WO2001082988A1

WO2001082988A1 - Phase separated particle preparation method for a porous framework used in the prostheses of tissue and organs

Info

Publication number: WO2001082988A1
Application number: PCT/IB2001/000633
Authority: WO
Inventors: Ping Hu; Feng Gao; Lingfei Jiang
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: AU2001246764A1; CN1275617A; CN1095497C

Abstract

The present invention relates to a phase-separated particle preparation method for a porous framework used in the prostheses of tissue and organs. First is to select the pore-forming agent and dissolve 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 to be quenched and then knocked out of the mould and dried. Then the freeze-dried preparation is dipped into deionized water or weak acid aqueous solution and vacuum dried again thereafter. Thus the porous framework of the present invention is prepared. The framework material prepared using the method of the present invention is high in porosity, good in biological compatibility and biologically degradable.

Description

Method for preparing porous scaffold for tissue and organ repair by phase separation particle filtration method TECHNICAL FIELD

The invention belongs to the field of biomedical engineering, and relates to a method for preparing a multi-hole scaffold for tissue and organ repair, in particular to a method for preparing a biodegradable porous scaffold as a cell growth carrier. Background technique

In recent years, tissue or organ defects or functional failures have posed great threats to human health and caused countless families a devastating blow. In clinical practice, the contradiction between the frequent occurrence of organ and tissue failure and the serious shortage of organs and tissues available for transplantation has increasingly become a bottleneck for improving people's health. In order to alleviate the above-mentioned contradiction, tissue engineering adopted a porous scaffold to culture a variety of implantable tissues and organs in vitro using bulk cells. The porous scaffold for tissue engineering should meet the following requirements: 1) the surface can make cells adhere and grow; 2) the polymer and its degradation products will not cause inflammation and toxic side effects after being implanted in the body; 3) the porous scaffold should have a three-dimensional structure Structure; 4) To provide sufficient space for extracellular regeneration, the porosity of the porous scaffold must not be less than 90%; 5) The polymer can be absorbed by the body immediately after the tissue regeneration is completed; 6) The degradation rate of the porous scaffold should be different from that of different tissues The rate of cell proliferation matches.

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; 3. The three-dimensional layering method 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. Make sure to write L The melt processing method is a method in which a pore former and a polymer are blended and extruded above a melting point of a polymer into a mold, and cooled to obtain a porous stent of a predetermined shape. The disadvantage of this method is that in the extruder, the melt The porosity has a large difference in density, so it is difficult to mix uniformly, and some polymers, especially biodegradable polymers, are easily thermally degraded during melt processing; 5. Phase separation method, which uses a solution mixture to cool to a solvent. Below the melting point, phase separation occurs, and the porous scaffold is obtained by vacuum drying. The disadvantage of this method is that the obtained pore diameter is generally below 100 microns, and it is difficult to control. 6. The high-pressure carbon dioxide method, which uses the formed The polymer is exposed to high-pressure carbon dioxide, and the carbon dioxide dissolved in the polymer is released under reduced pressure 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 for preparing a porous repair scaffold for tissues and organs by a phase separation particle filtration method. The method absorbs the advantages of the solution casting particle filtration method and the phase separation method, and solves the pore diameter of the simple phase separation method. The problem is too small, and the problem of particle settling of three-dimensional materials prepared by solution casting particle filtration method.

To achieve the above objective, the technical solution adopted by 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 to 500 microns. The pore-forming agent is sodium chloride, potassium chloride, potassium acetate, sodium bicarbonate, sodium carbonate, citric acid, and lemon. Potassium acid, etc.

2. A polymer selected from the group consisting of poly (3-hydroxybutyrate (PHB), 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (PHB-HH), polylactic acid (PLA), 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, In a solvent such as 2-dichloroethane, 1,4-dioxane-water mixture, and pyridine, the concentration is 5¾ ~ 30 (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. With stirring, pour the mixture in step 3 into a mold with a preset temperature and quench it. The quenching temperature is between -180 ° C ~ 11.8 ° C, so that the product is completely frozen;

5. Open the mold and freeze-dry the formed product. The freezing temperature is between -180 ° C ~ 11.8 ° C, the dry pressure is between 0.001325MPa ~ 0MPa, and the freeze-drying time is 2 ~ 4 days.

6. Soak the freeze-dried products in 5 into deionized water or weak acid, or immerse them in a weakly acidic aqueous solution, and then take out the products and soak them in deionized water. The soaking times of the deionized water or weak acid solution are respectively It 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, and is preferably 6-15%;

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 undergo precipitation and regular flow. The requirements vary, preferably 1: 15 ~ 1: 35; In the preparation method of the present invention, the quenching temperature in the step 4 is -170 ° C ~ 0 ° C, and the quenching time is 5 minutes. ~ 2 hours;

In the preparation method of the present invention, the freezing temperature of the product in step 5 is -170 ° C ~ 0 ° C;

In the method according to the present invention, a weakly acidic aqueous solution of hydrochloric acid in the step 6, the weak acid solution in a concentration of H + 2Μ ~ 1 (between Γ ⁴ Μ.

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 carried out in a vacuum drying box, the temperature is 37 ° C, the pressure is 0.001325MPa ~ OMPa, and it is dried for 24 to 48 hours. .

The scaffold material for tissue and organ repair prepared by applying the method of the present invention has the following characteristics and advantages:

(1) The porosity can reach more than 90%.

(2) The obtained scaffold material has good biocompatibility and is biodegradable.

(3) Directly preparing a three-dimensional scaffolding scaffold material, which solves the problem of particle sedimentation by solution casting particle filtration method.

(4) The size of the pores ranges from a few tens of meters to 300 microns, that is, there are large pores and small pores, which solves the problem that the pore size of the simple phase separation method is too small. In addition, the macropore-macropore, macropore-smallpore, and micropore-smallpore are connected to each other (as shown in Fig. 1 and Fig. 2), which is suitable for the growth of cells of various sizes and avoids the closed pores of the high pressure carbon dioxide method problem.

(5) High temperature operation is not required to avoid polymer degradation problems caused by melt processing. The tube of the drawings is to be explained

FIG. 1 is a scanning electron microscope image of a poly 3-hydroxybutyrate scaffold material prepared by the method of the present invention at a magnification of 100,000 times;

FIG. 2 is a scanning electron microscope image of a poly 3-hydroxybutyrate scaffold prepared by the method of the present invention at a magnification of 11 thousand times; FIG.

FIG. 3 is an external view of a poly 3-hydroxybutyrate stent material prepared by the method of the present invention; FIG. 4 is an appearance obtained based on a stent material of a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid prepared by the method of the present invention; Appearance of complex ear-shaped bracket. The best way to implement the invention

Example 1 1. Prepare a 10% (mass to volume) PHB solution in a beaker with 1 g of poly (3-hydroxybutyrate) (PHB), 10 ml of 1, ⁴ -dioxane at 55 ° C.

2. Add 15g of NaCl, where the diameter of NaCl is between 100 microns and 400 microns.

3. Stir at -10 ° C while stirring.

4. After freezing for 10 minutes, cut a sample of the desired shape.

5. Freeze-dry at -10 ° C for 4 days at 0.001325MPa ~ 0MPa.

6. Take out the product, immerse it in deionized water, change the water every 8 hours, and soak for 3 days.

7. Dry under vacuum for 1 day, the temperature is 37 ° C, and the pressure is 0.001325MPa ~ 0MPa, then the porous scaffold for tissue and organ repair of the present invention is obtained.

As shown in the poly 3-hydroxybutyrate stent material shown in Figures 1 and 2, it can be seen that there are both large holes and small holes in the stent material, and there are large holes-large holes, large holes-small holes, and small holes. -The pores communicate with each other, suitable for the growth of cells of various sizes. FIG. 3 is an external view of the poly 3-hydroxybutyrate stent material, which can be made into various external shapes according to actual needs. Example 2

1. Prepare a 15% (mass to volume) PLGA solution in a beaker with 1.5 g of lactic acid and glycolic acid copolymer (PLGA) and 10 ml of 1,4-dioxane at 60 ° C.

2. Add 22.5g KC1, where C1 is between 100 microns and 400 microns in diameter.

3. Pour into a mold with a preset temperature of 0 ° C while stirring and quench it.

4. After freezing for 20 minutes, open the mold to obtain a sample of the desired shape.

5. Freeze and dry at 0 ° C for 4 days at 0.001325MPa ~ 0MPa.

6. Take out the sample, immerse it in deionized water, change the water every 8 hours, and soak for 3 days.

7. Dry under vacuum for 1 day, the temperature is 37 ° C, and the pressure is 0.001325MFa ~ 0MFa, then the porous scaffold for tissue and organ repair of the present invention is obtained. Example 3

1. At 55 ° C, use 1.5 g of copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHB-HH) in a beaker, 10 ml of 1,4-dioxane-water mixture ( 1,4-dioxane, water volume ratio 87:13) Formulated to 15% (mass specific volume) Formulated FHB-HH solution.

2. Insert 22.5g KC1 into the mouth, where the diameter of KC1 is between 100 to 400 microns.

3. Pour into a mold cooled with liquid nitrogen (approximately -170 ° C) with stirring to quench.

5. Freeze-dry at 0 ° C, 0.001325MPa ~ 0MPa for 4 days.

FIG. 4 is an external view of an ear-shaped stent obtained based on the PHB-HH stent material of this embodiment. It can be seen that the use of the phase separation particle filtration method of the present invention makes it possible to obtain a stent with a complicated appearance. Example 4

1. At 60 ° C, select 1.5 grams of polylactic acid (PLA) and 10 ml of 1,4-dioxane in a beaker to prepare a 15% (mass to volume) PLA solution.

2. Add 22.5g NaCl, where the diameter of NaCl is between 100 microns and 400 microns.

3. Pour into a mold with a preset temperature of -15 ° C while stirring and quench it.

5. Freeze and dry for 4 days at -15 ° C and 0.001325MPa ~ 0MPa.

7. Vacuum dry for 1 day, the temperature is 37 ° C, and the pressure is 0.001325MPa ~ 0MFa, then the porous scaffold for tissue and organ repair of the present invention is obtained. Example 5

The polyhydroxybutyrate (PHB) scaffold material prepared in Example 1 was used to construct tissue-engineered artificial cartilage in the animal. The entire experiment was completed by the Institute of Basic Medicine of the Academy of Military Medical Sciences and Tsinghua University.

The articular cartilage of New Zealand young rabbits was taken, chondrocytes were isolated and cultured, and a certain number of chondrocytes were seeded on the PHB scaffold material prepared in Example 1 to form a cell-scaffold complex, which was then transplanted under the skin of the New Zealand rabbit's back At 8 and 12 weeks after surgery, samples were taken for histology and histochemical staining, and cartilage tissue formation was found in the complex. The I¾B scaffold material was biocompatible and could be used as an artificial extracellular for chondrocytes. Matrix material. The PHB porous scaffold material has a degree of hemolysis of <5%, which meets the requirements of hemolytic oak of biomaterials. The planting experiments showed that the material has no obvious inflammation and rejection, no tissue necrosis and fibrous encapsulation. . 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, and can obtain a porous scaffold with a certain degradation rate and uniformly adjustable pore diameter according to the requirements of the actual tissue and organ repair for the porous scaffold material, and The scaffold material with complex three-dimensional structure can be directly prepared, which effectively solves the problems of particle sedimentation and closed pores existing in the known technology. The porous scaffold prepared by the method of the invention has good histocompatibility. It is implanted under the skin of a living body, without obvious inflammation and rejection, without tissue necrosis and fibrous encapsulation, and can be used to build tissues in immune animals. The engineering bone has a good industrial application prospect.

Claims

Rights request

1. A method for preparing a porous scaffold for tissue and organ repair by a phase separation particle filtration method,

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

(1) A standard sieve is used to obtain a pore-forming agent having a particle diameter in the range of 50 zhenmi to 500 zhenmi. 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 to 30%, calculated as the ratio of the polymer mass to the solvent volume, wherein the biodegradable polymer is selected from the group consisting of poly3- Copolymers of hydroxybutyrate, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, polylactic acid, copolymers of lactic acid and glycolic acid, copolymers of 3-hydroxybutyric acid and 3-hydroxyacetic acid, and polyglycolic acid One or more of the group consisting of, the solvent is selected from the group consisting of chloroform, 1,4-dioxane, 1,2-dichloroethane, 1,4-dioxane-water mixture One of pyridine;

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

(4) With stirring, pour the mixture in step (3) into a mold with a preset temperature, and quench the temperature between -180 ° C ~ 11.8 ° C to completely freeze the product;

(5) Open the mold, freeze-dry the formed product, the freezing temperature is between -180 ° C ~ 11.8 ° C, the dry pressure is between 0.001325MPa ~ 0MPa, and the freeze-drying time is 2 ~ 4 days;

(6) Soak the frozen and dried product in step (5) in deionized water or weak acid, or immerse in a weakly acidic aqueous solution, and then take out the product and soak it in deionized water or weakly acidic The soaking time of the solution is 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 quenching temperature in the step 4 is -170 ° C ~ -10 ° C.

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

It is characterized in that the quenching time in the step (4) is 5 minutes to 2 hours.

6. The preparation method as described in claim 1, 2, 3, 4 or 5,

It is characterized in that the freezing temperature of the product in step (5) for freeze-drying is -170 ° C ~ -10 ° C.

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 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 foregoing requirements,

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

12. The preparation method according to claim 11,

It is characterized in that the vacuum drying conditions are a temperature of 37 ° C, a pressure of 0.001325 MPa to 0 MPa, and a drying time of 24-48 hours.