KR101386399B1 - Fabrication method of porous biodegradable polymer scaffolds using supercritical fluid-organic solvent system - Google Patents

Abstract

The present invention relates to a method for producing a porous biodegradable polymer support, characterized in that for producing a porous biodegradable polymer support by applying an organic solvent to a supercritical fluid process, according to the present invention And it is possible to manufacture a biodegradable polymer scaffold with improved pore uniformity and good internal connectivity, which is very useful for fabricating scaffolds for improving tissue regeneration including various physiologically active substances such as proteins.

Description

Fabrication method of porous biodegradable polymer scaffolds using supercritical fluid-organic solvent system

The present invention relates to a method for preparing a porous biodegradable polymer support using a supercritical fluid-organic solvent system and a porous biodegradable polymer support prepared thereby.

Tissue engineering is based on the concepts and techniques of existing scientific fields such as life sciences, medicine, and engineering to create and transplant substitutes for living tissues, thereby maintaining, enhancing, and restoring the function of living tissues to restore damaged tissues or organs to normal tissues. It is a new discipline to replace and reproduce.

Treatment of critical size bone defects includes autografts, allografts (grafting bones of others) or transplant procedures. However, the shortage of organ donors has implications for overcoming biorejection and disease risk and for developing implants. For tissue engineering research, it is important to prepare biodegradable polymer scaffolds that are similar to biotissues, often used with cells or bioactive compounds that produce new tissues. [Lavik E, Langer R. Tissue engineering: current state and perspectives . Appl Microbiol Biotechnol 2004; 65: 1-8].

In addition to mechanical and structural constraints, the design of the support must take into account a variety of conditions including material composition, deterioration properties and material surface properties. Processing technology is needed to produce a scaffold that can match the size and abnormal shape of bone defects, the scaffold should promote cell binding and growth, have a nontoxic biocompatibility that does not cause blood clotting or inflammatory reactions after transplantation, Once the transplanted cell has had sufficient function as a tissue, it must have biodegradability that degrades into non-toxic elements over time.

Biodegradable polymer materials such as polyamino acids, polyanhydrides, polycaprolactones, polyglycolides, polylactides, lactide / glycolide copolymers are mainly used as scaffolds for synthetic tissue engineering. . Synthetic biodegradable polymers, such as polylactic acid (PLA) and polylactic acid / glycolic acid copolymers (PLGA), are approved for use in the human body by the US Food and Drug Administration (FDA) and are used as supports. Is known to follow the normal metabolic pathway [Gopferich A. Mechanisms of polymer degradation and erosion. Biomaterials 1996; 17: 103-14.

Recently, various researchers have tried various methods to make a polymer as a support. Typically, salt leaching method in which single crystal salt is mixed and dried to dissolve the salt in water [Whang K, Thomas CH, Healy KE, Nuber G. Polymer 1995; 36: 837], phase separation [Chen VJ, Ma PX. Biomaterials 2004; 25: 2065-73], prototyping / solid pre-form structures [Hutmacher DW. Biomaterials 2000; 21: 2529. However, these methods generally have the disadvantage of forming a skin layer, closed pores, or controlling porosity and pore size. In addition, there is a disadvantage that the cytotoxicity may appear when the interconnection between the pores is low or when a large amount of organic solvent is used.

In order to overcome this limitation, supercritical carbon dioxide is used as a solvent for the polymer, and a polymer-supercritical carbon dioxide mixture is prepared, and then a porous polymer support is prepared using a supercritical fluid that forms pores as carbon dioxide is slowly released. A report on the method was published centering on Steven M. Howdle Group of the University of Nottingham, UK [Tai H, Howdle SM, Shakesheff KM. European cells and Materials 2007; 14: 64-77. However, it has been reported that a polymer having a high molecular weight or containing a specific monomer has a low solubility in supercritical carbon dioxide, and thus there is a limit in preparing a support. Since it becomes difficult, there is a problem that grafting of various materials is difficult.

Therefore, the problem to be solved by the present invention is to improve the solubility of the polymer in the supercritical fluid by introducing a supercritical carbon dioxide-organic solvent system (co-solvent system), high molecular weight polyester-based biodegradable polymer of 10 kD or more By using the present invention, there is provided a method of preparing a biodegradable polymer support having no toxicity, improved porosity and high porosity, and good interconnection between pores.

In addition, by using the improved solubility to prepare a porous support at a temperature lower than the biological temperature it is easy to introduce a bioactive material, a method of preparing a bioactive porous polymer support improved tissue regeneration by the slow release of the introduced bioactive material To provide.

In order to achieve the above object,

(a) placing the biodegradable polymer and organic solvent mixture in a mold, and then placing the mixture in a high pressure reactor and heating the reactor to room temperature-75 ° C .;

(b) injecting carbon dioxide such that the internal pressure of the reactor is 70-700 bar, and maintaining the internal pressure of the reactor such that the injected carbon dioxide penetrates into the polymer in a supercritical fluid state; And

It provides a method for producing a porous biodegradable polymer support comprising a; (c) exhausting the carbon dioxide in the reactor to reduce the pressure in the reactor to atmospheric pressure.

According to an embodiment of the present invention, the biodegradable polymer is polyamino acid, polyanhydride, polycaprolactone, polyglycolide, polylactide, polytrimethylenecarborate carbonate, polyorthoester, polyethylene oxide and their It may be selected from the group consisting of a copolymer.

According to another embodiment of the present invention, the organic solvent is dimethyl sulfoxide, chloroform, dichloromethane, dioxane, toluene, xylene, ethylbenzene, dichloroethylene, dichloroethane, trichloroethylene, chlorobenzene, dichlorobenzene, tetra Hydrofuran, dibenzyl ether, dimethyl ether, acetone, methyl ethyl ketone, cyclohexanone, acetophenone, methyl isobutyl ketone, isophorone, diisobutyl ketone, methyl acetate, ethyl formate, ethyl acetate, diethyl carbo Nate, diethyl sulfate, butyl acetate, diacetone alcohol, diethyl glycol monobutyl ether, decanol, benzene acid, stearic acid, tetrachloroethane, hexafluoroisopropanol, hexafluoroacetone sesquihydrate, aceto In the group consisting of nitrile, chlorodifluoromethane, trifluoroethane, difluoroethane and mixtures thereof Can be selected.

According to another embodiment of the present invention, the weight average molecular weight of the biodegradable polymer may be 50,000 Daltons to 800,000 Daltons.

According to another embodiment of the present invention, the step (c) is to discharge some of the carbon dioxide in the reactor to reduce the pressure inside the reactor to 70-200 bar, and again to reduce the carbon dioxide so that the pressure inside the reactor is 100-700 bar The step of injecting and maintaining the pressure may be repeated 2-5 times and then reduced to normal pressure.

According to another embodiment of the present invention, the time for maintaining the pressure inside the reactor in step (b) may be 5-600 minutes.

According to another embodiment of the present invention, the time to exhaust the carbon dioxide in the step (c) may be 1-600 minutes.

According to another embodiment of the present invention, the preparation method may control the porosity and pore size of the polymer support by adjusting the pressure inside the reactor of step (a) and the carbon dioxide exhaust time of step (c).

According to another embodiment of the present invention, the mixture of step (a) may further comprise a bioactive material, the bioactive material is a bioactive ceramics, inorganic materials including metals, peptides, growth factors, cytokines It may be selected from the group consisting of chemokines, proteins, enzymes, polysaccharides, carbohydrates, organic materials including hexane and mixtures thereof.

In addition, the present invention provides a porous biodegradable polymer support prepared according to the above method, wherein the porosity of the polymer support is in the range of 30-98%, characterized in that the average size of the pores is in the range of 10 to 1000 ㎛.

According to the present invention, by introducing an organic solvent system into the supercritical carbon dioxide foaming technology, the solubility of the polymer is improved, so that the biodegradable polymer support with improved porosity and porosity and good internal connectivity in the process of the biotemperature range can be manufactured. Do. In addition, the biodegradable polymer scaffold according to the present invention is very useful for the introduction of various bioactive substances such as growth factors, chemotactic factors, proteins, and sustained release thereof, thereby improving scaffolds for tissue engineering.

Figure 1a is a SEM image showing a porous biodegradable polymer support prepared according to Example 1 of the present invention,
1B is an external image of a porous biodegradable polymer support prepared according to Example 1 of the present invention.
1C is a graph showing porosity and pore size of the porous biodegradable polymer support prepared according to Example 1 of the present invention.
Figure 2a is a SEM image showing a porous biodegradable polymer support prepared according to Example 2 of the present invention,
Figure 2b is an appearance image of a porous biodegradable polymer support prepared according to Example 2 of the present invention.
FIG. 3 shows that horseradish peroxidase slowly decreases in a PLCL (Poly (Lactide-co-Caprolactone)) polymer support prepared using horseradish peroxidase (horseradish peroxidase) as a protein according to Example 3 of the present invention. This is a graph showing the emission.
4A to 4C are SEM images showing porous biodegradable polymer supports prepared by injecting carbon dioxide until the pressure of the reactor becomes 100 bar, 180 bar, and 270 bar, respectively, according to Examples 4 to 6. FIG.
5A to 5C are SEM images showing porous biodegradable polymer supports prepared by discharging carbon dioxide at 50 minutes, 100 minutes, and 200 minutes according to Examples 7 to 9, respectively.

Hereinafter, the present invention will be described in detail.

Carbon dioxide is a non-toxic, non-flammable, inexpensive reagent that can be used in high purity and at temperatures and pressures above the critical point (T _c = 31.1 ℃, P _c = 73.8 bar), carbon dioxide acts as a supercritical fluid with both gas and liquid properties.

Liquid-like densities of carbon dioxide increase the dissolving ability, while gas-like viscosities have a high diffusion rate, so when supercritical carbon dioxide is injected into an amorphous polymer, the glass transition temperature (T _g ), viscosity, interfacial tension and permeability of the polymer It is excellent and foam material is produced.

In the supercritical carbon dioxide method, it is essential to repeat the dipping step and the exhausting step. During immersion in the supercritical carbon dioxide, the glassy polymer is saturated in the supercritical carbon dioxide under an elevated pressure to lower the glass transition temperature (T _g ) of the polymer. In addition, the polymer becomes a soft rubber state, and when the pressure is reduced at a constant temperature, the equilibrium solution causes foam nucleation under reduced pressure, and the nucleus grows into pores.

The present invention relates to a method for preparing a porous biodegradable polymer support by introducing an organic solvent into the supercritical carbon dioxide foaming process, wherein the solubility of the polymer is improved by a small amount of organic solvent, In the process, it is possible to produce a porous biodegradable polymer support having improved porosity and porosity and having good internal connectivity.

The method for preparing a porous biodegradable polymer according to the present invention includes the following steps.

(a) placing the biodegradable polymer and organic solvent mixture in a mold, and then placing it in a high pressure reactor and heating the reactor to room temperature-75 ° C.,

(b) injecting carbon dioxide such that the pressure inside the reactor is 70-700 bar, and maintaining the pressure inside the reactor so that the injected carbon dioxide penetrates into the polymer in a supercritical fluid state,

(c) exhausting carbon dioxide in the reactor to reduce the pressure in the reactor to atmospheric pressure.

Thereafter, the present invention may further include drying the porous support prepared in the above step and removing residual organic solvent.

In general, in the supercritical fluid process using carbon dioxide foaming, it is possible to manufacture a porous polymer support having various pore sizes by controlling the temperature of the reactor in various ways. In particular, as the temperature is increased, the solubility of carbon dioxide is lowered but the polymer penetration rate is increased. It is possible to prepare a porous polymer support having pores.

However, when the temperature is higher than the biological temperature (around 37 ° C) or higher, it is difficult to introduce a biomaterial such as a protein, and the present invention solves this problem and prevents problems such as protein denaturation during biomaterial introduction. It is characterized by.

In addition, the present invention added a small amount of an organic solvent in order to further improve the polymer solubility for the production of a porous polymer support having the same physical properties even at a lower temperature than the conventional supercritical fluid process.

The organic solvent is not limited thereby by the scope of the present invention, but preferably dimethyl sulfoxide, chloroform, dichloromethane, dioxane, toluene, xylene, ethylbenzene, dichloroethylene, dichloroethane, trichloroethylene, chlorobenzene , Dichlorobenzene, tetrahydrofuran, dibenzyl ether, dimethyl ether, acetone, methyl ethyl ketone, cyclohexanone, acetophenone, methyl isobutyl ketone, isophorone, diisobutyl ketone, methyl acetate, ethyl formate, ethyl acetate , Diethyl carbonate, diethyl sulfate, butyl acetate, diacetone alcohol, diethyl glycol monobutyl ether, decanol, benzene acid, stearic acid, tetrachloroethane, hexafluoroisopropanol, hexafluoroacetone ce Succihydrate, acetonitrile, chlorodifluoromethane, trifluoroethane, difluoroethane and these It may be selected from the group consisting of a mixture.

The biodegradable polymer is selected from the group consisting of polyamino acids, polyanhydrides, polycaprolactones, polyglycolides, polylactides, polytrimethylenecarborate carbonates, polyorthoesters, polyethylene oxides, and copolymers thereof. It may be at least one species, and the weight average molecular weight (Mw) may be at least 100,000.

In the step of reducing the pressure in the reactor, the carbon dioxide in the reactor is partially discharged to reduce the pressure in the reactor to 70-200 bar, and the carbon dioxide is injected to maintain the pressure in the reactor to 100-700 bar, and the pressure is maintained. After repeating the step 2-5 times can be reduced to normal pressure.

In addition, the porosity and pore size of the finally formed porous polymer support may be controlled by adjusting the pressure inside the reactor by injecting carbon dioxide and the carbon dioxide exhaust time in the depressurization step.

The porosity of the porous biodegradable polymer support prepared according to the above method is 30-98%, and the average pore size is 10-1000 μm.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It will be clear to those who have knowledge.

<Examples>

Example 1 Porous Biodegradable Polymer Support Prepared by Introducing Organic Solvent Hexafluoroisopropanol in Supercritical Fluid Process

180 mg of the lactide / e-caprolactone copolymer having a weight average molecular weight (Mw) of 400,000 and a molar ratio of 1: 1 and hexafluoroisopropanol were mixed, and then allowed to stand at room temperature for about 10 hours.

The mixture was placed in a cylindrical Teflon mold 1 cm in diameter and placed in a high pressure reactor for a supercritical fluid process. After raising the temperature of the reactor to 35 ℃, using a high pressure pump was injected carbon dioxide until the pressure in the reactor to 270 bar. The carbon dioxide was allowed to penetrate into the polymer for 1 hour while maintaining the pressure.

Carbon dioxide was discharged at a flow rate of 1000 cc / min until the pressure of the reactor of 270 bar was 180 bar, and the process of injecting carbon dioxide to 270 bar was repeated three times. Hexafluoroisopropanol was removed through the washing process, and finally carbon dioxide was completely discharged at a flow rate of 1000 cc / min from 270 bar to atmospheric pressure.

After the supercritical fluid process, the reactant was dried in a vacuum oven for 3 days to remove residual organic solvent to prepare a support.

1A is an SEM image showing a porous biodegradable polymer support prepared according to Example 1, and FIG. 1B is an exterior image of the porous biodegradable polymer support prepared according to Example 1.

As shown in Figure 1c, it was confirmed that the porous biodegradable polymer support having an average pore size of about 200 ㎛, having a porosity of about 62%.

Example 2. Porous biodegradable polymer support prepared by introducing organic solvent dimethyl sulfoxide into a supercritical fluid process.

100 mg of a lactide / e-caprolactone copolymer having a weight average molecular weight (Mw) of 400,000 and a molar ratio of 1: 1 and 60 µl of dimethylsulfoxide were mixed and allowed to stand at room temperature for 20-30 minutes.

This mixture was placed in a 6 mm diameter cylindrical Teflon mold and placed in a high pressure reactor for a supercritical fluid process. After raising the temperature of the reactor to 35 ℃, using a high pressure pump was injected carbon dioxide until the pressure in the reactor to 270 bar. In this state, the carbon dioxide was allowed to penetrate into the polymer for 2 hours.

Thereafter, carbon dioxide was completely discharged at a flow rate of 1000 cc / min to atmospheric pressure. After the supercritical fluid process, the support was dried in a vacuum oven for 3 days to remove residual organic solvent.

2A and 2B are SEM images showing a porous biodegradable polymer support prepared according to Example 2, respectively, and are appearance images.

Example 3 Preparation of Poly (Lactide-co-Caprolactone) Support

Horseradish peroxidase was used as a model protein to prepare a PLC (Poly (Lactide-co-Caprolactone)) scaffold that releases bioactive substances slowly.

After dissolving 2 mg of horseradish peroxidase in 180 µl of hexafluoroisopropanol, the mixture was mixed with 180 mg of a lactide / e-caprolactone copolymer having a weight average molecular weight (Mw) of 400,000 and a molar ratio of 1: 1. It was left at room temperature.

The mixture was placed in a cylindrical Teflon mold 1 cm in diameter and placed in a high pressure reactor for a supercritical fluid process. After raising the temperature of the reactor to 35 ℃, using a high pressure pump was injected carbon dioxide until the pressure in the reactor to 270 bar. In this state, the carbon dioxide was allowed to penetrate into the polymer for 1 hour.

The carbon dioxide was discharged at a flow rate of 1000 cc / min until the pressure of the reactor at 270 bar was 180 bar, and the process of injecting carbon dioxide up to 270 bar was repeated three times. Hexafluoroisopropanol was removed through the washing process, and finally carbon dioxide was completely discharged at a flow rate of 1000 cc / min from 270 bar to atmospheric pressure.

After the supercritical fluid process, the support was dried in a vacuum oven for 3 days to remove residual organic solvent.

As shown in Figure 3, it was confirmed that the horseradish peroxidase can be slowly released for a long time.

Examples 4 to 6. When the pressure inside the reactor according to the carbon dioxide injection is changed

Prepared in the same manner as in Example 1, but prepared by varying the pressure of the reactor according to the first carbon dioxide injection to 100 bar, 180 bar, 270 bar. In order to prevent denaturation due to introduction of biomaterials such as proteins, the temperature was not changed during the process, and the decompression time of the reactor pressure was 50 minutes.

4A to 4C are SEM images showing a porous biodegradable polymer support prepared by injecting carbon dioxide until the pressure of the reactor becomes 100 bar, 180 bar, and 270 bar, respectively.

Examples 7 to 9. Different CO2 Exhaust Times for Reducing Pressure in the Reactor

It was prepared in the same manner as in Example 1, but prepared by varying the carbon dioxide exhaust time in the pressure reduction step inside the reactor. In order to prevent denaturation due to the introduction of biomaterials such as proteins, the temperature during the process was not changed, and the initial reactor pressure was 270 bar.

5A to 5C are SEM images showing porous biodegradable polymer supports prepared by exhausting carbon dioxide at 50 minutes, 100 minutes, and 200 minutes, respectively.

Claims (12)

(a) placing the biodegradable polymer and organic solvent mixture in a mold, and then placing the mixture in a high pressure reactor and heating the reactor to room temperature-75 ° C .;
(b) injecting carbon dioxide such that the internal pressure of the reactor is 70-700 bar, and maintaining the internal pressure of the reactor such that the injected carbon dioxide penetrates into the polymer in a supercritical fluid state; And
(C) exhausting the carbon dioxide in the reactor to reduce the pressure in the reactor to atmospheric pressure; manufacturing method of a porous biodegradable polymer support comprising a,
In the step (c), the carbon dioxide in the reactor is partially discharged to reduce the pressure in the reactor to 70-200 bar, and to inject carbon dioxide and maintain the pressure so that the pressure inside the reactor is 250-700 bar. Method for producing a porous biodegradable polymer support characterized in that the pressure is reduced to normal pressure after repeated five times. The method of claim 1,
The biodegradable polymer is selected from the group consisting of polyamino acids, polyanhydrides, polycaprolactones, polyglycolides, polylactides, polytrimethylenecarborate carbonates, polyorthoesters, polyethylene oxides and copolymers thereof Method for producing a porous biodegradable polymer support characterized in that. The method of claim 1,
The organic solvent is dimethyl sulfoxide, chloroform, dichloromethane, dioxane, toluene, xylene, ethylbenzene, dichloroethylene, dichloroethane, trichloroethylene, chlorobenzene, dichlorobenzene, tetrahydrofuran, dibenzyl ether, dimethyl ether, Acetone, methyl ethyl ketone, cyclohexanone, acetophenone, methyl isobutyl ketone, isophorone, diisobutyl ketone, methyl acetate, ethyl formate, ethyl acetate, diethyl carbonate, diethyl sulfate, butyl acetate, di Acetone alcohol, diethyl glycol monobutyl ether, decanol, benzene acid, stearic acid, tetrachloroethane, hexafluoroisopropanol, hexafluoroacetone sesquihydrate, acetonitrile, chlorodifluoromethane, trifluoro Porous, characterized in that selected from the group consisting of roethane, difluoroethane and mixtures thereof Method of producing a biodegradable polymer support. delete The method of claim 1,
Method for producing a porous biodegradable polymer support, characterized in that the weight average molecular weight of the biodegradable polymer is 50,000 Daltons to 800,000 Daltons. The method of claim 1,
The method of maintaining the pressure inside the reactor in the step (b) is 5-600 minutes, the method for producing a porous biodegradable polymer support. The method of claim 1,
Method for producing a porous biodegradable polymer support, characterized in that for exhausting carbon dioxide in step (c) is 1-600 minutes. The method of claim 1,
The method of manufacturing a porous biodegradable polymer support, characterized in that for controlling the porosity and pore size of the polymer support by adjusting the internal pressure of the reactor of step (a) and the carbon dioxide exhaust time of step (c). The method of claim 1,
The mixture of step (a) is a method for producing a porous biodegradable polymer support, characterized in that it further comprises a bioactive material. The method of claim 9,
The bioactive material is selected from the group consisting of bioactive ceramics, inorganic materials including metals, peptides, growth factors, cytokines, chemokines, proteins, enzymes, polysaccharides, carbohydrates, organic materials including hexane, and mixtures thereof. A method for producing a porous biodegradable polymer support. delete delete

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