected in the mixing part 400, and the two-step crosslinking occurs, and stabilization occurs in the body.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not construed as being limited by the following Examples.

Manufacturing example

Preparation of crosslinked hyaluronic acid derivatives

1 g of caustic soda was dissolved in 100 ml of distilled water to prepare a 1% (w / v) caustic soda aqueous solution. 10% (w / v) of hyaluronic acid (molecular weight: about 110 to 1.2 million daltons) was added thereto and homogenized at 900 to 1,000 rpm for 180 minutes at room temperature using a homogenizer to obtain hyaluronic acid hydrogel Gel. After 2.5% (v / v) of a crosslinking agent BDDE (1,4-butanediol diglycidyl ether) was added to the prepared hyaluronic acid hydrogel, the mixture was stirred at 900 to 1,000 rpm for 30 minutes using a homogenizer at room temperature, And incubated in a set incubator (Incubator) for 40 hours. The prepared basic crosslinked hyaluronic acid derivative hydrogel was adjusted to pH 6.5 to 7.5 with 1N hydrochloric acid solution. After the crosslinking reaction, the remaining crosslinking agent, BDDE, was placed in a 13,000 MWCO RC dialysis bag, and dialyzed with PBS (phosphate buffer saline) and distilled water for 2 days to remove residual crosslinking agent. The crosslinked hyaluronic acid derivative dialysis solution from which the crosslinking agent had been removed was lyophilized for 2 days to obtain a white crosslinked hyaluronic acid derivative and pulverized to a particle size of D ₅₀ 200 μm using a colloid mill.

Example  One

Preparation of double-crosslinked hyaluronic acid-chitosan complex hydrogel containing 0.01% (w / v) chitosan

4% (w / v) of a crosslinked hyaluronic acid derivative having a particle size D _{50 of} 200 μm prepared in Preparation Example 1 and 2% (w / v) of a crosslinked hyaluronic acid having a molecular weight of 250 to 260,000 daltons were dissolved in a buffer Mixed and homogenized at 900 to 1,000 rpm for 20 minutes at room temperature using a homogenizer to prepare an ideal anionic crosslinked hyaluronic acid hydrogel. A buffer solution containing 0.01% (w / v) of chitosan previously prepared was mixed and stirred at 900 to 1,000 rpm for 30 minutes using a homogenizer, and then aged for 12 hours in an incubator set at 30 DEG C to prepare an ideal hyaluronic acid-chitosan complex A hydrogel was obtained.

Example  2

Preparation of double-crosslinked hyaluronic acid-chitosan complex hydrogel containing 0.1% (w / v) chitosan

4% (w / v) of a crosslinked hyaluronic acid derivative having a particle size D _{50 of} 200 mu m produced by the above preparation example and 2 to 2% (w / v) of a crosslinked hyaluronic acid having a molecular weight of 250 to 260,000 daltons were dissolved in a buffer Mixed and homogenized at 900 to 1,000 rpm for 20 minutes at room temperature using a homogenizer to prepare an ideal anionic crosslinked hyaluronic acid hydrogel. A buffer solution containing 0.1% (w / v) of chitosan prepared in advance was mixed and stirred at 900 to 1,000 rpm for 30 minutes using a homogenizer, followed by aging for 12 hours in an incubator set at 30 DEG C to prepare an ideal hyaluronic acid-chitosan complex A hydrogel was obtained.

Example  3

Preparation of double-crosslinked hyaluronic acid-chitosan complex hydrogel containing 0.5% (w / v) chitosan

4% (w / v) of a crosslinked hyaluronic acid derivative having a particle size D _{50 of} 200 μm produced in the above preparation example and 2% (w / v) of hyaluronic acid (molecular weight of about 250 to 260 million daltons) And then homogenized at room temperature for 20 minutes at 900 to 1,000 rpm using a homogenizer to prepare an anionic crosslinked hyaluronic acid hydrogel. The mixture was mixed with a buffer solution containing 0.5% (w / v) of chitosan previously prepared and agitated at 900 to 1,000 rpm for 30 minutes using a homogenizer, followed by aging for 12 hours in an incubator set at 30 DEG C to prepare an ideal hyaluronic acid- A hydrogel was obtained.

Example  4

Preparation of double-crosslinked hyaluronic acid-chitosan complex hydrogel with modified 1,2-degree cross-linking conditions

5% (w / v) of a derivative having a particle size D _{50 of} 200 μm produced in the above Preparation Example and 1% (w / v) of crosslinked hyaluronic acid having a molecular weight of about 250 to 260 million daltons were dissolved in a buffer solution PBS: phosphate buffer saline), and homogenized at 900 to 1,000 rpm for 20 minutes at room temperature using a homogenizer to prepare a biphasic hyaluronic acid hydrogel. (PBS) containing 0.2% (w / v) of chitosan previously prepared was mixed, and the mixture was stirred at 900 to 1,000 rpm for 30 minutes using a homogenizer and then cooled to 30 ° C Biphasic hyaluronic acid-chitosan complex hydrogel was prepared by aging in a set incubator for 12 hours.

Comparative Example  One

Preparation of primary crosslinked ideal hyaluronic acid hydrogel

1 g of caustic soda was dissolved in 100 ml of distilled water to prepare a 1% (w / v) caustic soda aqueous solution. After adding 10% (w / v) of hyaluronic acid (molecular weight: about 250 to 260 million daltons), the mixture was homogenized at 900 to 1,000 rpm for 180 minutes using a homogenizer at room temperature to obtain hyaluronic acid hydrogel . After 2.5% (v / v) of a crosslinking agent BDDE (1,4-butanediol diglycidyl ether) was added to the hyaluronic acid hydrogel prepared above, the mixture was stirred at 900-1,000 rpm for 30 minutes at room temperature using a homogenizer, And incubated in a set incubator (Incubator) for 40 hours. The prepared basic crosslinked hyaluronic acid hydrogel was adjusted to pH 6.5 to 7.5 with 1 N hydrochloric acid solution. After the crosslinking reaction, the remaining crosslinking agent, BDDE, was placed in a 20,000 MWCO CE dialysis bag, and the remaining crosslinking agent was removed by dialysis against PBS (phosphate buffer saline) and distilled water for 2 days each. The crosslinked hyaluronic acid hydrogel dialysis solution from which the crosslinking agent had been removed was lyophilized for 2 days to obtain a white crosslinked hyaluronic acid solid. Preparation A mixture of 4% (w / v) of crosslinked hyaluronic acid and 2% (w / v) of crosslinked hyaluronic acid (molecular weight: about 250 to 260 million daltons) was mixed in a phosphate buffered saline (PBS) Biphasic hyaluronic acid hydrogel was prepared by agitating in an incubator (Incubator) set at 30 DEG C for 12 hours using a homogenizer at 900 to 1,000 rpm for 30 minutes and then stirring.

Comparative Example  2

Preparation of primary cross-linked single-phase hyaluronic acid hydrogel

1 g of caustic soda was dissolved in 100 ml of distilled water to prepare a 1% (w / v) caustic soda aqueous solution. 10% (w / v) of hyaluronic acid (molecular weight: about 110 to 1.2 million daltons) was added thereto and homogenized at 900 to 1,000 rpm for 180 minutes at room temperature using a homogenizer to obtain hyaluronic acid hydrogel Gel. After 2.5% (v / v) of a crosslinking agent BDDE (1,4-butanediol diglycidyl ether) was added to the hyaluronic acid hydrogel thus prepared, the mixture was stirred at 900 to 1,000 rpm for 30 minutes using a homogenizer at room temperature. 2% (w / v) lactic acid (molecular weight: about 250 to 260 million daltons) was added. After the addition, the gel was formed by aging in an incubator set at 30 DEG C for 40 hours. The prepared basic crosslinked hyaluronic acid hydrogel was neutralized by adding HCl-PBS solution, and the gel was swollen on the shaker for 48 hours. The gel was placed in a 13,000 MWCO RC dialysis bag and dialyzed in PBS pH 7.4 buffer solution for 4 days to prepare a monophasic hyaluronic acid hydrogel.

Final concentration (% by weight) Mixing ratio (weight ratio) Remarks Bridged hyaluronic acid derivative Non-crosslinked hyaluronic acid Chitosan Bridged hyaluronic acid derivative Chitosan Non-crosslinked hyaluronic acid Example 1 4.0% 2.0% (2.5 to 2.6 MDa) 0.01 400 One 200 Sincerity double bridge Example 2 4.0% 2.0% (2.5 to 2.6 MDa) 0.1 40 One 20 Sincerity double bridge Example 3 4.0% 2.0% (2.5 to 2.6 MDa) 0.5 8 One 4 Sincerity double bridge Example 4 5.0% 3.0% (2.5 to 2.6 MDa) 0.2 25 One 15 Sincerity double bridge Comparative Example 1 4.0% 2.0% (2.5 to 2.6 MDa) - 2 0 One Ideal Chitosan None Comparative Example 2 10.0% 2.0% (2.5 to 2.6 MDa) - 5 0 One Single phase chitosan None

Test Example 1 Test of hyaluronic acid-chitosan complex hydrogel Viscoelasticity test

The rheological properties of Examples 1 to 4 and Comparative Examples 1 to 2 prepared by the present invention were analyzed using a rheometer.

Analysis conditions of a rotating rheometer

(1) Test equipment: Rotational Rheometer (TA Instrument Ltd., DHR1)

(2) Frequency: 01 ~ 10Hz

(3) Temperature: 25 ± 1 ° C

(4) Strain: 1%

(5) Minimum torque oscillation: 10 nN * m

(6) Maximum torque: 150nN * m

(7) Torque resolution: 0.1nN * m

(8) Measuring geometry: 25 mm plate

(9) Measuring Gap: 1.0 mm

Frequency: 1.0Hz G '(Pa) G "(Pa) Tan (Delta) Complex Viscosity (Pa, S) Example 1 639 292 0.457 111 Example 2 711 305 0.429 123 Example 3 530 303 0.572 97 Example 4 614 270 0.439 106 Comparative Example 1 324 119 0.367 54 Comparative Example 2 75 22 0.293 12

From the above Table 2, it can be seen that Examples 1 to 4 of the present invention exhibit excellent elastic properties while having a higher compound viscosity than Comparative Examples 1 and 2. That is, the hyaluronic acid-chitosan complex hydrogel containing chitosan prepared according to the present invention exhibited excellent mechanical characteristics compared with Comparative Examples 1 and 2. It was confirmed that it has good overall elasticity and high elasticity, and excellent physical properties when mixed with chitosan. The results are shown in Table 2 and FIG.

Experimental Example 2: Analysis of particle size of double-crosslinked hyaluronic acid-chitosan complex hydrogel prepared by the present invention

The Particles and sizes of the hyaluronic acid-chitosan complex compositions of Examples 1 to 4 were measured using a HELOS Particle Size Analyzer. The results are shown in Table 3 and FIG.

Test Example  2: Hyaluronic acid-chitosan complex produced by the present invention Hydrogel Particle size analysis

The particle size and distribution of the hyaluronic acid-chitosan complex compositions of Examples 1 to 4 and Comparative Examples 1 and 2 prepared according to the present invention were measured using a HELOS Particle Size Analyzer. The results are shown in Table 2 and FIG.

Average particle size (D ₅₀ , 占 퐉) Example 1 193.63 Example 2 208.74 Example 3 198.75 Example 4 227.38 Comparative Example 1 222.38 Comparative Example 2 300.76

In Table 3, the average particle size of the anionic hyaluronic acid hydrogel and the chitosan-bound Examples 1 to 4 by one-step crosslinking were uniform, and the average particle size distribution of Examples 1 to 4 was uniform, Which is similar to the average particle size of the first stage crosslinked anionic hyaluronic acid hydrogel. That is, it can be seen from the above results that chitosan does not affect the hyaluronic acid particle size.

Test Example  3: The hyaluronic acid-chitosan complex composition prepared by the present invention Injection pressure  Measure

Injection pressure measurement was carried out using Push Pull Gauges to confirm the pressure output in the pre-field syringe of the injection pressure measurement of Examples 1 to 4 and Comparative Examples 1 and 2 manufactured by the present invention.

Injection pressure test analysis conditions

(1) Tester: Mechanical Force Gauges (Push Pull Gauges, IMADA)

(2) Test speed: 20 nm / min

(3) Measured displacement: 10 mm

(4) Measuring range: 5N (500gf) to 500N (50kgf)

(5) Test environment: 25 ± 2 ℃

Maximum injection pressure (N) Minimum injection pressure (N) The average inlet pressure ((N) Example 1 19.4 16.1 16.9 Example 2 19.1 15.9 17.4 Example 3 18.0 14.3 16.2 Example 4 21.3 15.3 17.9 Comparative Example 1 28.9 23.4 26.5 Comparative Example 2 22.3 17.1 19.2

Table 4 shows the measurement results of the injection pressure for the hyaluronic acid-chitosan complex hydrogel prepared according to the present invention. As can be seen from the results of the measurement results, the composition prepared by the present invention had a lower injection pressure than the comparative example. It was confirmed that the hyaluronic acid-chitosan complex hydrogel containing chitosan more than Comparative Examples 1 and 2 resulted in an excellent injection pressure, thereby increasing the softness of the gel, which is important in filler treatment.

Experimental Example  4: Measurement of enzyme resistance of hyaluronic acid-chitosan complex composition prepared by the present invention

Experiments were conducted to determine the amount of decomposition of each sample within the same period to test the biodegradation characteristics of the hydrogel particles by the enzyme. A pH 7.4 solution of PBS was used, and hyaluronidase, which degrades hyaluronic acid, was used as the enzyme used. Unit (1U) represents the enzyme activity and hyaluronase is defined as changing the absorbance of 0.330 at 600nm at 37 ° C per minute. In the experiment, each sample was added to each solution at a concentration of 0.1 g / 3 ml. PBS and 100 U / ml hyaluronase, mixed buffer solution, were used for the degradation. Each sample was weighed every 3 days in a shaking bath maintained at 37 ° C to determine the degree of degradation.

Fig. 6 shows the result of measuring the weight of the sample every 3 days and showing the degree of biodegradation by the enzyme. Polysaccharide is composed of a sugar bond between an aldehyde (-CHO) and an alcohol (-OH), a bond in which carbon 1 and carbon 4 are connected by an acetal bond, that is, a β-glycosidic bond. It serves to capture a specific moiety within the composition and break this bond. Enzymes degraded more quickly because enzymes that catalyze catalyzed hydrolysis faster in pure buffer solutions. The reason why hyaluronic acid-based particles are decomposed faster than chitosan-based particles is that because of the high degree of swelling of the particles due to the extreme hydrophilicity of the hyaluronic acid, the penetration of water is fast due to the low degree of crosslinking and the surface area of contact with the enzyme is high, do. Thus, a composition having a high degree of crosslinking has a lower decomposition rate than hyaluronic acid. That is, if the structure has a high degree of crosslinking and a dense surface, the penetration of water is relatively difficult and the decomposition rate becomes slow.

As shown in FIG. 6, the reaction proceeded under similar conditions, but the compositions of Comparative Examples 1 and 2 without chitosan were composed of only hyaluronic acid, and the enzyme decomposition was performed somewhat faster than the other compositions. As a result, It can be seen that the hyaluronic acid-chitosan complex composition of the Example is excellent in resistance to enzymes.

100; The first inlet
200: 2nd inlet
300; piston
400: mixing part

Claims (8)

Cross-linking homogenized hyaluronic acid gel and BDDE (1,4-butanediol diglycidyl ether) to obtain a crosslinked hyaluronic acid derivative;
Dialyzing the crosslinked hyaluronic acid derivative;
Lyophilizing the crosslinked hyaluronic acid derivative;
Pulverizing the lyophilized crosslinked hyaluronic acid derivative;
To the solution of the crushed crosslinked hyaluronic acid derivative in 2 to 10% (w / v), 1 to 10% (w / v) of crosslinked hyaluronic acid having a molecular weight of about 600,000 daltons to 3 million daltons was mixed with a phosphate buffer solution A chemical one-step crosslinking comprising obtaining an anionic crosslinked hyaluronic acid hydrogel; And
Chitosan complex hydrogel obtained by mixing the anionic hyaluronic acid obtained by the one-step crosslinking with the cationic chitosan gel to obtain a hyaluronic acid-chitosan complex hydrogel. ≪ / RTI >
The method according to claim 1,
Wherein the crosslinking hyaluronic acid derivative has a 1-step degree of crosslinking of 1 to 8%.
The method according to claim 1,
Wherein the freeze-drying is carried out at -80 to 70 캜 for 2 to 3 days.
The method according to claim 1,
Wherein the pulverized crosslinked hyaluronic acid derivative has a particle size of D ₅₀ 200 to 300 탆.
The method according to claim 1,
The two-step crosslinking was performed by mixing 0.01 to 0.5% w / v of cationic chitosan gel with 4% (w / v) anionic hyaluronic acid hydrogel obtained by the first step crosslinking into a buffer solution (PBS) to form hyaluronic acid - < / RTI > chitosan complex hydrogel of the present invention.
The method according to claim 1,
The one-step crosslinking
1 to 10% by volume of BDDE was added to 1 to 10% by volume of homogenized hyaluronic acid hydrogel, and the mixture was stirred at room temperature for 30 to 60 minutes at 900 to 1000 rpm using a homogenizer, and aged at 25 to 30 ° C for 30 to 60 minutes Obtaining a basic crosslinked hyaluronic acid derivative;
Adjusting the basic crosslinked hyaluronic acid derivative to pH 7.5 or less with 1N hydrochloric acid solution;
Dialyzing with buffer (PBS) and distilled water for 2 days each to remove the crosslinking agent BDDE;
Lyophilizing the dialyzed solution of the crosslinked hyaluronic acid derivative obtained by removing the crosslinking agent for 2 days;
Pulverizing the lyophilized crosslinked hyaluronic acid derivative; And
4% by volume of the ground crosslinked hyaluronic acid derivative and 2% by volume of non-crosslinked hyaluronic acid were mixed in a buffer solution, homogenized at 900 to 1000 rpm for 20 minutes at room temperature using a homogenizer, And then aging the solution to obtain a biosynthetic anionic hyaluronic acid hydrogel.
The method according to claim 1,
The two-step crosslinking
A buffer solution containing 0.01 to 0.5% (w / v) of cationic chitosan was mixed with the anionic hyaluronic acid hydrogel obtained by the first-step crosslinking, and the mixture was stirred at 900 to 1000 rpm for 30 to 60 minutes using a homogenizer, Lt; 0 > C for 10 to 12 hours to obtain a hyaluronic acid-chitosan complex.
A biocidal composition for tissue repair using the double crosslinking obtained by any one of claims 1 to 7.

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