JPH03272772A - Hollow microorganism cellulose, its manufacture, and artificial blood vessel made of cellulose - Google Patents

Abstract

PURPOSE:To provide an artificial blood vessel having excellent blood adaptability, durability and uniformity and enable substitution for a minor bore blood vessel by allowing it to contain cellulose produced by microorganisms. CONSTITUTION:Microorganisms producing cellulose are cultivated on the internal surface and/or external surface of a hollow carrier capable of dipping oxygen, such as cellophane, teflon, silicon, ceramic, unwoven cloth, fabric, etc. The The cellulose produced by the microoganism is composed alpha-cellulose having high crystallinity is very strong in the surface orientation property, has a very high strength, presents less deterioration in a vital organism, and impracticality excels in the vital organism adaptability.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a hollow cellulose made of cellulose produced by microorganisms, a method for producing the cellulose, and an artificial blood vessel made of the cellulose.

This hollow microbial cellulose, like other synthetic polymer materials, is used as an immobilization carrier for various enzymes, bacterial bodies, cells, etc., a tubular industrial material, a medical material, a chemical material, etc. For example, in the case of medical materials, they are used as substitutes for the ureters, trachea, digestive tract, lymphatic vessels, blood vessels, and other hollow organs in the body.

[Conventional technology and issues]

Conventionally, microorganisms, especially acetic acid bacteria, have been used to produce cellulose (hereinafter, cellulose produced by microorganisms is called "microbial cellulose").
It is widely known that these materials are produced and used in various industrial materials, medical materials, chemical products, etc.

In the technique disclosed in JP-A No. 62-36467, membrane cellulose produced by acetic acid bacteria is press-dried,
Alternatively, after being disintegrated, it is provided as a high mechanical strength material either alone or in a composite with other materials. JP-A-62-87099 discloses a method for producing bead-shaped microbial cellulose used as an immobilization carrier and the like. JP-A-61-221201 discloses a method for producing ultrafine particles by subjecting microbial cellulose to acid hydrolysis or the like. Also, JP-A-59-1
Publication No. 20159, Japanese Unexamined Patent Publication No. 152601/1983,
As disclosed in JP-A No. 01-50815, this microbial cellulose has very high biocompatibility, so it can be used in medical pads, artificial skin, cultured skin carriers, carriers for mass cell culture, and oral adhesives. It is said to be very good.

In the produced state, microbial cellulose has a network-like structure in which very thin (approximately 1100 nm in width or diameter) ribbon-like fibers made of highly crystalline and uniaxially oriented cellulose are intricately intertwined. I am doing it. This network-like structure contains a large amount of liquid in its voids and has a gel-like appearance. Liquid components, such as water, contained in the voids within this microbial cellulose structure exist as free water and are easily squeezed out when external force is applied. Since it is composed of a large number of ribbon-like fibers with extremely high crystallinity, it can withstand external forces such as tension even when wet.

Microbial cellulose has no difference in primary structure from cellulose of plant origin, but the so-called higher order structure such as the network mentioned above is not found in cellulose of plant origin and is unique to microbial cellulose. Yes, therefore,
It exhibits various unique properties, such as being strong despite being gel-like.

However, there are many problems when comparing microbial cellulose as a polymer material with other polymer materials widely used in the world. This is because thermoplastic polymer materials such as polyethylene and polyester have plasticity, so they can be made to the desired size or size without changing their physical properties by applying heat or adding a softening agent. Can be molded into shapes. It is also possible to dissolve it in a solvent or the like and mold it into various shapes, or to laminate it.

On the other hand, microbial cellulose does not have this kind of plasticity because it has a network structure as mentioned above, and when dissolved in a solvent, it loses the characteristics based on its higher-order structure, making it a cellulose of plant origin. There will be no difference.

When microbial cellulose is produced, it has a gel-like form. In other words, microbial cellulose is composed of thin fibers as mentioned above, and contains more than 95% of the liquid component by weight in the voids between the fibers. The liquid component in the voids is not molecularly restricted like in general polymer gels. For example, when the liquid component is water, most of it exists as free water in gel-like microbial cellulose, and it is possible to squeeze out the liquid component by simply pinching it with your fingers.

Because it has no plasticity and the liquid components it contains are not restricted, this gel-like material can be molded into the desired shape and size while retaining its original structure and properties. It was very difficult to do so. For example, until now it has been impossible to obtain long strips of several meters or hundreds of meters, spherical objects several centimeters in size, and hollow objects such as pipes. Therefore, there are naturally limitations regarding industrial use in terms of shape, size, and workability. For example, when used as a hollow fixed carrier or a hollow organ to be implanted in the human body, such as the ureter, trachea, digestive tract, lymphatic vessel, or blood vessel, sufficient strength is required. It was impossible to create artificial organs.

Looking at artificial blood vessels in particular among artificial organs, we have succeeded in replacing large-caliber arteries (with an inner diameter of more than 6 mm) with artificial blood vessels, but small-caliber arteries, which account for the majority of systemic blood vessels, Small-diameter artificial blood vessels that can replace arteries, veins, and capillaries have not yet been developed.

This is because the problem of occlusion due to thrombus formation in small-caliber artificial blood vessels has not been resolved. That is,
Artificial blood vessels that have been used clinically so far have simple structures such as polyester m-fiber woven or knitted fabrics, or polytetrafluoroethylene porous materials, and when transplanted into a living body, living tissue is It functions as an artificial blood vessel only after it is formed around that skeleton. Therefore, in small-caliber blood vessels, problems such as occlusion due to thrombus formation are likely to occur until the pseudo-living tissue is formed.

Thrombi block the flow of blood or move along with the blood flow, causing cerebral thrombosis, myocardial infarction, pulmonary infarction, etc., resulting in a serious crisis for the human body.

As mentioned above, when considering alternatives to small-caliber blood vessels, stricter control of thrombus formation is required until stable biological tissue is formed, and various approaches have been taken in various fields to ensure this is the national authority. There is. Typical examples include heparinization, urokinase immobilization, and plasma treatment.

[Problem to be solved by the invention]

An object of the present invention is to provide a hollow material that is excellent as an immobilization carrier, an industrial material, a medical material including artificial organs, a chemical material, etc.

Another object of the present invention is to provide an artificial blood vessel that has excellent biocompatibility and can be used to replace small-caliber blood vessels with an inner diameter of 5 mm or less.

[Means to solve the problem]

The above problem can be solved by culturing cellulose-producing microorganisms on the inner and/or outer surfaces of oxygen-permeable hollow carriers, such as cellophane, Teflon, silicone, ceramics, nonwoven fabrics, and textiles. Achieved by cellulose. In addition, cellulose produced by microorganisms is impregnated with a medium and, if necessary, further hardened.
This is achieved by a hollow microbial cellulose molded article obtained by cutting.

Cellulose produced by microorganisms has characteristics such as being composed of highly crystalline α-cellulose, having extremely strong surface orientation, extremely high strength, and little in-vivo deterioration. Excellent compatibility (especially blood compatibility).

Cellulose produced by microorganisms includes cellulose, heteropolysaccharides with cellulose as the main chain, and β and α
Contains glucan such as. In the case of heteropolysaccharides, components other than cellulose include hexoses, pentoses, and organic acids such as mannose, fructose, galactose, xylose, arabinose, rhamnose, and uronic acid. There are celluloses in which these polysaccharides exist as a single substance, and there are celluloses in which two or more types of polysaccharides coexist.

Microorganisms that produce such cellulose are not particularly limited, but include, for example, Acetobacter pasteurianus.
ATCC10821, aceti (A, aceti),
A, ransens, Sarcina ventriculi
, Bacterium chydylloides
xyloides), bacteria of the genus Pseudomonas, bacteria of the genus Agrobacterium, bacteria of the genus Rhizobium, and the like.

In order to produce and accumulate cellulose, the above-mentioned microorganisms may be used in accordance with a general method for culturing ordinary bacteria.

That is, it may be added to a conventional nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary, and cultured at a temperature of 20°C to 40°C.

The first method for producing hollow microbial cellulose is to transform cellulose-producing microorganisms into oxygen-permeable hollow objects,
For example, this is a method of culturing on the inner and/or outer surface of a hollow body made of cellophane, Teflon, silicone, ceramic, nonwoven fabric, textile, etc. Specifically, an object that is oxygen permeable and has a hollow shape (hereinafter referred to as a "hollow carrier"),
For example, cellophane, Teflon, silicon, ceramic,
A hollow carrier made of nonwoven fabric, textile, etc. is immersed in a culture solution,
Production bacteria and a medium are placed outside and/or inside a hollow carrier, and an oxygen-containing gas or liquid is introduced into the outside and/or inside for cultivation. If the hollow carrier is hydrophilic,
It is sufficient to simply attach a culture solution containing bacteria to the inner and/or outer surfaces thereof. By culturing using such an apparatus, gel-like cellulose with a thickness of 0.01 to 20 IO [11] is formed on the surface of the carrier. If there is any interaction between the cellulose-producing bacteria and/or the produced cellulose and the hollow carrier, and if the bacteria and/or cellulose are attached or adhered to the carrier, the interaction between the cellulose and the hollow carrier will eventually occur. A complex is formed. In addition, if the hollow carrier has holes of 0, 1 to 5 μm through which cellulose or bacteria can pass, the cellulose formed on one side and the opposite side of the hollow carrier may be connected to each other. be. As described above, a composite in which microbial cellulose is attached to the inner and/or outer surface of a hollow carrier is obtained. If the cellulose and the carrier are not attached or adhered to each other, a hollow molded article made of cellulose alone can be obtained by removing the hollow carrier after producing the cellulose.

Since the cellulose thus produced contains bacterial cells or medium components, it may be washed depending on the purpose.

Cleaning may be carried out using dilute alkali, dilute acid, organic solvent, hot water, etc. alone or in combination.

In addition to creating a composite in which cellulose is attached to a hollow carrier as described above, substances other than microbial cellulose are used as auxiliary materials, and these are integrally included in the hollow cellulose to form a composite. You can also do that. These auxiliary materials include granular and fibrous inorganic or organic materials such as alumina, glass, and crystalline cellulose, agar, dextran, polyacrylamide, polyvinylpyrrolidone, alginate, chitin, hyaluronic acid, curdlan, and polyacrylic acid. Water-soluble salts, heparin, sulfated polysaccharides, pullulan, carrageenan, glucomannan, cellulose derivatives, polyethylene glycol, polyvinyl alcohol, gelatin, collagen, laminin, fibronectin, keratin, silk hydrolyzate, polyamino acids, polyorganic acids, enzymes, etc. Examples include polymeric materials and other materials that form soluble or hydrophilic gels in neutral or polar solvents. By impregnating, laminating, or adsorbing these auxiliary materials onto hollow microbial cellulose, a composite material of hollow microbial cellulose and these materials can be obtained. Further, a composite material may be obtained in which a granular or gel-like auxiliary material is incorporated into the three-dimensional structure of microbial cellulose, or a fibrous auxiliary material is entangled with the structure of cellulose.

A second method for obtaining hollow microbial cellulose is to impregnate cellulose produced by microorganisms with a medium, further harden if necessary, and then cut. That is,
First, bacteria are inoculated into a container containing the medium described above, and static culture is performed to produce gel-like microbial cellulose on the surface of the medium.

This microbial cellulose is impregnated with a medium, and if necessary, it is hardened, frozen, and increased in viscosity to restrain the liquid components between the fibers that make up the microbial cellulose and prevent them from moving freely. Process.

Examples of media include polyhydric alcohols such as glycerin, erythritol, glyconopesorbitol, and maltitol, sugars such as glucose, caractose, mannose, maltose, and lactose, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, carboxymethylcellulose, agar, Natural and synthetic polymers such as starch, alginates, xanthan gum, polysaccharides, oligosaccharides, collagen, gelatin, and proteins, and water-soluble polar solvents such as acetonitrile, dioxane, acetic acid, and propionic acid are used. These media may be used alone or in a mixture of two or more.

Alternatively, it may be a solution containing an appropriate solute.

After impregnating the microbial cellulose with the medium, preferably
By freezing or solidifying the impregnated medium by cooling, or by inducing a suitable chemical reaction such as crosslinking to harden or increase the viscosity of the impregnated medium, the bond between the microbial cellulose fibers can be improved. The liquid component is restricted. This restriction of the liquid component makes it possible to easily cut the gel-like microbial cellulose using a common machine tool, such as a drill.

For example, to specifically explain the method of performing the above series of treatments using glycerin, the concentration ranges from 0.5% to 40% by weight.
After immersing gel-like microbial cellulose produced by static culture in an aqueous glycerin solution for several hours to several tens of hours, it is frozen at -80°C and then holed in the frozen state using a tool such as a cork polar. Process it into the desired shape by drilling it or cutting it with something like a knife. Thereafter, a hollow microbial cellulose molded article can be obtained by returning it to room temperature and immersing it in an appropriate solvent such as water or physiological saline to remove glycerin between the fibers.

When the cellulose produced in this manner is dried, the thin ribbon-like fibers that make up the gelled cellulose stick to each other through hydrogen bonds, resulting in a rigid film-like or plastic-like shape.

To adjust the hardness, the amount of hydrogen bonds derived from the hydroxyl groups of cellulose during drying is adjusted by adding a softening agent such as glycerin. Moreover, if freeze drying, critical point drying, drying after solvent replacement, etc. are performed to prevent hydrogen bonding during drying, it is possible to create a porous film that is different from a rigid film.

The third method for creating hollow microbial cellulose molded products is to prepare two glass tubes with different diameters, insert the smaller diameter glass tube into the larger diameter glass tube, and then create a cylinder between these glass tubes. Cellulose is produced in a cylindrical shape by culturing microorganisms in the gaps of the shape, which can be taken out as is.

The prepared hollow microbial cellulose molded product and its composite can be further shaped into a bellows shape or the like to make a molded product with good handling and flexibility.

The hollow microbial cellulose molded articles and composites thereof prepared in this way can be manufactured into various shapes as mentioned above, and therefore, like other synthetic polymer materials, various enzymes can be used. It can be used as an immobilization carrier for bacteria, cells, etc., hollow industrial materials, medical materials, chemical materials, etc.

When hollow microbial cellulose is used as an artificial blood vessel, it may be used as is to replace an in-vivo blood vessel, or it may be subjected to some pretreatment. As a pretreatment, it is possible to attach endothelial cells to the surface in advance, for example.

Hollow microbial cellulose has excellent biocompatibility, particularly blood compatibility, and has strong surface orientation and high mechanical strength. When this is used as an artificial blood vessel material, thrombus formation is suppressed, and it can be sufficiently replaced with small-diameter blood vessels with an inner diameter of 5 mm or less.

〔Example〕

Hereinafter, the present invention will be specifically explained based on Examples. In the examples, 1% is based on weight.

Example 1 Chouce o-su 5g/di, yeast extract (Difco)
0.5g/di, ammonium sulfate 0.5g/dl, monopotassium phosphate 0.3 g/dL magnesium sulfate heptahydrate 0.05
A medium with a composition of g/dl (pH 5,0) was incubated at 120°C for 20
After autoclaving for 1 minute, Acetobacter aceti subspicis xylinum
er acetisubspecies
m) (ATCC 10821) was inoculated at a concentration of 1×10′ cells/ml. Pour this liquid into a tube with an inner diameter of 5 mm and a length of 15 cm.
The cells were placed in a cellophane dialysis tube, tied and sealed at both ends, and cultured in air at 30°C for 3 weeks. A gel-like microbial cellulose with a thickness of about 2 ml was formed inside the dialysis tube. After collecting this, it was washed in a 2% hydroxide solution to remove bacterial cells and medium components. This was washed with excess water to obtain hollow gel-like microbial cellulose.

Example 2 The same method as Example 1 was applied to the inner diameter 3 [11111, outer diameter 4r]
nm.

A hollow gel-like microbial cellulose was obtained using a silicone tube with a length of 30 cm.

Example 3 A silicone tube with an inner diameter of 2 mm, an outer diameter of 3 mrn, and a length of 10 cm was placed in a culture medium inoculated with the bacteria described in Example 1. While applying a pressure of atmospheric pressure plus 0.1 atm inside the silicone tube, 10- Air was vented for 7 minutes. After two weeks, the microbial cellulose produced around the tube was collected and washed to obtain hollow microbial cellulose.

Example 4 A cylindrical cotton cloth with an inner diameter of 3 mm, a length of 10 cm, and a thickness of approximately 0.5 mm was placed in a container filled with a culture medium inoculated with bacteria to a depth of 30 cm as described in Example ↓. 50rrL of humidified sterile air per minute inside the cylindrical cloth.
It was flowed at a speed of 1. At this time, air was prevented from leaking from the surface of the hollow cloth. After two weeks, a gel-like microbial cellulose with a thickness of about 1 to 2 mm was formed on the inner and outer walls of the fabric. This was added to a 4% NaOH solution at 70°C for 3 hours.
Washed bacterial cells and medium components were removed by soaking for a period of time. A composite of cotton-like cloth and microbial cellulose was obtained.

Example 5 The composite of microbial cellulose and hollow cloth obtained in Example 4 was immersed in a 0.5% glycerin solution at room temperature for 5 hours.

This was dried to obtain a dried composite product in which cotton-like cloth and microbial cellulose were integrated.

Example 6 Inner diameter 3111 [11, length 10 cm, thickness approximately 0.511]
1 [1] A hollow cotton cloth made of 11 was placed in a container filled with a culture medium inoculated with bacteria to a depth of 30 cm as described in Example 1, and the inside of the hollow cloth was saturated with humidified air. Fluorinert FC-40 (manufactured by 3M) was flowed at a linear velocity of 3 cm per minute. After 2 weeks, gel-like microbial cellulose had formed on the inner and outer walls of the fabric to a thickness of about 1 to 2 u. The washed bacterial cells and medium components were removed by immersing this in a 4% NaOH solution at 70° C. for 3 hours. A composite of cotton-like cloth and microbial cellulose was obtained.

Example 7 Acetobacter aceti subspice xylinum (ATCC 10821) was added 1X to the medium described in Example 1.
It was inoculated at a concentration of 10' cells/-. This solution was placed in a double cylindrical test tube that had been autoclaved in advance, and cultured in air at 30 degrees for two months. A gel-like hollow microbial cellulose with a thickness of about 8 cm was produced on the surface of the culture solution. After collecting this, it was boiled for 1 hour in 10 times the amount of 2% sodium hydroxide solution. This boiling operation was repeated three times. Through this operation, bacterial cells and medium components were removed.

The hollow microbial cellulose after boiling was washed with excess water until the pH became neutral, and dried at 105 degrees for 4 hours to obtain a hollow microbial cellulose with a diameter of 1 mm.

Example 8 10 liters of the culture medium prepared according to the method of Example 7 was placed in a container of 30 cm square and 20 cm deep that had been autoclaved in advance, and the mixture was heated in air for 30 min.
The cells were cultured for 50 days at 30°C. A membrane-like cellulose with a thickness of about 3 centimeters was formed on the surface of the culture solution. After collecting this, 1
Boiling was carried out for 1 hour in 0 times the volume of 2% sodium hydroxide solution. This boiling operation was repeated three times. Through this operation, bacterial cells and medium components were removed. The membrane cellulose after boiling was washed with excess water until the pH became neutral. This is 1
After being immersed in a 0% glycerin solution for 10 hours, it was held at -80 degrees for 10 hours. This was molded using special thin-blade cork polar to a length of 20 cm, an inner diameter of 2 mm, and an outer diameter of 8 mm. This was boiled in distilled water for 3μ minutes. After drying this at 105 degrees for 4 hours,
Autoclave at 120 degrees for 30 minutes and add heparin 10/-
It was immersed in the solution for one day and night, and dried aseptically to obtain a composite of hollow microbial cellulose.

Example 9 Using the culture medium described in Example 1 and the double cylindrical test tube described in Example 7, Acetobacter bassullianus (Ace
tobacter pasturianus) ATCC
23769 was inoculated and cultured to obtain gel-like hollow microbial cellulose with a length of about 7 cm similar to that in Example 7. By washing and processing this in the same manner as in Example 7, hollow microbial cellulose with a diameter of 1.2 mm was obtained.

Example (0 seaucrose 5 g/dl, yeast extract 0.5 g/dl
, ammonium sulfate 0.5g/dl, potassium hydrogen phosphate 0.3g/dl
A culture medium (pH 5.0) containing 0.05 g/dl of magnesium sulfate was poured into a 200-volume Erlenmeyer flask and steam sterilized at 120° C. for 20 minutes to prepare a culture solution.

Next, this culture solution was poured into the cylindrical gap between the double glass tubes. After that, yeast extract 0.5g/cil and peptone 0.3g/d1 were added to this culture solution.
Acetobacter aceti subspice xylinum (ATCC 10g21) grown for 3 days at 30°C on a test tube slanted agar medium (pH 6,0) containing 2.5 g/dl of mannitol was inoculated and cultured at 30°C. .

After culturing for 30 days, dense white bacterial cellulose was formed between the double glass tubes.

The glass tube was removed, the cylindrical bacterial cellulose was taken out, and thoroughly washed with water to obtain an artificial blood vessel with an inner diameter of approximately 2 to 3 mm.

The blood compatibility (antithrombotic properties) of the artificial blood vessels created in this way was evaluated by blood vessel replacement experiments in mongrel dogs.In other words, the artificial blood vessels created from part of the inferior arteries and jugular veins of mongrel dogs. It was replaced and left in place for one month.Then, the replaced artificial blood vessel was taken out and the status of thrombus adhesion was observed.As a result, although a small amount of thrombus was observed at the suture site,
In all cases, there was almost no thrombus adhering to the inner surface of the blood vessel, indicating good patency.

〔Effect of the invention〕

The hollow microbial cellulose of the present invention is excellent as an immobilization carrier, an industrial material, a medical material including artificial organs, a chemical material, etc. In particular, it is possible to provide an artificial blood vessel that has excellent blood compatibility, excellent durability and uniformity, and can replace a small-caliber blood vessel with an internal diameter of 6 or less, for example.

Claims (1)

[Scope of Claims] 1. Hollow microbial cellulose containing cellulose produced by microorganisms. 2. The hollow microbial cellulose according to claim 1, which is in the form of a composite formed by adhering mainly to one or both of the inner and outer surfaces of the hollow carrier. 3. The hollow microbial cellulose according to claim 1, wherein the hollow microbial cellulose is in the form of a composite integrally containing other substances. 4. A method for producing hollow microbial cellulose, which comprises culturing cellulose-producing microorganisms on either or both of the inner and outer surfaces of an oxygen-permeable hollow carrier. 5. The method according to claim 4, wherein the oxygen-permeable hollow object is made of at least one selected from cellophane, Teflon, silicone, ceramic, nonwoven fabric, and woven fabric. 6. A method for producing hollow microbial cellulose, which comprises impregnating cellulose produced by microorganisms with a medium, further curing it if necessary, and then cutting it. 7. The method according to claim 6, wherein the medium is at least one selected from polyhydric alcohols, polysaccharides, proteins, natural and synthetic polymers, and polar solvents. 8. The method according to claim 6, wherein the medium is glycerin. 9. Artificial blood vessels mainly made of cellulose produced by microorganisms.