CN1168506C - Polyhydroxybutyrate/polyethylene glycol porous scaffold material and preparation method thereof - Google Patents

Abstract

The present invention relates to polyhydroxybutyrate/polyglycol porous scaffold material and a preparation method thereof. The present invention belongs to the field of biomedical material science engineering. The purpose of the present invention is to improve the hydrophilicity of PHB, and enhance the size of the hole diameter and the distribution controllability of PHB. The present invention adopts polyhydroxybutyrate and polyglycol as base material. A template method and a freeze-drying method are combined to prepare the polyhydroxybutyrate/polyglycol porous scaffold material. The factor of porosity of a scaffold is determined by the concentration of initial solution. The pre-freezing temperature also influences the average hole diameter and the hole diameter distribution. The polyhydroxybutyrate/polyglycol porous scaffold material of the present invention has good biocompatibility, hydrophile/dewatering equilibrium can be adjusted, the size of the hole diameter and the distribution can be controlled, and the requirements of cells with different types can be satisfied. Simultaneously, the mechanical strength and the biomass (cell) compatibility of porous material are ensured. The scaffold material is extremely suitable for the apposition growth of cartilage histiocyte.

Description

Polyhydroxybutyrate/polyethylene glycol porous scaffold material and preparation method thereof

Technical field

The invention belongs to a porous support used in the field of biomedical material science and engineering.

Background technique

Tissue engineering is an emerging discipline that uses the basic principles of engineering science and life science to research and develop biological substitutes to restore, maintain and improve tissue functions. The basic idea is: separate and cultivate cells in vitro, plant a certain amount of cells into a three-dimensional biomaterial skeleton with a certain shape, and continue to cultivate them, and finally form tissues and organs with a certain structure and implant them back into the body to achieve repair or repair. purpose of reconstruction. Polyhydroxybutyrate (PHB) is a high molecular polymer stored in the cells of microorganisms under unbalanced growth conditions, and is widely found in many prokaryotes in nature. PHB has many excellent biological and physical and chemical properties, such as: biodegradability, biocompatibility, piezoelectricity, optical activity, non-toxic, non-irritating, non-immunogenic and other special properties. These characteristics make it widely used in biomedicine, such as being used as drug release carrier tissue engineering scaffold material. At present, tissue engineering scaffold materials are mostly synthetic polyesters, such as polylactic acid (PLA), polyglycolic acid (PGA), etc., because they are synthetic materials, lack cell recognition signals, and their degradation products are acidic, and the pH pulse effect of the products To a certain extent, it causes aseptic inflammation; while PHB does not have such problems. PHB is a high molecular polymer synthesized by microorganisms. It has information contained in natural biological materials that is conducive to cell attachment or maintenance of differentiation functions, and its final degradation products It is 3-hydroxybutyric acid, which is a common metabolite in human blood and will not bring any toxic effects to the human body.

There are many applications of PHB in tissue engineering scaffold materials. At present, most of the researches are on cartilage, bone, skin, heart valve, blood vessel, nerve and other tissue engineering. However, pure PHB material has poor hydrophilicity, which is not conducive to the adhesion and growth of cells on the surface of the material. Polyethylene glycol is a non-toxic polymer approved by the US FDA for oral administration. It has excellent blood compatibility, excellent hydrophilicity, and no immunogenicity. It is widely used in the fields of pharmacy and biomedical materials. . If PEG is used to modify the PHB material, the hydrophilicity/hydrophobicity of the material can be well improved, and the blood compatibility of the surface of the material can be improved.

In addition, as a tissue engineering scaffold material, its pore size and distribution have an impact on cell adhesion and growth. If the pore size and distribution of the material can be controlled, the material can be adapted to the culture requirements of different cells. The scaffold material obtained by a single freeze-drying method has a large pore size and uneven distribution, while the present invention uses the freeze-drying method combined with the template method to artificially control and adjust the size and distribution of the pores, so that the obtained scaffold material can be applied to a variety of cells cultivation.

Contents of Invention

The purpose of the present invention is to improve the hydrophilicity of PHB, enhance the controllability of its pore size and distribution, while retaining its good

Good cytocompatibility to meet the needs of different types of cells.

The object of the present invention is achieved like this: polyethylene glycol and polyhydroxybutyrate are used as base materials, and the weight percentage is 1-7:10. The preparation method is as follows:

1. A certain amount of polyethylene glycol with a molecular weight of 4,000 to 20,000 and polyhydroxybutyrate with a molecular weight of 200,000 to 600,000 are dissolved in an organic solvent at a weight percentage of 1 to 7:10. 1. The solvent can be an organic solvent such as chloroform, dioxane, tetrahydrofuran, etc.—prepared to a solution of 3% to 15% by weight;

2. Preparation of template: Take a certain amount of template material—the templates used in this experiment include SPAN, TWEEN, AOT, etc.—heat and dissolve in deionized water to form a stable emulsion;

3. Mix the template emulsion with polyhydroxybutyrate/polyethylene glycol solution, and stir at high speed (500-1000 rpm) for 2-4 hours. After the solution is uniformly mixed and stable, transfer to a chemically stable and low-temperature-resistant A liquid level of 1-10mm height is formed in a flat-bottomed container;

4. Prefreeze the solution in a refrigerator at -10°C to -80°C for 8 to 24 hours; lower the temperature of the drying chamber of the freeze dryer to -40°C to -60°C, and move the petri dish into the drying chamber; temperature balance Finally, seal the drying room and turn on the air pump to keep the air pressure in the drying room less than 5000 Pascals for 24 to 72 hours, then raise it to normal pressure and heat to 40°C for 1 to 3 hours, then place the obtained porous material in a vacuum at 40°C Dry in a drying oven for 72 hours.

The porosity of the PHB porous scaffold material prepared by combining the template method and the freeze-drying method is determined by the concentration of the initial solution. The greater the initial concentration, the lower the porosity (see Figure 1); the pre-freezing temperature affects the average pore size and pore size Distribution, the lower the pre-freezing temperature, the smaller the pore size, and the higher the pre-freezing temperature, the larger the pore size (as shown in Figure 2).

The polyhydroxybutyrate/polyethylene glycol porous scaffold material of the present invention has low price, good biocompatibility, adjustable hydrophilic/hydrophobic balance, controllable pore size and distribution, and can adapt to the needs of different types of cells, while ensuring The mechanical strength of porous materials and the affinity of biomass, such as cells. The scaffold material is very suitable for attachment and growth of cartilage tissue cells.

Description of drawings

Figure 1. Microscopic electron microscope images of the pore size of the polyhydroxybutyrate/polyethylene glycol porous scaffold material at different pre-freezing temperatures.

Fig. 2 is a graph showing the influence of different initial concentrations on the porosity (%) of the polyhydroxybutyrate/polyethylene glycol porous scaffold material.

Detailed ways

1. Get the polyhydroxybutyrate 4g that molecular weight is 4 * ¹⁰⁵ and the polyethylene glycol 0.4g that molecular weight is 6000,

2. Put in 80ml of chloroform, stir at 40°C for 2 hours, transfer the solution to a 100ml volumetric flask after complete dissolution, and add chloroform solution to obtain 100ml polyhydroxybutyrate/polyethylene glycol chloroform solution (A); take Dissolve 0.12g of SPAN60 in 60ml of deionized water at 70°C to obtain a stable template emulsion (B); take 20ml of solution (A) and 0.5ml of (B) and mix together, stir at 700 rpm for 3 hours, and wait for the solution to mix After being uniform and stable, transfer to a 60mm glass petri dish, and place it in a -80°C low-temperature refrigerator for pre-freezing for 24 hours; control the temperature in the drying room of the freeze dryer to -50°C, and move the petri dish containing the pre-frozen body into the drying room; temperature After balancing, close the drying room and turn on the air pump, keep the air pressure in the drying room less than 50μatm, and keep it for 48 hours; then rise to normal pressure and heat to 40°C for 2 hours, then place the obtained porous material in a 40°C vacuum drying oven Dry for 72 hours.

3. Take 11g of polyhydroxybutyrate with a molecular weight of 2× ¹⁰⁵ and 7.7g of polyethylene glycol with a molecular weight of 4000, put them into 80ml of chloroform, stir at 40°C for 2 hours, and transfer the solution to 100ml after complete dissolution Volumetric flask, add chloroform solution, obtain 100ml polyhydroxybutyrate/polyethylene glycol chloroform solution (A); Get TWEEN60 0.12g and dissolve in 70 ℃ 60ml deionized water, obtain stable template emulsion (B); Take solution ( A) 20ml and (B) 0.5ml are mixed together and stirred at 700 rpm for 3 hours. After the solution is mixed evenly and stably, transfer it to a 60mm glass petri dish and place it in a -80°C low-temperature refrigerator for 24 hours. ;Control the temperature of the drying chamber of the freeze dryer to -50°C, move the petri dish containing the pre-frozen body into the drying chamber; after the temperature is balanced, close the drying chamber and turn on the air pump to keep the air pressure in the drying chamber less than 50μatm for 48 hours; then rise to After normal pressure, it was heated to 40° C. and baked for 2 hours, and then the obtained porous material was dried in a vacuum oven at 40° C. for 72 hours.

4. Take 8g of polyhydroxybutyrate with a molecular weight of 6× ¹⁰⁵ and 2g of polyethylene glycol with a molecular weight of 20,000, put them into 80ml of chloroform, stir at 40°C for 2 hours, and transfer the solution to a capacity of 100ml after complete dissolution Bottle, add chloroform solution, obtain 100ml polyhydroxybutyrate/polyethylene glycol chloroform solution (A); Take AOT 0.12g and dissolve in 70 ℃ 60ml deionized water, obtain stable template emulsion (B); Take solution (A ) 20ml and (B) 0.5ml are mixed together, stirred at 700 rpm for 3 hours, after the solution is uniformly mixed and stable, transferred to a 60mm glass petri dish, and placed in a -80°C low-temperature refrigerator for 24 hours to pre-freeze; Control the temperature of the drying room of the freeze dryer to -50°C, move the petri dish containing the pre-frozen body into the drying room; after the temperature is balanced, close the drying room and turn on the air pump to keep the air pressure in the drying room less than 50μatm for 48 hours; then rise to normal temperature. After pressing, it was heated to 40° C. and baked for 2 hours, and then the obtained porous material was dried in a vacuum oven at 40° C. for 72 hours.

Claims (4)

1, a kind of biomedical tissue engineering porous support materials is characterized by, it by molecular weight be 4000～20000 Polyethylene Glycol and molecular weight be 200000～600000 poly butyric ester with 1～7: 10 weight ratio constitutes.

2, a kind of preparation method of biomedical tissue engineering porous support materials is characterized in that it comprises the following steps:

(1) getting 1～7 part of molecular weight is that 4000～20000 Polyethylene Glycol and 10 parts of molecular weight are 200000～600000 poly butyric ester mix homogeneously, is dissolved in an amount of organic solvent, is mixed with percentage by weight and is 3～15% solution;

(2) get a certain amount of mould material heating and be dissolved in the deionized water, form stable emulsion;

(3) the template emulsion is mixed with polyhydroxybutyrate/polyglycol solution, stirred 2～4 hours with 500～1000 rev/mins speed, after treating that the solution mix homogeneously is stable, be transferred to do not react with solution and low temperature resistant Flat bottom container in form the liquid level of 1～10mm height;

(4) inserted in-10 ℃～-80 ℃ the cryogenic refrigerator pre-freeze 8～24 hours; Freezer dryer hothouse temperature is dropped to-40 ℃～-60 ℃, Flat bottom container is moved into hothouse; Close drying chamber after the hygral equilibrium, and unlatching air pump, keep hothouse air pressure less than 5000 Pascals 24～72 hours, rise to normal pressure post-heating to 35 ℃～45 ℃ baking the affected part after applying some drugs 1～3 hour then, the porous material that obtains was placed 35 ℃～45 ℃ vacuum drying oven inner dryings 72 hours again.

3, the preparation method of biomedical tissue engineering porous support materials according to claim 2 is characterized by, and described organic solvent is chloroform, dioxane or oxolane.

4, the preparation method of biomedical tissue engineering porous support materials according to claim 2 is characterized by, and described mould material is SPAN class, TWEEN class or AOT class.

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