CN1117587C - Manufacture of porous holder for repairing tissue and organ - Google Patents

Abstract

The invention relates to a preparation method of a porous scaffold for tissue and organ repair. First, a pore-forming agent is selected, a degradable polymer is dissolved in a solvent, and then the pore-forming agent is added to the solution, and the mixture is added to a mold, and the mold is closed. , dry, after the product is dry, remove the mold, then put it into a vacuum drying oven to dry, immerse the dried product in deionized water or acidic aqueous solution, and finally vacuum dry the product again to obtain the porous scaffold of the present invention . In the invention, by adjusting the particle diameter of the pore-forming agent and the concentration of the solution, a support with interconnected pores and different porosity requirements is obtained.

Description

Preparation method of porous scaffold for tissue and organ repair

The invention belongs to the field of biomedical engineering. Specifically relates to a method for preparing a porous scaffold for organ and tissue repair.

The loss or dysfunction of tissues and organs is one of the major hazards to human health and the leading cause of human disease and death. According to a data from the United States, millions of Americans suffer from loss and dysfunction of various tissues and organs every year. It requires 8 million operations per year to repair, with an annual hospital stay of between 40 and 90 million and an annual cost of more than $400 billion. my country is a country with a large population. The cases of tissue and organ loss or dysfunction caused by trauma and disease are the first in the world. Every year, there are millions of patients who need skin transplantation just because of burns.

With the development of life science, material science and related physical and chemical disciplines, people put forward a new concept—tissue engineering. It is a science that applies the principles of cell biology and engineering to research and develop biological substitutes for repairing and improving the structure and function of damaged tissues. The basic principle and method are to adsorb normal tissue cells cultured and expanded in vitro to a porous biomaterial with good biocompatibility and absorbed by the body to form a complex, and to implant the cell-biomaterial complex into body tissues and organs. In the lesion site, cells form new corresponding tissues and organs with shape and function during the process of biomaterials being gradually degraded and absorbed by the body, so as to achieve the purpose of repairing trauma and reconstructing functions.

Scaffold materials play a very important role in tissue engineering research, and it is the key to realize the industrialization of tissue engineering. The processing method of the scaffold material occupies an extremely important position in it. Scaffolds for tissue engineering require high porosity and interconnection between pores, because only in this way can cells enter the interior of the scaffold after implantation, and the tissue formed in the future can be uniform. The high porosity makes it easy for water, inorganic salts and other nutrients needed for cell growth to penetrate into the material, so that the internal cells can grow and reproduce well, and the quality and performance of the formed tissue are good. It is generally believed that the porosity of scaffolds for tissue engineering should be higher than 90%.

Extensive research has been done abroad on the preparation methods of scaffolds used in tissue engineering. So far, the preparation methods can be basically divided into 1. Non-woven fiber method, which has the advantage of high porosity, but it is difficult to maintain the predetermined shape after implantation in the body. 2. Solution casting, pore-forming agent filtration method. The content of the pore-forming agent used in this method is low, and because the solution is cast in the vessel, the pore-forming agent sinks, the pores are unevenly distributed, and the morphology of the upper and lower surfaces is different. 3. Three-dimensional layering method. A three-dimensional scaffold is formed by preparing a porous membrane and then bonding the layers together with a solvent. The process of this method is complex, and during the bonding process, the holes of the bonding part are closed, thereby forming an interface and making the internal shape of the material uneven. 4. Melt processing method. In this method, the porogen is blended with the polymer and extruded into the mold above the melting point of the polymer. Cooling yields a porous scaffold of predetermined shape. The disadvantage of this method is that in the extruder, due to the large difference in density between the melt and the pore-forming agent, it is difficult to mix uniformly. Moreover, some polymers, especially biodegradable polymers, are prone to thermal degradation during melt processing. 5. Phase separation method. This method employs cooling of a solution mixture below the melting point of the solvent, thereby causing phase separation. Then vacuum-dried to obtain a porous scaffold. The disadvantage of this method is that the obtained pore size is generally below 100 microns, and it is difficult to control. 6. High-pressure carbon dioxide method. This method employs exposing the shaped polymer to high pressure carbon dioxide. The carbon dioxide dissolved in the polymer is then released by decompression to form a porous scaffold. The disadvantage of this method is that the pores formed are closed.

There is no report on a new method for preparing porous scaffolds for tissue engineering in China. Existing methods are also directly copied from abroad.

The purpose of the present invention is to provide a method for preparing a porous scaffold for repairing tissues and organs with good operability and effectiveness. The method has good controllability and can meet the different needs of various tissue engineering. The basic principle of this method is to add a pore-forming agent during the molding process of the scaffold. After the product is formed, the pore-forming agent is extracted, and the space occupied by the original pore-forming agent forms the pores of the future scaffold.

The preparation method of the porous scaffold for tissue and organ repair proposed by the present invention comprises the following steps:

1. Pass through a standard sieve to obtain a pore-forming agent with a particle size in the range of 50 microns to 500 microns. The pore-forming agent is sodium chloride, potassium chloride, potassium acetate, sodium bicarbonate, sodium carbonate, citric acid, citric acid Potassium etc.

2. Copolymerization of poly 3-hydroxybutyrate, copolymer of 3-hydroxybutyric acid and 3-hydroxycaproic acid, polylactic acid, copolymer of lactic acid and glycolic acid, 3-hydroxybutyric acid and 3-hydroxyvaleric acid One or several of other biodegradable polymers such as polyglycolic acid and polyglycolic acid are dissolved in one of solvents such as chloroform, 1,4-dioxane, 1,2 dichloroethane, pyridine, etc. The concentration is between 5% and 30% (ratio of polymer mass to solvent volume).

3. Add the pore forming agent in step 1 to the solution described in step 2 at a ratio of 1:10 to 1:40 (mass ratio of polymer to pore forming agent) (the specific number matches the solution concentration item), Stir well. The porogen is present in such proportions that no settling and normal flow of the porogen occurs in the resulting homogeneous mixture.

4. Put the mixture into the mold, and close the mold under the pressure of 0-5MPa. Naturally dry in the air, the temperature is normal temperature.

5. After the product is dry, demould. Then put it into a vacuum drying oven for drying, the temperature is normal temperature, and the pressure is 0.005MPa～0MPa. The time is between 24 and 48 hours, so that all the solvents are evaporated cleanly.

6. Immerse the dried product in deionized water or acidic aqueous solution (the concentration of H ⁺ is between 2M and 10 ^-4 M) (the volume ratio of product to deionized water or acidic aqueous solution is between 1:50 and 1:200 between), change the deionized water or acidic water solution every 8 hours. The total soaking time is between 72 and 80 hours.

7. Carry out vacuum drying to the product again, the temperature is normal temperature, and the pressure is 0.01-0 MPa. The time is between 24 and 48 hours. If an acidic aqueous solution is selected in the above step 6, it needs to be soaked in deionized water for 72 to 80 hours, and the water should be replaced every 8 hours. Then this step is carried out again, and the porous scaffold with interconnected pores of the present invention is obtained.

The advantage of the present invention is that the desired pore size can be obtained by adjusting the particle size of the pore-forming agent. By adjusting the concentration of the solution, the content of the pore-forming agent required to meet the condition that the pore-forming agent will not precipitate is changed, thereby obtaining a scaffold with interconnected pores and different porosity requirements.

Description of drawings:

Fig. 1 adopts the scanning electron micrograph of the poly-3-hydroxybutyrate porous support surface that method of the present invention obtains

Fig. 2 adopts the scanning electron micrograph of the section of poly-3-hydroxybutyrate porous support material obtained by the method of the present invention

Fig. 3 adopts the scanning electron micrograph of the porous support section of the 3-hydroxybutyric acid and 3-hydroxycaproic acid copolymer obtained by the method of the present invention

Figure 4 adopts the different porosity obtained by the method of the present invention to obtain the appearance of the poly-3-hydroxybutyrate porous scaffold

Embodiments of the present invention are described below.

Embodiment one:

1. Sieve through a standard sieve to obtain sodium chloride particles with a particle size in the range of 200 to 400 microns.

2. Weigh 2.0 g of poly-3-hydroxybutyrate (PHB), and pour into 20 ml of chloroform. Heat in a water bath at 65°C for 30 minutes. The polymer is completely dissolved.

3. Add 60 grams of sodium chloride pore-forming agent with a pore size ranging from 200 microns to 400 microns to the above solution, and stir well to make it evenly mixed.

4. Pour the above homogeneous mixture into the mold, and close the mold under the pressure of 0.2MPa. At room temperature, dry for 48 hours.

5. Demoulding, put the formed product into a vacuum oven to dry, the pressure is 0.01MPa, and the time is 48 hours.

6. Soak the product in 200ml deionized water. Change the deionized water every 8 hours. The article was removed after 72 hours.

7. Put the product into the vacuum oven again to dry, the pressure in the vacuum oven is 0.01MPa, and the time is 48 hours. The article is taken out, so the porous scaffold has been made. The measured porosity is 92%, and the pore shape is shown in Figure 1 and Figure 2.

Embodiment two

1. Sieve through a standard sieve to obtain potassium acetate particles with a particle size in the range of 50 to 200 microns.

2. Weigh 2.0 g of a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (PHB-HH), pour into 20 ml of chloroform, and heat in a water bath at 65° C. for 30 minutes. The polymer is completely dissolved.

3. Add 60 grams of potassium acetate pore-forming agent with a pore size ranging from 50 to 200 microns to the above solution. Stir well to combine evenly.

The remaining steps are the same as the corresponding steps in Embodiment 1.

The article is taken out, so the porous scaffold has been made. The measured porosity is 93%, and the appearance of the product is shown in Figure 3.

Embodiment three:

1. Sieve through a standard sieve to obtain sodium bicarbonate particles with a particle size in the range of 300 to 500 microns.

2. Weigh 2.0 grams of polylactic acid and pour 40ml of chloroform into it. The polymer was completely dissolved by heating in a water bath at 65°C for 30 minutes.

3. Add 80 grams of sodium bicarbonate pore-forming agent with a pore size ranging from 300 microns to 500 microns to the above solution. Stir well to combine evenly.

4. Pour the above homogeneous mixture into the mold, and close the mold under the pressure of 0.1MPa. Dry under pressure for 48 hours at room temperature.

5. Demoulding, put the formed product into a vacuum oven to dry, the pressure is 0.01MPa, and the time is 48 hours.

6. Soak the product in 200ml of 0.5M hydrochloric acid, and replace the 0.5M hydrochloric acid every 8 hours. The article was removed after 72 hours.

7. Soak the product in 200ml deionized water. The deionized water was changed every 8 hours, and the products were removed after 72 hours.

8. Put the product into a vacuum oven to dry, the temperature is normal temperature, the pressure in the vacuum oven is 0.01MPa, and the time is 48 hours.

The article is taken out, so the porous scaffold has been made.

Embodiment four:

1. Sieve through a standard sieve to obtain citric acid particles with a particle size in the range of 50-200 microns.

2. Weigh 2.0 g of the copolymer of lactic acid and glycolic acid, and pour 10 ml of chloroform into it. Heat in a water bath at 65°C for 30 minutes. The polymer is completely dissolved.

3. Add 40 grams of citric acid pore forming agent with a pore size ranging from 50 to 200 microns to the above solution. Stir well to combine evenly.

The remaining steps are the same as the corresponding steps in Embodiment 1.

In this way a porous material is produced.

Embodiment five:

1. Sieve through a standard sieve to obtain sodium carbonate particles with a particle size in the range of 200 to 400 microns.

2. Weigh 2.0 g of the copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid, and pour 6.7 ml of chloroform into it. Heat in a water bath at 65°C for 30 minutes. The polymer is completely dissolved.

3. Add 20 grams of sodium carbonate pore-forming agent with a pore size ranging from 200 to 400 microns to the above solution. Stir well to combine evenly.

The remaining steps are the same as the corresponding steps in Embodiment 1.

The article is taken out, so the porous scaffold has been made.

Embodiment six:

1. Sieve through a standard sieve to obtain sodium chloride particles with a particle size in the range of 50 to 500 microns.

2. Weigh 2.0 g of the copolymer of 3-hydroxybutyric acid and 3-hydroxycaproic acid, and pour it into 25 ml of 1,4-dioxane. Heat in a water bath at 65°C for 30 minutes. The polymer is completely dissolved.

3. Add 70 grams of sodium chloride pore forming agent with a pore size ranging from 200 to 500 microns to the above solution. Stir well to combine evenly.

The remaining steps are the same as the corresponding steps in Embodiment 1.

The article is taken out, so the porous scaffold has been made.

Claims (1)

1. A method for preparing a porous scaffold for tissue and organ repair, characterized in that the method comprises the following steps: (1) sieve through a standard sieve to obtain a pore-forming agent with a particle diameter within the range of 50 microns to 500 microns, the pore-forming agent being any one of sodium chloride, potassium acetate, sodium bicarbonate, sodium carbonate, and citric acid; (2) Dissolving the biodegradable polymer in a solvent so that the concentration of the solution is 5% to 30%, wherein the polymer is a copolymer of poly 3-hydroxybutyrate, 3-hydroxybutyric acid and 3-hydroxycaproic acid any one or more of polylactic acid, copolymer of lactic acid and glycolic acid, copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid, the solvent is chloroform or 1,4-dioxane ; (3) Add the pore forming agent in step 1 to the solution described in step 2 according to the mass ratio of the polymer to the pore forming agent in a ratio of 1: 20 to 40, and stir evenly; (4) Add the mixture to the mold, close the mold under a pressure of 0-5 MPa, dry naturally in the air, and the temperature is normal temperature; (5) After the product is dried, remove the mold, then put it into a vacuum drying oven to dry, the temperature is normal temperature, the pressure is 0.005MPa-0Mpa, and the time is between 24-48 hours, so that all the solvents are evaporated clean; (6) Immerse the dried product in deionized water or acidic aqueous solution, the H ⁺ concentration in the acidic aqueous solution is between 2M and 10 ^-4 M, and the volume ratio of the product to deionized water or acidic aqueous solution is 1:50～ Between 1:200, replace deionized water or acidic aqueous solution every 8 hours, and the total soaking time is between 72 and 80 hours; (7) Vacuum dry the product again, the temperature is normal temperature, the pressure is 0.01～0Mpa, and the time is between 24～48 hours. If the acidic aqueous solution is selected in the above step 6, it needs to be soaked in deionized water for 72～ During 80 hours, the water is replaced every 8 hours, and then this step is performed to obtain the porous scaffold of the present invention.

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