WO2001082987A1

WO2001082987A1 - A preparation method for a porous framework used in the prostheses of tissue and organs

Info

Publication number: WO2001082987A1
Application number: PCT/IB2001/000632
Authority: WO
Inventors: Ping Hu; Lingfei Jiang; Feng Gao
Original assignee: Clonetic Technologies Ltd; Tsinghua University
Priority date: 2000-04-14
Filing date: 2001-04-17
Publication date: 2001-11-08
Also published as: WO2001082987A8; AU2001246763A1; CN1269247A; CN1117587C

Abstract

The present invention relates to a preparation method for a porous framework used in the prostheses of tissue and organs. First is to select the pore-forming agent into the degradable polymer in the dissolvent, then put the pore-forming agent into the polymer solution. After mixed even, the mixture is put into the mould and then closed in the mould and dried. After dried, the preparation is knocked out of the mould and dried in the vacuum drying oven. Then the dried preparation is dipped into deionized water or weak acid aqueous solution and dried again thereafter. Thus the porous framework of the present invention is prepared. In this invention, frameworks that require mutually-connected pores and different porosity is prepared by modulating the particle diameters of the pore-forming agent and concentration of the solution.

Description

Method for preparing porous scaffold for tissue and organ repair

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

Tissue and organ loss or dysfunction is one of the major hazards to human health, and it is also the leading cause of human disease and death. According to a data from the United States, millions of Americans suffer from various tissue and organ loss and dysfunction each year, and require 8 million operations to repair each year, with annual hospitalization days between 40-90 million. , Costing more than $ 400 billion annually. China is a country with a large population. The number of cases of tissue, organ loss or dysfunction caused by trauma and disease is the highest in the world. Every year, there are millions of patients who need skin transplantation because of burns.

With the development of life sciences, materials science, and related physics and chemistry disciplines, people have proposed a new concept—tissue engineering. It is a science that applies the principles of cell biology and engineering to the research and development of biological alternatives that repair and improve the scab and function of damaged tissues. The basic principle and method is to adsorb normal tissue cells expanded and cultured in vitro on a porous biomaterial with good biocompatibility and absorbed by the body to form a complex, and implant the cell-biomaterial complex into the body tissues and organ In the damaged part, the cells form new corresponding tissues and organs with morphology and functions during the process of biological materials being gradually degraded and absorbed by the body, so as to achieve the purpose of repairing wounds and reconstructing functions.

The scaffold material plays a very important role in tissue engineering research, and it is the key to the industrialization of tissue engineering. And the processing method of the stent material plays an extremely important role in it. Tissue engineering scaffolds need high porosity and pores communicate with each other, because only after the cells are implanted can they enter the inside of the stent, and the tissues formed in the future can be uniform. High porosity allows for water, inorganic salts and

Confirm with write L Other nutrients easily penetrate into the material, so that the internal cells can grow, reproduce, and form tissues with good quality and performance. It is generally believed that the porosity of tissue engineering scaffolds should be higher than 90%.

Extensive research on the preparation methods of scaffolds used in tissue engineering has been conducted abroad. 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, porogen Filtration method, the content of pore-forming agent used in this method is low, because the solution is cast into the device, which causes the pore-forming agent to sink, the pores are unevenly distributed, and the upper and lower surface morphologies are different; The porous membrane is then bonded to each other by a solvent to form a three-dimensional scaffold. The method is complicated, 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. This method is to blend the pore former and polymer into the mold above the melting point of the polymer, and cool it to obtain a porous stent of a predetermined shape. The disadvantage of this method is that the melt It has a large difference from the density of the pore-forming agent, so it is difficult to mix uniformly, and some polymers, especially biodegradable polymers, are melt-processed. Thermally degradable; 5, phase separation method, the method using the mixture solution was cooled to below the melting point of the solvent, thereby producing a phase separation, and then by vacuum drying 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, which uses exposing the polymer to high-pressure carbon dioxide, and then dissolves the polymer dissolved in the polymer under reduced pressure. Carbon dioxide is released to form a porous scaffold. The disadvantage of this method is that the pores formed are closed.

No new method for preparing porous scaffolds for tissue engineering has been reported in China. The existing methods are also copied directly from abroad. Disclosure of invention

An object of the present invention is to provide a method with good operability and effective preparation of a porous scaffold for tissue and organ repair. The method has good controllability and can meet the requirements of various tissue engineering projects. Same need.

The basic principle of this method is to add a porogen during the forming process of the stent. 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.

In order to achieve the object of the invention, the technical solution of the present invention is as follows:

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

2. It is selected from the group consisting of poly 3-hydroxybutyrate (FHB), copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (PHB-HH), polylactic acid (FLA), copolymer of lactic acid and glycolic acid ( PLGA), a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid, and one or more of biodegradable polymers such as polyglycolic acid are soluble in chloroform, 1,4-dioxane, 1, One of 2-dichloroethane, 1,4-dioxane-water mixture, pyridine and other solvents, the concentration is 5% ~ 30¾

(The ratio of polymer mass to solvent volume).

3. According to the ratio of 1: 10 ~ 1: 40 (mass ratio of polymer to pore-forming agent) (the specific number matches the solution concentration term), add the pore-forming agent in step 1 to the solution described in step 2, Stir well.

4. Add the mixture to the mold, close the mold under 0 ~ 5MPa pressure, and dry the product.

5. After the product is dry, release the mold, and further dry the product, so that all the solvents are evaporated.

6. Soak the dried product in deionized water or weakly acidic aqueous solution, or immerse it in the weakly acidic aqueous solution first, and then take out the product and soak it in deionized water. The soaking time of deionized water or weakly acidic solution is 70 ~ 80 hours.

7. Dry the product again to allow all solvents to evaporate.

In the preparation method of the present invention, the concentration of the biodegradable polymer varies with the type of pore-forming agent selected and the requirement of the pore size of the repaired tissue, It is preferably 6 to: 15%.

In the preparation method of the present invention, the mass ratio of the polymer to the pore-forming agent is selected so that the pore-forming agent in the obtained homogeneous mixture does not precipitate and flow normally. The requirements of the cell vary, preferably 1: 15 ~ 1: 35; in the preparation method of the present invention, the molding conditions of the mixture in a mold are at 0.;! The mold is closed under a pressure of ~ 2 MPa, and then naturally dry at room temperature.

In the preparation method of the present invention, the drying of the product in step 5 is performed in a vacuum drying box, the temperature is normal temperature, the pressure is 0.005 MPa ~ 0 MPa, and the time is between 24-48 hours.

In the method according to the present invention, in the step 6 is a weakly acidic aqueous solution of hydrochloric acid, H + weak acid solution of a concentration between 2M ~ 10- ⁴ M.

In the preparation method of the present invention, the volume ratio of the product to deionized water or weakly acidic aqueous solution in step 6 is between 1: 50 ~ 1: 200, and the deionized water or acidity is replaced every 5 ~ 8 hours. Aqueous solution.

In the preparation method of the present invention, the re-drying of the product in step 7 is performed in a vacuum drying box, the temperature is normal temperature, and the pressure is 0.01 to 0 MPa. Time is between 24-48 hours.

According to the above method of the present invention, a porous stent with pores communicating with each other can be obtained. The advantage is that the expected pore diameter can be obtained by adjusting the particle diameter of the pore-forming agent; the concentration of the solution can be adjusted to change the pore-forming agent. The pore-forming agent content required for this condition will not be precipitated to obtain stent with pores communicating with each other and having different porosity requirements; meanwhile, the stent obtained by the preparation method of the present invention has a higher porosity of more than 90% At the same rate, the product still meets the requirements for strength of the product, and has uniform hole formation, adjustable material degradation rate, and good product feel. Brief description of the drawings

Figure 1 shows the scanning electrons of the surface of a porous poly-3-hydroxybutyrate scaffold obtained by this method. Mirror image

FIG. 2 is a scanning electron microscope image of a cross section of a poly 3-hydroxybutyrate porous scaffold material obtained by this method;

3 is a scanning electron microscope image of a cross section of a porous scaffold of a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid obtained by this method;

Figure 4 shows the appearance of poly 3-hydroxybutyrate porous scaffolds with different porosities obtained by this method;

Fig. 5 is a diagram showing the effect of generating bone tissue in nude mice after the poly-3-hydroxybutyrate porous scaffold material of the present invention is implanted on bone marrow stromal cells. The best way to implement the invention

Example 1

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

2. Weigh 2.0 grams of poly 3-hydroxybutyrate (PHB), pour 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 sodium chloride pore-forming agent with a pore size in the range of 200 to 400 microns to the above solution, and stir well to make it mix well.

4. Pour the above homogeneous mixture into a mold and close the mold at a pressure of 0.2 MPa. Dry at room temperature for 48 hours.

5. Remove the mold, put the molded product into a vacuum oven and dry it under the pressure of O. OlMPa for 48 hours.

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

7. Put the product into the vacuum oven again and dry it. The pressure in the vacuum oven is O. OlMPa for 48 hours.

The article is removed, and the porous scaffold is made. The measured porosity was 92¾, and the pore morphology is shown in the scanning electron microscope images of Figs. 1 and 2. The surface of the material is still viewed from the cross section, and they all have relatively uniform and uniform pore diameters, which meets the basic requirements as a structural material for the stent. FIG. 4 shows the appearance of the poly 3-hydroxybutyrate porous scaffold obtained in this example. Example 2

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

2. Weigh 2.0 g of the copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (PHB-HH), pour 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 in the range of 50 to 200 metric meters to the above solution, and stir well to make it evenly mixed.

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

The article is removed, and the porous scaffold is made. After measuring the porosity of 93¾, the cross-section scanning electron microscope image of the porous scaffold material is shown in Fig. 3, which has a relatively uniform pore appearance. Example 3

1.Use a standard sieve to obtain sodium bicarbonate particles with a particle size ranging from 300 to 500 metric meters.

2. Weigh 2.0 grams of polylactic acid and pour 40ml of chloroform. 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 in the range of 300 μm to 500 μm to the above solution, and stir well to make it mix well.

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

5. Remove the mold, put the molded product into a vacuum oven and dry it under the pressure of O. O LMFa for 48 hours.

6. Soak the product in 200ml of 0.5M hydrochloric acid, replace 0.5M hydrochloric acid every 8 hours, and remove the product after 72 hours. 7. Soak the product in 200ml deionized water. Deionized water was changed every 8 hours, and the product was removed after 72 hours.

8. Dry the product in a vacuum oven at normal temperature. The pressure in the vacuum oven is 0.1 MPa and the time is 48 hours.

The article is removed, and the porous scaffold is made. Example 4

1. Sieve potassium citrate particles with a particle size in the range of 50 ~ 200 microns through a standard sieve.

2. Weigh 2.0 g of the copolymer of lactic acid and glycolic acid, pour 10 ml of chloroform, and 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 in the range of 50 ~ 200 microns to the above solution, and stir it thoroughly to make it evenly mixed.

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

In this way, a porous material has been made. Example 5

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

2. Weigh 2.0 g of the copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid, pour 6.7 ml of chloroform, and 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 in the range of 200 to 400 metric meters to the above solution, and stir well to make it evenly mixed.

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

The article is removed, and the porous scaffold is made. Example 6

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

2. Weigh 2.0 g of copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid and pour into 25ml The 1,4-dioxane was heated 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 in the range of 200 to 500 metric meters to the above solution, and stir well to make it mix well.

The remaining steps are the same as the corresponding steps in the first embodiment.

The article is removed, and the porous scaffold is made. Example Ί

The polyhydroxybutyrate (FHB) scaffold material prepared in Example 1 was used to construct tissue engineering bones in nude mice. The entire experiment was performed in cooperation with the Tissue Engineering Laboratory of Jaw Surgery, Fourth Military Medical University School of Medicine.

Experimental method: New Zealand rabbit skeletal bone was taken, bone marrow stromal cells were isolated, differentiated into osteoblasts, and the cells were collected and inoculated subcutaneously in nude mice with 4 × 10 OVml. Materials were taken at the 6th and 8th weeks, and the X-ray film was taken. Stained with HE and observed by trichromatic staining.

Experimental results: Bone tissue formation can be seen at the 6th week and the 8th week, as shown in Figure 5 is the effect of bone tissue formation at the 8th week. At the same time, the biocompatibility of the PHB porous scaffold material was evaluated. The results showed that the degree of hemolysis of PHB was less than 5%, which met the criteria for hemolysis of biomaterials. And the subcutaneous implantation experiments of the PHB porous scaffold showed no significant inflammation and rejection, no tissue necrosis and no The fiber encapsulation phenomenon indicates that the FHB material has good histocompatibility. Industrial applicability

The method for preparing a porous scaffold for tissue and organ repair according to the present invention has simple process and easy operation. According to the requirements of the porous scaffold material for actual tissue and organ repair, a porous scaffold with a certain degradation rate and adjustable pore diameter can be obtained. Moreover, the porous scaffold prepared by the method of the invention has good histocompatibility. It can be implanted under the skin of a living body, without obvious inflammation and rejection, without tissue necrosis and fibrous encapsulation, and can be used to construct tissues in immune animals. The engineering bone has a good industrial application prospect.

Claims

Profit requirements

1. A method for preparing a porous scaffold for tissue and organ repair,

It is characterized in that the method includes the following steps:

(1) A pore-forming agent having a particle size in the range of 50 zhen meters to 500 microns is obtained through a sieve sieve, and the pore-forming agent is selected from the group consisting of sodium chloride, potassium chloride, potassium acetate, sodium bicarbonate, sodium carbonate, Any one of citric acid and potassium citrate;

(2) dissolving the biodegradable polymer in a solvent to obtain a solution having a solution concentration of 5¾-30 in terms of the ratio of the mass of the polymer to the volume of the solvent, wherein the biodegradable polymer is selected from the group consisting of poly3-hydroxyl Composed of butyrate, copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, polylactic acid, copolymer of lactic acid and glycolic acid, copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid, and polyglycolic acid One or more of the group, the solvent is selected from the group consisting of chloroform, 1,4-dioxane, 1,2-dichloroethane, 1,4-dioxane-water mixture, one of p-pyridine;

(3) Add the pore-forming agent in the step (1) to the solution described in the step (2) according to the mass ratio of the polymer to the pore-forming agent of 1: 9 ~ 1: 40, and stir well. To obtain a mixture;

(4) adding the mixture to a mold, closing the mold under a pressure of 0 to 5 MPa, and drying the product;

(5) After the product is dry, release the mold, and further dry the product, so that all the solvents are evaporated.

(6) Soak the dried product in deionized water or weakly acidic aqueous solution, or immerse it in the weakly acidic aqueous solution first, and then take out the product and soak it in deionized water. The soaking time of the deionized water or weakly acidic solution is respectively 70 ~ 80 hours;

(7) Dry the product again to allow all solvents to evaporate.

2. The preparation method according to claim 1, It is characterized in that the concentration of the biodegradable polymer solution in the step (2) is 6-15%.

3. The preparation method as described in claim 1 or 2,

It is characterized in that the mass ratio of the polymer to the pore former is 1: 15 ~ 1: 35.

4. The preparation method as described in claim 1, 2 or 3,

It is characterized in that the molding condition of the mixture in the mold is that the mold is closed under a pressure of 0.1 to 2 MPa, and then naturally dry at room temperature.

5. The preparation method according to the requirements 1, 2, 3, 4 or 5,

It is characterized in that the drying of the product in the step (5) is performed in a vacuum drying box.

6. The preparation method according to claim 5,

It is characterized in that the conditions for the vacuum drying are temperature and normal temperature, pressure of 0.005 Mfa to 0 MPa, and time between 24 to 48 hours.

7. The preparation method according to any one of the above-mentioned requirements,

It is characterized in that the weakly acidic aqueous hydrochloric acid solution in the step (6).

8. The preparation method according to any one of the foregoing requirements,

Wherein said step (6) in a weak acid concentration of H ⁺ between 2M ~ 10- ⁴ M.

9. The preparation method according to any one of the above-mentioned requirements,

It is characterized in that the volume ratio of the product in the step (6) to deionized water or weakly acidic aqueous solution is between 1: 50 ~ 1: 200.

10. The preparation method according to any one of the foregoing requirements,

It is characterized in that the step (6) is to replace the deionized water or the acidic aqueous solution every 5 to 8 hours.

11. The preparation method according to any one of the above-mentioned requirements,

It is characterized in that the re-drying of the product in the step (7) is performed in a vacuum drying oven.

12. The preparation method according to claim 11,

It is characterized in that the vacuum dry conditions are temperature and normal temperature, a pressure of 0.01 to 0 MPa, and a time of 24-48 hours.