WO2008103017A1

WO2008103017A1 - Biodegradable porous composite and hybrid composite of biopolymers and bioceramics

Info

Publication number: WO2008103017A1
Application number: PCT/KR2008/001085
Authority: WO
Inventors: Hong Sung Kim
Original assignee: Pusan National University Industry-University Cooperation Foundation
Priority date: 2007-02-23
Filing date: 2008-02-25
Publication date: 2008-08-28

Abstract

The present invention relates to a biodegradable porous composite and a hybrid composite of biopolymers and bioceramics, more specifically, to a dual pore- structured biodegradable porous material having biocompatibility as well as biodegradability, which contains a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components; a dual pore-structured porous composite comprising a combination of the biopolymer and a domestic or wild silk fibroin; and a biodegradable hybrid composite comprising a combination of the biopolymer, the silk fibroin and a physiologically active bioceramic material.

Description

BIODEGRADABLE POROUS COMPOSITE AND HYBRID COMPOSITE OF BIOPOL YMERS AND BIOCERAMICS

TECHNICAL FIELD

The present invention relates to a biodegradable porous composite and a hybrid composite made of biopolymers and bioceramics, as well as preparation methods thereof. More specifically, the present invention relates to: a dual pore-structured biodegradable porous material, which contains a biopolymer having excellent biodegradability and bioactivity; a dual pore-structured porous composite comprising a combination of the biopolymer and domestic or wild silk fibroin; and a biodegradable hybrid composite comprising a combination of the biopolymer, the silk fibroin and a physiologically active bioceramic material; the use thereof; and preparation methods thereof.

BACKGROUND ART

In tissue engineering, the major functions of biomaterials are to provide a specific space for the growth of cells into tissues or organs and to provide a signal capable of physically and chemically controlling the proliferation and differentiation of cells, and thus the design of suitable scaffolds is of increasing importance. Materials for scaffolds are biomimetic materials, which can induce and promote the formation of new tissue and can be absorbed in vivo, and natural or synthetic materials have been considered as the scaffold materials.

However, synthetic biomaterials, developed to date, are mostly non-biodegradable metallic, ceramic or polymeric materials, which have low biocompatibility and biofunctionality. Thus, these synthetic biomaterials are merely simple materials substituting for the human body. Natural biomaterials, which are extracted from the human body or animals, have poor moldability in particular applications and are supplied in very small amounts, and thus the use thereof has been limited. Recently, synthetic polymeric materials have been developed, but these synthetic polymeric materials have shortcomings in that they cause inflammatory reaction due to low biocompatibility and are difficult to process compared to other biomaterials, thus resulting in low functionality. Accordingly, there is an urgent need for biomimetic materials having improved biocompatibility and biofunctionality and techniques for precisely processing the biomaterials.

Meanwhile, even though chitin and chitosan possess advantageous functions, including biocompatibility, antimicrobial properties and resorption properties, tissue scaffolds and biomembranes, manufactured therefrom, have physical or structural problems, when they are used as biomaterials. To solve these problems, studies on blends of chitosan and other polymers have been actively conducted.

Methods of blending chitosan with other polymers include blending anionic polymers and cationic chitosan into polymeric composites.

Silk fibroin is a linear protein consisting of 17 amino acids and has a /3-sheet structure because the main components thereof are alanine and glycine, which are simple nonpolar molecules. Thus, it serves as an enzyme-immobilizing substrate having excellent dynamic physical properties and has blood compatibility and dissolved oxygen permeability in a wet state. Accordingly, it has been considered as a biomaterial in various applications, including media for culturing from artificial skins or artificial blood vessels to mammalian cells.

Consequently, it is preferable to develop biopolymer composite materials, which are based on chitosan and fibroin for tissue regeneration and have suitable physical properties. When a chitosan/fibroin composite as a tissue scaffold is implanted in vivo, the adhesion and growth of cells injected therein will be activated due to the glucosamine and acetylglucosamine of chitosan, the induction of the surrounding cells thereinto will be promoted, and a space for the growth of new tissue will be provided due to the relatively rapid induction of degradation of the fibroin protein.

In related prior art, Korean Patent Publication No. 2003-0097691 discloses a functional material consisting of a composite of silk fibroin alone or a composite material made from domestic or wild silk fibroin and another secondary substance selected from among cellulose, chitin, chitosan, chitosan derivatives, wool keratin and polyvinyl alcohol. Also, Korean Patent Laid-Open Publication No. 2003- 0022425 discloses a method for preparing a composite, in which organic and inorganic phases are combined with each other without phase separation, the method comprising allowing a specific biodegradable polymer and a coupling agent to react with each other at a given ratio to prepare a coupled biodegradable polymer, adding a hydrolysable ceramic precursor and a water-soluble calcium salt to the coupled biodegradable polymer, and then subjecting the mixture to a sol-gel reaction.

Tissue regeneration refers to effectively regenerate tissue by providing a scaffold, when an organ or tissue in the body loses its function, or they are lost. Herein, the scaffold must be physically stable at the implanted site and have physiological activity capable of regulating tissue regeneration, and in addition, must be degraded in vivo without causing severe inflammatory reaction, after new tissue is formed. This scaffold for tissue regeneration is manufactured to have a three-dimensional porous structure using a polymer having a specific strength and shape and is molded in the form of sponge-like fibrous matrices or gels. Among them, the fibrous matrix-type scaffolds have a structure in which cells readily adhere and proliferate, because open pores having high porosity can be formed. However, when they are made of natural polymers, they have a very weak strength in an in vivo wet environment, and thus are readily degraded and shrunk, making it difficult to maintain the shape thereof. Also, when they are made of hydrophobic synthetic polymers, it is difficult to maintain a specific space only with the fibrous structure. For this reason, the fibrous matrix-type scaffolds mainly have a two-dimensional rod-type structure rather than a three-dimensional structure. However, in order for the scaffolds to have excellent cell regeneration ability, the scaffolds must be manufactured to have a three-dimensional structure with high porosity and suitable pore size. Accordingly, there is an urgent need to develop a natural material- based biomimetic biomaterial, which is biocompatible and, at the same time, biodegradable.

In addition, bone tissue regeneration refers to effectively generate tissue by providing bone cell scaffolds, when an organ or tissue in the body loses its function, or they are lost. As the scaffolds for bone tissue regeneration, porous scaffolds, made of polymers and bioceramics having a given strength and shape, have been used. However, for natural polymers, collagen is used to cause low adhesion to bioceramics and production cost is very high, thus making the general use thereof difficult. For synthetic polymers, non-degradable polymers such as polymethylmethacrylate are used for the adhesion and molding of bioceramics and have serious biological problems, including tissue isolation. Recently, scaffolds comprising biodegradable synthetic polymers, including polylactide, have been developed, but these scaffolds also have a problem in that the degradation products cause inflammatory reaction. Accordingly, there is an urgent need to develop biomimetic materials and biomaterials, which can be prepared in a three dimensional structure having high porosity and suitable pore size for bone tissue regeneration and have excellent biocompatibility and biodegradability.

Meanwhile, hydroxyapatite, which is the main component of natural bone, can form a direct chemical bond with the surrounding hard tissue due to its bioactivity, compatibility and osteoconductivity, and is used as a bone regeneration material and a bone graft for biomedical applications due to its nontoxicity, noninflammatory and non-immune properties. Hydroxyapatites extracted from animal bone tissue, which have been used as biomaterials in the prior art, has very limited applicability due to its high cost despite its biological utility. In comparison with this, a hydroxyapatite, which is obtained from shells or synthesized, is produced at a relatively low cost and may have a wide size distribution ranging from micrometers to nanometers, but it is difficult to use as a tissue engineering scaffold, because it is likely to be brittle in nature and has low biodegradation rate. Also, hydroxyapatite in the form of fine particles has shortcomings in that the particles are unstable when they are mixed with physiological saline or blood, and thus move from the implanted site to the surrounding tissue to damage healthy tissue.

Accordingly, the present inventors have made many efforts to develop a composite biomaterial and a composite scaffold, which have biocompatibility and biodegradability, and, as a result, have found that a porous composite consisting of fibroin and a biopolymer, which contains natural chitosan that is similar to an extracellular substrate, as a main component, has biodegradability and bioactivity as well as compatibility with a living body, and in addition, have found that a hybrid composite consisting of a combination of a biopolymer, fibroin and a bioceramic material is useful as a bone tissue scaffold, thereby completing the present invention.

SUMMARY OF INVENTION

Therefore, it is an object of the present invention to provide a dual pore-structured biodegradable porous material, which contains a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan or derivatives thereof, as main components, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof, as well as a preparation method thereof.

Another object of the present invention is to provide a dual pore-structured porous biodegradable composite, which comprises a combination of a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan or derivatives thereof, as main components, and a domestic or wild silk fibroin, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof, as well as a preparation method thereof.

Another object of the present invention is to provide a biodegradable hybrid composite, which contains a chitosan-based biopolymer, fibroin and bioceramic hydroxyapatite, as well as a preparation method thereof.

To achieve the above objects, in one aspect, the present invention provides a dual pore-structured biodegradable porous material, which contains a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof.

In another aspect, the present invention provides a dual pore-structured porous composite, which comprises a combination of a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, and domestic or wild silk fibroin, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof.

In still another aspect, the present invention provides a method for preparing a dual pore-structured biodegradable porous material, the method comprising the steps of: (a) obtaining an aqueous biopolymer solution by adding a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, to an acidic aqueous solution; (b) adding an aqueous silk fibroin solution to the aqueous biopolymer solution, and then bubbling the mixture solution by mechanical stirring, thus obtaining an aqueous composite solution having a liquid-gas (bubble) colloidal phase; (c) subjecting the aqueous biopolymer solution of step (a) or a mixed solution of the aqueous biopolymer solution of step (a) and the aqueous composite solution of step (b) to thermally- induced phase separation and solvent sublimation, thus obtaining a biodegradable porous material having a single pore structure consisting of regular anisotropic pores; and (d) partially dissolving the porous material to form irregular isotropic pores on the front surface or specific portion thereof and, at the same time, form a regular anisotropic pore structure on the inside surface or the back surface thereof, thus obtaining a biodegradable porous material having a dual pore structure.

In still another aspect, the present invention provides a method for preparing an acetylated biodegradable porous material, which comprises acetylating the dual pore-structured biodegradable porous material, prepared according to the above method, to control the acetylation degree of the porous material to 1-100%.

In still another aspect, the present invention provides a method for preparing a multilayer membrane, the method comprising compressing the dual pore-structured biodegradable porous material having irregular isotropic pores and regular anisotropic pores, thereby forming a multilayer membrane consisting of plural layers. Also, the present invention provides a multilayer membrane prepared according to said method, which consists of plural layers of the dual pore- structured biodegradable porous material.

In still another aspect, the present invention provides a scaffold for tissue engineering and a medical material for skin application, which contains, as an active ingredient, the dual pore-structured biodegradable porous material, the acetylated biodegradable porous material, prepared according to the above method, or the biodegradable multilayer membrane. In still another aspect, the present invention provides a membrane for tissue regeneration induction or barrier and a sustained-release carrier, which contain, as an active ingredient, the dual pore-structured biodegradable porous material, the acetylated biodegradable porous material, prepared according to the above method, or the biodegradable multilayer membrane.

In still another aspect, the present invention provides a biodegradable hybrid composite, which comprises a combination of (i) a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, (ii) a domestic or wild silk fibroin, and (iii) a bioceramic material having physiological activity, and has regular anisotropic pore structure. The present invention also provides a dual pore-structured biodegradable hybrid composite, which comprises a combination of (i) a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, (ii) a domestic or wild silk fibroin, and (iii) a bioceramic material having physiological activity, and has irregular isotropic pores on the front surface or specific portion thereof and a regular anisotropic pore structure on the inside surface or the back surface thereof.

In still another aspect, the present invention provides a method for preparing a biodegradable porous hybrid composite having a regular anisotropic pore structure, the method comprising the steps of: (a) adding a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, to an aqueous acidic solution containing a physiologically active bioceramic material dispersed therein, thus obtaining an aqueous solution of a biopolymer-bioceramic composite; (b) adding an aqueous silk fibroin solution to the bioceramic-biopolymer composite solution, and then bubbling the mixture solution by mechanical stirring, thus obtaining an hybrid composite solution having a liquid-gas (bubble) colloidal phase; and (c) subjecting the hybrid composite solution to thermally-induced phase separation and solvent sublimation, thus obtaining said biodegradable porous hybrid composite having a regular anisotropic pore.

In still another aspect, the present invention provides a method for preparing a dual pore-structured porous biodegradable hybrid composite, the method comprising partially dissolving the biodegradable porous hybrid composite having a regular anisotropic pores, thereby forming irregular isotropic pores on the front surface or specific portion thereof and, at the same time, forming a regular anisotropic pore structure on the inside surface or the back surface thereof.

In still another aspect, the present invention provides a method for preparing an acetylated biodegradable hybrid composite, the method comprising acetylating the biodegradable hybrid composite to control the acetylation degree of the hybrid composite to 1-100%.

In still another aspect, the present invention provides a method for preparing a multilayer membrane, the method comprising compressing the biodegradable hybrid composites, thereby forming a multilayer membrane consisting of plural layers of the porous biodegradable hybrid composite. Also, the present invention provides a multilayer membrane prepared according to said method, which consists of plural layers of the porous biodegradable hybrid composite.

In still another aspect, the present invention provides a method for preparing a nanofiber membrane, the method comprising the steps of: (a) obtaining an bioceramic-biopolymer composite aqueous solution by adding a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, to an acidic aqueous solution in which bioceramic having bioactivity is dispersed; (b) obtaining an hybrid composite solution having a liquid-gas (bubble) colloidal phase by adding an aqueous silk fibroin solution to the bioceramic-biopolymer composite aqueous solution, and then bubbling the mixture solution by mechanical stirring; and (c) forming a membrane consisting of nanofibers by removing bubbles from the hybrid composite solution, and then electrospinning.

In still another aspect, the present invention provides a tissue engineering scaffold and medical materials for filling cavities in tooth or bone matter, which contain the biodegradable hybrid composite as an active ingredient.

In yet another aspect, the present invention provides a membrane for tissue regeneration induction or barrier, which contains the biodegradable hybrid composite as an active ingredient.

Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows cross-sectional scanning electron micrographs of a composite according to the present invention [(a): a photograph of the non-uniform anisotropic pore structure of a chitosan/fϊbroin (80/20) composite; and (b): a photograph of the regular anisotropic multilayer structure of the chitosan/fibroin (80/20) composite].

FIG. 2 shows a scanning electron micrograph of a multilayer membrane according to the present invention.

FIG. 3 shows scanning electron micrographs of the anisotropic pore structure of a porous hybrid composite according to the present invention [(A): the porous wall surface of test group 11 (x200); (B): the porous wall surface of test group 14 (x200); (C): the porous wall surface of test group 16 (x200); (D): the porous wall surface of test group 16 (x lOOO); (E): the porous wall surface of test group 16 (x5,000); and (F): the porous wall surface of test group 18 (^χ2,000)].

FIG. 4 shows scanning electron micrographs of the anisotropic multilayer structure of a porous hybrid composite according to the present invention. In FIG. 4, photographs showing the comparison between the front surface and the back surface for the direction in which a solvent is sublimated the composite of (A): test group 11, (B): test group 13, (C): test group 16, (D): test group 14, (E): test group

17 (upper photograph: front surface (x70), lower photograph: back surface (^χ30)), and (F): the cross-section of test group 18 (*200).

FIG. 5 is a scanning electron micrograph showing the inventive multilayer membrane consisting of a plurality of layers compressed to each other.

DETAILED DESCRIPTION OF THE INVENTION,

AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a dual pore- structured biodegradable porous material, which contains a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof, as well as a preparation method thereof.

In another aspect, the present invention relates to a dual pore-structured porous composite, which comprises a combination of a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, and a domestic or wild silk fibroin, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof, as well as a preparation method thereof. In the present invention, polysaccharides containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, extracted from the shell of crustaceans such as crabs or synthesized of inorganic salts, and a fibroin extracted from domestic silk or wild silk, are used.

In the present invention, chitosan serves as an adhesive agent for biosubstrates and bioceramics by virtue of its biocompatibility, resorption, stopping blood flow, anti- infection, softness and adhesive properties, and contains more than 90 wt% of glucosamine and acetylglucosamine, and chitosan derivatives, containing glucosamine and acetylglucosamine, may also be used. In the present invention, chitosan can be used in an amount of 20-95 wt% based on the weight of the composite. An acidic solvent for dissolving chitosan may be selected from among acetic acid, formic acid, lactic acid and hydrochloric acid, and is preferably acetic acid. However, the present invention is not limited thereto, and according to circumstances, any suitably selected acidic solvent may be used in the present invention.

Also, fibroin serves as an enzyme-immobilizing substrate having excellent dynamic physical properties and has blood compatibility and dissolved-oxygen permeability in a wet state. As the fibroin in the present invention, fibroin extracted from domestic silk or wild silk may be used, in addition to silk, byproducts derived from domestic or wild silk production, silk fibers, silk products and silk fiber composites may also be used.

In order to prepare a dual pore-structured biodegradable porous material according to the present invention, a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, is first added to an acidic aqueous solution to obtain an aqueous biopolymer solution. Then, an aqueous silk fibroin solution is added to the aqueous biopolymer solution, and then bubbled by mechanical stirring to obtain an aqueous biopolymer composite solution having a liquid-gas (bubble) colloidal phase.

In the present invention, an acidic solvent for dissolving said biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, is selected from among acetic acid, formic acid, lactic acid and hydrochloric acid. Preferably, acetic acid is used. However, the present invention is not limited thereto, and according to circumstances, any suitably selected acidic solvent may be used in the present invention.

In the present invention, the aqueous fibroin solution is obtained by adding silk to a concentrated aqueous solution of a neutral salt, such as calcium chloride, calcium nitride, lithium bromide or lithium thiocyanate, and heating the silk solution. The heating temperature is preferably 25-70 ^°C , and more preferably 50-60 ^°C . If the heating temperature exceeds 70 ^°C , the molecular weight of the fibroin protein will be reduced, and the polymer properties of the material will be lost, thus resulting in a decrease in the moldability thereof. The heating time is preferably about 3-24 hours.

When the obtained composite solution is subjected to thermally-induced phase separation and solvent sublimation, a porous material having regular anisotropic pores can be obtained. When the obtained porous material is partially dissolved, it is possible to obtain a dual pore-structured porous material which has irregular anisotropic pores formed on the front surface or a specific portion thereof, and regular anisotropic pores formed on the inside surface or the back surface thereof

When the dual pore-structured porous composites, prepared in the present invention, are compressed, a multilayer membrane having a plurality of fine layers can be formed. In another aspect, the present invention relates to a method for preparing a multilayer membrane, the method comprising compressing the dual pore-structured porous materials, which have anisotropic pores and isotropic pores, to form a multilayered adhesive membrane, and relates to a biodegradable multilayer membrane in which the dual pore-structured porous materials prepared by the inventive method are finely adhered to each other in multi-layers.

The dual pore-structured biodegradable porous material according to the present invention is used to effectively support, induce, regenerate, culture or selectively block biological cells and tissues or to supply or exude physiologically active substances and pharmacological substances, and can be applied to scaffolds for tissue engineering, medical materials for skin application, membranes for tissue regeneration induction or barrier, sustained-release carriers, substrates for biological cell proliferation, etc.

In still another aspect, the present invention relates to a biodegradable hybrid composite, which comprises a combination of (i) a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, (ii) a domestic or wild silk fibroin, and (iii) a bioceramic material having physiological activity, and has regular anisotropic pore structure. The present invention also relates to a dual pore-structured biodegradable hybrid composite, which comprises a combination of (i) a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, (ii) a domestic or wild silk fibroin, and (iii) a bioceramic material having physiological activity, and has irregular isotropic pores on the front surface or a specific portion thereof and a regular anisotropic pore structure on the inside surface or the back surface thereof.

The inventive method for preparing the porous biodegradable hybrid composite having regular anisotropic pores comprises the steps of: (a) adding a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, to an aqueous acidic solution containing a physiologically active bioceramic material dispersed therein, thus obtaining an aqueous solution of a biopolymer-bioceramic composite; (b) adding an aqueous silk fibroin solution to the biopolymer-bioceramic composite solution, and bubbling the mixture solution by mechanical stirring, thus obtaining an aqueous hybrid composite solution having a liquid-gas (bubble) colloidal phase; and (c) subjecting the obtained hybrid composite solution to thermally-induced phase separation and solvent sublimation, thus obtaining a porous biodegradable hybrid composite having regular anisotropic pores.

When the porous hybrid composite, prepared according to the above method, is partially dissolved, a dual pore-structured porous biodegradable hybrid composite, which has irregular isotropic pores formed on the front surface or specific region thereof, and regular anisotropic pores formed on the inside surface or the back surface thereof, can be prepared.

That is, in order to prepare the biodegradable hybrid composite according to the present invention, hydroxyapatite is first uniformly dispersed in an acidic solution, and a glucosamine-based polysaccharide is then added thereto and dissolved therein. Then, an aqueous fibroin solution is added thereto, and the prepared hybrid composite solution is subjected to thermally-induced phase separation and solvent sublimation, thus preparing a porous hybrid composite having regular anisotropic pores. In addition, when the surface of the prepared porous hybrid composite having regular anisotropic pores is dissolved, a dual pore-structured biodegradable hybrid composite, which has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof, can be prepared. The dual pore structure of the hybrid composite according to the present invention has an irregular isotropic pore structure on the surface thereof and a regular anisotropic multilayered pore structure inside thereof, and consists of two different consecutive pore structures. When the porous hybrid composites are compressed, a multilayer membrane having a plurality of fine layers may be formed.

In the present invention, the physiologically active bioceramic material is preferably hydroxyapatite. Hydroxyapatite for use in the present invention is extracted from shells and is preferably used in an amount of 1-70 wt%, and more preferably 20-60 wt%, based on the total weight of the composite, for bone cell compatibility of the composite. That is, the contents of fibroin and bioceramic material in the composite are preferably 5- 100 parts by weight and 1-300 parts by weight, respectively, based on 100 parts by weight of the biopolymer.

In the present invention, the biodegradable hybrid composite is preferably in a form selected from the group consisting of a porous material, a film, a gel and a fiber.

The dual pore-structured porous composite and hybrid composite, prepared according to the present invention, have an irregular isotropic pore structure on the surface thereof and a regular anisotropic multilayer structure inside thereof and consist of two different consecutive phase structures.

The multilayer pore structure is formed due to the phase separation of the polymer solution in specific conditions. Specifically, when the temperature of the polymer solution becomes lower than the freezing point of a solvent, the crystallization of the solvent occurs, and the polymer phase is pushed to the front of the solvent crystals. The polymer extracted from the solvent crystal is agglomerated to form a continuous polymer-rich phase, and after the solvent crystals are sublimated, a multilayer pore structure having repeated gaps similar to the solvent crystals is formed. The repeated gaps vary depending on cooling rate and the concentration of the polymer, and the temperature gradient along the heat transfer direction in the frozen porous material is determined according to the time during which the porous material is maintained in a freeze-drying container before a vacuum process. For this reason, before the solvent is sublimated, the solvent crystals on the outside of the porous material melt, resulting in the collapse of phase separation on the surface or specific region of the porous material. As a result, the irregular isotropic pore structure is formed throughout the porous material or the partial region (including the surface in the heat transfer direction) of the porous material, depending on the maintenance time and temperature, and the regular anisotropic multilayer structure is formed throughout the porous material or the partial region (including the inside in the heat transfer direction) of the porous material.

The density, pore size and shape, and pore structure of the porous composite and hybrid composite according to the present invention can be controlled depending on the treatment condition including the content and concentration of each component, temperature, time, etc. Thus, the porous composite or hybrid composite according to the present invention may have an irregular isotropic single pore structure, a regular anisotropic multilayered single pore structure, or a dual pore structure in which the two pore structures are simultaneously formed on the surface in one direction and on the inside in the opposite direction.

The porous composite and hybrid composite according to the present invention may be acetylated. In this case, the glycosamine of polysaccharide (chitosan) is converted to acetylglycosamine, and the degree of acetylation means the conversion ratio polysaccharide into acetylglycosamine.

Glycosamine and acetylglycosamine have different biological effects in vivo. Thus, for example, if the content of glycosamine is high, the cell adhesion, absorption, antibacterial and reactive properties of the resulting composite will increase, but the tissue compatibility, biodegradability, resorption properties of the resulting composite will decrease, and if the content of acetylglucosamine increases, the opposite effects will occur. Accordingly in order to design a biomaterial most suitable for specific application, the degree of acetylation suitable for the relevant application must be considered. However, the reason why a hybrid composite, prepared using a chitosan having a glucosamine content of 97 wt%, in the following Example, was acetylated, is because an increase in the content of acetylglucosamine makes it difficult to form the composite into a desired shape.

Meanwhile, when bubbles are removed from the aqueous hybrid composite solution consisting of a uniform dispersion of glucosamine-based polysaccharide, fibroin and hydroxyapatite, and the resulting composite is electrospun, a membrane consisting of nanofibers can be prepared.

The biodegradable hybrid composite according to the present invention is used to effectively regenerate organs or tissues, when organs or tissues in the body lose their functions or they are lost. The porous hybrid composite useful for cell adhesion can be applied to a scaffold for tissue engineering, a membrane material for tissue regeneration induction or barrier, a sustained-release carrier, a substrate for biological cell proliferation, etc.

Examples

Hereinafter, the present invention will be described 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 biopolymer material having dual pore structure

1-1 : Preparation of aqueous chitosan solution Chitosan flakes were commercially purchased. 1 part by weight of the chitosan flakes were dissolved in 2 parts by weight of an aqueous acetic acid solution, until a uniform solution was formed, and the solution was neutralized with 2 parts by weight of NaOH solution to precipitate the chitosan. The precipitated chitosan was purified by washing it with deionized water and ethanol and vacuum drying. The purified chitosan had a deacetylation degree of 97% and a weight-average molecular weight of 400,000. 2 parts by weight of the purified chitosan was dissolved in 2 parts by weight of an aqueous acetic acid solution for 5 hours with slow stirring, thus preparing an aqueous chitosan solution.

1-2: Preparation of aqueous silk fibroin solution

After raw silk produced from Bombyx mori silkworms was subjected to the conventional scouring process, and then it was dissolved in a mixed solvent of 7M calcium chloride, water and ethanol, and then heated at 50 ^°C to obtain an aqueous fibroin solution. Because the fibroin solution contained, in addition to fibroin, a large amount of neutral salt ions resulting from the solvent, it was dialyzed against running water through a cellulose dialysis membrane for 5 days.

1-3: Preparation of porous material according to the present invention

The aqueous chitosan solution, prepared in Example 1-1, was uniformly mixed with the aqueous fibroin solution, prepared in Example 1-2, to have chitosan/fibroin weight ratios of 100/0, 90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 30/70, 20/80 and 10/90, thus preparing an aqueous solution containing chitosan alone, and mixed solutions of the aqueous chitosan solution and the aqueous fibroin solution.

The aqueous solutions were vigorously stirred to form dynamically induced liquid- gas colloids (bubble solutions), and the bubble solutions were placed in polyethylene terephthalate (PET) molds, which were then immediately transferred and sufficiently frozen in a freezer at -98 ^°C for 24 hours in order to induce liquid- liquid or solid-liquid phase separation. Then, the solutions were transferred and left to stand in a vacuum freeze dryer at room temperature for 10 minutes, followed by freeze drying. The solidified biopolymer composites were dried at a temperature of 60 °C and a pressure of 0.5 mmHg for at least 2 days to remove the remaining solvent, particularly acetic acid.

FIG. 1 shows a dual pore-structured porous composite structure having a chitosan/fibroin ratio of 80/20 (test group 3), among the composites prepared according to the above-described method. In FIG. 1, (a) shows the front surface having an irregular isotropic pore structure, and (b) shows the on the inside surface or the back surface having a regular anisotropic multilayer structure.

Also, as shown in Table 1 below, the porous biopolymer composites prepared as described above had a porosity of more than 80%, and in the case where the content ratio of any one component of the two biopolymers was 70 parts by weight, the porosity was more than 90%.

Table 1

Example 2: Shape of porous material

Scaffolds for tissue engineering must have high porosity and open pores in order to induce the adhesion and proliferation of cells and deliver a variety of nutrients and cell-activating substances. Such requirements have direct effects on the survival and three-dimensional growth of cells implanted in the scaffolds. The surfaces of the porous materials, prepared to have the contents shown in Example 1-3, were plated with platinum, and the surface configurations and pore shapes thereof were measured with a scanning electron microscope (SEM) (Hitachi S-3500N, Japan).

As a result, as shown in FIG. 1, various open pores having a size of less than 300μin were formed in the composite, and a multilayer pore structure, having an irregular isotropic pore structure and regular anisotropic channel-like gaps, was formed in the composite. This pore structure was formed due to solvent crystallization, phase separation, partial dissolution of solvent crystals, and solvent sublimation.

Accordingly, the composite could be formed to have a generally irregular isotropic single pore structure, a regular anisotropic multilayered single pore structure, or a dual pore structure in which the two pore structures were simultaneously formed on the surface in one direction and on the inside in the opposite direction, depending on the preparation conditions. In the present invention, the density, pore size and shape, and pore structure of the composite could be controlled depending on the content and concentration of each component, and process conditions including temperature, time, etc.

Example 3: Measurement of water absorption rate and weight loss of porous material

The inflammatory reaction of the cell scaffolds resulting from the temporary water absorption thereof is advantageous for the adhesion and growth of cells, because it increases pore size in a three-dimensional structure. Also, the solubility thereof in water is the important physical property of tissue engineering materials. In order to measure water absorption and solubility for the test groups 1 -4 among the porous materials prepared in Example 1-3, the porous materials were soaked in deionized water at 50 ^°C for 1 hour, and the measurement was carried out. The water absorption rate of the porous materials was calculated as the weight ratio of absorbed water to the dry weight of the porous material.

In the measurement results, the water absorption rate was increased with an increase in the content ratio of fibroin to chitosan, and the test group 4 was swollen, having an absorption rate up to 38 times and showed a weight loss of about 15 wt.%, suggesting that the porous material was clearly soluble.

Example 4: Acetylation

Proteoglycan, which is a main component forming an extracellular substrate and cell wall of the human body, consists of a number of glucosaminoglycan branches at the protein main chain, in which each of the glucosaminoglycans is formed of long chains consisting of repeated disaccharides having an acetylglucosamine group. The porous materials prepared in Example 1-3 are biopolymers consisting of a disaccharide monomer having 97% glucosamine group, and depending on the applications and characteristics thereof, glucosamine therein can be completely or partially converted to acetylglucosamine according to the following acetylation method.

The prepared chitosan/fibroin (80/20) composite was added to a treatment solution, obtained by dissolving acetic anhydride, in an amount corresponding to a desired anhydride/glucosamine molar ratio (1 /1), in 100 parts by weight of an aqueous solution of 1,2-propanediol. The resulting solution was acetylated for 24 hours with slow stirring. The acetylated composite was washed several times with deionized water and ethyl alcohol, and then dried. The degree of acetylation was calculated according to a colloidal titration method using 0.0025N polyvinyl sulfate potassium salt and toluidine blue-O. The degree of acetylation of the prepared biopolymer composite was 48%, and the shape thereof was kept. Also, the degree of acetylation could be controlled to 0-97% depending on the molar ratio of the treatment solution.

Example 5: Preparation of multilayer membrane

The porous material (test group 3), having a chitosan/fϊbroin content ratio of 80/20 and an isotropic multilayer structure, were swollen by absorbing deionized water therein. Then, pluralities of the porous layers were put one upon another, applying pressure at a pressure of 5-25 kg/mm² in a given direction using a press roller and, at the same time, were dewatered. Then, the multilayer structure was slowly dried under pretention, thus preparing a multilayer membrane consisting of several layers compressed against each other (FIG. 2).

Example 6: Preparation of hybrid composite of biopolymers and bioceramics

As a typical example of bioceramic material, hydroxyapatite powder, having an average particle size of about 2μm and a calcium/phosphorus molar ratio of 1.55, was dispersed in 2 wt% of an aqueous acetic acid solution at the content ratios shown in Table 2 below. Then, the dispersions were treated with ultrasonic waves for 20 minutes in order to uniformly disperse the hydroxyapatite particles.

In the ultrasonically treated hybrid composite solutions, 2 wt% of purified chitosan, having a weight-average molecular weight of 400,000 and a deacetylation degree of 97%, was dissolved and stirred for 5 hours. Also, fibroin was uniformly mixed with the dispersed solutions of hydroxyapatite/chitosan in an amount of 20 wt% od hydroxyapatite based on the weight of chitosan. The above-prepared aqueous hybrid composite solutions of a biopolymer derived from chitosan and fibroin, and hydroxyapatite were vigorously stirred to form dynamically induced liquid-gas colloids (bubble solutions). Then, the bubble solutions were placed in a polyethylene terephthalate (PET) mold and sufficiently frozen in a freezer at -98 ^°C in order to induce liquid-liquid or solid-liquid phase separation. Then, the solutions were left to stand in a vacuum freeze dryer for 5- 30 minutes, and then freeze-dried. The solidified hybrid composites were dried at 60 ^°C under the condition of reduced pressure at 0.5 mmHg for 2 days to remove the remaining solvent. The densities and porosities of the prepared porous hybrid composites are shown in Table 2 below.

Table 2

Example 7: Shape of hybrid composite

Scaffolds for tissue engineering must have high porosity and open pores in order to induce the adhesion and proliferation of cells and deliver a variety of nutrients and cell-activating substances. Such requirements have direct effects on the survival and three-dimensional growth of cells implanted in the scaffolds.

The surfaces of the test groups 11-18, prepared in Example 6, were plated with platinum, and the surface configurations and pore shapes thereof were observed with a scanning electron microscope (SEM) (Hitachi S-3500N, Japan).

As a result, as shown in FIG. 3, the porous hybrid composites had various open pores having a size of less than 200 μm, and hydroxyapatite particles having a size of 0.5-2 [M were uniformly dispersed on the pore wall due to the high viscosity of the polymer solution. When the content of hydroxyapatite was low, the hydroxyapatite particles were uniformly dispersed on the thin solid wall of the pores, and as the content of hydroxyapatite was increased, the polymer phase surrounded the hydroxyapatite particles to form partially mixed masses. In FIG. 3, (A), (B) and (C) are 20Ox electron micrographs showing the shapes of the test groups 11, 14 and 16, respectively, (D) and (E) are l,000x and 5,000x photographs of the test group 16, respectively, and (F) is a 2,00Ox photograph showing the wall surface of pores.

Also, as shown in FIG. 4, the hybrid composites prepared in Example 6 had a nonuniform isotropic pore structure and a regular anisotropic multilayer pore structure. In FIG. 4, (A), (B), (C) and (D) are photographs showing the structures of composites having biopolymer/hydroxyapatite weight ratios of 100/0, 80/20, 50/50 and 70/30, respectively, (E) shows the pore structure of the test group 17 having a biopolymer/hydroxyapatite weight ratio of 40/60 (upper photograph: the isotropic pore structure of the front surface in the direction in which a solvent is removed by sublimation; and lower photograph: an isotropic multilayer pore structure on the inside surface and the back surface), and (F) is a cross-sectional photograph of the test group 18 having a biopolymer/hydroxyapatite weight ratio of 30/70.

Example 8: Measurement of water absorption and weight loss of hybrid composite by synthetic body fluid

Synthetic body fluids (SBFs) were prepared by dissolving NaCl, NaHCO₃, KCl, K₂HPO₄ 3H₂O, MgCl₂-OH₂O, CaCl₂ and Na₂SO₄ in deionized water in the amounts shown in Table 3 below on the basis of the ionic component contents of body fluid, and the synthetic body fluids had a pH of 7.4 and were buffered with trishydroxymethylaminomethane and I M hydrochloric acid at 36.5 ^°C . Table 3 shows the comparison between the ionic concentrations of the synthetic body fluids used in the experiment and the ionic concentration of human blood plasma, and the synthetic body fluids used in the experiment had an ionic concentration similar to that of human blood plasma.

Table 3

The inflammatory reaction resulting from body fluid absorption of cell scaffolds is advantageous for the adhesion and growth of cells, because it increases the size of pores in a three-dimensional structure. Also, the stability of the cell scaffolds in body fluid is an important factor in the practical use of tissue engineering materials.

In order to examine the body fluid absorption and osmotic stability of the hybrid composites according to the present invention, the hybrid composites prepared in Example 6 were soaked in synthetic body fluids at 37 ^°C , and the water absorption rates thereof with time was measured.

As a result, the absorption rate of synthetic body fluids showed the highest value in the test group 13, and the hybrid composite of the test group 13 was swollen to the highest absorption rate corresponding to 25 times the weight thereof, and then started to decrease rapidly, and after 3 hours, the absorption rate was maintained at 15 times, which is about 60% of the highest absorption rate. After 2 days, the absorption rate started to increase slowly again, and after 1 week, it was stably maintained at 18 times, which is about 70% of the highest absorption rate. This change in absorption rate with time showed similar tendencies in all the hybrid composites. After 1 week, the weight loss was about 3 wt%, and then there was no weight loss during 4 weeks.

As the content of hydroxyapatite was increased, the test group 11 containing no hydroxyapatite showed an absorption rate up to 10 times for 1 hour, whereas the test groups 12 and 13 showed an absorption rate up to about 20 times. In the test groups 14-18 having higher hydroxyapatite contents, the absorption rate was reduced again due to a decrease in the porosities of the composites and a reduction in the hydrated polymer content. Particularly, the results of X-ray crystal analysis and SEM image analysis revealed that, in the test groups 16-18, the mixture of a amorphous polymer phase and highly crystalline hydroxyapatite started to be formed, and thus an absorption rate similar to that of the test group 11 was shown.

Example 9: Tensile strength of hybrid composite

A cell scaffold having high porosity for cell growth must have dynamic strength sufficient to maintain the shape thereof during tissue regeneration. Thus, in order to measure the dynamic strength, the tensile strengths according to hydroxyapatite contents of the test groups 11-18 prepared in Example 6 were measured with a universal material testing machane (United SSTM-I, USA) provided with a load cell of 5 kg.

In the measurement results, the tensile strength of the test group 11 containing no hydroxtapatite was 2.7 gf/mm². As the content of hydroxyapatite was increased, the tensile strength was slowly increased and showed a maximum of 4.5 gf/mm in the test group 15. On the contrary, in the case of the test group 16 containing hydroxyapatite in an amount higher than that in the test group 15, the tensile strength was significantly reduced. Generally, the stress transfer between a reinforcement material and a composite determines the dynamic properties of the reinforced polymer, and thus it was observed that the addition of hydroxyapatite to the biopolymer at a ratio of less than 50/50 led to an increase in the dynamic properties (tensile strength) of the composite. This result suggests that the hybrid composite of the present invention is suitable for use as a biomaterial.

Example 10: Protein permeability of hybrid composite

The adhesion and survival of cells are controlled by the continuous supply of extracellular components and serum proteins to the cell scaffold, and thus the protein permeability of the scaffold is a very important factor.

The protein permeability of the hybrid composites according to the present invention was measured in diffusion cell units connected in parallel at both sides of a sample seat having a diffusion area of 50 mm². Among the units, the donor cell was filled with 3 ml of a synthetic body fluid having 1 mg/ml of fluorescein isocyanate-conjugated fetal bovine albumin dissolved therein, and the opposite acceptor cell was filled only with 3 ml of synthetic body fluid. The solution in each of the cells was stirred, while it was placed in an incubator at 37 ^°C . The synthetic body fluid in the acceptor cell was collected at a given time interval, and the protein permeability thereof was measured by measuring the absorbance at 495 nm (the excitation wavelength of fluorescein isocyanate) using an UV spectrophotometer.

As a result, the protein permeability was almost linearly increased with the passage of time. In the case of the test group 16, the cumulative permeability for 1 day was 12 #g/mnf, and the cumulative permeability for 7 was about 30 μg/mirf. Thus, the cumulative permeability of the test group 16 was significantly increased compared to the permeability (1-5 μg/ws) of other test groups for 1 day. In comparison with this, the protein permeability of the test group 13, which showed the highest absorption in Example 8, was 1.5 /zg/mm²for 1 day, suggesting that the permeation of proteins in the hybrid composites was interfered by an increase in body fluid absorption and was effectively influenced by the pore structure of the composites. Such results suggest that the hybrid composites of the present invention have protein permeability sufficient to use as biomaterials.

Example 11: Acetylation of hybrid composites

Proteoglycan, which is a main component forming an extracellular substrate and cell wall of the human body, consists of a number of glucosaminoglycan branches at the protein main chain, in which each of the glucosaminoglycans is formed of long chains consisting of repeated disaccharides having an acetylglucosamine group. The composites prepared in Example 6 are biopolymers consisting of a disaccharide monomer having 97% glucosamine group, and depending on the applications and characteristics thereof, glucosamine therein can be completely or partially converted to acetylglucosamine according to the following acetylation method.

Among the hybrid composites prepared in Example 6, the hybrid composite of the test group 14 was added to a treatment solution obtained by dissolving acetic anhydride in an amount corresponding to a desired anhydride/glucosamine molar ratio (1/1) in 100 parts by weight of an aqueous solution of 1,2-propanediol. The resulting solution was acetylated for 24 hours with slow stirring. The acetylated composite was washed several times with deionized water and ethyl alcohol, and then dried. The acetylation degree of the treated hybrid composite could be controlled to about 50% while maintaining the shape thereof. Example 12: Preparation of multilayer membrane using hybrid composite

The composites of the test groups 11-18 having an anisotropic multilayer structure, prepared in Example 6, were swollen by absorbing deionized water therein. Then, pluralities of the porous layers were put one upon another in a given direction at a pressure of 5-25 kg/mm² using a press roller and, at the same time, were dewatered. Then, the multilayer structures were slowly dried under pretension, thus preparing multilayer membranes consisting of several layers compressed to each other (FIG. 5).

Example 13: Preparation of nano fiber membrane using hybrid composite

Each of aqueous solutions of biopolymers for preparing the composite of the test group 14, prepared in Example 6, was concentrated and mixed with each other to prepare an aqueous composite solution having a viscosity of about 800 poise, and bubbles in the composite solution were removed under reduced pressure. The aqueous composite solution was electrospun through a plurality of nozzles having a diameter of 100 μm, at a voltage of 25 kV at a spinning distance of 15 cm, thus preparing a nanofiber membrane consisting of nanofibers having an average diameter of 200 nm.

The experiment was carried out in various conditions, including a viscosity of 100- 2000 poise, a voltage of 10-60 kV, a spinning distance of 5-20, varying nozzle thicknesses, and varying spinning time. As a result, the shape and thickness of fibers forming the prepared nanofiber membrane were influenced mainly by the viscosity of the aqueous composite solution and the thickness of the nozzles, and the thickness and porosity of the membrane was proportional to the spinning time. In addition, the voltage magnitude, the current quantity, the spinning distance and the like influenced the conditions of membrane formation within a given range. INDUSTRIAL APPLICABILITY

As described in detail above, the present invention provides: a dual pore-structured porous material containing a biopolymer having excellent biodegradability and bioactivity; a dual pore-structured porous composite comprising a combination of the biopolymer and silk fibroin; and a biodegradable hybrid composite containing the biopolymer, fibroin and bioceramic material. The composites according to the present invention are advantageous for the adhesion and proliferation of cells, are effective for the supply of physiologically active substances and the migration of exudates, and have excellent bioactivity, biodegradability and biocompatibility. Thus, the composites according to the present invention can substitute for existing expensive medical biomaterials and have improved functionality.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS

What is Claimed is:

L A dual pore- structured biodegradable porous material, which contains a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof.

2. A dual pore-structured biodegradable porous composite, which comprises a combination of a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, and a domestic or wild silk fibroin, and has irregular isotropic pores on the front surface thereof and regular anisotropic pores on the inside surface or the back surface thereof.

3. A method for preparing a dual pore-structured biodegradable porous material, the method comprising the steps of: (a) obtaining an aqueous biopolymer solution by adding a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, to an acidic aqueous solution;

(b) adding an aqueous silk fibroin solution to the aqueous biopolymer solution, and then bubbling the mixture solution by mechanical stirring, thus obtaining an aqueous composite solution having a liquid- gas (bubble) colloidal phase;

(c) subjecting the aqueous biopolymer solution of step (a) or a mixed solution of the aqueous biopolymer solution of step (a) and the aqueous composite solution of step (b) to thermally-induced phase separation and solvent sublimation, thus obtaining a biodegradable porous material having a single pore structure consisting of regular anisotropic pores; and

(d) partially dissolving the porous material to form irregular isotropic pores on the front surface or specific portion thereof and, at the same time, form a regular anisotropic pore structure on the inside surface or the back surface thereof, thus obtaining a biodegradable porous material having a dual pore structure.

4. A method for preparing an acetylated biodegradable porous material, which comprises acetylating the dual pore-structured biodegradable porous material prepared by the method of claim 3, to control the acetylation degree of the porous material to 1-100%.

5. A method for preparing a multilayer membrane, the method comprising compressing the dual pore-structured biodegradable porous material having isotropic pores and anisotropic pores prepared by the method of claim 3, thereby forming a multilayer membrane consisting of plural layers.

6. A biodegradable multilayer membrane, prepared by the method of claim 5, which consists of plural layers of the dual pore- structured biodegradable porous material.

7. A scaffold for tissue engineering, which contains, as an active ingredient, the dual pore-structured biodegradable porous material of claim 1 or 2, the acetylated biodegradable porous material prepared by the method of claim 4, or the biodegradable multilayer membrane of claim 6.

8. A medical material for skin application, which contains, as an active ingredient, the dual pore-structured biodegradable porous material of claim 1 or 2, the acetylated biodegradable porous material prepared by the method of claim 4, or the biodegradable multilayer membrane of claim 6.

9. A membrane for tissue regeneration induction or barrier, which contain, as an active ingredient, the dual pore- structured biodegradable porous material of claim 1 or 2, the acetylated biodegradable porous material prepared by the method of claim 4, or the biodegradable multilayer membrane of claim 6.

10. A sustained-release carrier, which contain, as an active ingredient, the dual pore-structured biodegradable porous material of claim 1 or 2, the acetylated biodegradable porous material prepared by the method of claim 4, or the biodegradable multilayer membrane of claim 6.

11. A substrate for biological cell proliferation, which contain, as an active ingredient, the dual pore-structured biodegradable porous material of claim 1 or 2, the acetylated biodegradable porous material prepared by the method of claim 4, or the biodegradable multilayer membrane of claim 6.

12. A biodegradable hybrid composite having regular anisotropic pore structure, which comprises a combination of:

(i) a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, (ii) a domestic or wild silk fibroin, and (iii) a bioceramic material having physiological activity.

13. A dual pore- structured biodegradable porous hybrid composite having irregular isotropic pores on the front surface or a specific protion thereof and a regular anisotropic pore structure on the inside surface or the back surface thereof, which comprises a combination of;

14. The biodegradable hybrid composite according to claim 12 or 13, wherein the physiologically active bioceramic material is hydroxy apatite.

15. The biodegradable hybrid composite according to claim 12 or 13, wherein the contents of fibroin and bioceramic material in the composite are 5-100 parts by weight and 1-300 parts by weight, respectively, based on 100 parts by weight of the biopolymer.

16. The biodegradable hybrid composite according to claim 12 or 13, which is in a form selected from the group consisting of a porous material, a film, a gel and a fiber.

17. A method for preparing a biodegradable porous hybrid composite having a regular anisotropic pore structure, the method comprising the steps of:

(a) adding a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, to an aqueous acidic solution containing a physiologically active bioceramic material dispersed therein, thus obtaining an aqueous solution of a biopolymer-bioceramic composite;

(b) adding an aqueous silk fibroin solution to the bioceramic-biopolymer composite solution, and then bubbling the mixture solution by mechanical stirring, thus obtaining an hybrid composite solution having a liquid-gas (bubble) colloidal phase; and

(c) subjecting the hybrid composite solution to thermally-induced phase separation and solvent sublimation, thereby obtaining a biodegradable porous hybrid composite having a regular anisotropic pores

18. A method for preparing a dual pore-structured biodegradable porous hybrid composite, the method comprising partially dissolving the biodegradable porous hybrid composite having a regular anisotropic pores prepared by the method claim 17, thereby forming irregular isotropic pores on the front surface or a specific

5 portion thereof and, at the same time, forming a regular anisotropic pore structure on the inside surface or the back surface thereof.

19. A method for preparing an acetyl ated biodegradable hybrid composite, the method comprising acetylating the biodegradable hybrid composite of claim 12 or

10 13, to control the acetylation degree of the hybrid composite to 1-100%.

20. A method for preparing a multilayer membrane, the method comprising compressing the biodegradable hybrid composites of claim 12 or 13, thereby forming a multilayer membrane consisting of plural layers of the porous 5 biodegradable hybrid composite.

21. A multilayer membrane prepared by the method of claim 20, which consists of plural layers of the porous biodegradable hybrid composite. 0

22. A method for preparing a nano fiber membrane, the method comprising the steps of:

(a) obtaining an bioceramic-biopolymer composite aqueous solution by adding a biopolymer containing glucosamine and acetylglucosamine derived from chitin, chitosan, or derivatives thereof, as main components, to an acidic aqueous 5 solution in which bioceramic having bioactivity is dispersed;

(b) adding an aqueous silk fibroin solution to the bioceramic-biopolymer composite aqueous solution, and then bubbling the mixture solution by mechanical stirring, thus obtaining an hybrid composite solution having a liquid-gas (bubble) colloidal phase; and 0 (c) removing bubbles from the hybrid composite solution, and then electrospinning, thus forming a membrane consisting of nanofibers.

23. A tissue engineering scaffold, which contain the biodegradable hybrid composite of any one claim among claims 12-16, as an active ingredient.

24. A medical material for filling cavities in tooth or bone matter, which contain the biodegradable hybrid composite of any one claim among claims 12-16, as an active ingredient.

25. A membrane for tissue regeneration induction or barrier, which contains the biodegradable hybrid composite of any one claim among claims 12-16, as an active ingredient.