KR100452221B1 - Injectable soft tissue prosthetic composition inducing autologus tissue formation - Google Patents

Abstract

The present invention relates to a method for manufacturing a prosthetic material injected into the soft tissues of the body to complement the anatomical shape, atelocollagen and hyaluronic acid that acts as a structure of autologous tissue, fibrinogen ) When the complex solution and thrombin are mixed and pluronic compound is injected, the method hardens in the body and is then replaced with autologous tissue in the body over time with regeneration of magnetic cells.

Description

Injectable soft tissue prosthetic composition inducing autologus tissue formation

The present invention is to provide a prosthetic composition for injection into the soft tissue of the body to complement the anatomical shape.

Injecting a mixture of atelocollagen, hyaluronic acid, and fibrinogen complex, which serve as a structure of autologous tissue, with thrombin, it hardens in the body and is then replaced by autologous tissue in the body over time with regeneration of magnetic cells.

All soft tissues of the human body are maintained anatomically by extracellular matrix including glycosaminoglycans and major proteins such as collagen and elastin. In general, in the case of anatomical deformation of soft tissues due to congenital or pathological factors, synthetic polymer materials have been used as prosthetic materials to restore their shape. Representative material is silicone, which mainly uses solids with a molecular weight of 300,000 or more, and some methylmethacrylic acid polymers are also used. Although they are excellent in biocompatibility, they tend to cause fibrosis in the body over time because they exist as permanent foreign substances in the body. Meanwhile, a method of inducing integration with autologous tissues by injecting extracellular matrix components such as collagen or hyaluronic acid alone has been devised, but collagen injected into the body is degraded by collagen degrading enzymes, and in the case of hyaluronic acid, surrounding tissue cells Because it is degraded according to the change in cell cycle, it disappears within 6 months after insertion into the body, requiring re-treatment.

In general, collagen is an extracellular matrix that attracts and directly binds to cell invasion and thus plays an important role in maintaining tissue tension and flexibility because it has a high strength of binding to moisture as well as a structure. Removing the telopeptides present at both ends of the collagen molecule having a size of about 2.4 nanometers in diameter has the advantage of not causing an immune response in the human body. The atelo collagen thus prepared is acid soluble in trace amounts of hydrochloric acid or acetic acid. And so on. Collagenase is hampered by EDTA, dithiothrenol, cyteine, cortisone, theophyline, and the like. Hyaluronic acid is a type of glycosaminoglycans (GAG), but unlike conventional GAGs that bind to proteins and exist in the form of proteoglycans, they can be used alone. Upon absorption, they form a gel-like structure in which fibrous proteins can be easily filled. Fibrinogen is a globular glycoprotein present in plasma, a substance that coagulates while forming fibrin, an amorphous solid, when it reacts with thrombin, an enzyme that hydrolyzes between arginine and glycine, at fifty-five degrees Celsius. Fibrin has the property of forming a gel by swelling in a weakly acidic (pH 6.0-6.8) solution. Pluronic, on the other hand, is a substance used for gelation when producing tissue culture media. It has excellent tissue compatibility as a non-degradable substance in vivo. Especially, it is easily soluble at low temperatures, but changes to gel at room temperature. I have it.

The present invention relates to a method for manufacturing a prosthetic material injected into the soft tissues of the body to complement the anatomical shape, atelocollagen and hyaluronic acid that acts as a structure of autologous tissue, fibrinogen ) When a complex solution and thrombin are mixed and pluronic compound is injected, the self-organized injectable soft tissue that hardens in the body and then is replaced by autologous tissue in the body over time with regeneration of magnetic cells. In providing a prosthetic composition.

1 is a graph comparing the degree of degradation by collagen degrading enzyme of the composition of the present invention. FIG. 2 is a scanning electron micrograph after drying the composition of the present invention.

3 is a photograph showing a comparative experiment of cell affinity of the present composition.

The present invention is to provide a self-organizing infusion soft tissue complement composition that is completely different from the conventional foreign material injection.

The present invention provides a structural substrate having excellent biocompatibility using atherocollagen and hyaluronic acid that have been treated with non-immunized collagen, which is a major component of the soft tissue in the body, and when injected into the body, crosslinking in the body is caused by binding of fibrinogen and thrombin. As a result of the formation of a fibrous structure, firm structural substrates are delayed in the body by biodegradation enzymes combined with collagenase inhibitors, and Pluron retains its initial form, thus producing a material that complements the anatomical form of soft tissues. It is devised for the purpose of describing the following specific methods as examples.

In the present invention, atelo collagen is dissolved in 0.5 to 2% by weight in water, and then hyaluronic acid is dissolved in water to 0,5 to 2% by weight. Combine the two solutions and add 0.2-10% by weight of the deophylline solution.

Alkali is added to the solution thus prepared to adjust the pH to 6.0-8.0. At this time, an alkali selected from potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate and the like may be used.

5-50 mg of fibrinogen is added to the pH-adjusted solution.

Approximately 125 units / mg solution is prepared by diluting 1000 units / mg of thrombin with 40 mM calcium chloride solution.

Separately, 1 to 10% by weight of a solution of Pluronic was prepared in water.

In about 20 ml of this pluronic solution is mixed 5-50 mg of thrombin.

The floronic solution is mixed with the thrombin solution and refrigerated at 4 degrees Celsius or less.

Next, the present invention will be described in more detail by way of examples.

EXAMPLE

Example 1

1. Add 2 mL of 1N acetic acid solution to 100 ml of distilled water at 2-8 degrees Celsius, add 1 g of atelocollagen, and dissolve completely with stirring at 200-400 rpm.

2. Add 1 g of hyaluronic acid to 100 ml of distilled water at room temperature and dissolve completely with stirring at a speed of 100-200 rpm.

3. At 2-8 degrees Celsius, add the solution prepared in 1 to the solution prepared in 2, then add 5-10 ml of 0.5% theophyline anhydrate solution with stirring at a rate of 60-100 rpm.

4. Adjust to pH 7.4 using 2N sodium hydroxide solution in solution prepared at 3 at room temperature.

5. Add 10-20 mg of fibrinogen at room temperature with stirring at a rate of 100-200 rpm to the solution prepared in 4.

6. Dilute 1000 units / mg of thrombin with 40 mM calcium chloride solution to prepare a 125 units / mg solution.

7. A 2-5% Pluronic solution is prepared by adding Pluronic powder while stirring 20 ml of distilled water at a speed of 100-200 rpm at 2-8 degrees Celsius.

8. Mix 10-20 mg of thrombin at 2-8 degrees Celsius to 2-5% Pluronic solution prepared in 7.

9. Mix the solution prepared in 8 to the solution prepared in 6 at 2-8 degrees Celsius and then stir for 12 hours at a speed of 60-100 rpm.

10. Refrigerate the prepared solution at 4 degrees Celsius or less.

Next, the present invention will be described in more detail as an experimental example.

Experimental Example 1

Comparison of the Degree of Degradation by Collagen Degrading Enzymes of the Compositions of the Present Invention

The composition of the present invention was divided into several groups according to the concentration of the crosslinking agent, and the degree of degradation by collagen degrading enzyme was compared. As a reference material, a specimen without any treatment was selected and used. After a certain time, the amount of collagen decomposed by the collagenase was measured using the Newman & Logan method to measure the degree of crosslinking.

The results are shown in FIG.

In the first degree

"non-treated" means untreated atelocollagen-hyaluronic acid solution,

"50 mM EDC treated" means a 50mmol ethyldimethylcarbodiimide treated atelocollagen-hyaluronic acid solution,

"0.25M EDTA treated" means 0 25 molar EDTA treated atelocollagen-hyaluronic acid solution,

"0.25M EDTA / 0.5% theophyllin treated" means a solution of 0.25 mole EDTA and 0.5% deophylline treated atelocollagen-hyaluronic acid,

"50 mM edc / 0.5 theophyllin treated" means 50 mmol EDC and 0.5% deophylline double treated atelocollagen-hyaluronic acid.

As shown in FIG. 1, atelocollagen dissolution by collagenase cannot be observed in the case of atelocollagen-hyaluronic acid complex solution double-treated with 50 mmol EDC and 0.5% deophylline. Deophylline may interfere with the action of collagenase.

Experimental Example 2

Viscosity Determination of Chemically Treated Colorogen-Haluronic Acid Solutions

The material prepared according to the present invention was divided into a group treated with deophylline, a group treated with EDTA, and a group not treated with a certain amount, and measured using a viscosity meter (Brookfielf Programmable CV-II + Viscometer).

The results are shown in Table 1 below.

Table 1:

As confirmed in Table 1, the atelocollagen-hyaluronic acid composite solution treated with deophylline has a viscosity corresponding to about 5/6 times of the untreated solution.

Experimental Example 3

The composition of the present invention was dried and observed by scanning electron microscope.

The results are shown in A and B of FIG.

A is a deophylline treated atelocollagen-hyaluronic acid complex solution,

B is a solution complexed with Pruron after adding fibrin source and thrombin to Solution A. Due to the coagulation of fibrinogen and thrombin, the atelocollagen-hyaluronic acid fibrous substance of A is in close contact with floronics to form a salvage body.

Experimental Example 4

Comparison of cell affinity of the composition

In the cytotoxicity test, L929 fibroblasts were cultured at a constant concentration, and then the composition of the present invention was subjected to cytotoxicity test by indirect extraction.

The results are shown in FIG.

In Figure 3,

A is negative control-polyethylene film,

B is a positive control polyurethane film,

C is a dry film of the composition prepared by the present invention.

There was no cell adhesion on the surface of the negative control group, and cell adhesion could be observed on the surface of the positive control group. In the case of the composition of the present invention, since the cells are adhered and the solute has proliferated to the inside, it can be seen that it has excellent cell affinity.

The self-organizing injection-type soft tissue prosthesis composition prepared according to the present invention, when delivered to the body through subcutaneous injection or intramuscular injection, can be arbitrarily given a form within 30 minutes required for the gelation of pluronic and provided non-immunity. Using atelocollagen and hyaluronic acid, it provides a structural structure with excellent biocompatibility, induces adhesion and growth of surrounding cells and induces regeneration of autologous tissues, and crosslinks in the body by combining fibrinogen and thrombin. As a solid structural substrate is formed, a synergistic effect can be obtained on the inhibition of body degradation of atelocollagen treated with collagenase activity inhibitor, and thus the body degradation is suppressed, and the initial form provided by in vivo non-degradable pluronic The anatomical shape corrected by the regenerated autologous tissue can be maintained.

Claims (2)

An implantable prosthesis composition in a soft tissue comprising a mixture of atelocollagen, hyaluronic acid, fibrinogen, thrombin, and deophylline in water. The method according to claim 1, wherein 0.1 to 5 g of atelocollagen, 0.1 to 5 g of hyaluronic acid, 0.01 to 0.1 g of deophylline, 0.001 to 0.2% by weight of fibrinogen, and 50 to 1000 units of thrombin are added and dissolved in water, and the pH is about neutral. Injectable prosthetic composition in the soft tissue prepared by adjusting to 100ml the whole.