KR102067187B1 - 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 tissue repair biocomposite having excellent shape holding power and good injection power, and a biocomposite prepared therefrom, and more particularly, to perform a one-step low-crosslinking crosslinking reaction to improve injection power and reduce side effects. The injection pressure is lowered to facilitate the injection, and the ionic two-step crosslinking of the one-step crosslinked body and the cationic biocompatible polymer crosslinker proceeds in the body, thereby providing excellent injection strength and excellent shape retention through double crosslinking. The present invention relates to a method for producing a repaired biocomposite and a biocomposite prepared therefrom.

Description

TECHNICAL FIELD OF THE INVENTION A method for preparing a biocomposition for tissue repair using double crosslinking and a biocomposition prepared therefrom {A PREPARING METHOD OF TISSUE REPAIR TREATMENT COMPOSITION USING THE TWO STEP CROSS-LINKING AND THE COMPOSITION THEREFROM}

The present invention relates to a method for producing a tissue repair biocomposite having excellent shape holding power and good injection power, and a biocomposite prepared therefrom, and more particularly, to perform a one-step low-crosslinking crosslinking reaction to improve injection power and reduce side effects. The injection pressure is lowered to facilitate the injection, and the ionic two-stage crosslinking of the crosslinking agent using the one-step crosslinked body and the cationic biocompatible polymer proceeds in the body, thereby providing excellent injection power and double shape retention. The present invention relates to a method for producing an excellent tissue repair biocomposite and a biocomposite prepared therefrom.

The skin consists of the epidermis and the dermis. The epidermis serves to protect the skin from external stimuli, and the dermis is the layer that determines skin condition. In addition, the epidermis is composed of ceramide and the dermis is composed of hyaluronic acid, collagen and elastin. The dermis is a layer that is 0.1 ~ 0.3mm below the skin, and the thickness is about 0.6 ~ 4mm. As the aging progresses, the dermis becomes thinner. Meanwhile, hyaluronic acid, which is one of the constituents of the dermis, is filled between collagen and elastin fibers, and as aging progresses, hyaluronic acid disappears and causes wrinkles.

As the skin ages, many wrinkles appear around the face due to gravity and exposure to sunlight. Recently, as the interest in beauty increases, the demand for improving such wrinkles gradually increases, and patients tend to solve cosmetic needs such as wrinkle improvement by using a biomaterial for tissue repair having a low burden and a short downtime. Accordingly, various types of tissue repair biomaterials are being researched and developed, and the demand thereof is gradually increasing.

Representative examples include hyaluronic acid, and hyaluronic acid (hereinafter, may be abbreviated as "HA") has a linear repeating unit consisting of N-acetyl-D-glucosamine and D-glucuronic acid, as shown in Formula 1 below. It is a biopolymer material that is connected to, and in the form of hyaluronic acid derivatives, it has been used for various purposes such as anti-adhesion films or gels after surgery, implants for wrinkle improvement, and molding aids.

N is an integer of 1 or more.

Conventional hyaluronic acid (HA) tissue repair biomaterials are cross-reacted products using a single-component chemical crosslinking agent such as BDDE (1,4-Butanediol diglycidyl ether) to increase the viscosity and elasticity in order to extend the retention period. Has been developed. However, when crosslinking with synthetic chemicals such as BDDE, there are side effects due to three types of unreacted crosslinking agents.

On the other hand, the tissue repair biomaterials for improving facial wrinkles are composed of two types, monophasic and biphasic. The biomaterial for tissue repair in the form of a single phase is a homogeneous gel form and has the advantage of allowing a soft and delicate shape to be injected during the procedure, but has a disadvantage of low shape retention. In comparison, the biphasic tissue repair biomaterial has a merit of increasing the volume of the skin during the procedure in which the gel gel is mixed with the hyaluronic acid solution and maintaining the shape. However, it has a disadvantage in that the injection force is higher than the single phase-type tissue repair biomaterial for injection.

Therefore, there is a continuing need for a tissue repair biocomposite having a low injection pressure, which is easy to inject and has an excellent shape-keeping ability.

Republic of Korea Patent Application No. 2010-0055556 "Method for preparing cationic polymer / hyaluronic acid microbeads and metal ions chelate cationic polymer / hyaluronic acid microbeads"

It is an object of the present invention to provide a method for producing a tissue repair biocomposite having excellent morphology while having low injection force through two-stage double crosslinking.

Still another object of the present invention is to provide a tissue repair biocomposition having a low injection force and excellent shape maintenance ability through two-step double crosslinking.

The present invention provides a method for preparing a biocomposition for hyaluronic acid-chitosan composite hydrogel tissue repair having excellent morphology while having low injection force through two-stage double crosslinking.

Still another object of the present invention is to provide a hyaluronic acid-chitosan composite hydrogel tissue repair biocomposition having a low injection force and excellent morphology through two-step double crosslinking.

The hyaluronic acid-chitosan composite hydrogel tissue repair biocomposition according to the present invention has a two-step crosslinking, that is, a low-cost crosslinking composition in vivo, which has an excellent injection power and excellent shape retention.

The first picture of Figure 1 is a view showing the state 1 to 4 and purified water 5 and the addition of chitosan after dissolving hyaluronic acid in a basic solution, the second picture was reacted at pH 3.5, pH 4.0, pH 5.0, pH 6.5 in order Is a diagram showing the status,
Figure 2 is a photograph showing the state color change according to the chitosan concentration added to the hyaluronic acid solution at room temperature,
Figure 3 is a photograph showing the state color change according to the chitosan concentration added to the hyaluronic acid solution at 40 ℃,
4 is a graph showing the rheological properties of the Examples and Comparative Examples using a rheometer,
5 is a particle size analysis graph of the hyaluronic acid-chitosan composite hydrogel and Comparative Examples 1 to 2 compositions of Examples 1 to 4 in order;
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 composite composition of the present invention.

The present invention

1 to 10% by volume of BDDE (1,4-Butanediol diglycidyl ether) is added to 1 to 10% by volume of the homogenized hyaluronic acid hydrogel, and then stirred for 30 to 60 minutes at 900 to 1000 rpm using a homogenizer at room temperature, followed by 25 to Aging at 30 ° C. for 30 to 60 minutes to obtain a basic crosslinked hyaluronic acid derivative;

Adjusting the basic crosslinked hyaluronic acid derivative to less than pH 7.5 with 1N hydrochloric acid solution;

Dialysis with PBS and distilled water for 2 days to remove the crosslinker BDDE;

Lyophilizing the crosslinked hyaluronic acid derivative dialysis solution for 2 days after removing the crosslinker;

Grinding the lyophilized crosslinked hyaluronic acid derivative; And

4% by weight of the pulverized crosslinked hyaluronic acid derivative and 2% by weight of uncrosslinked hyaluronic acid were mixed with PBS (Phosphate buffer saline), homogenized at 900 to 1000 rpm for 20 minutes using a homogenizer at room temperature, and set to 25 to 35 ° C. A first step crosslinking with a degree of crosslinking of 1 to 8 mol%, comprising the step of aging in an incubator for 12 hours to obtain a biphasic anionic hyaluronic acid hydrogel; And

4% by weight of anionic hyaluronic acid hydrogel obtained by the first step crosslinking and 0.01 to 0.5% by weight of cationic chitosan gel were mixed in PBS, stirred at 900 to 1000 rpm for 30 to 60 minutes using a homogenizer, and then 30 ° C. 10 to 12 hours in the incubator set to a method for producing a tissue repair biocomposite using a double cross-linking, characterized in that it comprises an electrostatic two-stage cross-linking to obtain a hyaluronic acid-chitosan composite hydrogel

In addition, the present invention provides a tissue composition for biological repair prepared by the above method.

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

The biological composition for tissue repair according to the present invention, that is, a hyaluronic acid-chitosan composite hydrogel, is obtained by double crosslinking, and is a single-step crosslinking by chemical crosslinking, and an electrostatic charge of anionic hyaluronic acid hydrogel and cationic chitosan gel. It is characterized in that obtained through the two-step crosslinking by attraction.

The hyaluronic acid-chitosan composite hydrogel tissue repair method for preparing a biocomposition of the present invention will be described in detail step by step.

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

Preparation of crosslinked hyaluronic acid derivative (one step crosslinking)

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

After the addition of the cross-linking agent BDDE (1,4-Butandiol diglycidyl ether) 2.5% (v / v) to the prepared hyaluronic acid hydrogel using a homogenizer at room temperature and stirred for 30 minutes at 900 to 1,000rpm incubator set to 20 to 40 ℃ The reaction is carried out for 30 to 40 hours. When reacting at the temperature and time it is preferable to obtain 1 to 8 mol% of the low-crosslinking to be desired in the present invention.

In the case of a tissue composition for tissue repair, it is common to increase crosslinking to about 20 mol% in order to increase shape retention. If the degree of crosslinking is high, the morphology is good when injected into the body, but it is difficult to inject and causes necrosis, disproportionation or dizziness. There is a possibility to cause it. In the present invention, by reducing the degree of crosslinking it is possible to minimize the side effects due to high crosslinking.

The prepared basic crosslinked hyaluronic acid derivative is adjusted to pH 6.5 to 7.5 with 1N hydrochloric acid solution. As confirmed in FIG. 1, the reason for maintaining the pH at 6.5 to 7.5, more preferably at least pH 6.5 is that the product is excellent in transparency.

In order to remove the remaining crosslinking agent BBDE after the crosslinking reaction, dialysis is carried out with a buffer solution (PBS: Phosphate buffer saline) and distilled water for 2 days to remove the remaining crosslinking agent. The crosslinked hyaluronic acid derivative dialysis solution from which the crosslinking agent was removed is lyophilized for 2 days to obtain a white crosslinked hyaluronic acid derivative and then ground.

At this time, the particles of the pulverized crosslinked hyaluronic acid derivative 200-300 micrometers is preferable.

Anionic  Cross-linked hyaluronic acid Hydrogel  Produce

4% by weight of the prepared crosslinked hyaluronic acid derivative and 2% by weight of uncrosslinked hyaluronic acid (molecular weight: about 250 to 2.6 million daltons) were mixed in a buffer solution, homogenized at 900 to 1,000 rpm for 20 minutes using a homogenizer at room temperature. , Aged 12 hours in an incubator set to 30 ℃ to prepare anionic biphasic hyaluronic acid hydrogel. 2 and 3 show the state of the hyaluronic acid hydrogel at room temperature and 40 ℃ can be confirmed that the best quality and appearance of the product at room temperature.

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

4% by weight of the pulverized crosslinked hyaluronic acid derivative prepared in one-step crosslinking and 2% by weight of uncrosslinked hyaluronic acid (molecular weight: about 250 to 2.6 million daltons) are mixed in a buffer solution and used at a room temperature using a homogenizer at 20 to 900 to 1,000 rpm. Homogenize for a minute to produce Biphasic anionic hyaluronic acid hydrogel.

Here, a buffer solution containing 0.01 to 0.5% by weight of chitosan prepared in advance was mixed, stirred for 30 minutes at 900 to 1,000 rpm using a homogenizer, and then aged in an incubator set at 30 ° C. for 12 hours to produce anionic hyaluronic acid and cationic chitosan. To prepare a hyaluronic acid-chitosan composite hydrogel bound by electrostatic attraction.

2 and 3 show the state of the hyaluronic acid-chitosan composite hydrogel at room temperature and 40 ℃ it can be seen that the chitosan concentration of 0.001 to 1.0% by weight product quality and appearance is the best.

When explaining the injection method of the hyaluronic acid-chitosan composite hydrogel produced by the present invention as follows.

Anionic crosslinked hyaluronic acid hydrogel prepared by one-step crosslinking was injected into the first inlet 100 using the double filler injector shown in FIG. 7, and cationic chitosan gel was injected into the second inlet 200. After the piston 300 is pushed and injected, the composition in the first inlet and the second inlet is collected in the mixing unit 400, and then, 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 limited to the following Examples.

Production Example

Preparation of Crosslinked Hyaluronic Acid Derivatives

1 g of caustic soda was dissolved in 100 ml of distilled water to prepare an aqueous 1 wt% caustic soda solution. Hyaluronic acid (molecular weight: about 110 to 1.2 million daltons) was added to 10% by weight, and then homogenized at room temperature using a homogenizer (homogenizer) for 180 minutes at 900 rpm to prepare a hyaluronic acid hydrogel. . After adding 2.5% by weight of the cross-linking agent BDDE (1,4-Butandiol diglycidyl ether) to the prepared hyaluronic acid hydrogel using a homogenizer at room temperature and stirred for 30 minutes at 900 ~ 1,000 rpm (Incubator) ) For 40 hours. The prepared basic crosslinked hyaluronic acid derivative hydrogel was adjusted to pH 6.5-7.5 with 1N hydrochloric acid solution. In order to remove the remaining crosslinking agent BDDE after the crosslinking reaction, the crosslinking agent was placed in a 13,000 MWCO RC dialysis bag, and the remaining crosslinking agent was removed by dialysis with a buffer solution (PBS: phosphate buffer saline) and distilled water for 2 days. The crosslinked hyaluronic acid derivative dialysis solution from which the crosslinking agent was removed was lyophilized for 2 days to obtain a white crosslinked hyaluronic acid derivative, which was ground to a particle size of 200 μm using a colloid mill.

Example  One

Preparation of 0.01 wt% chitosan-containing double crosslinked hyaluronic acid-chitosan composite hydrogel

Pulverized particle size prepared in Preparation Example 1 The ideal anionic crosslinked hyaluronic acid was mixed with 4% by weight of 200 µm cross-linked hyaluronic acid derivative and 2% by weight of uncrosslinked hyaluronic acid molecular weight of 2.5 to 2.6 million daltons in a buffer solution and homogenized at 900 to 1,000 rpm for 20 minutes using a homogenizer at room temperature. Lonic acid hydrogels were prepared. Here, the buffer solution containing 0.01 wt% of chitosan prepared in advance was mixed and aged for 30 hours at 30 ° C. after stirring at 900 to 1,000 rpm using a homogenizer to obtain an ideal hyaluronic acid-chitosan composite hydrogel. It was.

Example  2

Preparation of a Double-crosslinked Hyaluronic Acid-Chitosan Composite Hydrogel Containing 0.1 wt% Chitosan

Pulverized particle size prepared by the preparation example The ideal anionic crosslinked hyaluronic acid was mixed with 4% by weight of 200 µm cross-linked hyaluronic acid derivative and 2% by weight of uncrosslinked hyaluronic acid molecular weight of 2.5 to 2.6 million daltons in a buffer solution and homogenized at 900 to 1,000 rpm for 20 minutes using a homogenizer at room temperature. Lonic acid hydrogels were prepared. Here, the buffer solution containing 0.1 wt% of chitosan prepared in advance was mixed and aged for 30 hours in an incubator set at 30 ° C. after stirring for 30 minutes at 900 to 1,000 rpm using a homogenizer to obtain an ideal hyaluronic acid-chitosan composite hydrogel. It was.

Example  3

Preparation of a Double-crosslinked Hyaluronic Acid-Chitosan Composite Hydrogel Containing 0.5 wt% Chitosan

Pulverized particle size prepared by the preparation example 4% by weight of a 200 μm cross-linked hyaluronic acid derivative and 2% by weight of uncrosslinked hyaluronic acid (molecular weight: about 250 to 2.6 million daltons) were mixed in a buffer solution and homogenized at 900 to 1,000 rpm for 20 minutes using a homogenizer at room temperature. Anionic crosslinked hyaluronic acid hydrogels were prepared. A buffer solution containing 0.5% by weight of chitosan prepared in advance was mixed and aged for 30 hours in an incubator set at 30 ° C. after stirring for 30 minutes at 900 to 1,000 rpm using a homogenizer to obtain an ideal hyaluronic acid-chitosan composite hydrogel. It was.

Example  4

Preparation of double-crosslinked hyaluronic acid-chitosan composite hydrogels with modified first and second crosslinking conditions

Pulverized particle size prepared by the preparation example 5 wt% of a 200 μm derivative and 1 wt% of uncrosslinked hyaluronic acid (molecular weight: about 2.5 to 2.6 million daltons) are mixed in a buffer solution (PBS: phosphate buffer saline), and a homogenizer is used at room temperature. A biphasic hyaluronic acid hydrogel was prepared by homogenizing for 20 minutes at rpm. Here, a pre-prepared chitosan containing 0.2% by weight of a buffer solution (PBS: phosphate buffer saline) was mixed, and stirred for 30 minutes at 900 ~ 1,000 rpm using a homogenizer (incubator) The biphasic hyaluronic acid-chitosan composite hydrogel was prepared by aging 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 an aqueous 1 wt% caustic soda solution. 10% by weight of hyaluronic acid (Hyaluronic acid, molecular weight: about 2.5 to 2.6 million daltons) was added and homogenized at 900 to 1,000 rpm using a homogenizer (homogenizer) at room temperature for 180 minutes to prepare hyaluronic acid hydrogel. After adding 2.5% by weight of the cross-linking agent BDDE (1,4-Butandiol diglycidyl ether) to the prepared hyaluronic acid hydrogel using a homogenizer at room temperature for 30 minutes at 900 ~ 1,000 rpm and incubator set to 30 ℃ (Incubator ) For 40 hours. The prepared basic crosslinked hyaluronic acid hydrogel was adjusted to pH 6.5-7.5 with 1N hydrochloric acid solution. In order to remove the remaining crosslinking agent BDDE after the crosslinking reaction, the crosslinking agent was placed in a 20,000 MWCO CE dialysis bag, and dialysis was carried out with a buffer solution (PBS: phosphate buffer saline) and distilled water for 2 days to remove residual crosslinking agent. The crosslinked hyaluronic acid hydrogel dialysis solution from which the crosslinker was removed was lyophilized for 2 days to obtain a white crosslinked hyaluronic acid solid. 4% by weight of the pulverized crosslinked hyaluronic acid and 2% by weight of uncrosslinked hyaluronic acid (molecular weight: about 2.5 to 2.6 million daltons) are mixed in a buffer solution (PBS: phosphate buffer saline), and a homogenizer is used. Biphasic hyaluronic acid hydrogels were prepared by aging for 12 hours in an incubator set at 30 ° C. after stirring for 30 minutes at 900 to 1,000 rpm.

Comparative example  2

Preparation of Primary Crosslinked Monophasic Hyaluronic Acid Hydrogel

1 g of caustic soda was dissolved in 100 ml of distilled water to prepare a 1% (w / v) caustic aqueous solution. Hyaluronic acid (molecular weight: about 110 to 1.2 million daltons) 10% (w / v) was added thereto, and then homogenized at 900 to 1,000 rpm using a homogenizer at room temperature for 180 minutes to hyaluronic acid hydro Gels were prepared. After adding 2.5% by volume of the crosslinking agent BDDE (1,4-Butandiol diglycidyl ether) to the prepared hyaluronic acid hydrogel, using a homogenizer at room temperature, the mixture was stirred at 900 to 1,000 rpm for 30 minutes, and then hydrated hyaluronic acid (molecular weight: About 2.5-2.6 million daltons) 2% by volume was added. After addition, the mixture was aged for 40 hours in an incubator set at 30 ° C. to form a gel. The prepared basic crosslinked hyaluronic acid hydrogel was neutralized by the addition of HCl-PBS solution and the gel expanded over a shaker over 48 hours. The gel was placed in a 13,000 MWCO RC dialysis bag and subjected to dialysis for 4 days in PBS pH7.4 buffer to prepare a monophasic hyaluronic acid hydrogel.

Final concentration (% by weight) Mixing ratio (weight ratio) Remarks Cross-linked hyaluronic acid derivative Non-crosslinked hyaluronic acid Chitosan Cross-linked hyaluronic acid derivative Chitosan Non-crosslinked hyaluronic acid Example 1 4.0% 2.0% (2.5-2.6MDa) 0.01 400 One 200 Ideal Castle Bridge Example 2 4.0% 2.0% (2.5-2.6MDa) 0.1 40 One 20 Ideal Castle Bridge Example 3 4.0% 2.0% (2.5-2.6MDa) 0.5 8 One 4 Ideal Castle Bridge Example 4 5.0% 3.0% (2.5-2.6MDa) 0.2 25 One 15 Ideal Castle Bridge Comparative Example 1 4.0% 2.0% (2.5-2.6MDa) - 2 0 One No abnormal chitosan Comparative Example 2 10.0% 2.0% (2.5-2.6MDa) - 5 0 One No single phase chitosan

Test Example 1 Hyaluronic Acid-Chitosan Composite Hydrogel Viscoelastic test

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

Analysis Condition of Rotational Rheometer

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

(2) Frequency: 01 ~ 10Hz

(3) Temperature: 25 ± 1 ℃

(4) Strain: 1%

(5) Minimum torque oscillation: 10nN * 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

It can be seen from Table 2 that Examples 1 to 4 of the present invention exhibit excellent elasticity characteristics while having a higher composite viscosity than Comparative Examples 1 to 2. That is, compared with Comparative Examples 1 and 2, the hyaluronic acid-chitosan composite hydrogel containing chitosan produced by the present invention showed excellent mechanical properties. It has high overall viscosity and at the same time good elastic properties, it was confirmed to have excellent physical properties when mixed with chitosan and the results are shown in Table 2 and FIG.

Experimental Example 2: Particle size analysis of the double cross-linked hyaluronic acid-chitosan composite hydrogel prepared according to the present invention

Produced by the present invention Particles were measured using a HELOS Particle Size Analyzer to confirm particle size and distribution of the hyaluronic acid-chitosan composite compositions of Examples 1 to 4. The results are shown in Table 3 and FIG.

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

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

Average particle size (㎛) 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

Looking at the average particle size of Examples 1 to 4 combined with anionic hyaluronic acid hydrogel and chitosan by one-step crosslinking in Table 3, the average particle distribution of Examples 1 to 4 appeared evenly, which did not contain chitosan. Similar to the average particle of one step crosslinked anionic hyaluronic acid hydrogel. In other words, it can be seen that the chitosan does not affect the hyaluronic acid particle size.

Test Example  3: hyaluronic acid-chitosan composite composition prepared according to the present invention Injection pressure  Measure

Injecting pressure measurement of Examples 1 to 4 and Comparative Examples 1 and 2 prepared by the present invention was carried out using a push pull gauge to determine the extrusion force in a pre-filled syringe.

Injection pressure test analysis conditions

(1) Test equipment: Mechanical Force Gauges (Push Pull Gauges, IMADA)

(2) Test speed: 20nm / min

(3) Measuring displacement: 10mm

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

(5) Test environment: 25 ± 2 ℃

Injection pressure (N) Injection pressure (N) Average injection 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 results of measuring the injection pressure of the hyaluronic acid-chitosan composite hydrogel prepared according to the present invention. As can be seen from the table in the above 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 composite hydrogel containing chitosan than Comparative Examples 1 and 2 exhibited excellent injection pressure, thereby increasing the softness of the gel, which is important in the filler procedure.

Experimental Example  4: measurement of enzyme resistance of hyaluronic acid-chitosan composite composition prepared according to the present invention

In order to test the biodegradation properties of the hydrogel particles by enzymes, an experiment was conducted to determine the amount of degradation of each sample within the same period. PBS was used as a pH 7.4 solution, and hyaluronic acid (hyaluronidase) was used to decompose hyaluronic acid. Unit (1U) represents the enzyme activity, and hyaluronase is defined as changing the absorbance of 0.330 at 600 nm at 37 ° C. per minute. In the experiment, each sample was placed in each solution at a concentration of 0.1 g / 3 ml. As a solvent to decompose, a buffer solution mixed with PBS and 100 U / ml hyaluronicase was used. Each sample was weighed every 3 days in a shaking bath maintained at 37 ℃ to determine the degree of degradation.

Figure 6 is the result of measuring the weight of the sample every three days showing the degree of biodegradation by the enzyme. Polysaccharide is composed of β-glycosidic bond, that is, a molecule in which water molecules are lost between aldehyde (-CHO) and alcohol (-OH) between sugar molecules and carbon 1 and 4 are connected to acetal bonds. It captures certain parts of the composition and serves to break this bond. The catalyzed enzyme accelerated the hydrolysis faster in pure buffer, so the group containing the enzyme degraded faster. The reason hyaluronic acid-based particles decompose faster than chitosan-based particles is due to the extreme hydrophilicity of hyaluronic acid. do. Therefore, the composition having a high degree of crosslinking was slower in decomposition rate than hyaluronic acid. In other words, if the crosslinking degree is high and the surface has a dense structure, the penetration of water is relatively difficult, resulting in a slow decomposition rate.

As shown in FIG. 6, the reaction proceeded under similar conditions, but the compositions of Comparative Examples 1 and 2 without chitosan consisted of hyaluronic acid only, so that the enzymatic degradation is somewhat faster than other compositions. As a result, produced by the present invention It can be seen that the hyaluronic acid-chitosan composite composition of Examples is excellent in resistance to enzymes.

100; 1st entrance
200: second entrance
300; piston
400: mixing part

Claims (8)

1-10% by volume of BDDE (1,4-Butanediol diglycidyl ether) is added to 1-10% by volume of the homogenized hyaluronic acid hydrogel, followed by stirring at 900-1000 rpm for 30 to 60 minutes using a homogenizer at room temperature. Aging at 30 ° C. for 30 to 60 minutes to obtain a basic crosslinked hyaluronic acid derivative;
Adjusting the basic crosslinked hyaluronic acid derivative to less than pH 7.5 with 1N hydrochloric acid solution;
Dialysis with PBS and distilled water for 2 days to remove the crosslinker BDDE;
Lyophilizing the crosslinked hyaluronic acid derivative dialysis solution for 2 days after removing the crosslinker;
Grinding the lyophilized crosslinked hyaluronic acid derivative; And
4% by weight of the pulverized crosslinked hyaluronic acid derivative and 2% by weight of uncrosslinked hyaluronic acid were mixed with PBS (Phosphate buffer saline) and homogenized at 900 to 1000 rpm for 20 minutes using a homogenizer at room temperature, and set to 25 to 35 ° C. A first stage crosslinking with a degree of crosslinking of 1 to 8 mol%, comprising the step of aging in an incubator for 12 hours to obtain a biphasic anionic hyaluronic acid hydrogel; And
4% by weight of anionic hyaluronic acid hydrogel obtained by the first step crosslinking and 0.01 to 0.5% by weight of cationic chitosan gel were mixed in PBS, stirred at 900 to 1000 rpm for 30 to 60 minutes using a homogenizer, and then 30 ° C. 10 to 12 hours in an incubator set to a method for producing a tissue repair biocomposite using a double crosslinking, characterized in that it comprises an electrostatic two-stage crosslinking to obtain a hyaluronic acid-chitosan composite hydrogel.
delete delete The method of claim 1,
Particle size of the pulverized cross-linked hyaluronic acid derivative is a method for producing a tissue composition for tissue repair using a double crosslinking, characterized in that 200 to 300㎛.
delete delete delete Biological composition for tissue repair using double crosslinking obtained by claim 1.

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