JP2003284767A - Scaffolding material for system engineering and artificial blood vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a porous scaffolding material for systems engineering, which can easily and stably organize the take and culture of cells, and an artificial blood vessel small in diameter and good in rate of patency. <P>SOLUTION: The scaffolding material for system engineering is made of a thermoplastic resin formed in a porous three-dimensional network structure having communicating property that an average pore diameter is 100-650 μm and an apparent density is 0.01-0.5 g/cm<SP>3</SP>, and the artificial blood vessel is composed of the scaffolding material. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a porous scaffold for tissue engineering, which enables easy cell engraftment and culture and stable organization, and an artificial blood vessel using the scaffold. . INDUSTRIAL APPLICABILITY The scaffold material for tissue engineering and the artificial blood vessel of the present invention are useful not only for basic research in biotechnology, but also for medical materials used as artificial skeleton base materials in replacement medicine by artificial organs and regenerative medicine by tissue engineering. Particularly, by utilizing the property of vascular endothelial cells engrafting on the entire inner wall surface, it is useful for an artificial blood vessel having a good patency rate even if the inner diameter is 6 mm or less.

[0002]

2. Description of the Related Art Conventionally, as scaffold materials for tissue engineering, there are materials such as polystyrene petri dish and polyester mesh coated with an extracellular matrix such as collagen, which are widely used mainly for monolayer culture. Was there. Culture forms other than the monolayer culture include spheroid forms by shaking culture and embedded culture using collagen gel. In particular, embedded culture using collagen gel should be cultured in vivo (in vivo), That is, it is possible to grow cells in a three-dimensional morphology, and it has become possible to perform basic research on cell function, which was insufficient in monolayer culture.

On the other hand, as a conventional artificial blood vessel, a tube made of a mesh made of polyester resin or PTFE resin has been put to practical use for a long time, and research is being advanced with a view to reducing the diameter and improving the patency rate. The mainstream technologies that have been studied to date are those that use segmented polyurethane tubing, which has a proven record as an antithrombotic material, and artificial blood vessel materials that have an antithrombotic substance such as heparin immobilized on the surface using graft chains. Is.

[0004]

The collagen gel for embedding culture is not a porous structure such as a three-dimensional network structure, and cell engraftment cannot be uniformly obtained on the entire surface or distribution of engraftment. There is a problem that can not be adjusted. As a method for producing a porous material having a three-dimensional network structure, a salt-containing method or a bubble method is known, but it is difficult to strictly and arbitrarily adjust the pore diameter and the pore density, and an appropriate tertiary A scaffold material composed of the original net-like structure has not been obtained yet.

Further, the cell engraftment structure obtained by the collagen gel embedding culture has no physical strength in the collagen gel itself, which is a scaffold material, and can be used for evaluation of cell function, but it is mechanical. Loaded applications, such as
It could not be used for artificial blood vessels.

On the other hand, although an artificial blood vessel as a substitute for an autologous blood vessel has already been widely clinically applied, since a small-caliber artificial blood vessel has poor patency, a coronary artery bypass surgery,
As for the small-diameter blood vessels required for peripheral artery reconstruction, autologous vein transplantation is currently used. Even if only anti-thrombogenicity is pursued as in the current mainstream technology for reducing the diameter, only the pseudointimal membrane made of fibrous collagen is formed on the inner wall of these conventional artificial blood vessels, and even the formation of endothelium As a result, the patency rate of the small-diameter artificial blood vessel is low. In addition, since there are no holes through which cells can enter the inner wall, even if pannus invades from the anastomosis, it floats without adhering to the inner wall, which causes obstruction in many cases.

The present invention has been achieved in view of the above problems of the prior art, and is composed of a homogeneous porous body having a three-dimensional network structure, and cells are uniformly engrafted on the entire inner surface of the porous structure. A scaffold material for tissue engineering to obtain,
It has excellent physical strength and maintains a high patency rate for a long time even when applied not only to basic research in the field of tissue engineering, but also to artificial blood vessels, especially artificial blood vessels with a small diameter of 6 mm or less. An object of the present invention is to provide a scaffold material for tissue engineering capable of forming an artificial blood vessel that can be used, and an artificial blood vessel using the scaffold material for tissue engineering.

[0008]

The scaffold material for tissue engineering of the present invention is a scaffold material for tissue engineering made of a thermoplastic resin, and the thermoplastic resin has an average pore size of 100 to 100.
650 μm, apparent density 0.01-0.5 g / cm
³ to form a porous three-dimensional network structure having connectivity.

The scaffold material for tissue engineering of the present invention is
Since it has a porous three-dimensional network structure of the thermoplastic resin having the above-mentioned specific average pore size and apparent density, cells and collagen suspension can easily penetrate into the pores of this porous three-dimensional network structure. Therefore, cells can be uniformly seeded over the entire porous three-dimensional network structure, for example, it is also possible to obtain an artificial peritoneum composed of two layers of mesothelial cells and fibroblasts, and glycoprotein in peritoneal dialysis. It can be expected to be used for analysis of silation mechanism and basic examination of dialysis.
When this scaffold for tissue engineering is used as an artificial blood vessel, it is possible to allow vascular endothelial cells to exist on the inner wall of the artificial blood vessel, obstruction does not easily occur, and as a result, an artificial blood vessel with a small diameter can be realized. It is possible.

The artificial blood vessel of the present invention is made of such a scaffold material of the present invention, and has a high patency even with a small diameter of 6 mm or less, and is suitable for coronary artery bypass surgery, peripheral artery reconstruction, etc. It can be effectively applied.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the scaffold material for tissue engineering and the artificial blood vessel of the present invention will be examined in detail.

The three-dimensional network structure made of a thermoplastic resin which constitutes the scaffold for tissue engineering of the present invention has an average pore diameter of 100 to 650 μm and an apparent density of 0.01 to.
A porous three-dimensional network structure of 0.5 g / cm ³ , which is continuous, that is, has continuous porosity, may be used. Even if the entire structure from the inner wall to the outer wall has a similar structure, It may be different near the outer wall. Further, the average pore size or the apparent density may be partially changed, for example,
The average pore diameter gradually changes from the inner wall toward the outer wall,
It may have so-called anisotropy.

The porous three-dimensional network structure made of this thermoplastic resin has an average pore diameter of 100 to 650 μm and an apparent density of 0.01 to 0.5 g / cm ³ , but a preferable average pore diameter is 100 to 400 μm. Preferably 100 to 3
It is 00 μm. 0.01-0.5 as apparent density
Within the range of g / cm ³ , cell engraftment is good, excellent physical strength is maintained, and elastic properties similar to those of a living body are obtained, but preferably 0.01 to 0.2 g / cm ^3. , And more preferably 0.01 to 0.1 g / cm ³ .

Further, in the concept of average pore size, the distribution of pore size is preferably monodisperse, and it is desirable that the contribution of pores having a pore size of 150 to 300 μm, which is an important pore size for cell invasion, is high. When the contribution rate of pores having a pore diameter of 150 to 300 μm is 10% or more, preferably 20% or more, more preferably 30% or more, further preferably 40% or more, particularly preferably 50% or more, cells easily invade, and Since the invading cells easily adhere and grow, it is effective for use as a scaffold material and an artificial blood vessel.

The contribution rate of pores having a pore diameter of 150 to 300 μm in the average pore diameter of the porous three-dimensional network means the pore diameter of 150 to 300 μm with respect to the total number of pores in the method for measuring the average pore diameter in Example 1 described later. Refers to the ratio of the number of holes.

With such a porous three-dimensional network structure having an average pore size, apparent density and pore size distribution, cells / collagen suspension culture solution and the like easily permeate into pores and cells adhere to the porous structure layer. It is possible to obtain a good scaffold material that is easy to grow. Therefore, when this is formed into a tubular shape, cells can be engrafted over the entire area from the inner wall to the outer circumference, so that an artificial blood vessel with a low risk of blockage and a high patency rate can be realized.

As the thermoplastic resin constituting the scaffold material for tissue engineering of the present invention, polyurethane resin, polyamide resin, polylactic acid resin, polyolefin resin, polyester resin, fluororesin, acrylic resin, methacrylic resin and their derivatives are used. These may be exemplified, and these may be used alone or in combination of two or more, but are preferably polyurethane resins, and among them, the segmented polyurethane resin is antithrombotic and physical properties. It is preferable because an excellent artificial blood vessel can be obtained in terms of the above.

The segmented polyurethane resin is synthesized from three components of a polyol, a diisocyanate and a chain extender, and has an elastomeric property due to a block polymer structure having a so-called hard segment portion and a soft segment portion in the molecule, and thus such a segment is used. The scaffold material and artificial blood vessel obtained by using the modified polyurethane resin have an SS curve that is elastically mechanically similar to that of a living blood vessel (high elasticity in a low blood pressure region and low elasticity, and a low blood pressure region in a high blood pressure region). It is also possible to form a tubular structure exhibiting lower compliance and high elasticity), and it has excellent antithrombotic properties and physical properties.

If the thermoplastic resin is hydrolyzable or biodegradable, the thermoplastic resin is gradually decomposed and absorbed after transplantation of an artificial blood vessel into a living body, and is finally made of resin while leaving engrafted cells. It is also possible to exclude the base material itself from the living body.

In the porous three-dimensional network structure composed of such a thermoplastic resin, collagen type I, collagen type II, collagen type III, collagen type IV, atelo type collagen, fibronectin, gelatin, hyaluronic acid, From heparin, keratanic acid, chondroitin, chondroitin sulfate, chondroitin sulfate B, copolymer of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, copolymer of hydroxyethyl methacrylate and methacrylic acid, alginic acid, polyacrylamide, polydimethylacrylamide and polyvinylpyrrolidone One or more selected from the group consisting of may be retained, and further, fibroblast growth factor, interleukin-1, tumor growth factor β, epidermal growth factor and diploid fibroblasts. One or more cytokines selected from the group consisting of cell growth factors may be retained, and further embryonic stem cells, vascular endothelial cells, mesodermal cells, smooth muscle cells, peripheral vascular cells and medium One or more cells selected from the group consisting of skin cells may be attached. The embryonic stem cells may be differentiated.

Further, in the scaffold material for tissue engineering of the present invention, it is possible to provide fine pores in the skeleton itself made of the thermoplastic resin that constitutes the porous three-dimensional network structure layer. Such fine pores make the skeleton surface not a smooth surface but a complex uneven surface, and it is also effective for retaining collagen, cell growth factor, etc. As a result, it is possible to improve cell engraftment is there. However, the fine pores in this case are not introduced into the concept of calculating the average pore diameter of the porous three-dimensional network structure portion in the present invention.

The shape of the scaffold material for tissue engineering of the present invention is not particularly limited, but for example, in the case of a tubular structure, it can be used as an artificial blood vessel.

In this case, the tubular structure has an inner diameter of 0.3 to
Outer diameter 0.4 to 20.0 mm at 15.0 mm, preferably inner diameter 0.3 to 10.0 mm and outer diameter 0.4 to 15.0 m
m, more preferably 0.3 to 6.0 mm inside diameter and 0.1 mm outside diameter.
4 to 10.0 mm, particularly preferably 0.3 to 2.5 inner diameter
It is preferable that the outer diameter is 0.4 to 10.0 mm in mm, particularly preferably the inner diameter is 0.3 to 1.5 mm and the outer diameter is 0.4 to 10.0 mm. Even with such artificial blood vessels, a high patency rate can be maintained over a long period of time.

The artificial blood vessel of the present invention made of the scaffold material of the present invention may have its outer side coated with another tubular structure, and by providing such a coating layer, the scaffold of the present invention is provided. Prevents blood from leaking for a certain period of time after transplantation when the density of impregnation of the hold material with collagen or the like is low or when the wall thickness of the scaffold material is thin, allowing sufficient cell adhesion and engraftment. It is also possible to give an auxiliary effect such that the substance is absorbed by the living body and disappears when the possibility of leak is low. The tubular structure for coating is not particularly limited, for example, chitosan,
Polylactic acid resin, polyester resin, polyamide resin, polyurethane resin, fibronectin, gelatin, hyaluronic acid, keratanic acid, chondroitin, chondroitin sulfate, chondroitin sulfate B, a copolymer of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, hydroxyethyl methacrylate A tube formed of one or more selected from the group consisting of a copolymer of methacrylic acid, alginic acid, polyacrylamide, polydimethylacrylamide, polyvinylpyrrolidone, cross-linked collagen and fibroin can be mentioned. The thickness of the tubular structure for coating (difference between outer diameter and inner diameter) is preferably about 5 to 500 μm.

The artificial blood vessel of the present invention is novel in that it has a high patency even if it has a small diameter, which cannot be achieved by the prior art, and can secure a stable blood flow. However, it has a large diameter, for example, an inner diameter of 6 mm. There is no problem even if it is applied to those exceeding.

An example of a method for producing a porous three-dimensional network structure composed of the thermoplastic polyurethane resin constituting the scaffold material or the tubular structure of the artificial blood vessel of the present invention will be given below. The method for producing the thermoplastic resin structure having the original network structure is not limited to the following method. Further, according to the following method, it is possible to manufacture a thermoplastic resin substrate having a three-dimensional network structure of various shapes required as a scaffold material for tissue engineering such as a planar substrate.

In order to produce a porous three-dimensional network structure composed of a thermoplastic polyurethane resin, first, a polyurethane resin, a water-soluble polymer compound described below as a pore-forming agent, and an organic solvent which is a good solvent for the polyurethane resin. A polymer dope is prepared by mixing with a solvent. Specifically, a polyurethane resin is mixed with an organic solvent to form a uniform solution, and then a water-soluble polymer compound is mixed and dispersed in this solution. Examples of the organic solvent include N, N-dimethylformamide, N-methyl-2-pyrrolidinone, and tetrahydrofuran. However, the organic solvent is not limited to this as long as the thermoplastic polyurethane resin can be dissolved. It is also possible to melt the polyurethane resin by the action of heat without using it and mix the pore-forming agent therein.

As the water-soluble polymer compound as a pore-forming agent, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
Examples thereof include alginic acid, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, etc., but not limited to these as long as they are uniformly dispersed with a thermoplastic resin to form a polymer dope. Depending on the type of thermoplastic resin, it is also possible to use a lipophilic compound such as phthalate ester or paraffin, or an inorganic salt such as lithium chloride or calcium carbonate, instead of the water-soluble polymer compound. It is also possible to promote the formation of secondary particles at the time of solidification, that is, the formation of the skeleton of the porous body, by utilizing a crystal nucleating agent for polymers.

The polymer dope produced from the thermoplastic polyurethane resin, the organic solvent and the water-soluble polymer compound is then immersed in a coagulation bath containing a poor solvent for the thermoplastic polyurethane resin, and the organic solvent and the organic solvent are added to the coagulation bath. The water-soluble polymer compound is extracted and removed. By removing a part or all of the organic solvent and the water-soluble polymer compound in this manner, a porous three-dimensional network structure material composed of a polyurethane resin can be obtained. As the poor solvent used here,
Examples include water, lower alcohols, and low carbon number ketones. The coagulated polyurethane resin may be finally washed with water or the like to remove the residual organic solvent and pore-forming agent.

[0030]

EXAMPLES The present invention will be described in more detail below with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples unless it exceeds the gist.

(Example 1) Thermoplastic polyurethane resin (Miractran E980PNA manufactured by Nippon Miractolan Co., Ltd.)
T) is N-methyl-2-pyrrolidinone (Kanto Chemical Co., Inc.,
Dissolver (about 2, for peptide synthesis reagent, NMP)
(000 rpm) at room temperature to give a 5.0% solution (w / w). About 1.0 kg of this NMP solution
Was weighed and placed in a planetary mixer (manufactured by Inoue Seisakusho, 2.0 L, PLM-2 type), and methylcellulose (manufactured by Kanto Chemical Co., Inc., reagent, 25 cp grade) equivalent in weight to the polyurethane resin was added at 40 ° C. for 20 minutes. Mix and then continue stirring for 10 minutes at 20 mmHg (2.
The pressure was reduced to 7 kPa) for defoaming to obtain a polymer dope.

An inner diameter of 3.5 mmφ and an outer diameter of 4.6 m prepared with a filter paper for chemical experiments (manufactured by Toyo Roshi Kaisha, for qualitative analysis, No. 2).
Tube molding consisting of a cylindrical paper tube with mφ and a length of 60 mm, a core rod made of SUS440 with a diameter of 1.2 mmφ, and a cylindrical polypropylene resin stopper that can fix this core rod to the center of the paper tube. The polymer dope was injected and injected into the jig using a 23-gauge needle, which was then tightly plugged and then placed in refluxing methanol to continue refluxing for 72 hours to The polyurethane resin was coagulated by extracting and removing the NMP solvent. At this time, the methanol was exchanged with a new liquid at any time while maintaining the reflux state. After 72 hours, the tube forming jig is transferred from the refluxed methanol into a methanol bath at room temperature without being dried, and the contents are taken out from the tube forming jig in the bath and washed in purified water in the Japanese Pharmacopoeia for 72 hours. Methyl cellulose, methanol and residual NMP
Was removed by extraction. For cleaning water, fresh liquid was supplied as needed. This was depressurized at room temperature for 24 hours (20 mmHg (2.7 kP
a)) It was dried to obtain a scaffold material having a tubular porous three-dimensional network structure that can be used as an artificial blood vessel, which is an embodiment of the present invention.

1 to 4 show this scaffold material as a scanning electron microscope (SEM, JMS-5800 manufactured by JEOL).
LV) or stereomicroscope (VH-630 manufactured by Keyence Corporation)
It is an image photographed in 0). From FIGS. 1 to 4, the obtained scaffold material substrate has a pore diameter of about 200 μm,
The inner diameter is 1.2 mmφ, the outer diameter is 3.2 mmφ, and the inside of the structure (Fig. 2), the inner wall surface layer (Fig. 3) and the outer peripheral surface layer (Fig. 4) are almost the same structure as a porous three-dimensional net structure, It can be seen that is a homogeneous porous body.

The average pore size and the apparent density of the obtained scaffold material were measured by the following methods. In the measurement of the average pore size and the apparent density, the sample was cut at room temperature using a double-edged razor (made by Feather, high stainless steel).

[Measurement of Average Pore Diameter] Individual holes on the same plane were taken using a photograph of a plane (cut surface) of a sample cut with a double-edged razor using a stereomicroscope (VH-6300 manufactured by Keyence Corp.). Image processing as a figure surrounded by the skeleton of the three-dimensional network structure (the image processing device is L
UZEX AP was used, and the image capturing CCD camera was LE N50 from Sony. ), And the area of each figure was measured. This was taken as the true circle area, and the diameter of the corresponding circle was determined and used as the hole diameter. As a result of ignoring the fine holes formed in the skeleton of the porous body and measuring only the communicating holes on the same plane, the average hole diameter was measured to be 169 ± 55 μm. At the same time, the contribution of the pore size of 150 to 300 μm in the pore size distribution was measured to be 71.2%, and it was confirmed that the porous body was mainly composed of pores of a size effective for cell adhesion.

[Measurement of Apparent Density] A sample cut with a double-edged razor to a length of about 10 mm was projected by a projector (Nikon, V-
The volume was obtained from the dimensions obtained by the measurement in 12), and the weight was divided by the volume. The result was 0.077 ± 0.0.
It was calculated to be 02 g / cm ³ .

The three-dimensional network structure, which is a feature of the present invention, is a structure having excellent hole-to-hole communication. The water permeability, which is an index of this communication, was evaluated as follows.

[Evaluation of Water Permeability] First, one end of a sample cut into a length of 10 mm in the same manner as described above was tightly plugged, and an opening at the other end gave an inner diameter of 0.3 mmφ and an outer diameter of 1.2 mm.
A needle having a diameter of 40 mm and a length of 40 mm was adjusted and inserted so that the effective permeation surface of the tubular structure of the sample was 0.50 mm in tube length. A silicon tube with a length of 50 mm and a diameter of 5 mmφ and a needle containing 25 g of water, a diameter of 20 mmφ and a length of 9
A 0 mm gator was connected and the permeability of distilled water was measured at 25 ° C. The water permeability is 13.47 ± 0.33g / 60 seconds,
It was 24.64 ± 0.35 g / 120 seconds. The water permeation rate in the unloaded state without wearing this sample was 13.70.
From / 60 seconds and 24.87 / 120 seconds, it was confirmed that this scaffold material was a highly conductive three-dimensional network structure having good water permeability.

Example 2 Bovine blood vessel-derived smooth muscle cells (cell density: 6 × 10 ⁶ cells / mL) in DMEM (medium component) (containing 10% FCS (fetal calf serum)) and collagen type I solution ( A 0.3% acidic solution (manufactured by Koken) was mixed in an equal amount while cooling on ice to prepare a suspension of smooth muscle cells (cell density: 3 × 10 ⁶ cells / mL).

One end of the tubular porous three-dimensional mesh structure scaffold material (inner diameter: 1.2 mmφ, outer diameter: 3.2 mmφ, length: 2 cm) produced in Example 1 was bound with a clamp and the other end. Further, the suspension of smooth muscle cells (1 mL) was injected until it exudes from the side wall of the tubular structure of the scaffold material. All the injection operations were performed on ice, and the tubular structure was filled up with a collagen solution containing smooth muscle cells by repeating several times. Then, the clamp was removed, and 1.2 made of SUS440 was attached to the center of the tubular structure made of scaffold material.
The cells were passed through a mmφ mandrel and cultured at 37 ° C. in an incubator to obtain a porous three-dimensional network structure material containing cells.

The cross-sectional tissue image observed by an optical microscope after culturing the porous three-dimensional network structure material containing cells thus obtained for 3 days is shown in FIG. From FIG. 5, it can be seen that cells are distributed over the entire surface inside the manufactured structural material. It was observed that the internal structure of the cell-containing structure was engrafted without necrosis even after additional culture for 1 week (FIG. 6).

Example 3 The scaffold material having a tubular porous three-dimensional network structure produced in Example 1 (inner diameter: 1.
One end of 2 mmφ, outer diameter: 3.2 mmφ, length: 2 cm) was bound with a clamp, and a collagen type I aqueous solution (0.15% by weight) was injected from the other end to fill the inside of the structure with the collagen solution. After that, the clamp was removed, and a 1.2 mmφ mandrel made of SUS440 was passed through the center of the tubular structure of the scaffold material, and the collagen solution was gelated by keeping it in the incubator at 37 ° C. A tubular structure filled with collagen gel was prepared.

The abdominal aorta of a rat was peeled off by about 3 cm, the both ends were bound with clamps to block the blood flow, and then the central part of the artery was cut and the ends were joined by the tubular structure. When the blood flow was restarted after removing the clamp, pulsation occurred and it functioned as an artificial blood vessel (Fig. 7). When this artificial blood vessel was taken out after one week and the luminal surface of the tubular tissue body was observed, no thrombus was attached to the luminal surface, and it was extremely smooth (FIG. 8).

(Comparative Example 1) Thermoplastic polyurethane resin (Miractran E980PNA manufactured by Nippon Miractolan Co., Ltd.)
T) is tetrahydrafuran (made by Wako Pure Chemical Industries, TH
It was heated and dissolved in F) at 60 ° C. to obtain a 5.0% solution (weight / weight). 12 g of NaC was added to 16 mL of this THF solution.
l particles (particle size of 100 to 200 μm by sieving treatment)
To prepare a suspension in which A 1.2 mmφ mandrel made of SUS440 was dipped in this suspension, dried, and a tube-like film was formed around the mandrel with polyurethane containing NaCl particles. After drying it thoroughly,
It was thoroughly washed with ion-exchanged water to dissolve and remove the NaCl embedded in the tube. This was dried under reduced pressure (20 mmHg (2.7 kPa)) at room temperature for 24 hours to obtain a porous tubular structure having an inner diameter of 1.2 mmφ and an outer diameter of 3.2 mmφ.

Example 1 for this porous tubular structure
When the average pore size and the apparent density were measured by the same method as described above, the average pore size was 121 ± 65 μm, but the pore size was 1
The contribution rate of the holes of 50 to 300 μm was 31.8%.
Moreover, the apparent density is 0.086 ± 0.004 g / cm ^3.
Met.

According to the appearance observation by SEM, in Example 1, the surface layer and the inside have the same three-dimensional network structure, whereas in this comparative example, a dense layer is formed in the surface layer portion. (FIG. 9), the surface layer part and the inside are completely different structures, and the internal structure is a three-dimensional network structure in which spherical holes are gathered and the wall of the hole penetrates at the part where adjacent holes are in contact with each other. Not (Fig. 10).

The water permeability measured by the same method as in Example 1 was 11.22 ± 0.46 g / 60 seconds, 20.08.
The value was ± 0.96 g / 120 seconds, which was lower than that of Example 1. It was presumed that this was due to the low connectivity of the holes in the surface layer and the influence of the dense layer existing in the surface layer.

Comparative Example 2 A suspension of smooth muscle cells prepared in the same manner as in Example 2 (cell density: 3 × 10 ⁶ cells)
s / mL) is the porous tubular structure produced in Comparative Example 1 (inner diameter: 1.2 mmφ, outer diameter: 3.2 mmφ, length: 2
cm) and injecting it in the same manner as in Example 2 and then culturing to obtain a tubular structural material containing cells.

The tubular structure material containing cells thus obtained was cultured for 3 days and then observed with an optical microscope. From FIG. 11, it can be seen that almost no cells exist inside the manufactured structural material, but only on the inner wall surface.

[0050]

As described in detail above, according to the present invention, a tissue-engineering scaffold that is composed of a homogeneous porous body having a three-dimensional network structure and in which cells can be uniformly engrafted on the entire inner surface of the porous structure. As a hold material, it has excellent physical strength, and not only basic research in the field of tissue engineering, but also artificial blood vessels, especially,
A scaffold material for tissue engineering capable of constructing an artificial blood vessel capable of maintaining a high patency rate for a long period of time even when applied to an artificial blood vessel having a small diameter of 6 mm or less,
An artificial blood vessel using the scaffold material for tissue engineering is provided.

[Brief description of drawings]

FIG. 1 is an SEM image (20 times) of the entire tubular structure of the scaffold material manufactured in Example 1.

FIG. 2 is a stereoscopic microscope image (100 ×) of the internal fine structure of the tubular structure of the scaffold material manufactured in Example 1.

FIG. 3 is a SEM image (20 times) of the surface layer of the inner wall of the tubular structure made of the scaffold material manufactured in Example 1.

FIG. 4 is an SEM image (20 times) of the outer surface layer of the tubular structure of the scaffold material manufactured in Example 1.

5 is an optical microscope image (10 ×) of the porous three-dimensional network structure material containing cells produced in Example 2 after 3 days of culture.
Is.

FIG. 6 is an optical micrograph (× 10) showing that internal tissues were engrafted on the entire surface even after additional culture for 1 week in Example 2.

FIG. 7 is a photograph of a scene in which blood flow is secured by an artificial blood vessel and pulsation occurs in Example 3.

FIG. 8 is a photograph showing that no thrombus is formed inside the artificial blood vessel one week after the transplantation in Example 3.

9 is an S of the surface layer portion of the annular structure manufactured in Comparative Example 1. FIG.
It is an EM image (50 times).

FIG. 10 is an SEM image (50 times) of a fine structure inside the annular structure manufactured in Comparative Example 1.

FIG. 11 is an optical microscope cross-sectional image (10 times) of the tubular structure material containing cells produced in Comparative Example 2 after 3 days of culture.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4C081 AB13 AC03 BA12 BA13 DA03                       DB03 DB05                 4C097 AA15 BB01 CC03 DD02 EE02                       EE03 EE08 FF05

Claims (27)

[Claims] 1. A scaffold material for tissue engineering made of a thermoplastic resin, wherein the thermoplastic resin has an average pore diameter of 100 to 6
50 μm, apparent density of 0.01 to 0.5 g / cm ³ ,
A scaffold material for tissue engineering, which is characterized by forming a porous three-dimensional network structure having continuity. 2. The scaffold for tissue engineering according to claim 1, wherein the porous three-dimensional network structure has an average pore diameter of 100 to 400 μm and an apparent density of 0.01 to 0.5 g / cm ^3. Hold material. 3. The scaffold material for tissue engineering according to claim 2, wherein the porous three-dimensional network structure has an average pore diameter of 100 to 300 μm. 4. The apparent density of the porous three-dimensional network structure according to claim 1, which is 0.01 to
A scaffold material for tissue engineering, which is 0.2 g / cm ³ . 5. The scaffold material for tissue engineering according to claim 4, wherein the porous three-dimensional network structure has an apparent density of 0.01 to 0.1 g / cm ³ . 6. The pore size 1 according to claim 1, wherein the average pore size of the porous three-dimensional network structure is 1.
A scaffold material for tissue engineering, characterized in that the contribution of pores of 50 to 300 μm is 10% or more. 7. The scaffold material for tissue engineering according to claim 6, wherein the contribution ratio of pores having a pore diameter of 150 to 300 μm in the average pore diameter of the porous three-dimensional network structure is 20% or more. 8. The scaffold material for tissue engineering according to claim 7, wherein the contribution ratio of pores having a pore diameter of 150 to 300 μm in the average pore diameter of the porous three-dimensional network structure is 30% or more. 9. The scaffold material for tissue engineering according to claim 8, wherein the contribution ratio of pores having a pore diameter of 150 to 300 μm in the average pore diameter of the porous three-dimensional network structure is 40% or more. 10. The scaffold material for tissue engineering according to claim 9, wherein the contribution ratio of pores having a pore diameter of 150 to 300 μm in the average pore diameter of the porous three-dimensional network structure is 50% or more. 11. The thermoplastic resin according to claim 1, wherein the thermoplastic resin is a polyurethane resin, a polyamide resin, a polylactic acid resin, a polyolefin resin, a polyester resin, a fluororesin, an acrylic resin and a methacrylic resin, and derivatives thereof. A scaffold material for tissue engineering, which is one or more selected from the group consisting of: 12. The scaffold material for tissue engineering according to claim 11, wherein the thermoplastic resin is a polyurethane resin. 13. The scaffold material for tissue engineering according to claim 12, wherein the polyurethane resin is a segmented polyurethane resin. 14. The collagen type I, the collagen type II, the collagen type III, the collagen type IV, the atelo type collagen, the fibronectin, the gelatin according to any one of claims 1 to 13. , Hyaluronic acid, heparin, keratanic acid, chondroitin, chondroitin sulfate, chondroitin sulfate B, copolymer of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, copolymer of hydroxyethyl methacrylate and methacrylic acid, alginic acid, polyacrylamide, polydimethylacrylamide And a scaffold material for tissue engineering, which retains one or more selected from the group consisting of polyvinylpyrrolidone. 15. The porous three-dimensional network structure according to claim 14, further comprising fibroblast growth factor, interleukin-1, tumor growth factor β, epidermal growth factor and diploid fibroblast growth factor. 1 or 2 selected from the group consisting of
Scaffold material for tissue engineering, characterized by holding more than one species. 16. The scaffolding material for tissue engineering according to claim 15, which is cell-adhered to the porous three-dimensional network structure portion. 17. The method according to claim 16, wherein the cells are one or more selected from the group consisting of embryonic stem cells, vascular endothelial cells, mesodermal cells, smooth muscle cells, peripheral vascular cells and mesothelial cells. A scaffold material for tissue engineering, which is characterized by: 18. The scaffold material for tissue engineering according to claim 17, wherein the embryonic stem cells are differentiated. 19. The scaffold material for tissue engineering according to claim 1, wherein the scaffold material has a tubular structure. 20. The tubular structure according to claim 19, wherein the inner diameter is 0.3 to 15.0 mm and the outer diameter is 0.4 to 20.
A scaffold material for tissue engineering, which has a thickness of 0 mm. 21. The tubular structure according to claim 20, wherein the inner diameter is 0.3 to 10.0 mm and the outer diameter is 0.4 to 15.
A scaffold material for tissue engineering, which has a thickness of 0 mm. 22. In claim 21, the tubular structure has an inner diameter of 0.3 to 6.0 mm and an outer diameter of 0.4 to 10.0.
A scaffold material for tissue engineering, which is characterized by being mm. 23. The tubular structure according to claim 22, wherein the tubular structure has an inner diameter of 0.3 to 2.5 mm and an outer diameter of 0.4 to 10.0.
A scaffold material for tissue engineering, which is characterized by being mm. 24. The tubular structure according to claim 23, having an inner diameter of 0.3 to 1.5 mm and an outer diameter of 0.4 to 10.0.
A scaffold material for tissue engineering, which is characterized by being mm. 25. An artificial blood vessel comprising the scaffold material according to any one of claims 1 to 24. 26. The artificial blood vessel according to claim 25, wherein the outside of the scaffold material is covered with another tubular structure. 27. The tubular structure for coating the outside of the scaffold material according to claim 26, wherein chitosan, polylactic acid resin, polyester resin, polyamide resin, polyurethane resin, fibronectin, gelatin, hyaluronic acid, keratanic acid, chondroitin , Chondroitin sulfate, chondroitin sulfate B, copolymer of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, copolymer of hydroxyethyl methacrylate and methacrylic acid, alginic acid, polyacrylamide, polydimethylacrylamide, polyvinylpyrrolidone, crosslinked collagen and fibroin An artificial blood vessel, which is a tube formed of one or more selected from the group consisting of: