WO2003075974A1

WO2003075974A1 - Artificial digestive tract

Info

Publication number: WO2003075974A1
Application number: PCT/JP2003/002896
Authority: WO
Inventors: Yasuhiko Shimizu; Tatsuo Nakamura; Yoshio Hori
Original assignee: Tapic International Co., Ltd.
Priority date: 2002-03-12
Filing date: 2003-03-12
Publication date: 2003-09-18
Also published as: AU2003213326A1; TW200304837A

Abstract

It is intended to provide an artificial digestive tract which would not impose any serious burden to a patient, is free from any problems in supply and can be safely used for clinical purposes. An artificial digestive tract characterized by having a layer 30 made of a bioabsorbable material on the outer face of a layer 20 made of a gelled collagen containing at least one member selected from among nutrients, proliferation stimulants, differentiation inducers, antibiotics and antifungal agents.

Description

Specification

Artificial digestive tract technical field

The present invention relates to a substitute organ capable of restoring the function of the gastrointestinal tract by replenishing a defect in the artificial gastrointestinal tract, particularly the esophagus, stomach, small intestine, large intestine, and bile duct. Background art

If the gastrointestinal tract is damaged or removed due to various diseases, trauma, or surgical procedures, the site needs to be reconstructed, but if possible, direct fusion is performed.

However, the reconstruction range is naturally limited, and if the defect site is widespread, reconstruction is performed using other gastrointestinal tracts, such as the stomach tract, small intestine, and large intestine. Because of this, surgical procedures (skin incision, thoracotomy, laparotomy, etc.) are required outside the original surgical field, making surgery more invasive.

When a large amount of gastrointestinal tract is lost due to various diseases such as short bowel syndrome, there is no longer any gastrointestinal tract that can be used for reconstruction. It is impossible to do so, and there are problems such as loss of life due to malnutrition and the need to live at home under cardiovascular nutrition.

In addition, in biliary reconstruction with biliary tract alteration, such as biliary jejunostomy using the small intestine, the function of the papillary sphincter is due to the fact that the biliary tract is directly connected to the small intestine without the intervention of the fur nipple. As a result, complications such as reflux of the contents of the small intestine into the biliary tract and ascending cholangitis due to bacterial infection resulting therefrom may be present.

Many artificial gastrointestinal tracts using tubes made of synthetic polymers have been tried so far, but since these are foreign substances to the living body, they are all rejected from the living body after a certain period of time, and Not reached.

Attempts have also been made to transplant organs for the small intestine, but since the gastrointestinal tract is the part where food passes through and directly contacts the outside world, it is difficult to regulate immunosuppression, unlike kidney and liver transplants. Not yet common. Moreover, transplantation medicine has rejection The problem of having to administer immunosuppressive drugs for a lifetime to avoid infection and the problem of having an organ donor is essential. In particular, the shortage of donors is a serious problem.

In order to solve these problems, a self-regeneration-guided artificial gastrointestinal tract made of a biodegradable and absorbable material, which is replaced and absorbed by its own tissue after application to a living body, has also been attempted (Japanese Patent Application Laid-Open No. 20-210). However, the production process of the base layer is complicated, the regeneration speed of the self-tissue is slow, and the stenosis of the digestive tract is partially observed. There was still room for improvement. Disclosure of the invention

The present invention has been made to improve such a situation, and does not impose a great burden on the patient, has no supply problems, and can be used clinically safely. The purpose is to provide.

The present invention relates to an artificial gastrointestinal tract, comprising a layer 20 made of gelled collagen, which contains at least one of nutrients, growth stimulating factors, differentiation inducing factors, antibiotics, and antifungal agents. It is characterized by having a layer 30 made of a biodegradable and absorbable material on the outer surface.

Here, the layer made of gelled collagen (hereinafter referred to as a base layer) is a solution in which a basic buffer is added to a cold collagen hydrochloride solution, the cold collagen hydrochloride solution is neutralized, and heated to 37 ° C. Means a layer composed of a collagen solution gelled by In short, the collagen solution loses its fluidity and solidifies, specifically, an agar-like, elastic, uniform hydrated gel with a certain hardness and shape, and the collagen molecules are They are connected to each other to form a three-dimensional network structure, and the space is filled with liquid such as water. This layer serves as a scaffold for self-tissue regeneration (hereinafter, referred to as a collagen matrix or simply as a matrix), and at the same time directly and self-regenerates a self-tissue containing at least one kind contained therein. Alternatively, it also functions as a functional material capable of locally retaining a substance that supports indirectly, for example, a nutrient substance, a growth stimulating factor, a differentiation inducing factor, an antibiotic, an antifungal agent, or the like for a desired period of time. Here, the self-organizing tissue regeneration supporting substance converts a plurality of species into a single base layer. They may be contained, or each layer of the base layer composed of a plurality of layers may contain a different one. Further, as a form to be contained, the base layer may be entirely contained, or a part thereof may be contained therein and a part thereof may be contained on the surface thereof. ―

'Brief description of the drawings

FIG. 1 is a longitudinal sectional view schematically showing the wall structure of an artificial digestive tract (tubular) of the present invention.

FIG. 2 is a diagram showing the state of angiogenesis in a regenerated tissue in Test Example 3 and Comparative Test Example 3.

Where each code is

20 Layer consisting of gelled collagen (base layer)

20 'Layer composed of microfibrillated collagen (base layer equivalent part)

30 Layer made of biodegradable and absorbable material (outer layer)

40 0 Layer made of digestive juice-resistant material (inner layer)

, Respectively. BEST MODE FOR CARRYING OUT THE INVENTION

As a raw material of the collagen constituting the base layer, various types of conventionally used collagens such as neutral solubilized collagen, acid solubilized collagen, alkali solubilized collagen, and enzyme solubilized collagen can be used. Enzyme-solubilized collagen treated with enzymes, such as, for example, bepsin, trypsin, chymotrypsin, papain, pronase, is preferred. This is because telopeptide, which is an antigen group in the collagen molecule, is reliably removed, and the antigenicity is almost eliminated.

The origin of these collagens is not particularly limited, and type I collagen obtained by extracting and purifying from skin, bone, cartilage, tendons, organs, and the like of animals such as sea lions, pigs, night egrets, sheep, lignaroos, birds, and fish Alternatively, a mixture of type I collagen and type III collagen can be used. In some cases, human recombinant collagen may be used. The thickness of the base layer is 5 to 20 s, preferably 5 mm. If the thickness is more than 20 mm, it is difficult to sew locally, and if it is less than 5 mm, the layer absorbs too quickly and cannot maintain its shape until the self-organization has been regenerated From.

On the other hand, the constituent materials of the layer 30 (hereinafter referred to as the outer layer) made of a biodegradable and absorbable material include polyglycolic acid, polylactic acid, a copolymer of glycolic acid and lactic acid, and lactate and ε-force prolactone. And polydioxanone, and a copolymer of dalicholate and trimethylene monoponate. The outer layer prevents the collagen matrix from dissipating due to physical external forces such as friction until the self-tissue regenerates, and a barrier that prevents excessive invasion of inflammatory cells such as fibroblasts and macrophages into the collagen matrix. It also plays a role and provides the collagen matrix with an optimal environment for self-tissue regeneration and differentiation. In addition, the presence of this layer prevents the contraction of the base layer during the process of self-tissue regeneration, contributes to improved suturing at the time of surgery, and suppresses the occurrence of wrinkles due to tearing or bending of the collagen matrix. I can do it.

A specific use form of this layer 30 is a nonwoven sheet or tube. In addition, this non-woven sheet is usually formed by fibrosis (melt spinning) of the material, stretching → knitting (tubular knitting), non-woven fabric (folding of the knitted material and needle punching) → consolidation (hot pressing). It is manufactured through each operation (the tube may be obtained by molding the sheet thus manufactured into a cylindrical shape).

The artificial digestive tract of the present invention may further have a layer 40 (hereinafter, referred to as an inner layer) made of a material having digestive juice resistance on the inner surface of the base layer 20. Here, as a material having digestive juice resistance, a silicone tube (commonly used as a silicone tent in the medical field) and a silicone sheet can be cited. This layer functions as a septum for isolating the lumen (or inner surface) of the collagen matrix from contact with food or digestive fluid, and keeps the lumen in a predetermined shape until the self-tissue is regenerated. (Accordingly, this material is required to have not only digestive juice resistance but also appropriate stiffness. Note that this layer 40 is removed after self-tissue regeneration). Therefore, this layer is assembled together with the base layer 20 and the outer layer 30 from the stage of distribution as a product. It may be in a combined form or combined with the first two before applying to the patient.

Next, how to make an artificial gastrointestinal tract is generally performed as follows.

(1) Preparation of neutral collagen solution

About 1 N hydrochloric acid solution of cold collagen (pH = about 3, collagen concentration: preferably about

By adding a basic buffer (sodium hydrogen carbonate and HEPES dissolved in sodium hydroxide) to 0.5 to 3% by weight, particularly preferably about 0.5 to 1% by weight, the collagen hydrochloride solution is added. To neutral.

(2) Introduction of self-organizing regeneration support substances

A predetermined amount of a self-tissue regeneration supporting substance is dissolved in the collagen neutral solution with stirring. Here, self-tissue regeneration supporting substances include nutrient substances such as amino acids, bimin, glucose, electrolytes, etc .; growth stimulating factors and differentiation inducing factors such as basic and acidic fibroblast growth factor (bFGF), vascular Endothelial cell growth factor (VEGF), steroids, retinoic acid and the like; prophylactic agents for infections, for example, antibiotics, antifungal agents and the like. Two or more of these self-tissue regeneration supporting substances may be used as desired. In some cases, fibroblasts, epithelial cells, muscle cells and their stem cells (such as mesenchymal stem cells) collected from their own digestive tract, skin, and oral cavity may be added.

(3) Formation of base layer 20

A sheet-shaped outer layer material is laid on the bottom of a bat capable of horizontally storing the biodegradable and absorbable material (hereinafter referred to as the outer layer material) constituting the outer layer 30, and the self-prepared therein is prepared by the above operation. A predetermined amount of a neutral solution of collagen containing a tissue regeneration supporting substance (in some cases, further containing the autologous cells and / or their stem cells) is poured, and then the collagen neutral solution is poured at about 37 ° C. Heat to C to gel the neutral collagen solution. In about 10 minutes after the heating, a material integrally having the base layer 20 made of gelled collagen on the upper surface of the outer layer 30 is formed. Here, prior to the application of the outer layer material, it is preferable that the surface thereof, that is, the surface in contact with the base layer 20 be subjected to treatment such as plasma discharge and ozone irradiation. This is because the material is given hydrophilicity and can be integrated with the base layer.

(4) Application of inner layer The base layer 20 having the outer layer 30 on the sheet shape obtained in the previous step is wound around the inner layer 40 having a predetermined diameter, for example, the outer surface of a silicone tube, and stitched into a tubular shape (surgical thread) By using this, a human gastrointestinal tract consisting of the inner layer 40—the base layer 20—the outer layer 30 is produced. Further operations such as storage in a container for market distribution and sterilization may be performed according to a conventional method.

As described above, the method for producing an artificial digestive tract of the present invention has been described using the method using the sheet-like base layer 20 and the outer layer 30. However, the artificial digestive tract can also be produced using a tube-like outer layer material. In that case, the collagen solution may be poured in the above step (3) as follows.

“Tube having an inner diameter corresponding to the outer diameter of the base layer 20-a tube as a mold material, and a rod-shaped body having an outer diameter corresponding to the inner diameter of the base layer 20 (consisting of the material constituting the inner layer 40) It is convenient to use a tube), and a predetermined amount of a self-tissue regeneration supporting substance (in some cases, the above-mentioned substance) is placed in a space between the rod-shaped body and the tube as the mold. (Including further autologous cells and / or their stem cells). Here, the outer layer material may be attached to the inner wall surface of the tube as a mold before pouring, or the outer layer material itself may be used as a tube as a mold. Prior to application of the outer layer material, it is preferable that the surface of the outer layer material in contact with the base layer 20 is subjected to a hydrophilic treatment such as plasma discharge or ozone irradiation. is there. In addition, after the tubular base layer 20 is formed by the above-mentioned modification (to be precise, after the collagen which becomes the base layer 20 is gelled), the tubular outer layer material is provided on the outside thereof. What is necessary is just to cover on the outer surface of the base layer 20. In the gastrointestinal tract of the present invention, the base layer 20 is required to have a function capable of retaining the self-tissue regeneration supporting substance contained therein for a desired period. A simple method of verifying the function is exemplified by a fluorescent marker bead (a brand name of Bebson's Dickinson's Caribrite bead; trade name. The bead is made of polymethyl methacrylate having a diameter of 6 m). The base layer containing 10, 000 Zml-substrates is placed in PBS with gentle stirring, and the residual ratio of beads in the substratum is checked at predetermined intervals. The method is illustrated. If the remaining ratio of the beads remains at a predetermined level even after a predetermined elapsed time. If it has, the base layer is determined to have the desired self-tissue regeneration supporting substance holding function. If the base layer has the function of holding the self-tissue regeneration supporting substance, it is formed in the base layer at the initial stage of contact with PBS under in vitro conditions in which the gelled collagen as the base layer constituent material is not water-soluble. Because it does not occur other than the release of the beads through the physical channels that would be performed. Incidentally, the base layer of the artificial gastrointestinal tract of the present invention shows a residual rate of 80% or more even after 24 hours. In addition, in the base layer composed of microfibrillated collagen (in which the beads were introduced by impregnating the bead suspension with the same amount as the base layer composed of gelled collagen), 5 hours At the time of the survey, the surviving rate is less than 20%. Test example

Test example 1 (artificial small intestine 1)

Hydrochloric acid solution of enzyme-solubilized collagen (pH = 3.0, collagen concentration = 0.5% by weight), 5-fold concentrated Du1becco modified Eag1e medium, basic buffer

_{(N aHC0 3: 2. 2 g} , HEPES: 4. by melt-in the 8 g 0. 05MNaOH those with 100ml) of 7: 2: 1 in Ri by the mixing in a ratio of (volume ratio) An aqueous collagen solution was obtained. In order to prevent infection, the neutral collagen solution contains 100 μm / ml of penicillin as an antibiotic, 100 g / ml of streptomycin and 0.25 g / ml of amphotericin B as an antifungal agent (where ml is , Which is that of a neutral collagen solution).

On the bottom, a sheet made of Gunze's PGA nonwoven fabric with an area density of 30 to 35 g / m ² , a fineness of about ^2.5 denier, and a thickness of 0.15 orchid (the surface was previously irradiated with plasma. Is placed almost flat, and the neutral collagen solution is poured into the vat. Then, the neutral collagen solution is poured at about 37 ° C. for 10 minutes. After heating, an outer layer 30 having a base layer 20 (thickness: about 5 mm) made of gelled collagen formed thereon was obtained.

Cut the small intestine of an 8- to 15-kg beagle dog (nine dogs), join the cut end of the cut small intestine on the anal side to the jejunal cutaneous fistula that formed the colostomy, and then cut the small intestine 50 mm on the anal side The silicone tube 40, that is, the inner layer (inner diameter: Ι Οιηιικ thickness: 0.5 marauder), is inserted into each end of the autologous small intestine after the excision (one of the artificial digestive tract and autologous small intestine). The burlap was about 5 nun), and a single knot was sutured with the autologous small intestine using 3-0 proline suture. The outer layer with the base layer was wound around the outer periphery of the silicone tube so that the base layer 20 was on the inside, and the ends were sewn together to apply the artificial digestive tract of the present invention to the small intestine defect. One end of the previously cut small intestine, that is, the end on the stomach side, communicates with the lumen of the autologous small intestine at the anal side from the suture portion between the artificial digestive tract and the autologous small intestine. (The junction between the artificial digestive tract and the autologous small intestine is shown in the figure).

One month after the operation, the silicone tube was removed endoscopically. Observation with a gastrointestinal endoscope revealed regeneration of the autologous small intestine maintaining the lumen. In addition, shrinkage of the base layer 20 made of gelled collagen with the progress of regeneration was not observed. Histological observation confirmed that the regenerated small intestinal wall had submucosal tissue consisting mainly of connective tissue. Three months after surgery, regeneration of the mucosal layer was observed on the layer of regenerated submucosa (histological observation). Comparative test example 1 (artificial small intestine 2)

A tube made of PGA non-woven fabric on the inner surface of a tube (tube made of Teflon ^R. Inner diameter: about 52IM) in which a silicone tube (inner diameter: 10 bandages, thickness: 0.5I1) is arranged almost concentrically in the inner cavity. (Danze surface density: 30-35 g / m ² , fineness: about ^2.5 denier, thickness: 0.15 thigh A sheet of PGA non-woven fabric is formed into a tube with an inner diameter of about 52 mm. A molded product—one end surface corresponding to the longitudinal direction of the tube is stitched with surgical thread and its surface is made hydrophilic by plasma irradiation), and a tube made of PGA nonwoven fabric is attached to the inner surface. An enzyme-solubilized collagen solution of collagen (pH = 3.0, collagen concentration = 1.0% by weight) was introduced into the space between the tube and the silicone tube. Freeze the introduced collagen hydrochloride solution (about -20t: x 24Hr), lyophilized in vacuum (about 80 ° CX24Hr), and subjected to thermal dehydration cross-linking treatment (about 130 ° CX24Hr) under vacuum, silicone tube, inner layer 40 and fine fibers An artificial gastrointestinal tract was obtained, which was a composite comprising a base layer equivalent portion 20 'made of activated collagen and a tube made of a PGA nonwoven fabric, that is, an outer layer 30.

The small intestine of a 9 to 15 kg beagle dog weighing 8 to 15 kg was cut, and the anal end of the cut small intestine was joined to a jejunocutaneous fistula that formed an ostomy, and the small intestine was placed on the anal side. Before the application, the bandits infiltrated the artificial gastrointestinal tract (the base layer-equivalent portion 20 'with autologous blood before application). It was confirmed that the various trophic factors contained in the blood further promoted the regeneration of autologous tissue. (The same applies to the following.) Replace both ends of the inner layer 40 of the artificial gastrointestinal tract with each end of the autologous small intestine after resection, and the inner layer and the autologous small intestine overlap by about 5 bands. After inserting both ends of the base layer-equivalent portion 20 ′ having the outer layer 30 thereon so as to wrap them, the ends of the autologous small intestine are brought into contact with each end of the autologous small intestine, and then the autologous small intestine and the human digestive tract And a single knot suture with 3-0 proline suture. In substantially the artificial gut of Test Example 1 the same. Here, to antibiotics and antifungal agents of the same type such that the same conditions as in Test Example 1 to said test animal). One end of the previously cut small intestine, that is, the end on the stomach side, is designed so that their lumen communicates with the autologous small intestine on the anal side of the artificial gastrointestinal tract and the autologous small intestine. Joined.

One month after the operation, the silicone tube was removed endoscopically. Observation with a gastrointestinal endoscope showed regeneration of the autologous small intestine maintaining the lumen. Histological observation confirmed that the regenerated small intestinal wall had submucosal tissue consisting mainly of connective tissue. Three months after the operation, regeneration of the mucosal layer was observed on the layer of regenerated submucosa (histological observation). Test example 2 (artificial stomach-1)

The anterior wall of the stomach of 40 beagle dogs (3 animals) weighing 8 to 15 kg was resected at 40 thighs x 40 thighs, and a silicone sheet 40 (thickness: 1.0 mm, size: 5) was cut at the defect. 0 mm X

5 0 Marauders. Finally, the artificial gastrointestinal tract of the present invention (more precisely, the inner layer of the sheet) is sewn from the mucous membrane side of the stomach (using 3-0 proline suture), and then the defective portion is tested. In the outer layer 30 (size: 40 IM X 4 O mm) on which the base layer 20 made of the gelled collagen prepared in 1 is formed, the base layer faces and contacts the inner layer. An artificial gastrointestinal tract (exactly a sheet) was applied by covering the serosal side as described above and suturing its end to the anterior wall (using 3-0 proline suture).

One month after the operation, the silicone sheet was removed endoscopically. Observation with a gastrointestinal endoscope revealed regeneration of the autologous stomach wall. Also, no shrinkage of the base layer 20 made of gelled collagen with the progress of regeneration was observed. Histological observation confirmed that the regenerated stomach wall had submucosal tissue consisting mainly of connective tissue. Three months after the operation, regeneration of the mucosal layer was observed on the regenerated submucosal tissue layer (histological observation). Comparative test example 2 (artificial stomach—2)

The anterior wall of the stomach of 4 to 8 kg of beagle dogs (3 dogs) weighing 4 to 15 kg was resected by 4 O mm X 4 O mm, and a silicone sheet 40 (thickness: 1.0 hall, size: 5 O mm X 50 mm. The artificial gastrointestinal tract (to be more precise, the inner layer of a sheet) is sewn from the mucous membrane side of the stomach, and then the defective part is put on the silicone sheet 40 ( Thickness: 1.0 dragon, size: 50 mmX 50 iMi) is sewn from the mucous membrane side of the stomach (using a 3-0 proline suture), and the defect is made of microfibrillated collagen Outer layer 30 with base layer equivalent part 20 ′ formed on it. (Instead of a tube made of PGA non-woven fabric, use a sheet 30 of 40 mm X 40 mm made of PGA non-woven fabric to form a tube as a mold. Instead, the sheet made of the PGA non-woven fabric is placed almost flat on the bottom-stored in a bat that can be stored, and placed almost concentrically in the cavity of the mold. In the same manner as in Comparative Test Example 1 except that a silicone tube was not used), the portion corresponding to the base layer was coated from the serosa side so that the portion corresponding to the inner layer was opposed to and contacted with the inner layer. An artificial gastrointestinal tract (exactly a sheet shape) was applied by stitching to the front wall (using 3-0 proline suture) (where the same conditions as in Test Example 2 were applied to the test animal). The same antibiotics and antifungals were administered). In addition, in this artificial gastrointestinal tract, autologous blood was infiltrated into the base layer equivalent part 20 'before application.

One month after the operation, the silicone sheet was removed endoscopically. Observation with a gastrointestinal endoscope revealed regeneration of the autologous stomach wall. Histological observation confirmed that the regenerated stomach wall had submucosal tissue consisting mainly of connective tissue. Three months after the operation, regeneration of the mucosal layer was observed on the layer of regenerated submucosa (histological observation). As described above, the artificial gastrointestinal tract of the present invention does not require a special treatment, such as freezing, freeze-drying, and even heat dehydration crosslinking, for the collagen constituting the base layer 20 as in the conventional example. It was found that the shape was maintained, and the tissue regeneration ability equivalent to that of the conventional example was exhibited even without performing autologous blood infiltration into the base layer. Test example 3 (artificial esophagus 1)

After removing about 50 mm of the facial esophagus of three 8-beagle dogs (3 dogs), after excision of both ends of the silicone tube 40, the inner layer (inner diameter: 15 mm, thickness: 0.5 recitation) Was inserted into each end of the esophagus (the overlap between the artificial gastrointestinal tract and the esophagus was approximately 5 mm), and a single knot suture was performed between the esophagus and the autoesophagus with 3-0 proline suture. . The outer layer 30 with the base layer 20 prepared in Test Example 1 so that the base layer 20 is on the outer periphery of the silicone tube (however, the neutral collagen solution before gelation used in this test example includes: The artificial gastrointestinal tract of the present invention was applied to the esophageal defect by wrapping a wound around which the basic fibroblast growth stimulating factor 1b FGF- was added in an amount of 1 gZml, and sewn the ends together. The mode of connection with the autoesophagus is the same as in Test Example 1).

One month after the operation, the silicone tube was removed endoscopically. Gastrointestinal endoscopy showed regeneration of the autologous esophagus while maintaining the lumen. In addition, shrinkage of the base layer 20 made of gelled collagen with the progress of regeneration was not observed. Histological observation confirmed that the regenerated esophagus had submucosal tissue consisting mainly of connective tissue, and that a large number of capillaries were newly formed in the regenerated tissue (97 ± 17.0 / Field of view-microscope). The number of angiogenesis is represented by the number of new blood vessels per visual field of a microscope observed by immunohistochemical staining using an anti-factor VIII antibody (the same applies hereinafter). Three months after the operation, regeneration of the mucosal layer was observed on the regenerated submucosa layer (histological observation). Comparative Test Example 3 (Artificial Esophagus—2)

Approximately 50 mm of the cervical esophagus of 8 to 15 kg beagle dogs (3 dogs) was excised, and the portion was changed to a silicone tube 40 with an inner diameter of 15 thighs and a PGA nonwoven fabric. The diameter of the tube 30 was adjusted to the diameter corresponding to the diameter of the silicone tube (the thickness of the base layer equivalent part 20 ': the thickness of the 20 layer remained unchanged), and the test example was applied to the base layer equivalent part 20' made of microfibrillated collagen. Artificial gastrointestinal tract obtained in the same manner as in Comparative Test Example 1, except that basic fibroblast growth stimulating factor-b FGF— was included so that the same conditions as at 3 (at the start of the animal experiment) were used. (The junction between the artificial digestive tract and the autologous esophagus was the same as in Test Example 3). In addition, a method for incorporating bFGF into the portion corresponding to the base layer is to drop bFGF (concentration: 10 ^ g / ml) dissolved in PBS onto the portion corresponding to the base layer so as to have a volume of 1 gZml—corresponding to the portion corresponding to the base layer. Then, it was left to stand at room temperature for 1 hour.

One month after the operation, the silicone tube was removed endoscopically. Gastrointestinal endoscopy showed regeneration of the autologous esophagus while maintaining the lumen. Histological observation confirmed that the regenerated esophageal wall had submucosal tissue consisting mainly of connective tissue. The number of new blood vessels in the regenerated tissue at this time was 17 ± 2.6. Three months after the operation, regeneration of the mucosal layer was observed on the regenerated submucosal tissue layer (histological examination).

Test example 4 (artificial esophagus 3)

100,000 Zml of fibroblasts collected from their own oral mucosa and proliferated outside the body were added to the neutral collagen solution before gelation, and the luminal surface of the base layer 20 of the artificial digestive tract was added to the neutral collagen solution. Epithelial cells collected from the oral mucosa and grown outside the body adhered at a rate of 1000 cells / cm ² (specifically, the base layer was left overnight in a growth medium for the epithelial cells). An animal experiment was performed in the same manner as in Test Example 3 except that the experiment was performed. The fibroblasts and epithelial cells were cultured according to the following procedure (the same applies hereinafter).

1) fibroblast

The oral submucosa minced into 5mm square, was added to the DM E (Dulbecco 's modified eagle ) medium 20ml plus 10% © shea fetal serum and cultured in flasks 80cm ² (37 ° (: under 5% C_〇 _2). the medium was changed every 2-3 days, increasing only adherent cells Proliferate (suspended cells are eliminated from the system when the medium is changed). The grown adherent cells are passaged as necessary, and used when the required number of cells has been obtained.

2) epithelial cells

The collected oral mucosa was placed in dispase (3 OU / ml) at 4 ° C overnight to detach only the epithelial cells, and this was placed in a 0.25% trypsin solution (37 ° C).

Shake for 15 minutes to dissociate the individual cells into pieces. The cells thus obtained are cultured in an 8 Ocm 2 collagen-coated flask together with 20 ml of a bloodless medium for human keratinocytes manufactured by Gibco (37 ° C, 5% C% ₂ ). The medium is replaced every 2-3 days, and only adherent cells are grown (suspended cells are eliminated from the system when the medium is replaced). The grown adherent cells are passaged as necessary, and used when the required number of cells has been obtained.

One month after the operation, the silicone tube was removed endoscopically. Gastrointestinal endoscopy showed regeneration of the autologous esophagus while maintaining the lumen. Histological observation confirmed that the regenerated esophageal wall had not only the submucosa but also the epithelium, that is, the mucosal layer. The number of new blood vessels in the regenerated tissue at this time was 103 ± 14.8 fields / microscope. Comparative Test Example 4 (Artificial Esophagus 4)

An animal experiment was performed in the same manner as in Test Example 3 except that an artificial digestive tract containing no basic fibroblast growth stimulating factor—bFGF— was used in the base layer 20 of the artificial digestive tract. One month after the operation (when the silicon tube was removed), the number of new blood vessels in the regenerated tissue was 51 soil 6.6Z field-microscope. Test example 5 (artificial esophagus 5)

An animal experiment was performed in the same manner as in Test Example 4 except that an artificial digestive tract containing no basic fibroblast growth stimulating factor—bFGF—in the base layer 20 of the artificial digestive tract was used. One month after the operation (when the silicone tube was removed), the number of new blood vessels in the regenerated tissue was 14 ± 2.9 Z-field-one microscope. Comparative test example 5 (artificial esophagus 6)

Animal experiments were performed in the same manner as in Comparative Test Example 3 except that an artificial digestive tract that did not include basic fibroblast growth stimulating factor-bFGF-was used in the base layer equivalent part 20 'composed of microfibrillar collagen. did. One month after the operation (when the silicone tube was removed), the number of new blood vessels in the regenerated tissue was 15 ± 1.8 fields per microscope. For the artificial esophagus, the results of observation of the number of new blood vessels in the regenerated tissue at one month after the operation are summarized in the table below.

〔table 1〕

Note) * 1: GC is composed of gel collagen, SC is microfibrous collagen

Indicates that it consists of Lagen.

* 2: The symbols in the columns of bFGF and cells indicate whether (+) force is contained or not (one) in the base layer. As shown in the table, although the basic fibroblast proliferation stimulating factor-bFGF— was similarly contained in the base layer 20 or the base layer equivalent portion 20 ′, the artificial digestion tube of the present invention (Test Example 3) was used. ) Shows significantly more new blood vessels in the regenerated tissue than the conventional artificial gastrointestinal tract (Comparative Test Example 3), indicating that the effect is extremely high. Fig. 2 shows the state of each tissue in Test Example 3 and Comparative Test Example 3 (the upper figure is that of Comparative Test Example 3, and the lower figure is that of Test Example 3. In the figure, the elliptical shape with black dyeing is shown) The one in the lower part of the figure is clearer.)

Regarding the stenosis of the regenerated gastrointestinal tract, In the case of containing both cells and bFGF, it was confirmed by gastrointestinal endoscopy that the frequency and extent of the group of the present invention were smaller than those of the conventional group. I have.

The self-tissue regeneration-guided artificial gastrointestinal tract (composite consisting of inner layer, base layer, and outer layer) that regenerates the defect has been described, but the scaffolding function and support function for self-tissue regeneration are the artificial gastrointestinal tract. Since it is provided by a composite consisting of a base layer and an outer layer, the composite consisting of the base layer and the outer layer is applied so as to cover the periphery of the anastomotic portion during gastrointestinal surgery. It can be used as a gastrointestinal anastomosis cover material that has the function of inducing the wound healing ability inherent in the living body and inducing angiogenesis. The invention's effect

The artificial gastrointestinal tract of the present invention can be stably supplied without being influenced by the source thereof, and after application in a living body, remains until the autologous digestive tract regenerates, thereby exhibiting the action of promoting regeneration of self-tissue. However, it gradually decomposes and absorbs and replaces the self-tissue, so it can be used safely without inflammation. Also, since the regenerating tissue is the autologous digestive tract, there is no need to take immunosuppressive drugs as in transplantation, and there is no fear of rejection. Furthermore, since it induces regeneration of the autologous gastrointestinal tract, it is extremely useful for treating people who have lost a large amount of gastrointestinal tract due to short bowel syndrome or various diseases for which there was no conventional treatment. It enables the reconstruction of biliary tracts that pass through the papilla of Fata, which was previously impossible, and is a gospel for many patients who have suffered from ascending cholangitis due to biliary tract alterations. In addition, since it is not necessary to collect the intestinal tract for reconstruction, unnecessary skin incisions can be reduced, and the invasiveness of the operation can be reduced, thereby reducing the pain associated with the patient's operation. Furthermore, the base layer constituting the artificial gastrointestinal tract of the present invention can be prepared by a simpler operation than a conventional artificial gastrointestinal tract, and since these operations do not include operations in a high-temperature atmosphere, Even a substance which is weak to heat or a chemical substance can be used as a self-organization regeneration supporting substance contained in the base layer without lowering its activity or survival rate.

Claims

The scope of the claims

1. A layer made of biodegradable and absorptive material is formed on the outer surface of a layer made of gelled collagen containing at least one of nutrients, growth stimulating factors, differentiation inducing factors, antibiotics, and antifungal agents. Having an artificial digestive tract.

2. The artificial digestive tract according to claim 1, further comprising a layer made of a material having digestive juice resistance on the inner surface of the layer made of the gelled collagen.

3. The biodegradable and absorbable material is polydaricholic acid, polylactic acid, a copolymer of dalicholic acid and lactic acid, a copolymer of lactic acid and ε-force prolactone, polydixanone, and a mixture of dalicholic acid and trilic acid. The artificial gastrointestinal tract according to claim 1 or 2, wherein the artificial digestive tract is a nonwoven fabric material made of a material selected from the group of copolymers with methylene carbonate.

4. The artificial digestive tract according to claim 3, wherein the biodegradable absorbent material has an areal density of 30 to 35 g / V.

5. The artificial gastrointestinal tract according to claim 1, wherein the material having digestive juice resistance is a tubular material or a film-like material made of a silicone resin.

6. The artificial gastrointestinal tract according to any one of claims 1 to 5, wherein the layer made of the gelled collagen has a bead retention rate of 80% or more (at the time of 24 hours).