CN102961781B - A kind of preparation method of tissue engineering bracket material - Google Patents

Abstract

The invention provides a kind of preparation method of tissue engineering bracket material, comprising: a) bio-compatible polyester is mixed with non-polar solven, obtain the first solution; B) inversion of phases solvent is added in described first mixed solution, obtain the second solution; C) described second solution is placed in mould, removes described non-polar solven and inversion of phases solvent, dry solute by liquid-gas phase or with liquid-solid phase conversion process.Obtain tissue engineering bracket material.The method preparing tissue engineering bracket material provided by the invention, makes tissue engineering bracket have higher porosity having on good mechanical property basis.

Description

A preparation method of tissue engineering scaffold material

technical field

The invention relates to the field of preparation of polymer materials, in particular to a preparation method of a tissue engineering scaffold material,

Background technique

Tissue engineering scaffold material is a polymer material that can partially or completely replace human tissues and organs. Because of its good biocompatibility, non-toxic and harmless, it has been widely used in organ replacement, organ transplantation, clinical surgery and other fields.

For tissue engineering scaffold materials, in addition to requiring high porosity, there are also strict requirements on pore size. If the pores are too small, cells cannot enter the pores or hinder cell reproduction and expansion. If the pores are too large, cells are not easy to adhere and lose their function. The role of the scaffold. Now there are many different preparation methods for tissue engineering scaffolds, including molding method, gas foaming method, solution casting method, particle filtration method (Mikos A.G. Polymer35, 1994), frozen phase separation and so on. These methods have achieved varying degrees of success, but there is generally a problem of poor connectivity between holes, as shown in Figure 1. Ma P.X. is equal to the preparation process of the biodegradable polymer scaffold with a well-defined interconnected spherical pore network prepared by the method of particle fusion reported by Tissue Eng7:23, 2001. Although the penetration between the pores is increased, the pores of this method The channel diameter and channel density are difficult to control, and the mechanical strength of the scaffold is poor.

The microstructure of a good three-dimensional porous scaffold needs to have high porosity (>90%), good connectivity and good mechanical strength. A scaffold with high porosity can provide more space for cell growth, and good mechanical strength can provide mechanical support for cell growth. Some evidence suggests that pore connectivity and porosity are equally important for bone tissue ingrowth, especially in the early stages of bone tissue regeneration and infiltration. Because the channel between porous materials is the main way for the entry of cells, blood vessels, nutrients and the elimination of metabolites; and the pore size of the scaffold also affects cell viability and tissue regeneration. Scaffolds with different pore sizes are suitable for different types of cells, allowing them to enter and adhere in large quantities. The pore size of 5-15 μm is suitable for fibroblasts, 20-125 μm is suitable for adult skin tissue, and 100-350 μm is suitable for bone tissue. Therefore, many scholars focus on how to prepare porous scaffolds suitable for cell and tissue growth.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing tissue engineering scaffold material, so that the tissue engineering scaffold has higher porosity on the basis of good mechanical properties.

In order to solve the above technical problems, the invention provides a preparation method of tissue engineering scaffold material, comprising:

a) mixing the biocompatible polyester with a non-polar solvent to obtain a first solution;

b) adding a phase inversion solvent into the first mixed solution to obtain a second solution;

c) placing the second solution in a mold, removing the non-polar solvent and phase inversion solvent through a liquid-gas phase or liquid-solid phase inversion process, and drying the solute; obtaining a tissue engineering scaffold material.

Preferably, step c) is specifically:

c1) placing said second solution in a mould;

c2) The non-polar solvent is converted into a gas phase by natural evaporation, decompression, heating, etc. and then extracted;

c3) Adding a polar solvent to the mold to remove the phase inversion solvent, drying the solute, and obtaining a tissue engineering scaffold; the polar solvent is ethanol or water.

Preferably, it also includes adding a pore-forming agent to the second solution obtained in step b) to obtain a third solution.

Preferably, the method further includes adding an active agent to the third mixed solution to obtain a fourth solution, and subjecting the fourth solution to step c).

Preferably, the biocompatible polyester is selected from one or more of polylactic acid, polyglycolide, polylactide, glycolide-lactide copolymer, and polyε-caprolactone.

Preferably, the non-polar solvent is selected from one or more of chloroform, dichloromethane, 6-fluoroisopropanol and n-hexane.

Preferably, the phase inversion solvent is selected from one or more of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, dimethylacetamide, acetone and tetrahydrofuran.

Preferably, the pore forming agent is selected from crystal calcium chloride, sodium chloride, magnesium chloride, potassium chloride, sodium phosphate, potassium phosphate, calcium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid One or more of dipotassium hydrogen, sodium hydrogen phosphate, sucrose and glucose.

Preferably, the mass ratio of the pore-forming agent to the biocompatible polyester is 1:5-10.

Preferably, the active agent is polyethylene glycol.

The invention provides a method for preparing a scaffold material for tissue engineering, using biocompatible polyester as the main material, first mixing the biocompatible polyester with a non-polar solvent to obtain a first solution, and then adding the biocompatible polyester to the A phase inversion solvent is added to the first solution to obtain a second solution, and finally the first solution and the second solution are removed, and the solute is dried to obtain a tissue engineering scaffold material. The preparation method provided by the invention utilizes the phase inversion method to prepare tissue engineering scaffold materials, which is different from the molding method, liquid casting method and gas foaming method in the prior art. Or volatilization will form a certain void in the material, and removing the phase inversion solvent with a polar solvent will transform the biocompatible polyester solution with the solvent as the continuous phase into a three-dimensional large biocompatible polyester with the continuous phase Molecular network gel, biocompatible polyester is converted from a continuous phase solvent system of polymer materials to a three-dimensional network structure macromolecular gel solute with polymer materials as the continuous phase, and the gel solute can be controlled by controlling the ratio of raw materials. The voids and porosity in the glue, after the solute is dried, the tissue engineering scaffold material can be obtained. The method provided by the invention can control the porosity and voids of the product, so that the product not only has an appropriate pore diameter and porosity, but also ensures the mechanical properties of the product.

Description of drawings

The electron micrograph of the tissue engineering scaffold material prepared by the method provided by the embodiment of the present invention 1 in Fig. 1;

Fig. 2 is the electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 2 of the present invention;

Fig. 3 is an electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 3 of the present invention;

4 is an electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 4 of the present invention;

Fig. 5 is an electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 5 of the present invention;

Fig. 6 is an electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 6 of the present invention;

Fig. 7 is an electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 7 of the present invention;

Figure 8 is an electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 8 of the present invention;

Fig. 9 is an electron micrograph of the tissue engineering scaffold material prepared by the method provided in Example 9 of the present invention.

Detailed ways

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with the examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention rather than limiting the patent requirements of the present invention.

A kind of preparation method of tissue engineering scaffold material provided by the present invention comprises:

a) mixing the biocompatible polyester with a non-polar solvent to obtain a first solution;

b) adding a phase inversion solvent into the first mixed solution to obtain a second solution;

c) placing the second solution in a mold, removing the non-polar solvent and phase inversion solvent through a liquid-gas phase or liquid-solid phase inversion process, and drying the solute. Obtain tissue engineering scaffold materials.

In order to solve the problems of the prior art and make the tissue engineering scaffold material have high strength and controllable porosity and void size, the present invention uses a phase inversion method to prepare the first biocompatible polyester for preparing the tissue engineering scaffold material by dissolving solution, and then add a phase inversion solvent to the first solution to obtain a second solution. According to the present invention, during the phase inversion process, the separation of the gas phase and the liquid phase or the liquid phase and the liquid phase occurs due to the change of the solubility of the polyester in the solvent, and the scaffold material is solidified and formed. Holes are formed, and the second phase solution has a slight dissolution effect on the scaffold material, and can form micropores and space channels between the pores of the scaffold. The scaffold material prepared by this method has high porosity and high spatial connectivity, which is beneficial for cells to crawl inside the scaffold and transport nutrients.

According to the present invention, the selection of the biocompatible polyester is based on the premise that the material will not cause severe rejection when used in the human body, and has a certain mechanical strength. Biodegradability. According to the present invention, the biocompatible polyester is preferably one or more of polylactic acid, polyglycolide, polylactide, polyglycolide-lactide copolymer, polyε-caprolactone or the above poly Hydroxyapatite modified product of ester; more preferably polylactic acid, polylactide glycolide copolymer, polylactic acid modified by hydroxyapatite, polylactide, most preferably polylactic acid, glycolide lactide Ester copolymer, hydroxyapatite modified polylactic acid. The biocompatible polyester used in the present invention can be effectively dissolved in the non-polar solvent to form a uniform first solution during the preparation of the tissue engineering scaffold material.

According to the present invention, the non-polar solvent is preferably one or more of chloroform, dichloromethane, 6-fluoroisopropanol and n-hexane, more preferably chloroform, dichloromethane or n-hexane, most preferably chloroform . Non-polar solvents have better solubility for polyester. The mass volume ratio of the biocompatible polyester to the non-polar solvent is 1%-20%, more preferably 1%-10%.

In order to obtain a tissue engineering scaffold material with controllable pore size and porosity, it is necessary to control the ratio of the non-polar solvent and the desired inversion solvent. According to the present invention, the volume ratio of the non-polar solvent and the phase inversion solvent is preferably 1~ 20:1; the selection of phase inversion solvent is also a key factor, the desired inversion solvent selected by the present invention is selected from N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, dimethylacetamide , one or more of acetone and tetrahydrofuran, more preferably N,NO-dimethylformamide, tetrahydrofuran, N-methylpyrrolidone.

In order to further improve the formation of micropores in the tissue engineering scaffold material, the present invention preferably uses natural drying when removing the solvent, and after volatilizing the non-polar solvent, wash the second solution with a polar solvent to remove the remaining non-polar Sexual solvent and want to transform the solvent to dissolve out. The solute is obtained by filtration, and then the solute is preferably washed with water and dried to obtain a tissue engineering scaffold material. Specific steps are as follows:

c1) placing said second solution in a mould;

c2) The non-polar solvent is converted into a gas phase by natural evaporation, decompression, heating, etc. and then extracted;

c3) Adding a polar solvent to the mold to remove the phase inversion solvent, drying the solute, and obtaining a tissue engineering scaffold; the polar solvent is ethanol or water.

According to the present invention, the polar solvent is preferably water or ethanol, the usage amount of the polar solvent is preferably 500-1000mL, and the number of times of washing with water is preferably 2-5 times.

In order to adjust the porosity and pore size of the tissue engineering scaffold material, it is also preferred to add a pore-forming agent in the second solution, the pore-forming agent can effectively provide more micropores in the conversion process, and the porosity of the micropores is Proportional to the amount of pore forming agent added. According to the present invention, the pore-forming agent is selected from crystal calcium chloride, sodium chloride, magnesium chloride, potassium chloride, sodium phosphate, potassium phosphate, calcium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, One or more of dipotassium hydrogen phosphate, sodium hydrogen phosphate, sucrose and glucose, more preferably sucrose and glucose. According to the present invention, the mass ratio of the pore-forming agent to the biocompatible polyester is preferably 1:5-10.

On the basis of adding the pore-forming agent to the second solution, it is also preferable to add an active agent to the second solution to improve the pore-forming efficiency of the pore-forming agent. According to the invention, the active agent is preferably polyethylene glycol.

The preparation method provided by the invention utilizes the phase inversion method to prepare tissue engineering scaffold materials, which is different from the molding method, liquid casting method and gas foaming method in the prior art. Or volatilization will form a certain void in the material, and removing the phase inversion solvent with a polar solvent will transform the biocompatible polyester solution with the solvent as the continuous phase into a three-dimensional large biocompatible polyester with the continuous phase Molecular network gel, biocompatible polyester is converted from a continuous phase solvent system of polymer materials to a three-dimensional network structure macromolecular gel solute with polymer materials as the continuous phase, and the gel solute can be controlled by controlling the ratio of raw materials. The voids and porosity in the glue, after the solute is dried, the tissue engineering scaffold material can be obtained. The method provided by the invention can control the porosity and voids of the product, so that the product not only has an appropriate pore diameter and porosity, but also ensures the mechanical properties of the product.

Example 1:

Dissolve polyester polymer polylactic acid (PLA) in chloroform; add DMF with a volume ratio of 3:2 to chloroform into the polymer solution; wait for the polymer solution to evaporate at room temperature for several days to form a relatively dry solid; The block was placed in three-distilled water with a negative pressure of 10 MP and washed several times until there was no solvent or porogen in the aqueous solution after washing; it was dried in vacuum, and the morphology of the scaffold was detected by field emission scanning electron microscopy (ESEM). Figure 1.

Example 2

Dissolve polyester polymer polylactic acid (PLA) in chloroform; add DMF with a volume ratio of 2:3 to chloroform to the polymer solution; add a particle size of 200 μm with a mass ratio of 9:1 to PLA in the mold sucrose granules; pour the dual-solvent polymer solution into the mold filled with sucrose granules; wait for the polymer solution to evaporate at room temperature for several days to form a relatively dry solid; put the solid block into three-distilled water with a negative pressure of 10MP and wash it several times , until there is no more solvent or porogen in the aqueous solution after washing; vacuum drying, and field emission scanning electron microscopy (ESEM) to detect the morphology of the scaffold. Figure 2.

Example 3

Dissolve polyester polymer polylactic acid (PLA) in chloroform; add DMF with a volume ratio of 3:2 to chloroform to the polymer solution; add a particle size of 200 μm with a mass ratio of 9:1 to PLA in the mold sucrose granules; pour the dual-solvent polymer solution into the mold filled with sucrose granules; reduce the pressure to convert the non-polar solvent into a gas phase and extract it to form a relatively dry solid; put the solid block into three-distilled water with a negative pressure of 10MP Repeated washing several times until there is no more solvent or porogen in the aqueous solution after washing; vacuum drying, and field emission scanning electron microscopy (ESEM) to detect the morphology of the scaffold. Figure 3.

Example 4.

Dissolve polyester polymer polylactic acid (PLA) in chloroform; add DMF with a volume ratio of 2:3 to chloroform into the polymer solution; dissolve PEG with a mass ratio of 1:2 to PLA in the polymer solution Add sucrose particles with a particle size of 200 μm in a mass ratio of 9:1 to PLA in the mold; pour the dual-solvent polymer solution into the mold containing the sucrose particles; wait for the polymer solution to evaporate at room temperature for several days to form a relatively dry Solid; put the solid block in three-distilled water and wash it several times under a negative pressure of 10 MP until there is no solvent or porogen in the aqueous solution after washing; dry it in vacuum, and detect the morphology of the scaffold by field emission scanning electron microscope (ESEM). Figure 4.

Example 5.

Dissolve polyester polymer polylactic acid (PLA) in chloroform; add DMF with a volume ratio of 2:3 to chloroform into the polymer solution; dissolve PEG with a mass ratio of 1:1 to PLA in the polymer solution Add sucrose particles with a particle size of 200 μm in a mass ratio of 9:1 to PLA in the mold; pour the dual-solvent polymer solution into the mold containing the sucrose particles; heat the polymer solution to 37°C to accelerate the gasification of chloroform, and wait for The polymer forms a relatively dry solid; the solid block is placed in three-distilled water and washed several times under a negative pressure of 10 MP until there is no solvent or porogen in the aqueous solution after washing; vacuum drying, field emission scanning electron microscopy (ESEM) to detect the shape of the scaffold appearance. Figure 5.

Example 6.

Dissolve polyester polymer polylactic acid (PLA) in chloroform; add DMF with a volume ratio of 2:3 to chloroform into the polymer solution; dissolve PEG with a mass ratio of 2:1 to PLA in the polymer solution Add sucrose particles with a particle size of 200 μm in a mass ratio of 9:1 to PLA in the mold; pour the dual-solvent polymer solution into the mold containing the sucrose particles; wait for the polymer solution to evaporate at room temperature for several days to form a relatively dry Solid; put the solid block in three-distilled water and wash it several times under a negative pressure of 10 MP until there is no solvent or porogen in the aqueous solution after washing; dry it in vacuum, and detect the morphology of the scaffold by field emission scanning electron microscope (ESEM). Figure 6.

Example 7.

Dissolve the polyester polymer polyglycolide-lactide copolymer (PLGA) in chloroform; add DMF with a volume ratio of 2:3 to chloroform to the polymer solution; add DMF with a mass ratio of 1:1 to PLGA PEG was dissolved in the polymer solution, and sucrose particles with a particle size of 200 μm in a mass ratio of 9:1 to PLGA were added to the mold; the dual-solvent polymer solution was poured into the mold containing the sucrose particles; the polymer solution was cooled at room temperature Evaporate for several days to form a relatively dry solid; put the solid block in three-distilled water and wash it repeatedly at a negative pressure of 10MP until there is no solvent or porogen in the aqueous solution after washing; vacuum-dry and detect with a field emission scanning electron microscope (ESEM) Stent morphology. Figure 7.

Example 8.

The composite material containing 1% hydroxyapatite polylactic acid (1%HA/PLGA) was dissolved in chloroform; DMF with a volume ratio of 2:3 to chloroform was added to the polymer solution; the mass ratio to PLGA was 2:1 PEG was dissolved in the polymer solution, and NaCl particles with a particle size of 200 μm in a mass ratio of 5:1 to PLGA were added to the mold; the dual-solvent polymer solution was poured into the mold containing NaCl particles; Evaporate at room temperature for several days to form a relatively dry solid; put the solid block in three-distilled water and wash it repeatedly at a negative pressure of 10MP until there is no solvent or porogen in the aqueous solution after washing; vacuum drying, field emission scanning electron microscope (ESEM) Check the shape of the scaffold. Figure 8.

Example 9.

Dissolve polyester polymer polylactic acid (PLA) in chloroform; add DMF with a volume ratio of 1:4 to chloroform to the polymer solution; add a particle size of 200 μm with a mass ratio of 9:1 to PLA in the mold sucrose granules; pour the dual-solvent polymer solution into the mold filled with sucrose granules; wait for the polymer solution to evaporate at room temperature for several days to form a relatively dry solid; put the solid block into three-distilled water with a negative pressure of 10MP and wash it several times , until there is no more solvent or porogen in the aqueous solution after washing; vacuum drying, and field emission scanning electron microscopy (ESEM) to detect the morphology of the scaffold. As shown in Figure 9.

Mechanical test results:

The three-point bending Flexural strength (Mpa) test is carried out by INSTRON1121 universal material testing machine.

The Compressive strength (Mpa) test is carried out by INSTRON1121 universal material testing machine.

A kind of catalyst for Michael addition reaction provided by the present invention and the preparation method of a kind of nitro fatty aldehyde have been introduced in detail above, have applied concrete example in this paper and explained principle and implementation mode of the present invention, above embodiment The description is only used to help understand the method and core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements and modifications can be made to the present invention without departing from the principle of the present invention. These improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (5)

1. a preparation method for tissue engineering bracket material, is characterized in that, comprising:

A) bio-compatible polyester is mixed with non-polar solven, obtain the first solution;

B) inversion of phases solvent is added in described first mixed solution, obtain the second solution; Also comprise to step b) add perforating agent in described second solution that obtains, obtain the 3rd solution;

C) described 3rd solution is placed in mould, removes described non-polar solven and inversion of phases solvent, dry solute by liquid-gas phase or with liquid-solid phase conversion process; Obtain tissue engineering bracket material; Step c) be specially:

C1) described 3rd solution is placed in mould;

C2) described non-polar solven is converted into gas phase by natural evaporation, decompression, mode of heating and is extracted;

C3) in described mould, add polar solvent and remove described inversion of phases solvent, dry solute, obtains tissue engineering bracket; Described polar solvent is ethanol or water; Described non-polar solven be selected from chloroform, dichloromethane, 6-fluorine isopropyl alcohol and normal hexane one or more;

Inversion of phases solvent be selected from DMF, dimethyl sulfoxide, N-Methyl pyrrolidone and dimethyl acetylamide one or more.

2. preparation method according to claim 1, is characterized in that, also comprises and add activating agent in described 3rd mixed solution, obtain the 4th solution, and described 4th solution is carried out step c) operation.

3. the preparation method according to claim 1 ~ 2 any one, is characterized in that, described bio-compatible polyester be selected from polylactic acid, PGA, Vicryl Rapide, poly-epsilon-caprolactone one or more.

4. the preparation method according to claim 1 ~ 2 any one, it is characterized in that, described perforating agent be selected from crystal calcium chloride, sodium chloride, magnesium chloride, potassium chloride, sodium phosphate, potassium phosphate, calcium phosphate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, dibastic sodium phosphate, sucrose and glucose one or more.

5. preparation method according to claim 2, is characterized in that, described activating agent is Polyethylene Glycol.