WO2008130068A1

WO2008130068A1 - Method for preparing a porous polymer scaffold using dry ice

Info

Publication number: WO2008130068A1
Application number: PCT/KR2007/001972
Authority: WO
Inventors: So Hee Yun; Hyun Sook Park; Song Sun Chang
Original assignee: Modern Cell & Tissue Technologies Inc.
Priority date: 2007-04-23
Filing date: 2007-04-23
Publication date: 2008-10-30

Abstract

Disclosed is a method of preparing a porous polymer scaffold, which enables the size and shape of pores, which are formed in the preparation of the porous scaffold using a freeze drying method, to be uniform throughout the three-dimensional structure of the porous scaffold. More specifically, disclosed is a method for preparing a porous polymer scaffold which can be used as a scaffold for three-dimensional cell culture, a tissue engineering scaffold or a wound dressing, the method being characterized in that it comprises, in addition to the steps of the prior freeze-drying method, a step of mixing a polymer solution with dry ice to form a slush-like material.

Description

Method for preparing a porous polymer scaffold using dry ice

Technical Field

The present invention relates to a method of preparing a porous polymer scaffold, which enables the size and shape of pores, which are formed in the preparation of the porous scaffold using a freeze drying method, to be uniform throughout the three-dimensional structure of the porous scaffold. More specifically, the present invention relates to a method for preparing a porous polymer scaffold which can be used as a scaffold for three-dimensional cell culture, a tissue engineering scaffold or a wound dressing, the method being characterized in that it comprises, in addition to the steps of the prior freeze-drying method, a step of mixing a polymer solution with dry ice to form a slush-like material.

Background Art

Recently, tissue engineering, a field of biotechnology, has been rapidly developed due to an effort to reconstruct damaged biological tissue using tissue prepared in laboratories, and a new approach, defined as such tissue engineering, has raised a lot of interest. Tissue engineering is an applied study that utilizes the basic concepts and techniques of life science and engineering to understand the relationship between the structure and function of biological tissue and make a biological tissue substitute for transplantation, thereby to maintain, improve or restore the function of the human body.

As a need to develop artificial organs or regenerate tissue using such tissue engineering is greatly increased, many studies have been attempted to form tissues or organs by attaching only necessary cells to various natural or synthetic polymer materials and transplanting the polymer materials in vivo. An ideal polymer scaffold for use for this purpose should be made of a nontoxic, biocompatible material, which does not cause blood coagulation or inflammatory reaction after transplantation, and should have mechanical properties which can sufficiently support the growth of cells. Also, it should be in the form of a porous scaffold to which cells readily adhere and in which a sufficient space between cells is ensured such that oxygen or nutrients are easily supplied through the diffusion of body fluids, and angiogenesis readily occurs such that cells successfully grow and differentiate. Moreover, cells are basically cultured in a two-dimensional environment, but three-dimensional scaffolds are required to culture the cells in the form of tissues or organs. Such scaffolds have innumerable pores, and thus they should be able to attach cells to the inside and outside thereof and should have an opened structure in order to receive nutrients required for the growth of cells and to discharge waste matter.

Typical methods for preparing such three-dimensional porous polymer scaffolds include: a solvent-casting and particle-leaching technique comprising mixing a polymer with single-crystal salt particles, drying the mixture and then immersing the dried material to leach the salt particles (A. G. Mikos et al., Polymer, 35, 1068 (1994)); a gas forming technique comprising expanding a polymer with CO₂ gas (L. D. Harris et al., J. Biomed. Mater. Res., 42, 396 (1998)); a thermally induced phase separation technique including immersing a polymer-containing solvent in a non-solvent to make the polymer porous (C. Schugens, et al., J. Biomed. Mater. Res., 30, 449 (1996)); and a freeze- drying method comprising dissolving a polymer in a solvent to prepare a polymer solution and then freeze-drying the polymer solution with liquid nitrogen (K. Whang, Polymer, 36, 837 (1995)).

The solvent-casting and particle-leaching technique uses a large amount of salt particles and adopts a method of controlling pores by controlling the size of the salt particles, but has a disadvantage in that the salt particles can adversely affect cells when the subsequent complete removal thereof is not achieved. Also, the gas forming technique has a problem in that the uniformity of structures can be reduced because it is difficult to control pores. Moreover, in the freeze-drying method, which is one of the most widely used methods, when the polymer solution is cooled, the phase separation between the solvent and the solute will occur due to the difference in solubility therebetween, while the solvent will form crystals, so that the solute will make a scaffold, and the solvent will forms pores. Thus, it is possible to control pores depending on the concentration and cooling temperature of the polymer solution. However, during the cooling of the polymer solution, a temperature gradient can be formed between the surface and inside of the solution, resulting in different pore shapes. Thus, a porous scaffold having non-uniform and closed pores can be prepared.

Accordingly, the present inventors have made many efforts to overcome the problems occurring in the prior art and, as a result, have found that a porous polymer scaffold having uniform pores throughout the three-dimensional structure thereof can be prepared by mixing a polymer solution with dry ice to make a slush-like material, cooling the slush-like material such that the surface and inside of the slush-like material are rapidly cooled at the same temperature and cooling rate, and then dry-freezing the cooled material, thereby completing the present invention.

Disclosure of Invention

Technical Problem

It is therefore a main object of the present invention to provide a method for preparing a three-dimensional porous scaffold, which can clearly overcome the structural problems of the scaffolds prepared according to the prior methods and has suitable and uniform pore size and porosity such that the adhesion, differentiation and growth of cells can be promoted.

Another object of the present invention is to provide a porous scaffold prepared according to said method, as well as a cell culture scaffold, a tissue engineering scaffold or a wound dressing, which comprise said porous scaffold.

Technical Solution

According to one aspect of the present invention, there is provided a method for preparing a porous polymer scaffold, the method comprising the steps of: a) dissolving a polymer in a solvent; b) adding dry ice to the polymer solution; c) stirring the mixture to form a slush-like material; and d) placing the slush-like material in a mold, followed by freeze drying.

The present invention improves the prior method of preparing a porous polymer scaffold using the freeze-drying method. Specifically, in the prior method of preparing a porous polymer scaffold using the freeze-drying method, a temperature gradient is formed between the surface and inside of a polymer solution according to the transfer direction of cold air, and thus a porous scaffold, having different pore shapes and nonuniform and closed pores, can be prepared (see FIG. 1). However, according to the preparation method according to the present invention, a porous polymer scaffold having uniform pores throughout the three-dimensional structure thereof can be prepared by mixing a polymer solution with dry ice to make a slush-like material, cooling the slush-like material such that the surface and inside of the slush-like material are rapidly cooled at the same temperature and cooling rate, and then dry-freezing the cooled material. As the polymer for use in the inventive preparation method of the porous polymer scaffold, any artificial/natural biodegradable, or non-degradable polymer may be used as long as it can be dissolved in a solvent and prepared into a porous scaffold by freeze drying. For example, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, poly(lactic acid), poly(glycolic acid)(PGA), poly(lactic-co-glycolic acid)(PLGA), poly( ε -carprolactone), poly(anhydrides), polyorthoesters, polyviniyalcohol, poly(ethyleneglycol), polyurethane, polyacrylic acid, poly(N-isopropyl acrylamide), poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) copolymer(Pluronic™), a copolymer thereof, or a mixture thereof can be used as the polymer. Preferably, the polymer in the step a) is either a synthetic biodegradable polymer selected from the group consisting of poly glycolic acid (PGA), poly lactic acid (PLA), and poly(DL-lactic- co-glycolic acid) (PLGA), or a natural biodegradable polymer selected from the group consisting of chitosan, collagen and inorganic hydroxyapatite, a copolymer thereof, or a mixture thereof. In the present method for preparing a porous polymer scaffold, conventional freeze-drying methods known in the art can be used except that a step of adding dry ice which is characteristic in the present invention is added (see Development of biodegradable porous scaffolds for tissue engineering. Materials Science and Engineering C 17 (2001)63-69; Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. Biomaterials 25(2004)1077-1086; Effects of hydroxyapatite in 3D chitosan-gelatin polymer network on human mesenchymal stem cell construct develpoment. Biomaterials 27 (2006) 1859-1867; ECharacterization of emulsified chitosan-PLGA matrices formed using control led-rate freezing and lyophilization technique. J Biomed Mater Res A. 2006 Nov;79(2):418-30.; In vivo biocompatibility and degradation behavior of elastic poly(L-lactide-co-ε-caprolactone) scaffolds. Biomaterials 25 (2004) 5939-5946, etc.). In the inventive preparation method of the porous polymer scaffold, the solvent for dissolving the polymer may be selected from among solvents known in the art, depending on the kind of polymer. For example, for chitosan, collagen or gelatin, acetic acid can be used as the solvent, and for polyvinyl alcohol), ultrapure water can be used as the solvent. Also, for aliphatic polyesters such as PGA, PLA or PLGA, an organic solvent such as methylene chloride (CH₂CI₂) or chloroform can be used. Preferably, the polymer is chitosan, and the corresponding solvent is an aqueous acetic acid solution. The chitosan has a very high affinity for human cells, and thus when it is applied to an affected part, it will promote healing, because it is analgesic, has a moisture absorption property, promotes the formation of skin cells and shows antibiotic activity, and it will be naturally degraded in vivo.

The inventive method for preparing the porous polymer scaffold preferably further comprise, after the step (a), a step of mixing the polymer solution with an emulsifier, preferably one selected from the group consisting of butanol, pentanol, hexanol and octanol. The addition of the emulsifier can form fine pores. This is because the solute, the solvent 1 and the solvent 2 (emulsifier such as butanol) are separated in different ways during phase separation, such that the solvent 2 (butanol) in the aqueous acetic acid solution containing chitosan is frozen to have small size, and thus large pores and fine pores are formed. The emulsifier such as butanol also acts to facilitate the formation of the slush-like material according to the present invention. In the inventive method for preparing the porous polymer scaffold, the step b) preferably comprises breaking dry ice into small pieces. This allows the polymer solution to be uniformly cooled and promotes the formation of the slush-like material. However, it is not necessary to break dry ice into small pieces, and during the process of mixing a lump of dry ice with the polymer solution, the distribution of dry ice changes and the size thereof naturally decreases due to sublimation.

In the inventive method for preparing the porous polymer scaffold, the stirring in the step c) is preferably carried out when the solution starts to freeze, thus forming a soft slush-like material.

In the inventive method for preparing the porous polymer scaffold, the step d) preferably further comprises, after placing the slush-like material into the predetermined container or mold, a step of centrifuging the slush-like material to remove bubbles. In the centrifugation step, bubbles formed by mixing and the sublimation of dry ice can be removed. The porosity and pore size of the porous polymer scaffold can be controlled by changing centrifugal force (turning force). In the inventive method for preparing the porous polymer scaffold, the step d) preferably comprises freezing the slush-like material in a deep freezer, followed by drying in a freeze dryer.

According to another aspect of the present invention, there is provided a porous polymer scaffold prepared according to the inventive preparation method, which has uniform pores throughout the three-dimensional structure thereof. The porous polymer scaffold prepared according to the preparation method of the present invention has uniform pore size distribution throughout the three-dimensional structure thereof and has a specific surface area significantly larger than that of a polymer scaffold prepared according to the prior method. The three-dimensional porous polymer scaffold, having suitable porosity and good interconnection between pores, provides a surface area required for the adhesion of cells and ensures a space, which is required for the regeneration of extracellular matrixes and for effective mass transfer for smooth supply of oxygen and nutrients in ex vivo culture. Also, the three-dimensional porous polymer scaffold is preferably a porous scaffold having a structure in which pores having a size in a specific range are uniformly distributed to maintain a given mechanical strength.

The porous polymer scaffold of the present invention preferably further comprises drugs required for wound healing, for example, antibiotics or antiinflammatory drugs, or growth factors required for cell culture, for example, bFGF, VEGF, TGF-beta or insulin. Preparation methods of scaffolds containing such growth factors are well known in the art to which the present invention pertains (see VEGF scaffolds enhance angiogenesis and bone regeneration in irradiated osseous defects. J Bone Miner Res. 2006 May; 21 (5):735-44; Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels, etc.). According to still another aspect of the present invention, there is provided a scaffold for three-dimensional cell culture, which comprises the porous polymer scaffold according to the present invention. The cell culture scaffold according to the present invention may further comprise the above-described growth factors.

According to further still another aspect of the present invention, there is provided a tissue engineering scaffold comprising the porous polymer scaffold according to the present invention. Examples of the tissue engineering scaffold include scaffolds for tissue regeneration, such as artificial skins, artificial bones and artificial joints.

According to further still another aspect of the present invention, there is provided a wound dressing comprising the porous polymer scaffold according to the present invention. The wound dressing of the present invention may further comprise a drug required for wound healing.

Brief Description of the Drawings

FIG. 1 shows the transfer direction of cold air in the preparation of a porous polymer scaffold according to the prior freeze drying method (left), and shows nonuniform pores in the resulting scaffold (right).

FIG. 2 is a schematic diagram showing a process of preparing a porous polymer scaffold using a slush method according to the present invention. FIG. 3 shows an SEM photograph of the three surfaces of a porous scaffold prepared according to the slush method of the present invention (Example 1), an SEM photograph of the three surfaces of a porous scaffold prepared according to the prior freeze-drying method (Comparative Example 1), and an SEM photograph of the three surfaces of a porous scaffold having fine pores formed using only an emulsifier without dry ice (Comparative Example 2).

FIG. 4 is an SEM photograph showing the enlarged cross section of each of porous scaffolds prepared in Example 1 of the present invention, Comparative Example 1 and Comparative Example 2. FIG. 5 is a photograph showing the comparison of the absorption time of an aqueous toluidine blue solution between porous scaffolds prepared in Example 1 of the present invention, Comparative Example 1 and Comparative Example 2.

FIG. 6 is a graphic diagram showing the comparison of tensile strength between Example 1 of the present invention, Comparative Example 1 and Comparative Example 2.

FIG. 7 is a photograph of HDF cells, which were stained with H&E after they were cultured on the porous scaffold prepared according to the method of Example 1 of the present invention.

FIG. 8 is a photograph of HDF cells, which were stained with H&E after they were cultured on the porous scaffold prepared according to the method of Comparative Example 1.

Best Mode for Carrying Out The Invention

Hereinafter, the present invention will be descried in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1 : Preparation of porous scaffold using dry ice 2 g of chitosan was dissolved in 100 ml of 1 % acetic acid aqueous solution. The chitosan solution was filtered, if necessary, to remove impurities. 40 g of 1 % acetic aqueous solution was added to and uniformly mixed with 100 g of the chitosan solution, and 10 g of an emulsifier was added thereto. Herein, as the emulsifier, it is possible to use penthanol, hexanol or octanol, and preferably buthanol. The resulting solution had a chitosan concentration of 1 % and a butanol concentration of 10% and became a suspension. Dry ice was added to the prepared solution. Herein, if necessary, the dry ice can be broken into small pieces in order to facilitate processing. When the solution started to freeze, the solution was well stirred to form a soft slush-like material. Then, the slush-like material was placed in a predetermined container or mold, and if necessary, subjected to a process such as centrifugation to remove bubbles. Then, the slush-like material was frozen in a deep freezer, and then dried in a freeze dryer overnight. Then, the molded porous material was washed several times with alcohol. For the washing process, 100% alcohol, 70% alcohol and 50% alcohol can be sequentially used. After the remaining acetic acid was completely removed with alcohol, the porous material was washed with triple distilled water, frozen again in a deep freezer and then dried in a freeze dryer overnight, thus completing a porous chitosan scaffold. FIG. 2 is a schematic diagram showing the process of preparing the porous polymer scaffold using the slush method according to the present invention. In addition to chitosan, a polymer such as PGA, PLA or PLGA can be used together with dry ice in order to prepare a porous scaffold according to the prior known freeze drying method (see Development of biodegradable porous scaffolds for tissue engineering, Materials Science and Engineering C 17 (2001), pp. 63-69).

Comparative Example 1 : Preparation of porous scaffold using prior freeze drying method

A porous scaffold was prepared using chitosan in the same manner as in Example 1 , except that the process of adding dry ice was eliminated.

Comparative Example 2: Preparation of porous scaffold using emulsifier A porous scaffold was prepared without using dry ice in the same manner as in Comparative Example 1 , except that an emulsifier was added to form fine pores.

Example 2: Observation with scanning electron microscope (SEM)

The pore sizes and shapes of the porous scaffolds prepared in Example 1 of the present invention, Comparative Example 1 and Comparative Example 2 were observed with a scanning electron microscope (ZEOL) at 10 kV. FIG. 3 shows an SEM photograph at 20Ox magnification of the three surfaces of a porous scaffold prepared according to the slush method of the present invention (Example 1), an SEM photograph at 20Ox magnification of the three surfaces of a porous scaffold prepared according to the prior freeze-drying method (Comparative Example 1), and an SEM photograph at 20Ox magnification of the three surfaces of a porous scaffold having fine pores formed using only an emulsifier without dry ice (Comparative Example 2). As shown in FIG. 3, in the case of Comparative Example 1 prepared according to the prior method, the front surface (top surface), back surface (bottom surface) and side surface(cross section) thereof show different pore shapes. In the case of Comparative Example 2 prepared using the emulsifier without dry ice, small pores are formed between walls due to the emulsifier, because the solution is separated into water, chitosan and the emulsifier during phase separation. However, the pores of the side surface(cross section) and the pores of the bottom surface show different shapes. On the contrary, in the case of Example 1 of the present invention, prepared using the slush method, all crystals are uniformly formed to form uniform pores, and all the surfaces have an opened structure due to small pores between walls. FIG. 4 is an SEM photograph showing the enlarged cross section of each of porous scaffolds prepared in Example 1 of the present invention, Comparative Example 1 and Comparative Example 2. As shown in FIG. 4, Comparative Example 1 has large pores and non-uniform pore distribution, and Comparative Example 2 has uniform pore distribution, but has a layered pore structure, which can adversely affect cells. On the contrary, Example 1 of the present invention has uniform pores throughout thereof. From the results of the above-describe SEM observation, it can be seen that the porous material prepared using the slush method of the present invention has uniform pores through the three- dimensional structure thereof. Thus, the porous scaffold of the present invention will have improved functions by overcoming the shortcomings of the prior method.

Example 3: Measurement of specific surface area with porosimeter

The specific surface area and average pore diameter of the porous scaffold prepared according to each of the methods of Example 1 of the present invention 1 , Comparative Example 1 and Comparative Example 2 were measured with a mercury porosimeter (Pascal 140+440, Thermo Finnigan). The measurement results are shown in Table 1 below. From the results in Table 1 , it can be seen that the specific surface area of Example 1 is significantly larger than those of Comparative Examples 1 and 2 due to uniform pores. A larger surface area of a porous material means that the porous material has a higher function. Thus, the porous material of Example 1 has an increased area for contact with cells, and thus has an excellent function as a cell culture scaffold. In addition, it has a small contact angle such that liquid drops can be absorbed into the surface thereof, and thus it will also be highly useful as a wound dressing.

[Table 1]

Example 4: Measurement of water absorption

In order to test the water absorption of the porous scaffold prepared according to each of the method of Example 1 of the present invention, Comparative Example 1 and Comparative Example 2, the three completely dried samples, each having a size of 200 mm x 200 mm, were weighed, and then simultaneously immersed in 100 ml of triple distilled water. After 30 seconds, the samples were taken out, left to stand on a dried Petri dish for 10 seconds and shaken 2-3 times to confirm that no water drops fell, and the weight thereof was measured. The measurement results are shown in Table 2 below. From the results in Table 2, the porous material of Example 1 , prepared using the slush method, showed the highest water absorption. This is considered to be because the pores of the porous material were all opened due to uniform pores, and the increase in the surface area thereof led to the increase in water absorption. Also, the porous material of Example 1 , prepared using the slush method showed the shortest water absorption time. [Table 2]

Furthermore, in order to compare the water absorption rates of the three samples, a toluidine blue solution was dropped onto each of the scaffolds, and after 1 second, the spreading thereof until absorption was photographed. FIG. 5 is a photograph showing the comparison of the absorption time of an aqueous toluidine blue solution between the porous scaffolds prepared in Example 1 of the present invention, Comparative Example 1 and Comparative Example 2. The horizontally spread diameter of the toluidine blue solution was the largest in the case of Comparative Example 1 and the smallest in the case of Example 1. This is considered to be because of the difference in absorption time. That is, in the case of Comparative Example 1 , the absorption rate of the toluidine blue solution into the sample was slow, and thus the toluidine blue solution was widely spread in the transverse direction, and in the case of Example 1 , the absorption rate of the toluidine blue solution into the sample was fast, and thus the toluidine blue solution was narrowly spread in the transverse direction. In view of the fact that a wound dressing can show good effects when it should rapidly absorb various liquids released from wounds and should be provided with a wet environment, the scaffold of the present invention will be useful as a wound dressing.

Example 5: Measurement of tensile strength The porous scaffold prepared according to each of the methods of Example 1 of the present invention, Comparative Example 1 and Comparative Example 2 was measured with the lnstron universal tester. For this purpose, each of the samples was cut to a size of 3 cm x 5 cm. A length of 1 cm from each of the upper and lower end of each sample was clamped by a grip, such that each sample was mounted onto the tensile tester in a size of 3 cm x 3 cm. The tensile strength of each sample was measured four times at a speed of 0.1 mm/sec. The measurement results are shown in Table 3 below. FIG. 6 is a graphic diagram showing the comparison of tensile strength between Example 1 of the present invention, Comparative Example 1 and Comparative Example 2. As shown in Table 3 and FIG. 6, the porous material of Comparative Example 1 showed a high standard deviation in tensile strength. The porous material of Comparative Example 2, having fine pores, showed a low standard deviation in tensile strength, but had low tensile strength. The porous material of Example 1 , prepared using the slush method, showed the lowest standard deviation and the highest tensile strength. This is considered to be because the porous material of the present invention had large surface area and uniform pore distribution, and thus showed high tensile strength compared to the porous materials prepared according to the prior methods. Accordingly, the porous material of the present invention can be used in various applications and shows improved physical properties, compared to the porous materials prepared according to the prior methods. [Table 3]

Example 6: Cell culture experiment

In order to examine the cell culture properties of the porous scaffold prepared according to the slush method of the present invention, primarily cultured human dermal fibroblast cell (HDFC) were cultured on the chitosan sheet prepared according to the slush method. Then, the cultured cells were stained with H&E and photographed with an optical microscope to determine the cell culture properties of the porous scaffold. FIG. 7 is a photograph of HDF cells, which were stained with H&E after they were cultured for 3 weeks on the porous scaffold prepared according to the method of Example 1 of the present invention. As can be seen in FIG. 7, the HDF cells reached the inside of the porous scaffold, because opened pores were formed throughout the three-dimensional structure of the porous scaffold. FIG. 8 is a photograph of HDF cells, which were stained with H&E after they were cultured for 7 days on the porous scaffold prepared according to the method of Comparative Example 1. In FIG. 8, the arrow indicates the frame direction of the scaffold, and the downward direction from the upper surface does not coincide with the upward direction from the lower surface. As can be seen in FIG. 8, cells grew in the frame direction of the scaffold, but the upward movement direction of the cells did not coincide with the downward movement direction of cells, and thus the cells did not reach the inside of the scaffold, the area of parts of the scaffold, in which the cells were not present, was large, and the cells were distributed mainly on the surfaces.

Industrial Applicability

As described above, as shown in the SEM photograph, the porous material prepared through the prior freeze-drying method has a problem in that the size and shape of pores formed in the porous material are different between the surface and inside of the porous material. The method of the present invention can solve the above problem and prepare a porous material in which uniform and opened pores are formed. The present invention provides a method for preparing a porous material through freeze drying, in which a polymer solution for forming the porous material is rapidly cooled before it is frozen, such that the temperature gradient occurring in the freezing process is minimized, and thus pores are uniformly distributed in the porous material. This improves the problem of the prior freeze drying method, in that pores formed in the porous material are different between the surface and inside of the porous material due to the difference in freezing temperature between the surface and inside of the porous material. Thus, the present invention allows pores to be formed uniformly throughout the three-dimensional structure of the porous material. The porous material prepared according to the present invention shows high specific area, increased water absorption and excellent strength, and thus is highly useful as a wound dressing and a scaffold. Also, the inventive porous material can overcome the limitations of the prior porous materials, caused by low physical properties, because it has increased strength. In addition, the porous scaffold prepared according to the method of the present invention can be used not only as a tissue engineering scaffold, but also in all industrial applications which can good effects due to porosity, for example, wound dressings.

Claims

1. A method for preparing a porous polymer scaffold, the method comprising the steps of:

a) dissolving a polymer in a solvent;

b) adding dry ice to the polymer solution;

c) stirring the mixture to form a slush-like material; and d) placing the slush-like material in a mold, followed by freeze drying.

2. The method of Claim 1 , wherein the polymer in the step a) is either a synthetic biodegradable polymer selected from the group consisting of poly glycolic acid (PGA), poly lactic acid (PLA) and poly(DL-lactic-co-glycolic acid) (PLGA), or a natural biodegradable polymer selected from the group consisting of chitosan, collagen and inorganic hydroxyapatite, a copolymer thereof, or a mixture thereof.

3. The method of Claim 2, wherein the polymer is chitosan, and the solvent is an aqueous acetic acid solution.

4. The method of Claim 1 , which further comprises, after the step a), a step of adding and mixing an emulsifier with the polymer solution.

5. The method of Claim 4, wherein the emulsifier is selected from the group consisting of butanol, pentanol, hexanol and octanol.

6. The method of Claim 1 , wherein the step b) comprises breaking the dry ice into small pieces.

7. The method of Claim 1 , wherein the stirring in the step c) is performed when the solution starts to freeze, so as to make a soft slush-like material.

8. The method of Claim 1 , wherein the step d) further comprises, after placing the slush-like material in the mold, a step of centrifuging the slush-like material to remove bubbles.

9. The method of Claim 1 , wherein the step d) comprises freezing the slush-like material in a deep freezer, followed by drying in a freeze dryer.

10. A porous polymer scaffold prepared according to the method of any one Claims 1 to 9, which has uniform pores throughout the three-dimensional structure thereof.

11. The porous polymer scaffold of Claim 10, which further comprises a drug or a growth factor.

12. A scaffold for three-dimensional cell culture, which comprises a porous polymer scaffold according to Claim 10.

13. A tissue engineering scaffold comprising a porous polymer scaffold according to Claim 10.

14. A wound dressing comprising a porous polymer scaffold according to Claim 10.