WO2006129673A1

WO2006129673A1 - Material for tissue reconstruction and utilization of the same

Info

Publication number: WO2006129673A1
Application number: PCT/JP2006/310806
Authority: WO
Inventors: Yoshiaki Kuriu; Yuen Nakase; Akeo Hagiwara; Hisakazu Yamagishi; Eiji Kurihara; Junji Hamuro
Original assignee: Arblast Co., Ltd.
Priority date: 2005-05-30
Filing date: 2006-05-30
Publication date: 2006-12-07

Abstract

A material which is applicable over a broad scope and can promote the reconstruction of a damaged tissue while preventing it from adhesion (i.e., a material for tissue reconstruction); a method of producing the same; a method of using the same; and so on. A material for tissue reconstruction is produced by using amnion.

Description

Specification

Materials for tissue reconstruction and their use

Technical field

[0001] The present invention relates to a new use of amniotic membrane in the medical field. Specifically, the present invention relates to the use of amniotic membrane as a tissue reconstruction material. The tissue reconstruction material provided by the present invention is used for repairing / reconstructing tissue damaged due to disease or surgery.

Background art

[0002] Regenerative medicine refers to necrosis / injury. When the function of a tissue is impaired due to disease, it is transplanted to the damaged site using cells of itself, another person, or another animal, and the normal function of the tissue is restored. It is medical treatment aimed at regaining. In regenerative medicine, new trials of cell culture have been actively conducted for the purpose of treating various diseases such as corneal epithelial diseases, endothelial diseases, and retinal diseases in the ophthalmic field, and epidermal diseases in the dermatological field. And Such demand for regenerative medicine is expected to increase in the future.

[0003] In the human body, with the exception of blood cells and floating cells in ascites, almost all cells are adhered to the extracellular matrix typified by collagen. Only proliferate and differentiate. When a living tissue is severely damaged, the extracellular matrix is lost as well, so tissue regeneration will not occur just by supplementing the damaged tissue with cells. Therefore, it is preferable to transplant a carrier as an alternative to the extracellular matrix alone into the damaged tissue to promote growth from the remaining normal cells, or to transplant the cells seeded on the carrier together with the carrier. Carriers used for such purposes are required to have the following properties: (1) do not interfere with the regeneration of biological tissues' organs (preferably induce tissue regeneration) and (2) have tissue compatibility. The Substances that have been used as carriers to date include: (1) Substances derived from biological components: collagen, fibronectin, laminin, proteodarican, etc. (2) Artificial components: plastic, nylon, polydaricholic acid, ceramics (3) Hybrid substances of biological components and artifacts, etc., and are used for the reconstruction of valves, bones, cartilage, blood vessels, etc. Since tissues in vivo have different physical properties such as caloric pressure, weight, and flexibility, it is important to select a reconstruction carrier suitable for the tissue intended for reconstruction. [0004] The surface of organs of the abdominal organs such as the liver, small intestine, and large intestine are covered with serosal epithelial cells, which are representative of mechanical and chemical stimuli caused by surgical operation of the intestinal tract and peritoneum during open surgery. In the course of various diseases, serosal epithelial cells can easily fall off and damage organs. When the serosa is damaged, the damaged site coalesces with surrounding tissues or organs and frequently forms adhesions. When adhesion occurs, it causes ileus and adhesive ileus in the gastrointestinal surgery field. In the gynecological field, pelvic adhesions after surgery such as ovarian sac, uterine fibroids, and ectopic pregnancy can block the fallopian tube and cause infertility. These complications not only reduce the effectiveness of the original treatment, but in some cases require additional surgical operations, increasing the burden on the physician and patient. It has been discussed for many years to establish a method to reduce the adhesion formation of damaged tissues by inducing the serosal structure of damaged intra-abdominal organs to be reconstructed into normal tissues. In general, the phenomenon of adhesion formation is also a result of the normal wound healing process, and when tissue reconstruction is promoted, adhesion formation is also promoted. On the other hand, the formation of adhesions should be avoided as much as possible, but the adhesion and the natural healing process are closely related, and if the adhesion is suppressed, the natural healing will be delayed and the therapeutic effect may be reduced. Known empirically. Due to the above circumstances, there is no report that the organization reconstruction has been promoted and adhesion formation has been suppressed at the same time.

[0005] So far, research aimed at preventing adhesion rather than tissue reconstruction has preceded, and various materials have been proposed as a result. Sepra Finorem (Genzym), Surgegenore (Johnson and Johnson), Gelfoam (Apjon), Interseed, Chondron (Kaken Pharmaceutical), Xelan (Shionogi), Veriblast ( ZLB Behring), Tissir (Baxter), Bolhir (Ye Blood Research Institute), Octopus (Apenteis), etc. Of these, the most utilized substance is seprafilm, a bioabsorbable anti-adhesive agent consisting of sodium hyaluronate and carboxymethylcellulose. Sepurafilm acts as a physical noria that blocks the damaged tissue from physical contact with surrounding tissues and suppresses adhesions. Its application is achieved by inserting seprafilm after surgery.

Other anti-adhesive agents are also known, such as polysaccharides, chondroitin sulfate, cellulose, collagen, gelatin, or substances that are reconstituted by mixing these ingredients! / Speak. Disclosure of the invention

Problems to be solved by the invention

[0006] The anti-adhesive agents that have been used so far do not have the effect of actively reconstructing the tissue, and when applied, it is accompanied by a delay in spontaneous healing due to the prevention of adhesions, which is effective in reducing adhesions. However, it has a negative effect on tissue reconstruction, and the healing of damaged tissue is delayed. Therefore, it cannot be applied to areas where wound healing needs to be achieved as early as possible, such as at the anastomosis. In addition, Sepurafilm is generally fragile and easily breaks, so it requires skill. In addition, since it has the property of easily gelling when wet, it cannot be used in an area where it gets wet before application to the wound. For example, application to the dorsal peritoneum is impossible because it gels before application, and the application area is limited to the area under the wound, making it difficult to apply to complex sites. In addition, it has the disadvantage that it cannot be used under laparoscopic surgery. On the other hand, chondron, xeron, etc. have a risk of promoting bacterial infection and cannot be used in cases where infection is suspected. Also, there was a report that anaphylactic shock and edema occurred, which was not convenient. Veriplast, Tissir, Bolheal, Tacocomb, etc. have the effect of completely preventing postoperative hemostasis and suppressing adhesion caused by blood clot and exudate. Therefore, there is a problem that the effect is limited without actively preventing adhesions.

The present invention is applicable to a wide range of areas such as gastroenterological surgery, obstetrics and gynecology, and thoracic surgery, and is capable of promoting the reconstruction of damaged tissue while preventing adhesion (tissue reconstruction material) and Its purpose is to provide a method for its production and a method for its use.

Means for solving the problem

[0007] In order to achieve the above object, the present inventors focused on amniotic membrane and examined its suitability as a tissue reconstruction material. As a result, in an experiment using the cecal serosa disorder model, it was found that covering the damaged area with amniotic membrane prevented adhesion and promoted tissue reconstruction. That is, it became clear that the amniotic membrane has both an adhesion prevention function and a tissue reconstruction function. As a result of this, various cases that need to promote wound healing while suppressing adhesion formation (especially early wound healing such as reconstruction of tissue peritoneum after anastomosis) It was found that amniotic membrane is effective for patients who require It was also found that amniotic membrane is very thin and flexible, and has sufficient strength, making it suitable for application to organ surfaces. After obtaining the above findings, prepare amniotic membranes that differ in the presence or absence of epithelial cell layers and treatment methods in order to find the optimal form of amniotic membrane when used as a transplant material for tissue reconstruction. The prevention effect and the organizational restructuring effect were compared. As a result, the following knowledge was obtained.

(1) It is important to maintain the structure of the basement membrane in order to exhibit adhesion prevention and tissue reconstruction functions. Moreover, if the basement membrane structure is highly maintained, the adhesion prevention effect is higher in the absence of the epithelial cell layer. This means that even amniotic membrane that does not contain an epithelial cell layer can be used as a material for tissue reconstruction. The amniotic membrane from which the epithelial cell layer has been removed is extremely safe without any problems such as immune rejection caused by epithelial cells.

(2) Even an amniotic membrane that has been subjected to a freezing treatment or a freeze-drying treatment can exhibit excellent adhesion prevention effects and tissue reconstruction effects. This means that it can be used in a frozen state or a dried state that is easy to handle and excellent in storage stability.

(3) By allowing the epithelial cell layer to remain, it is considered that cross-linking of the dense layer (interstitial) constituent protein components accompanying γ-ray irradiation can be prevented. Therefore, if an amniotic membrane in which the epithelial cell layer is left is used, sufficient sterilization treatment such as γ-ray treatment can be performed while maintaining its function.

The present invention provides the following configurations based on the above knowledge or achievement. That is, the present invention relates to a tissue reconstruction material composed essentially of amniotic membrane.

The tissue reconstruction material of the present invention is preferably used for the reconstruction of an organ or a surface tissue disorder of an organ due to surgical invasion. In one aspect of the present invention, the organ or surface tissue of the abdomen, chest, or pelvis, or the surface tissue of the abdominal cavity, chest cavity, pelvic cavity, oral cavity, nasal cavity, ear cavity, or throat cavity, or ocular tissue Tissue reconstruction materials used for reconstruction are provided.

The tissue reconstruction material of the present invention is in one aspect! It is frozen or dry. In another embodiment, it is in a lyophilized state. In one embodiment of the present invention, the tissue reconstruction material is constructed of amniotic membrane from which the epithelial cell layer has been removed.

In one embodiment of the present invention, a tissue reconstruction material is constructed of amniotic membrane in which collagen IV, collagen VII, and laminin 5 as basement membrane components are detected with the same strength as that of untreated amniotic membrane.

In a preferred embodiment of the present invention, a tissue reconstruction material is constructed using human amniotic membrane! Speak. In one embodiment of the present invention, the tissue reconstruction material is constructed of amniotic membrane in which an adhesive component adheres to the surface of the amnion on the chorionic membrane side. The adhesive component here is preferably fibrinogen and thrombin, or fibrinogen, thrombin and caprotun. In one embodiment of the present invention, the surface of the amniotic membrane is the amniotic membrane coated with a bioabsorbable material. The material for tissue reconstruction is built!

[0009] As another aspect, the present invention provides a method for producing a tissue reconstruction material. The production method of the present invention comprises the following steps (1) to (3), that is, (1) a step of preparing a biomedical separated amniotic membrane, (2) a step of freezing or drying the amniotic membrane, and (3) optional The step includes a step of sterilizing the amniotic membrane.

In one embodiment of the present invention, the drying process in step (2) is a freeze-drying process. In another embodiment of the present invention, before the step (2), a step (step (a)) of removing the epithelial layer from the amniotic membrane leaving at least a part of the basement membrane is performed. Step (a) is preferably the following steps (al) to (a-3): (a-1) a step of freeze-thawing the amnion; (a-2) trypsin on the amniotic membrane after the freeze-thaw treatment. And (a-3) washing the amniotic membrane after the trypsin treatment.

In one aspect of the present invention, step (b) before or after step (2), that is, (b) a step of attaching an adhesive component to the chorion side of the amniotic membrane is performed.

[0010] The present invention further provides a tissue reconstruction method characterized by using amniotic membrane as a main component of the tissue reconstruction material, and covering the surface of the amnion with a damaged surface tissue.

Brief Description of Drawings

FIG. 1 is a diagram for explaining a method for fixing an amniotic membrane. (A) A pair of frames sandwich the amniotic membrane, (b) Frame Sandwich the amniotic membrane with a flat plate member.

圆 2] This is a table that summarizes the uses (main application sites, examples of application methods, preferred amniotic membrane forms, main purposes) of tissue reconstruction materials.

圆 3] A table summarizing the remaining state of epithelial cell layer and basement membrane components in amniotic membrane under various treatment conditions. * 1 + +: About 100 to 10% of epithelial cells remain, +: About 10 to 2% of epithelial cells remain, Shi: About 2% of epithelial cells remain, 1: No epithelial cells remain. * 2 +: Fresh and fresh · Residual to the same extent as the amnion with epithelium.

IV 4] A table summarizing the remaining state of dense layer components in amniotic membrane under various treatment conditions. * 2 +: Raw fresh · Remains to the same extent as the amniotic membrane with epithelium, but it remains, but the detected amount is significantly reduced.

圆 5] Cryopreserved, amnion with epithelium (A) HE stained image and immunostained image. The signal of each antibody is green in the immunostained image. Cell nuclei are displayed in red.

[Fig. 6] Cryopreservation · Epithelial-free amnion (B) is an HE-stained image and an immunostained image. The signal of each antibody is green in the immunostained image. Cell nuclei are displayed in red.

圆 7] Freeze-dried, with epithelium · HE-stained and immunostained images of γ-ray treated amniotic membrane (C). The signal of each antibody is green in comparison with the immunostained image. Cell nuclei are displayed in red.

[Fig. 8] HE stained image and immunostained image of fresh fresh epithelial amniotic membrane (F). The signal of each antibody is green on the immunostained image. Cell nuclei are displayed in red.

圆 9] Raw fresh, 10% trypsin treatment, γ-ray-untreated amniotic membrane (J) HE and immunostained images. The signal of each antibody is green in the immunostained image. Cell nuclei are displayed in red.

[FIG. 10] HE stained image and immunostained image of raw fresh, 10% trypsinized, and γ-treated amniotic membrane (Κ). The signal of each antibody is green in the immunostained image. Cell nuclei are displayed in red.

圆 11] A table showing the remaining amount of matrix membrane components in various amniotic membranes compared with fresh amnion with epithelium. +: Remains to the same extent as the control. : Although the protein remains, the detection intensity is significantly lower than the control (judgment by visual inspection). ―: Protein is detected I can't get out.

圆 12] A table showing the remaining amount of dense layer components in various amniotic membranes compared with fresh fresh 'epithelial amniotic membranes. +: Remains to the same extent as the control. : Although the protein remains, the detection intensity is significantly lower than the control (judgment by visual inspection). ―: Protein cannot be detected.

[13] Figure 13 shows the state of the cecal tissue one week after the amniotic coating. In the group that does not cover the amniotic membrane, it can be seen that the cecum and abdominal wall, testicular fat, small intestine and omentum are highly adhered. Fresh fresh · 10% trypsin treatment · γ-ray treated amniotic membrane has adhesions between the cecum, omentum and testicular fat. On the other hand, no adhesion was observed in the cryopreserved / epithelial-coated group. In addition to the results of the group frozen-preserved · covered with epithelial amniotic membrane, the results of the group cryopreserved · coated with epithelial-free amniotic membrane, and the results of the group freeze-dried 'with epithelium · coated with γ-ray treated amniotic membrane Also shown.

FIG. 14 shows a HE-stained image of the cecal tissue 4 weeks after the amniotic membrane coating. A layer of squamous epithelial cells (stained with hematoxin) is present on the outermost layer of the cecum on the abdominal side (lower side in the figure), which becomes mesothelial cells. In the group coated with amniotic membrane, mesothelial cells are present in the same manner as in the normal intestinal tract, and the serosa is reconstructed. In addition to the results of the cryopreserved 'epithelial amniotic membrane group, the results of the cryopreserved, epithelial-free amniotic membrane group and the freeze-dried · epithelial · y-ray treated amniotic membrane group were also combined. Showed.

[15] Figure 15 shows HE-stained images of cecal tissue 1 day to 4 weeks after cryopreservation and epithelial coating. The upper side of the figure is the mucosal layer of the small intestine lumen, and the lower side is the abdominal side. The white layer on the abdominal side is the amniotic membrane. It can be seen that the amniotic membrane is degraded over time and the muscle layer is regenerated.

[16] Fig. 16 shows HBME-1 (mesothelial cell marker) -stained images of the cecal tissue 1 day to 4 weeks after epithelial coating with cryopreservation. The upper side of the figure is the mucosal layer of the small intestinal lumen, and the lower side is the abdominal side. Over time, mesothelial cells attach to the amniotic membrane and the serosa is reconstructed.

[FIG. 17] FIG. 17 is a table summarizing the adhesion score and the presence or absence of serosal reconstruction of amniotic membrane after various treatments. BEST MODE FOR CARRYING OUT THE INVENTION

[0012] (Terminology)

In this specification, “tissue reconstruction material” refers to a material used for reconstruction (regeneration) of a living tissue. The “tissue reconstruction material” of the present invention is suitably used in a treatment aimed at reconstructing an organ or a surface tissue disorder caused by surgical invasion. The “tissue reconstruction material” of the present invention is particularly suitable as a material for reconstructing a surface tissue in which adhesion occurs in a normal healing process. As used herein, the term “tissue reconstruction” typically means restoring a damaged portion of a surface tissue to a normal state, but does not restore the surface thread and tissue of an organ or organ. It is also used as a term that includes the recovery of the organ to a normal state by preventing it (for example, preventing re-adhesion after detachment of the fallopian tube and returning it to a normal state). .

[0013] Suitable examples of tissues to be reconstructed according to the present invention include organs or organs in the abdomen, chest, or pelvis (stomach, large intestine, small intestine, cecum, duodenum, heart, lung, fallopian tube, rectum, liver, Surface tissue of the egg nest, uterus, etc.) or surface tissue of the abdominal cavity, chest cavity, pelvic cavity, oral cavity, nasal cavity, ear cavity, or throat cavity, or ocular tissue. Therefore, the tissue reconstruction material of the present invention can be used in the fields of digestive surgery, obstetrics and gynecology, thoracic surgery, oral surgery, otolaryngology, and ophthalmic surgery. The tissue reconstruction material of the present invention is particularly suitable as a material for reconstructing an organ in the abdomen, chest, or pelvis or the surface tissue of the organ, or the abdominal cavity, chest cavity, or surface tissue. However, the present invention can be widely applied in areas involving surgical operations even outside these areas. Details of the application site and application method of the tissue reconstruction material of the present invention will be described later (C. Column of application site and application method of tissue reconstruction material).

[0014] A. Composition of tissue reconstruction materials

The first aspect of the present invention relates to a tissue reconstruction material. In the tissue reconstruction material of the present invention, amniotic membrane is used as a main constituent. In a typical embodiment, the tissue reconstruction material of the present invention is substantially composed solely of amniotic membrane.

As a result of the study by the present inventors, it has been clarified that the amniotic membrane has an action of promoting the reconstruction of the tissue while preventing adhesion, as shown in Examples described later. This characteristic of amniotic membrane By utilizing this property, the tissue reconstruction material of the present invention can exhibit a good tissue reconstruction effect. In addition, the high transparency and toughness of the amniotic membrane makes the tissue reconstruction material of the present invention excellent in transparency and strength. Sarakuko is a material for tissue reconstruction that has higher biocompatibility and lower immunogenicity due to the high biocompatibility and low immunogenicity of amniotic membrane.

[0015] (Derived from amniotic membrane)

The “amniotic membrane” is a membrane that covers the outermost layers of the uterus and placenta in mammals, and is composed of a basement membrane and an epithelial layer formed on a collagen-rich parenchyma. Amniotic membranes such as humans, monkeys, chimpanzees, pigs, horses, lions, etc. can be used. Among them, it is preferable to use human amniotic membrane. This is because it is advantageous in terms of safety, including immunogenicity and viral infection.

[0016] (Amnion condition)

In one embodiment of the present invention, amniotic membrane from which the epithelial cell layer has been removed is used. The amniotic membrane from which the epithelial cell layer has been removed is extremely safe because there are no problems such as immune rejection caused by epithelial cells. The amnion from which the epithelial layer has been removed can be confirmed by examining that the tissue reconstruction material of the present invention does not contain cells of the amnion epithelial layer.

On the other hand, the tissue reconstruction material of the present invention may be constructed using amniotic membrane with the epithelial layer remaining. By leaving the epithelial layer of the amniotic membrane, sufficient sterilization treatment such as γ-ray treatment can be performed at the production stage, and safety can be improved.

[0017] The tissue reconstruction material of the present invention is provided in a wet state (for example, in a state of being immersed in a solution), a frozen state, or a dry state (including a semi-dry state). If it is frozen or dried, it is easy to handle and has excellent storage stability. If it is in a dry state, it can be stored at room temperature (eg, about 10 ° C to about 35 ° C). In other words, it is not necessary to manage in a frozen or refrigerated environment before use, and handling (storage, transportation, etc.) becomes easy. However, even in a dry state, it may be frozen or refrigerated as necessary.

In particular, in the case of freeze-dried state, in addition to excellent handling, it adheres well to the affected area when applied (infiltrates on the affected area surface and exerts adhesive force), so sutures after application are fundamental. (However, a suturing process may be performed to further ensure adhesion to the affected area!). If sewing is not necessary, the burden on the patient and doctor is greatly reduced. Preferably, a frozen state having an epithelial layer, a dry state having an epithelial layer (lyophilized state), a wet state having no epithelial layer, a frozen state having no epithelial layer, or a dry state having no epithelial layer ( The tissue reconstruction material of the present invention is constructed using amniotic membrane in a lyophilized state. In the present specification, a frozen amniotic membrane having an epithelial layer is also referred to as “cryopreserved amniotic membrane with epithelium”, and a lyophilized amniotic membrane having an epithelial layer is also referred to as “freeze-drying / amniotic membrane with epithelium”. A frozen amniotic membrane that does not have an epithelial layer is called “cryopreserved, epithelial-free amnion”, and an amnion that does not have an epithelial layer is called “freeze-dried / epithelial-free amnion”.

[0018] As shown in Examples described later, it was suggested that retention of the structure of the basement membrane is important for the amniotic membrane to exhibit adhesion prevention and tissue reconstruction functions. Therefore, a preferred embodiment of the present invention is characterized in that an amniotic membrane in which the basement membrane components (collagen IV (1, 2, 2 and 5), collagen VII, laminin 5) remain is used.

Whether or not a basement membrane component remains can be assayed by performing immunostaining using the component as a detection target. In one embodiment of the present invention, at least one, preferably a plurality, and all of these components are detected. In addition, the strength (preferably about the same, strength) of these untreated amniotic membranes (that is, amniotic membranes that have been separated from the living body and have not been subjected to freezing or the like treatment) are significantly different from those detected. Preferably, the component is detected.

On the other hand, it is preferable to use amniotic membrane in which the dense layer components (collagen I, III, V, fibronectin) remain. The residual state of the dense layer component can be assayed by immunostaining in the same manner as in the basement membrane component described above.

[0019] (Use of reconstructed amniotic membrane)

The tissue reconstruction material of the present invention can also be constituted using the reconstructed amniotic membrane. Specifically, for example, amniotic membrane that has been once decomposed by homogenizer, ultrasonic wave, or enzymatic treatment and reconstructed into a membrane shape can be used. It is preferable to use a homogenizer as the treatment method. This is because it is expected to keep the minute structure of the basement membrane relatively high. The condition (rotation speed) of the homogenization process is, for example, 3000rpn! ~ 50000rpm, preferably 10000rpm ~ 40000rpm, more preferably <30000rpm.

[0020] (Thickness)

By using amniotic membrane, the tissue reconstruction material of the present invention is formed into a very thin sheet. Can be made. The tissue reconstruction material of the present invention is prepared to a thickness of 10 m to 500 m, for example. Thus, versatility increases by being a very thin sheet form. The tissue reconstruction material of the present invention may be constructed using amniotic membrane from which a part of the dense layer on the chorionic membrane side (for example, about 10 μm to 30 μm) is removed, or a bioabsorbable material For example, the thickness may be about 100 μm to 500 μm.

[0021] (Use of adhesive components)

The tissue reconstruction material of the present invention is typically applied so as to cover the site (injured part) to be reconstructed with the chorionic side of the amniotic membrane facing down. In other words, the chorion side of the amniotic membrane becomes the adhesive surface. An adhesive component can be attached to the chorionic side of the amniotic membrane in order to enhance the adhesion. If the adhesiveness is improved in this way, it becomes possible to obtain a sufficient adhesive force without stitching, thereby simplifying the surgical procedure. In this specification, an amniotic membrane having an epithelium and having an adhesive component attached thereto is also referred to as “adhesive component attachment / amniotic membrane”, and an amniotic membrane having no epithelium and having an adhesive component attached thereto is referred to as “adhesive component attachment / epithelium”. It is also called “no amniotic membrane”.

[0022] As the adhesive component, for example, fibrinogen and thrombin can be used. When these components are used, when the tissue reconstruction material of the present invention is transplanted, fibrinogen is first specifically hydrolyzed by thrombin to produce fibrin, and then fibrin is polymerized and stabilized. It becomes a fibrin clot and exhibits an adhesive action. As will be described later, the tissue reconstruction material of the present invention is prepared through a process of attaching fibrinogen and thrombin to the amniotic membrane surface to an appropriate state (for example, a dry state or a wet state). Therefore, it is expected that some fibrinogen fibrin will form during the production process and depending on whether Z or its final state is used. When fibrin or a fibrin clot produced by such a cause is attached, it can be said that fibrinogen and thrombin are substantially used as adhesive components.

[0023] The origin of fibrinogen and thrombin is not particularly limited. For example, fibrinogen and thrombin can be prepared using blood such as humans, monkeys, chimpanzees, horses, horses, hidges, and pigs as materials. Further, as fibrinogen and thrombin, recombinants (recombinants) obtained using cultured cells (eg, CHO cells or COS cells) may be used. Fibrinogen and thrombin of human origin (especially human-derived thread and change) It is preferable to use it. It is also an advantageous force in terms of safety, including immunogenicity. In view of the point that stable quality can be used and the problem of infection, it is particularly preferable to use a recombinant.

It is particularly preferable to use fibrinogen and thrombin derived from the blood of a patient (recipient) who receives the transplantation of the tissue reconstruction material of the present invention. This is because there is no risk of inducing immune rejection caused by these adhesive components.

The origins of fibrinogen and thrombin are not necessarily the same. For example, human blood-derived fibrinogen and sushi blood-derived thrombin can be used in combination.

[0024] The amount of fibrinogen and thrombin attached is not particularly limited. For example, it is possible to set the amount of adhered Fuiburino one Gen range of 0.1mg~50mg per amnion lcm ^2. Similarly, the adhesion amount of thrombin can be set in the range of 0.5 μmg to 10 mg per 1 cm ² of amniotic membrane.

In setting the amount of fibrinogen and thrombin attached, the adhesive strength is considered first. That is, it is necessary to set the adhesion amounts of these components so that the expected adhesion amount can be obtained. On the other hand, if the amount of fibrinogen or thrombin attached is too large, the problem is that it tends to induce an immune reaction or angiogenesis, depending on the origin of the fibrinogen used.

Fuiburino one Gen amniotic lcm ² per 0.5~20mg Preferred range of coating weight, more preferably amniotic lcm ² per 0.5mg~10mg as a range, the amnion 1 cm ² per 0.5Mg～6mg (specifically a more preferred range For example, about 0.5 mg, about 1 mg, and about 2 mg). Similarly amniotic lcm Preferred range of coating weight of thrombin ² per 1 G～lmg, further amnion lcm ² per 5 g~200 g as preferred range, more preferably amniotic lcm ² per 10 g~100 g (specifically as a range For example, about 10 μg, about 20 μg, and about 30 g) can be mentioned.

In one embodiment of the present invention, aprotun is used as an adhesive component in addition to fibrinogen and thrombin. Aprotune inhibits the fibrin clot formed by the action of thrombin from being lysed by plasmin. Therefore, by using Aprotune together The decomposition of the fibrin clot can be suppressed, and the adhesive strength can be maintained or enhanced.

The origin of aprotinin is not particularly limited. For example, aprochons derived from the spleen such as ushi, horse, hidge, pig, monkey and chimpanzee can be used. You can also use recombinant (recombinant) aprotunes obtained using cultured cells (eg CHO cells or COS cells). Considering the point that stable quality can be used and the problem of infection, it is preferable to use recombinants.

When aprotinin is used, the amount of adhesion is not particularly limited. For example, the amount of aprotinin attached can be set in the range of 0.1 KIU to 200 KIU per lcm ² of amniotic membrane. Amniotic lcm ² per 1 as an adhesion amount of the preferred range of § Purochun KIU～100 KIU, further amnion lcm ² per 1 KIU～20 KIU as good preferable range, amnion lc m ² per KIU～10 KIU as more preferred range (Specific examples include about 1 KIU, about 2 KIU, and about 3 KIU). If the amount of aprotun is too large, not only will the production cost increase, but the risk of side effects due to the immunogenicity of aprotinin itself will increase. On the other hand, if the amount of aprochun is too small, there is a risk that the effect of aprochon, that is, inhibiting the degradation of the fibrin clot, may not be exhibited.

In various applications, fibrin clots are used as adhesives. In such applications, it is common to use aprotune together. As a result of the study by the present inventors, it has been found that the tissue reconstruction material of the present invention can provide sufficient adhesion to a living body without using aprochun. The fact that aprochon is not required is not only because the structure is simplified and it is advantageous in terms of production and cost, but it is not necessary to consider side effects due to the immunogenicity of aprochun itself. Means.

(Reinforcement with bioabsorbable material)

The strength of the tissue reconstruction material of the present invention can be increased by coating the chorionic side of the amniotic membrane with a bioabsorbable material. As a bioabsorbable material used for such a purpose, it is preferable to employ a material that is decomposed and absorbed earlier than amniotic membrane. For example, polydaractin 910, gelatin, collagen, polylactic acid and the like can be suitably used as the bioabsorbable material here. The form of the bioabsorbable material used for reinforcement is not particularly limited. For example, a bioabsorbable material molded into a mesh or sheet, with the amnion chorion side The amniotic membrane is reinforced by covering with. The amniotic membrane may be either wet or dry in the reinforcement process. However, in the final form of the product, it is preferable that the amniotic membrane is in a dry state. This is because when it is in a dry state, it is excellent in operability and storage. In addition, this

And, amniotic membrane with reinforcement is also called “hybrid amniotic membrane”!

[0027] (Form of provision)

The tissue reconstruction material of the present invention is provided, for example, in a state of being stored in a container such as glass or plastic, or in a state of being packaged using a transparent film, a light shielding sheet, or the like. Preferably, the tissue reconstruction material of the present invention is provided packaged so as to be practically free of contact with oxygen. In a powerful form, high quality can be maintained over a long period of time without deterioration of quality due to oxygen. Examples of the “substantially no contact with oxygen” include a state in which the container is evacuated, a state in which the container is filled with nitrogen (replaced with nitrogen), or a state in which the container is hermetically packaged with a film or sheet. be able to. The tissue reconstruction material of the present invention is usually sterilized in advance.

[0028] B. Manufacturing method of tissue reconstruction material

A second aspect of the present invention relates to a method for producing a tissue reconstruction material using amniotic membrane, and includes the following steps (1) to (3).

(1) Bio-power Steps to prepare the separated amniotic membrane

(2) Step of freezing or drying amniotic membrane

(3) As an optional step, sterilizing the amniotic membrane

[0029] (1) Step of preparing amniotic membrane (Step (1))

The amniotic membrane used in this step is preferably human amniotic membrane. For example, human amnion can also collect force such as human fetal membrane and placenta obtained as a postpartum at delivery. Specifically, a human amniotic membrane can be prepared by treating and purifying an integral body consisting of human fetal membrane, placenta and umbilical cord obtained immediately after delivery. As a method for preparing such human amniotic membrane, a known method such as the method described in JP-A-5-56987 can be employed. That is, the amniotic membrane can be peeled off from the fetal membrane obtained at the time of delivery, the remaining tissue can be removed by physical treatment such as ultrasonic washing and enzyme treatment, and the human amniotic membrane can be prepared through an appropriate washing step.

[0030] The amniotic membrane prepared in step (1) can be frozen and stored. Freezing human amniotic membrane For example, it can be carried out in a liquid in which equal volumes of DMEM (Dulbecco's modified Eagle's medium) and glycerol are mixed at a volume ratio of -80 ° C. The operability can be improved by cryopreserving, and the antigenicity can be expected to decrease. In addition, even when long-distance transportation is required, quality degradation is reduced. Convenience is improved by freezing in this way. The period of cryopreservation is, for example, 1 day to 2 years, preferably less than 1 year, and more preferably less than 6 months. The temperature for cryopreservation can be set, for example, within a range of 20 ° C to 1180 ° C. It is preferable to store frozen at approximately -80 ° C because of its low quality degradation and the ability to use a general-purpose freezer! /.

This cryopreservation step is performed as necessary. For example, the amnion after collection may be stored refrigerated rather than frozen and used for subsequent processing.

[0031] (2) Freezing treatment or drying treatment step (Step (2))

In this step, the amniotic membrane is frozen or dried. This treatment results in an amniotic membrane that has excellent storage stability and is easy to handle. As shown in the Examples below, the amniotic membrane that was cryopreserved after collection exhibited better anti-adhesion action than the amniotic membrane that was cryopreserved after collection. In other words, it was found that the anti-adhesion effect is enhanced by the freezing treatment, and the amniotic membrane becomes a more preferable material for tissue reconstruction. Therefore, it is preferable that the amniotic membrane is subjected to a freezing treatment in order to enhance the tissue reconstruction effect. The amniotic membrane can be frozen under the same conditions and method as in step (2) above.

[0032] On the other hand, according to the drying treatment, the amniotic membrane is very highly preserved and convenient. In addition, it is expected that the affinity (adhesiveness) of the amniotic membrane to the living tissue will be improved by the change of the surface form accompanying drying. It is preferable to dry the amniotic membrane by freeze-drying. This is because a decrease in the flexibility of the amniotic membrane can be suppressed. In addition, freeze-drying treatment is preferable from the viewpoint of maintaining the structure of the basement membrane component of the amniotic membrane.

[0033] In the lyophilization process, the boiling point generally ranges from about -20 ° C (107 Pa, 0.8 Torr) to about -50 ° C (4 Pa, 0.

In a low atmospheric pressure environment (vacuum) such as 03 Torr), moisture is removed by sublimation from a frozen and solidified sample (eg, frozen at about -40 ° C). According to the freeze-drying process, it can be dehydrated uniformly from the inside, and a high degree of dryness can be realized, so that it can be dried while maintaining its original function and form. In addition, freeze-drying process 1 It has the following characteristics: 2. Little deterioration during processing, 2. Easily sterilized, 3. Obtained dry product with excellent restorability, 4. Obtained dry product with excellent preservability.

[0034] The freeze-drying treatment can be performed by a freeze-dryer equipped with a vacuum chamber, a cooling and heating device, and an exhaust device (cold trap and vacuum pump). Numerous freeze-drying apparatuses are on the market, and in the step (ii) of the present invention, an arbitrarily selected one of these intermediate forces can be used. The processing conditions can be set based on the instruction manual attached to the device to be used. In that case, the size of the sample to be subjected to the drying treatment, the degree of dryness, etc. can be taken into consideration. Dryness can be set, for example, so that water activity (AW) is less than 0.5

In one embodiment of the present invention, before step (2), “step of removing epithelial layer from amniotic membrane leaving at least part of basement membrane (step (a))” is performed.

In this step, the epithelial layer is removed, but at this time, at least a part of the epithelial layer remains without removing the basement membrane. Such a treatment is performed by, for example, a manual peeling method, an enzyme peeling method using trypsin or the like, a mechanical peeling method, or a peeling method in which these treatments are arbitrarily combined. Preferably, adhesion between cells constituting the epithelial layer is loosened in advance with EDTA or proteolytic enzyme, and then repelling with a cell scraper or the like is performed. However, such pretreatment (treatment with EDTA or the like) is preferably performed under conditions that do not destroy the structure of the basement membrane that mediates adhesion of the epithelial layer to the parenchyma. For example, treatment with dispase under general conditions (for example, dispase added to 1.2 IU and allowed to react at 37 ° C for 1 hour) will damage not only the epithelial layer but also the basement membrane. When such pretreatment is performed, the basement membrane having the original function cannot be left. The important point in the step (a) of the present invention is to remove the epithelial layer while leaving at least a part of the basement membrane in a state in which its original function is maintained.

The presence of the basement membrane can be confirmed by detecting components characteristic of the basement membrane (collagen IV 1, α2, and α5), collagen VII, and laminin 5).

It is preferable to minimize the influence on the dense layer accompanying the removal of the epithelial layer. This is to prevent a decrease in strength. The degree of damage to the dense layer is determined by the components characteristic of the dense layer (colla It can be confirmed by detecting (gen I, III, V, fibronectin).

[0036] In order to prevent damage to the basement membrane accompanying the removal of the epithelial layer, it is preferable to remove the epithelial layer in the following steps (a1 to a-3). Prior to removing the epithelial layer, it is preferable to fix the prepared amniotic membrane to the frame. Fixing the amniotic membrane with a frame makes it easier to handle. Figure 1 shows a specific example of how to fix the amniotic membrane to the frame. In the example of Fig. La, two frames (1, 2) of the same shape are used. The amniotic membrane 10 is fixed by holding the edges of these two frames. Fix the amniotic membrane in an expanded state. In the example of Fig. Lb, the amniotic membrane 10 is fixed using the frame 3 and the plate-like member 4. First, the amniotic membrane 10 is spread on the plate-like member 4. At this time, the epithelial side of the amniotic membrane 10 is turned up. Subsequently, the upper frame 3 of the amniotic membrane 10 is placed, and the edge of the amniotic membrane 10 is sandwiched between the plate-like member 4 and the frame 3. As a result, only the epithelial side of the amniotic membrane 10 is exposed. Therefore, when the trypsin treatment described later is performed, the trypsin solution can be brought into contact only with the epithelial side of the amniotic membrane (for example, the trypsin solution is added inside the frame 3). As a result, trypsin treatment can be performed without causing any influence on the parts other than the epithelium (the amnion dense layer and the basement membrane). That is, it is possible to protect the amnion basement membrane and the like from the action of trypsin while allowing trypsin to act on the epithelium of the amniotic membrane.

[0037] (Freeze-thaw treatment: Step a-1)

In this step, the amniotic membrane is once frozen and then thawed. This freezing and thawing process makes it easier for the amniotic epithelial layer to peel off during subsequent trypsin treatment. This is thought to be due to the loosening of the adhesive state (bonded state) between the amniotic epithelial layer and the basement membrane.

A freezing temperature of about 20 ° C to about 80 ° C can be used. In consideration of the fact that a sufficient frozen state can be obtained and a general-purpose freezer can be used, it is preferable to freeze at about 80 ° C. On the other hand, a melting temperature of about 4 ° C to about 50 ° C can be employed. Preferably the melting temperature is about 37 ° C.

It is preferable to repeat the freeze-thaw treatment. By repeatedly performing the treatment, the effect of the freeze-thaw treatment that the epithelium is easily detached in the subsequent trypsin treatment is enhanced. However, if it is repeated more than necessary, it is also expected to adversely affect parts other than the epithelium. Therefore, for example, it is preferable to repeatedly perform freeze-thaw treatment in the range of 2 to 4 times. As a result of the study by the present inventors, it was found that the freeze temperature was -80 ° C and the freeze temperature was 37 ° C. It was found that the necessary and sufficient effect can be obtained by performing the treatment twice. From this knowledge, it can be said that the freeze-thaw treatment is preferably performed twice under the conditions of a freezing temperature of -80 ° C and a thawing temperature of 37 ° C.

The conditions (freezing temperature and thawing temperature) for each time when the freeze-thaw treatment is repeatedly performed may be the same, partly different, or different from each other. However, from the viewpoint of operability, it is preferable that the conditions are the same each time.

[0038] (Trypsin treatment: Step a-2)

In this step, the amniotic membrane after the freeze-thaw treatment is treated with trypsin. Trypsinization is performed by bringing a trypsin solution into contact with the amniotic membrane. For example, a trypsin solution having a trypsin concentration of about 0.01% (w / v) to about 0.05% (w / v) can be used. Preferably, a trypsin solution having a trypsin concentration of about 0.02% (w / v) is used. If the trypsin concentration of the trypsin solution is too low, the action of trypsin will not be fully exerted. On the other hand, if the trypsin concentration is too high, trypsin can act well on the amniotic epithelium, while trypsin also acts on the amnion dense layer and the basement membrane, which may damage the part.

As a treatment method for amniotic membrane, a treatment method using dispase can be considered in addition to a treatment method using trypsin. Examination of dispase treatment revealed that the amnion epithelium was not detached when a dispase solution with a low concentration of dispase, such as 1 U or 10 U, was observed, but detachment of the epithelium was observed only when a solution with a concentration of 100 U or higher was used. It was done. However, the dense layer of amniotic membrane was damaged to the extent that it could be visually discerned, and the network structure in the dense layer was partially unrolled and roughened. In addition, collagen VII, which is one of the basement membrane components, was damaged and was unable to leave the basement membrane intact. Thus, dispase is generally unsuitable as a solution to be used for epithelial detachment, in which damage to the amnion dense layer and basement membrane is generally larger than that of trypsin solution.

Many trypsins are commercially available such as those derived from ushi, porcine, and human. For example, Trypsin-EDTA (Invitrogen) and trypsin 1: 250 (Sigma) can be preferably used.

[0039] A chelating agent is usually added to the trypsin solution, but the chelating agent is not essential. As a chelating agent, EDTA, NTA, DTPA, HEDTA, GLDA, etc. can be used. Any combination of these may be used. The chelating agent is added, for example, to a concentration of about O.lmM to about 0.6 mM.

[0040] It is preferable to carry out trypsin treatment under conditions where only the amnion epithelial side is in contact with the trypsin solution. This is to protect the action force of trypsin on parts other than the amniotic epithelium. For example, immerse only the amnion epithelium side in a trypsin solution, do not add trypsin solution to the amnion epithelium side, apply it, and block the amnion chorion side to avoid contact with the solution. Thereafter, only the amnion epithelial side can be brought into contact with the trypsin solution by, for example, immersing it completely in a trypsin solution. As described above, if amniotic membrane (frame-fixed amniotic membrane) fixed in advance to the frame as shown in Fig. Lb is used, only the epithelial side of the amniotic membrane is exposed, so for example, the frame-fixed amniotic membrane is immersed in a trypsin solution. It is possible to contact only the amnion epithelium side with the trypsin solution. This method also has the advantage that the trypsin treatment can be performed by a simple operation of immersing the frame-fixed amniotic membrane. Even when the amniotic membrane fixed to the frame is used in this way, if the entire frame is immersed in a trypsin solution, only the epithelial side portion of the amniotic membrane is immersed in the trypsin solution (for example, the epithelium of the amniotic membrane). (You can dip it in a trypsin solution with the side down), add the trypsin solution in the frame, or apply the trypsin solution to the amnion epithelial side to infect only the epithelial side of the amniotic membrane with the trypsin solution. .

[0041] The trypsin treatment time (contact time of the trypsin solution) is, for example, about 5 minutes to about 60 minutes. It is preferably about 10 minutes to about 20 minutes, more preferably about 15 minutes. If the treatment time is too short, trypsin cannot be sufficiently exerted, resulting in insufficient removal of the amniotic epithelium. On the other hand, if the treatment time is too long, trypsin may also act on the basement membrane and dense layer of the amniotic membrane to damage the part.

The temperature condition of the trypsin treatment is, for example, about 25 ° C. to about 42 ° C. so that trypsin works well.

It is preferred to keep the amniotic membrane stationary while contacting the trypsin solution. It is also the force that the trypsin solution is thought to penetrate through the basement membrane and the dense layer.

The trypsin treatment can be performed in a plurality of times. [0042] (Washing: Step a-3)

After contacting the trypsin solution by the above method, the amniotic membrane is washed. This washing removes the attached trypsin solution and simultaneously removes the amniotic epithelium (epithelial cells). For example, leave it in a liquid with an appropriate flow (for example, flowing water), shake it in a suitable liquid (for example, shake up and down), or apply ultrasonic waves while immersed in a suitable liquid. The amniotic membrane after trypsinization is washed by adding. Examples of the liquid used for washing include physiological saline, phosphate buffer, pure water, and DMEM.

[0043] The washed amniotic membrane may be refrigerated or frozen until use. For example, it can be stored in a state of being immersed in a storage solution containing glyceride (for example, 50% glycerol-containing DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL)).

[0044] On the other hand, in one embodiment of the present invention, before or after step (2), (b) a step of attaching an adhesive component to the chorion side of the amniotic membrane is performed. When step (a) is performed, step (b) is usually performed after step (a).

For example, fibrinogen and thrombin can be used as the adhesive component. In the following, the case where these adhesive components are used will be described.

[0045] It is preferable to dry the amniotic membrane prior to the adhering operation of the adhesive component. By using the amniotic membrane in a dry state, an adhesive component such as fibrinogen can be better adhered. Examples of the drying treatment include freeze drying, air drying, vacuum drying, and reduced pressure drying. Among them, it is preferable to employ freeze-drying. This is because the flexibility of the amniotic membrane is unlikely to decrease in the case of freeze-drying.

[0046] The adhesion of fibrinogen and thrombin to the surface of the amniotic membrane is performed individually or simultaneously. The attachment method is not particularly limited. Examples of the attaching method include a method of applying, dripping, or spraying the solution of the component to be attached to the surface of the amniotic membrane, or a method of immersing the amniotic membrane in the solution of the component to be attached. Further, fibrinogen itself (or thrombin itself) or fibrinogen (or thrombin) dissolved in a suitable solvent and added to the surface of the amniotic membrane (sprayed) to fibrinogen (or Can also be attached to the amniotic membrane surface.

Preferably, a mixture of these two components is prepared and applied to the mixture, dripping, etc. Therefore, fibrinogen and thrombin are simultaneously attached to the amniotic membrane surface. A specific example of a method for attaching these two components simultaneously will be described below.

First, a fibrinogen solution is prepared. Specifically, fibrinogen is dissolved in a solvent (solvent) such as ethanol (for example, 94% ethanol) to a desired concentration. In addition to ethanol, alcohols such as absolute ethanol, isopropanol, and methanol, and acetone can be used as the solvent. On the other hand, prepare a thrombin solution separately by the same procedure. As the solvent in this case, for example, ethanol (for example, 99.5% ethanol), alcohols such as absolute ethanol, isopropanol and methanol, acetone, and the like can be used.

[0047] Next, the fibrinogen solution and the thrombin solution prepared by the above procedure are mixed. Using the mixed solution thus obtained, application or dropping onto the amniotic membrane is performed as described above. As described above, when the fibrinogen solution and the thrombin solution are mixed and the adhesion operation is performed using the mixed solution, it is preferable to take care not to increase the amount of water in the mixed solution. If the amount of water in the mixed solution increases, a reaction between fibrinogen and thrombin occurs before the attachment operation, which hinders the attachment operation. In order to obtain good adhesion after transplantation, it is preferable that fibrinogen and thrombin adhere to the amniotic membrane in a state where they do not act in advance. Considering the above points, it is preferable to employ a water-soluble solvent with a small amount of water and a volatile solvent as a fibrinogen solvent and a thrombin solvent.

[0048] Application and dripping of a fibrinogen solution and a thrombin solution or a mixture of fibrinogen and thrombin are typically performed uniformly over the entire area of the amniotic membrane surface. This can be done only on partial areas (for example, on multiple areas at intervals like spots, or only on the periphery), or by applying a shade to the amount of adhesion.

[0049] In the above-described method, fibrinogen and thrombin are simultaneously attached. The respective components may be attached in separate steps. That is, fibrinogen attachment and thrombin attachment may be performed in two steps. However, because the operation can be simplified and fibrinogen and thrombin can be attached in a more uniform dispersion state, the fiber It is preferable to perform the adhesion operation in one step using a mixture of rinogen and thrombin. Fibrinogen and thrombin can be prepared from blood according to a conventional method. Recombinant fibrinogen or the like can also be used, and in this case, it can be prepared by a conventional method from a culture solution or a cell disruption solution of an appropriate cultured cell. A commercially available fibrinogen or the like may be used. For example, human-derived fibrinogen can be purchased from Paxter. Similarly, human-derived thrombin can be purchased from Baxter.

[0050] In addition to fibrinogen and thrombin, aprotun may be attached to the surface of the amniotic membrane. That is, in this embodiment, the step of attaching aprotune (step b-1) is further performed. Aprochung can be attached by the same means and procedure as fibrinogen and the like. That is, aprochon can be adhered to the amniotic membrane surface by application, dripping, spraying, dipping, etc. using aprotonic solution. Aprotun solution can be prepared by dissolving aprotun in a sodium chloride solution (eg, 0.85% solution), potassium chloride solution, calcium chloride solution, magnesium chloride solution, or the like. Aprotune can be prepared from the spleen of ushi according to a conventional method. Recombinant aprotune can also be used, and in this case, it can be prepared by a conventional method from a culture solution or cell disruption solution of an appropriate cultured cell. In addition, a commercially available aprotune may be used. For example, aprochun derived from Ushi can be purchased from Bayer Yakuhin.

It is also possible to carry out the step of attaching caprotun alone. Preferably, it is carried out simultaneously with the step of attaching fibrinogen and thrombin. The adhesion operation of the adhesive component is simplified as a whole. In addition, fibrinogen, thrombin, and aprotune can adhere to the amnion surface in a more uniformly dispersed state. For example, by preparing a mixture of fibrinogen, thrombin, and aprotune and applying it, these three components can be attached simultaneously to the amniotic membrane. The order of mixing these three components is not particularly limited.

[0051] Before or after step (2), a step of covering the chorion side of the amniotic membrane with a bioabsorbable material may be performed. This treatment can increase the strength of the amniotic membrane. Bioabsorbability Examples of the material include polydaractin 910, gelatin, collagen, polylactic acid and the like.

[0052] (3) Sterilization step (Step (3))

By performing sterilization, the risk of contamination with bacteria can be minimized. Amnion can be sterilized by EOG (ethylene oxide gas), UV (ultraviolet light), γ-ray treatment, etc. Among these, it is preferable to employ y-ray sterilization. This is because there is little decrease in the physical properties of the amniotic membrane. The dose in 0-line sterilization is, for example, 2 kGy to 50 kGy, preferably 10 kGy to 30 kGy, and more preferably 15 kGy to 25 kGy.

[0053] It is preferable that the sterilization treatment is performed in a state where the amniotic membrane after the series of treatments is stored in a container or packaged with a film or a sheet. Therefore, it is preferable to implement a step of storing the amniotic membrane in a container or the like prior to the sterilization treatment. It is preferable to store or wrap the amniotic membrane in a state where there is substantially no contact with oxygen. This is because degradation of quality is suppressed and long-term storage is possible.

[0054] C. Application site and application method of tissue reconstruction material

The uses of the tissue reconstruction material of the present invention can be broadly classified into the following three types based on the application method and application purpose.

(1) Covering tissue reconstruction materials (Application method) · Organization reconstruction (purpose of application) type

This is a method (use method) aimed at reconstructing the injured tissue surface by applying tissue reconstruction material on the organ surface, peritoneum surface, etc. Specific examples of this use are the following 1 1, 1 4, 1-5, 1-6.

(2) Covering tissue reconstruction material (application method), anti-adhesion (application purpose) mold

It is a usage (use method) aimed at suppressing adhesion formation with surrounding tissues by applying amniotic membrane to the organ surface, peritoneum surface, etc. Specific examples of this use are the following 2-1, 3-1, 3-2, 3-3.

(3) Indwelling material for tissue reconstruction (application method), anti-adhesion (purpose of application) type

This is a method of use that suppresses the formation of adhesions by placing an amniotic membrane at a site where adhesions are highly formed. Specific examples of the application are the following 1 2, 1 3 and 2-2. Many conventional anti-adhesive agents take this form of use. For example, Sepura film is 1 2 It is the same usage pattern. However, Sepurafilm cannot be used in the following usage modes 1−3, 2−2.

The following are specific examples of uses for tissue reconstruction materials (see Figure 2).

1. Application in the field of digestive surgery

1-1. Reconstruction of damaged organs and application methods for adhesion prevention

When various types of surgery are performed, organs are slightly damaged during the operation. If the injured area loses the serosa structure and is not reconstructed early after surgery, it will cause adhesions to form between organs, which may be detrimental to the underlying function. Such problems can be solved by utilizing the ability to reconstruct amniotic membrane and prevent adhesions. Specifically, the surface of the injured organ is covered with a tissue reconstruction material to reconstruct the tissue and prevent adhesion. For such purposes, tissue reconstruction materials constructed with cryopreservation / amniotic membrane with epithelium, cryopreservation / amniotic membrane without epithelium, freeze-drying, amniotic membrane with epithelium, adhesion component adhesion, amnion without epithelium can be suitably used. Tissue reconstruction materials constructed with dry amniotic membranes (e.g., freeze-dried / epithelial amniotic membrane, freeze-dried / non-epithelial amniotic membrane) are preferable because they are easy to handle, but they are frozen in areas where the carrier needs flexibility such as the heart. It is also possible to select a material for tissue reconstruction constructed with a preserved amniotic membrane (eg, cryopreserved amniotic membrane with epithelium, cryopreserved non-epithelial amnion). The application method is to directly cover the damaged area of the organ with the tissue reconstruction material so that the amnion basement membrane faces the abdominal side when the surgery is completed. Then, fix as necessary. A suture such as bicyclyl can be used for fixation. When using a tissue reconstruction material constructed with a dry amniotic membrane, a high binding force can be expected, so that a fixing process such as suturing can be omitted. A high bond strength can be expected in the same way when using a tissue reconstruction material constructed using an adhesive component. As described above, it is preferable that the tissue reconstruction material is fixed to the application site without separately performing a fixing process such as suturing. This is because the operation becomes complicated when sutures are performed, and there is a possibility that inflammation is induced and adhesion formation is promoted. In particular, the use of a tissue preservation material constructed of cryopreserved epithelial amniotic membrane is particularly preferred because it can exert a high binding force to the application site and does not cause the problem of foreign body reaction due to adhesive components. Yeah.

By the above operation, reconstruction of the serosa structure can be expected in the early postoperative period. • Prevention of adhesion is achieved.

[0056] 1-2. Injured organs Application method for preventing adhesions between wounds

After various surgical procedures, adhesions may form between the abdominal organs and the wound. When the abdominal organs adhere to the wound, the organs are physically fixed, and the motility of the organs weakens, causing the lumen to close and cause paralytic bowel obstruction. Such problems can be solved by utilizing the ability of amnion to prevent adhesions. For this purpose, a tissue reconstructing material constructed of cryopreserved amniotic membrane with epithelium, cryopreserved / non-epithelial amniotic membrane, freeze-dried / amniotic membrane with epithelium, reinforced hybrid amniotic membrane, etc. can be used. Since sufficient strength is required, it is preferable to use a tissue reconstruction material constructed with a dry amniotic membrane. It is a dry amniotic membrane that is constructed with a hybrid amniotic membrane with reinforcement. It is even more preferred to use tissue reconstruction materials. For example, the application method is as follows. When the surgery is complete, insert tissue reconstruction material directly under the wound and leave it in place. At this time, the tissue reconstruction material is applied so that the basement membrane side of the amniotic membrane is the abdominal cavity side and the chorionic membrane side is the abdominal wall side. After application, it may be fixed by suturing or the like, but it is preferable to leave it without fixing.

By the above operation, it is possible to prevent adhesion between the organ and the wound after the operation.

[0057] 1-3. Application to prevent pelvic floor adhesions

In the pelvis, there are extra-abdominal organs such as the rectum and the uterus where part of the organ surface is not covered by the peritoneum. When such an extra-abdominal organ is operated, there is a region where the peritoneum does not exist in the pelvis, the small intestine falls into the pelvic floor, and the pelvic wall and the small intestine form an adhesion. Once adhesions are formed, peeling is often difficult. Precautionary measures are therefore important. Pelvic floor adhesions can be prevented using the amniotic serosa reconstruction ability. For such purposes, tissue reconstruction materials constructed of cryopreserved 'amniotic membrane with epithelium, cryopreserved amniotic membrane without epithelium, freeze-dried amniotic membrane with epithelium, reinforced hybrid amniotic membrane, etc. can be suitably used. The properties required for tissue reconstruction materials are the same as in 1-2. The application method is as follows, for example. At the end of the surgery, insert tissue reconstruction material into the pelvic floor and lightly press against the peritoneum to cover it. At this time, the tissue reconstruction material is applied so that the basement membrane side of the amniotic membrane is the abdominal cavity side and the chorion side is the peritoneum side. After application, suture, etc. Although it is possible to fix by, it is better to just cover without fixing.

By the above operation, prevention of adhesion at the pelvic floor after the operation can be achieved.

[0058] 1 4. Application method for wall side peritoneum reconstruction

The parietal peritoneum is lost due to multiple operations and peritoneal diseases such as abdominal wall hernia. Amniotic membrane can be used as a carrier for filling the defective abdominal wall. For this purpose, a tissue reconstructing material constructed by cryopreservation / amnion with epithelium, cryopreservation / amnion without epithelium, lyophilization / amnion with epithelium, adhering adhesion, amnion without epithelium can be suitably used. If the peritoneal defect is widespread and severe, it is preferable to use a tissue reconstruction material constructed with cryopreserved 'amniotic membrane with epithelium' from the viewpoint of strength. Application takes place at the end of the surgery. First, the tissue reconstruction material is placed on the abdominal wall defect area and covered. Cover the abdominal wall defect region with tissue reconstruction material so that the basement membrane side of the amniotic membrane is on the abdominal side. Thereafter, the tissue reconstruction material may be fixed using a suture or the like. When a tissue reconstruction material constructed of amniotic membrane with an adhesive component attached is used, fixation may be achieved with the adhesive component. The above operation can be expected to rebuild the abdominal wall.

[0059] 1 5. Application to suppression of peritoneal dissemination metastasis

Peritoneal dissemination is a case in which gastric cancer, colon cancer, and ovarian cancer progress, and cancer cells are released from the tissue and metastasize to the body cavity through pleural effusion or ascites. Although the prognosis of cancer associated with peritoneal dissemination is extremely pleasing, a method that suppresses extremely bad metastasis is preferred, but no effective method is known at present. There is a possibility that metastasis can be suppressed by utilizing the properties of amniotic membrane. The omentum, diaphragm, and mesentery are known as sites where cancer cells frequently metastasize. Metastasis suppression can be achieved by forcing these milk spots together with a tissue reconstruction material to form noria. For this purpose, tissue preservation materials constructed with cryopreserved amnion with epithelium, cryopreservation, amnion without epithelium, freeze-drying, amnion with epithelium, adherent adhesion, amnion without epithelium can be used. Therefore, it is preferable to use a tissue reconstructing material constructed with freeze-dried epithelial and amniotic membrane. As an application method, for example, the application site is covered with a tissue reconstruction material so as to wrap the tissue. Alternatively, the tissue reconstructing material may be fixed to the application site by applying a part to the part and stitching, or by using an adhesive component adhered to the amniotic membrane. When the tissue reconstruction material was applied, cancer progressed and metastasized Even after it has occurred, and sowing has occurred! /, No! /, Even at the stage! /, (Preliminary use).

[0060] 1 -6. Application to prevention of recurrent adhesions

After performing laparotomy, the intestines or the intestine and the abdominal wall are bent due to adhesions, resulting in dysfunction and dysfunction. Even after surgery to release the adhesions, serosal defects occur in adhesive tissues, especially in highly inflamed and scarred tissues. Amnion can be used to prevent postoperative adhesion recurrence and to repair and reinforce the serosa defect. For this purpose, it is possible to use a tissue reconstructing material constructed of cryopreserved amniotic membrane with epidermis, cryopreserved, epithelial-free amniotic membrane, freeze-dried, amniotic membrane with epithelium, adherent adhesion, non-epithelial amnion. In view of easy handling, it is preferable to use a tissue reconstructing material constructed with lyophilized / epithelial amniotic membrane. As an application method, for example, the intestine is wrapped in a tube shape with a tissue reconstruction material at the stage where adhesions are physically separated after laparotomy. At this time, the tissue reconstruction material is applied so that the basement membrane side of the amniotic membrane becomes the abdominal cavity side. After this, although it is usually fixed, the organ and amniotic membrane may be sutured using a suture such as bicyclyl, or the amnion may be sutured together (amniotic membrane becomes tubular) Alternatively, it may be left without performing suturing. When a tissue reconstruction material constructed of amniotic membrane with an adhesive component attached is used, fixation may be achieved with the adhesive component. It is preferable that the tissue reconstruction material is fixed to the application site without performing a fixing process such as suturing separately. This is because the operation becomes complicated when sutures are performed, and inflammation may be caused and adhesion formation may be promoted. In particular, it can be said that the use of a tissue reconstruction material constructed with a dry amniotic membrane is particularly preferable because it can exhibit a high binding force to the application site and does not cause a problem of a foreign body reaction due to an adhesive component. By the above operation, recurrence of adhesion can be prevented, and the reconstruction of the serosa structure can be expected early after the operation.

[0061] 2. Application in obstetrics and gynecology

2- 1. Application to fallopian tube obstruction

When the fallopian tube adheres to the peritoneum, the fallopian tube is blocked and the egg cannot pass through, causing infertility. Amnion can be used to prevent oviduct adhesions. Organization for this purpose The application method of the reconstruction material is as follows, for example. Remove the adhering part and perform normal tubal fistula formation. Cover the area of the fallopian tube with tissue reconstructive material before closing after surgery. For this purpose, it is possible to use tissue reconstruction materials constructed of cryopreserved 'amniotic membrane with epithelium, cryopreserved amniotic membrane without epithelium, freeze-dried / amniotic membrane with epithelium, adherent adhesion, amnion without epithelium. Since it is simple, it is preferable to use a tissue reconstruction material constructed with freeze-dried / epithelial amniotic membrane. As an application method, for example, the application site is covered with a tissue reconstruction material so as to wrap the tissue. In addition, place a part on the part and suture it, or fix the tissue reconstruction material to the application site with an adhesive component attached to the amniotic membrane.

[0062] 2- 2. Application to prevent pelvic floor adhesions

The uterus in the pelvis is an extra-abdominal organ, and about 50% of the organ surface is not covered by the peritoneum. Therefore, when hysterectomy is performed, a peritoneal defect occurs, which causes adhesion formation between the pelvic floor and the small intestine. Amnion can be used to prevent pelvic floor adhesions. For this purpose, the form and application method of the tissue reconstruction material used are the same as in 1-3.

[0063] 3. Application in ophthalmology

3- 1. Application to glaucoma surgery

Glaucoma is a disease in which the optic nerve is damaged, the visual field narrows, and visual acuity is reduced. In the treatment of glaucoma, surgery to form a new aqueous humor drainage system by trabeculectomy is performed, but after the operation, the sclera and conjunctiva adhere to each other, and there is a case where the therapeutic effect cannot be expected. The use of amniotic membrane is considered effective for this problem. Specifically, after normal trabeculectomy, tissue reconstruction material is inserted under the conjunctiva. For this purpose, an amnion constructed with cryopreserved amniotic membrane with epithelium, cryopreserved, amnion without epithelium, freeze-dried, amnion with epithelium, adhesion component adhesion, amnion without epithelium, etc. can be used. Therefore, it is preferable to use a tissue reconstructing material constructed of freeze-dried-epidermis amniotic membrane. After the application, the tissue reconstruction material may be fixed to the application site with a suture or the like.

[0064] 3- 2. Application to Ryukyu adhesion

Ryukyu adhesion is a disease in which scarring occurs from the eyelid conjunctiva to the eyeball, causing adhesion between the eyelid and the eyeball. When Ryukyu adhesions occur, the surface of the eye is often damaged extensively It often recurs after the attached tissue is removed. It is thought that Ryukyu adhesion can be suppressed using amniotic membrane. As a specific application method, for example, after removing the adhesion between the eyelid and the eyeball, the scarred conjunctival tissue is peeled, the sclera is exposed, and the tissue is reconstructed. For this purpose, tissue reconstruction materials constructed with cryopreserved 'amniotic membrane with epithelium, cryopreserved amniotic membrane without epithelium, freeze-dried, amniotic membrane with epithelium, adhesion component adhesion, amnion without epithelium, etc. can be used. For reasons such as simplicity, it is preferable to use a material for tissue reconstruction that is constructed with an amnion without an adhesive skin. When the tissue reconstruction material is used, fixation to the application site is achieved mainly by adhesive components. The part covered with the tissue reconstruction material can be on the heel side or the sclera.

[0065] 3-3. Application to recurrent pterygium

A pterygium is a disease in which the conjunctival tissue grows abnormally, and the proliferating tissue adheres to the cornea, causing astigmatism and decreased visual acuity. Amniotic membrane is considered effective against the disease. As a specific application method, for example, the pterygium tissue is peeled off and the sclera is exposed, and then covered with a tissue reconstruction material. For this purpose, tissue reconstruction materials constructed with cryopreserved 'amniotic membrane with epithelium, cryopreserved amniotic membrane without epithelium, freeze-dried, amniotic membrane with epithelium, adherent adhesion, amnion without epithelium etc. can be used. For reasons such as easy handling, it is preferable to use a tissue reconstruction material constructed with an amnion with no epithelium. When the tissue reconstruction material is used, fixation to the application site is achieved mainly by adhesive components.

Example 1

[0066] 1. Examination of detachment condition of amniotic epithelial cell layer

1-1. Relationship between trypsin concentration and epithelial cell layer removal

Amniotic membranes were collected at the time of cesarean section in the operating room after giving sufficient informed consent with the obstetrician and gynecologist in advance for pregnant women scheduled for cesarean section without systemic complications. The operation was careful of cleanliness, and a special gown was worn after hand washing according to the surgical operation. Before delivery, a clean bat for collecting amnion and physiological saline for washing were prepared. After delivery, the placenta tissue was transferred to a vat and the amnion tissue was manually detached from the placenta. The area where the adhesion between the amniotic membrane and the placenta was strong was removed with scissors. Put 20 ml of stock solution into a 50 ml sterile tube Each amnion collected in 1 was placed and labeled one by one, and then stored in a refrigerator at _80 ° C. As a preservation solution, 50% sterilized glycerol in DMEM (Dulbecco'S Modofied Eagle Medium: GI BCOBRL) was used.

[0067] The amniotic membrane treatment was performed in the order of (1) washing, (2) chorionic detachment, and (3) trimming. In all processes, it is preferable to operate in a clean draft. Use containers and equipment that have been sterilized. Petri dishes should be sterilized and discarded (disposable). The type was used. Blood components adhering to the collected amnion were removed while washing with physiological saline, and further washed with a sufficient amount of physiological saline (0.005% ofloxacin added). Next, the amniotic membrane was transferred to a sufficient amount of phosphate buffer (PBS), and the chorion was manually detached. When the chorion could no longer be detected visually, it was divided into sizes of about 3 x 3 cm using scissors.

Each 1 cc of the stock solution was put into a 2 cc sterilized cryotube, and each collected amnion was labeled and stored in a -80 ° C refrigerator. As a preservation solution, 50% sterilized glyceride in DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL) was used.

[0068] The amniotic membrane cryopreserved after collection was thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish. Immerse this amniotic membrane in 0.2% (Condition 1), 0.1% (Condition 2), 0.05% (Condition 3), 0.02% (Condition 4), 0.01% (Condition 5) trypsin solution, and visually check the front of the amniotic membrane Treatment was performed at 37 ° C until the epithelium of the skin was detached. After thoroughly washing with PBS, lml stock solution was put into a 2ml sterilized cryotube, and each amnion was put one by one and stored at 80 ° C. 50% sterilized glycerol in DMEM was used as the stock solution. In order to examine the remaining epithelial cells or proteins in this treated amniotic membrane, immunostaining and HE staining were performed according to the following procedure.

[0069] (1) Immunostaining method

First, each amniotic membrane obtained by the above-mentioned predetermined treatment was cut into a size of 1.5 X I.5 cm, embedded in an OCT compound, and frozen at 80 ° C. to obtain a frozen specimen. This specimen was cryopreserved (CM1900 Leica) with a thickness of 8 m, cut in a direction perpendicular to the amnion surface, and mounted on a slide glass to prepare a frozen section. Using this frozen section, immunostaining was performed according to the following procedure and conditions. 1. Acetone fixation 5 minutes 2. PBS wash 30 minutes 3. PBS / 3% BSA blocking 15 minutes 4. Primary antibody 1 hour 5. PBS wash 30 minutes 6. PBS / 3% BSA blocking 15 minutes, 7. Secondary antibody 1 hour, 8. PBS wash 30 minutes, 9. Enclosed.

The encapsulated sample was observed under a fluorescence microscope (Leica DMIRB).

The antibodies used were as follows, and the dosage was according to the manufacturer's manual. Collagen I (Collagen I): LSL LB-1190, Collagen III (Collagen III): LSL LB1300, Collagen IV (Collagen IV) : LSL LB-1407, Collagen V (Collagen V): LSL LB1581, Collagen VII (Collagen VII): Chemicon MAB1345, Laminin-5 (Chemicon MAB19562, Fibronectin (bronectin): LSL LB-1021.

[0070] Collagen IV, VII and laminin 5 are expressed in the amnion basement membrane layer, and collagen I, III, V and fibronectin are expressed in the dense layer. Therefore, the remaining of the amnion basement membrane and dense layer can be observed by immunostaining with each antibody. In addition, in this experiment, PI staining is also performed, so that the presence or absence of amniotic epithelial cells can be simultaneously determined.

[0071] (2) HE staining method

When amnion is stained with hematoxin, amnion epithelial cells and dense nucleated cells are stained. On the other hand, when it is dyed with eosin, the dense layer is dyed. Therefore, according to HE staining, the presence or absence of epithelial cell detachment and the presence or absence of damage to the dense layer can be determined. The HE staining method was as follows. First, a frozen section of amniotic membrane was prepared in the same manner as in immunostaining. This frozen section was used for HE staining under the following procedure and conditions.

1.10% formalin solution 5 minutes, 2.flowing water wash 15 minutes, 3.hematoxin solution 10 seconds, 4.flowing water wash 15 minutes, 5.eosin solution 10 seconds, 6.flowing water wash 15 minutes, 7.70% ethanol 10 seconds, 8.90 % Ethanol 10 seconds, 9.95% ethanol 10 seconds, 10.100% ethanol 10 seconds, 11.100% xylene 10 seconds, 12.100% xylene 30 minutes, 13. Enclosed.

The encapsulated sample was observed under an optical microscope (Olympus BX50).

[0072] As a result, (condition 1) was treated for 3 hours, (condition 2) was treated for 3 hours, (condition 3) was treated for 4 hours, (condition 4) was treated for 5 hours, and (condition 5) was treated for 12 hours. It was possible to detach the cells.

[table 1] Trypsin concentration Processing time required for epithelial detachment

0.2% (Condition 1) 3 hours

0.1% (Condition 2) 3 hours

0.05% (Condition 3) 4 hours

0.02% (Condition 4) 5 hours

0.01% (Condition 5) 12 hours Next, the basement membrane components (collagen IV, VII, laminin 5) remain in the same degree as the control fresh fresh 'epidermis amniotic membrane (F) by immunostaining. confirmed. As shown in Fig. 3, in (Condition 1) and (Condition 5), the basement membrane components were completely undetectable. In (Condition 2), (Condition 3), and (Condition 4), Collagen IV has a lower detection intensity than that of the control (Fresh and Epithelial amnion: F), and Collagen VII and Laminin-5 cannot be detected. I helped.

On the other hand, for the dense layer components (collagen I, collagen III, collagen V, fibronectin), as shown in Fig. 4, although the detection sensitivity of fibronectin is decreased, any one of (conditions 1) to (condition 5) As for the control, the same level of survival as in the fresh fresh epithelial amniotic membrane (F) was confirmed.

From the above results, it was shown that amniotic epithelial cells can be detached by treatment with trypsin 0.2% to 0.01%. When the amniotic membrane is subjected to an enzyme treatment, it is preferable that the basement membrane component of the amniotic membrane remains in the tissue reconstruction / adhesion prevention application. The basement membrane components Collagen VII and Laminin 1 were strong in any condition, so it was judged that the five conditions from (Condition 1) to (Condition 5) were not appropriate for the treatment of amniotic membrane. . When these five conditions are compared, (Condition 2) to (Condition 4) are preferred. This is because the basement membrane component collagen IV remains. Among them, (condition 4), that is, treatment with 0.02% trypsin is most preferable. This is because the concentration of trypsin is low, and it is considered that the influence on the dense layer is small.

1-2. Relationship between trypsin treatment time and removal state of epithelial cell layer

After collection, the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish. This amniotic membrane is immersed in 0.02% trypsin solution and treated for 30 minutes depending on the treatment time. The reaction was performed under the conditions of (condition 6), 1 hour treatment (condition 7), 2 hour treatment (condition 8), 3 hour treatment (condition 9), and 5 hour treatment (condition 10). The treatment temperature was 37 ° C under all conditions. After thorough washing with PBS, it was stored at 80 ° C. In order to investigate the remaining epithelial cells or proteins of the treated amniotic membrane, immunostaining and HE staining were performed according to the procedure described in 11.1.

[0074] When the remaining amniotic epithelial cells were examined, about 90% of cells in (Condition 6), about 50% in (Condition 7), about 10% in (Condition 8), and about 1% of cells in (Condition 9) (Condition 10), almost all epithelium was detached and wrinkled.

Next, it was confirmed by immunostaining whether the basement membrane components (collagen IV, VII, laminin 1) remained in the same extent as the fresh fresh epidermis amniotic membrane (F). As shown in Fig. 3, in (Condition 6), it was confirmed that the basement membrane components remained as much as the control (F)! Under (Condition 7), a decrease in the detection intensity of collagen VII and laminin-5 was observed. Under (Condition 8), collagen VII could not be detected, and the detection intensity of collagen IV and laminin 5 decreased. Under (Condition 9) and (Condition 10), collagen VII and laminin-5 could not be detected, and a decrease in the detection intensity of collagen IV was observed.

On the other hand, for the dense layer components (collagen I, collagen III, collagen V, fibronectin), as shown in Fig. 4, the detection sensitivity of fibronectin is decreased, but any of (condition 6) to (condition 10) amniotic membrane As for the control, the same level of survival as the fresh fresh epithelial amniotic membrane (F) was confirmed.

From the above results, when treating with a trypsin 0.02% solution, the treatment time was preferably 1 hour or less, more preferably 30 minutes or less in order to keep the basement membrane component remaining. However, epithelial detachment could not be achieved in the treatment time under the condition that the amniotic membrane was simply immersed in trypsin solution. For this reason, it seemed necessary to provide another step after trypsin treatment to detach the epithelium.

[0075] 1-3. Effect of shaking during trypsin treatment on removal of epithelial cell layer

The amniotic membrane was immersed in a trypsin solution (37 ° C) in the same procedure as in 1-2. and shaken for 30 minutes (condition 11) or 1 hour (condition 12). Shaking is expected to increase the number of trypsin molecules in contact with the amniotic epithelial cells and detach the epithelium. Wash thoroughly with PBS After storage, it was stored at 80 ° C. For the purpose of examining the remaining epithelial cells or proteins in this treated amniotic membrane, HE staining and immunostaining were performed according to the procedure described in 11.1.

As a result of examining the remaining of the amniotic epithelial cells, about 10% remained in (Condition 11), and almost all epithelium was detached in (Condition 12).

Next, it was confirmed by immunostaining whether the basement membrane components (collagen IV, VII, laminin 1) remained in the same extent as the fresh fresh epidermis amniotic membrane (F). As shown in Fig. 3, a decrease in the detection intensity of collagen VII was observed under (Condition 11). Under (condition 12), collagen VII could not be detected, and a decrease in the detection intensity of collagen IV and laminin-5 was observed.

On the other hand, for the dense layer components (collagen I, collagen III, collagen V, fibronectin), fibronectin was not detected in both (condition 11) and (condition 12) as shown in FIG.

From the above results, it was possible to detach epithelial cells by treatment with trypsin 0.02% for 1 hour by shaking compared to 1-2. However, collagen VII and fibronectin could not remain, and collagen IV and laminin 5 were partially degraded. From these results, it was determined that shaking was not appropriate during trypsin treatment.

1 -4. Effect of washing treatment on removal of epithelial cell layer

Immerse the amniotic membrane in trypsin solution in the same manner as in 1-2 (37 ° C), leave it for 30 minutes (condition 13) and 1 hour (condition 14), then leave the amniotic membrane in running water for 20 minutes The epithelial cells were physically peeled off. After thorough washing with PBS, it was stored at -80 ° C. In order to examine the remaining of the epithelial cells or proteins of this treated amniotic membrane, HE staining and immunostaining were performed according to the procedure described in 1-1.

When the remaining amniotic epithelial cells were examined, about 5% remained in (Condition 13), and almost all epithelium was detached in (Condition 14).

Next, it was confirmed by immunostaining whether the basement membrane components (collagen IV, VII, laminin 1) remained in the same extent as the fresh fresh epidermis amniotic membrane (F). As shown in Fig. 3, a decrease in the detection intensity of collagen VII was observed under (Condition 13). (Condition 14) , Collagen VII could not be detected, and the detection intensity of laminin-5 decreased.

On the other hand, for the dense layer components (collagen I, collagen III, collagen V, fibronectin), although the detection sensitivity of fibronectin is decreased as shown in Fig. 4, the control is fresh amnion with epithelium (F). It was confirmed that collagen I, III, and V remained at the same level.

From the above results, epithelial cells could be detached in (Condition 14). However, collagen VII could not remain, and laminin 5 and part of fibronectin were degraded. On the other hand (condition 13), although collagen VII was partially decomposed, collagen IV and laminin 5 remained in the same degree as in control (F). Since the remaining epithelial cells are a part, it has been shown that it is possible to achieve both epidermal detachment and basement membrane component survival by pretreating amniotic membrane and shifting to trypsin treatment.

1-5. Effects of freeze-thaw treatment and washing treatment on removal of epithelial cell layer

The amniotic membrane was frozen and thawed twice in advance by allowing it to stand at 80 ° C for 30 minutes and at 37 ° C for 30 minutes. The amniotic membrane was immersed in trypsin solution in the same procedure as in 1-2 (37 ° C), and allowed to stand for 5 minutes (condition 15), 15 minutes (condition 16), and 30 minutes (condition 17). At this time, in order to minimize damage to the dense layer, trypsin solution was contacted only on the epithelial cell side. The amniotic membrane was left in running water for 20 minutes to physically peel off the epithelial cells. After thorough washing with PBS, it was stored at -80 ° C. In order to investigate the presence of the epithelial cells in the treated amniotic membrane and the remaining protein, HE staining and immunostaining were performed according to the procedure described in 1-1.

When the remaining amniotic epithelial cells were examined, about 5% remained in (Condition 15), and almost all epithelium was detached in (Condition 16) and (Condition 17).

Next, it was confirmed by immunostaining whether the basement membrane components (collagen IV, VII, laminin-1) remained in the same extent as the fresh fresh epithelial amnion (F) as a control. As shown in Fig. 3, it was confirmed that (Condition 15) and (Condition 16) remained almost the same as (F). Under (Condition 17), a decrease in the detection intensity of collagen IV and collagen VII was observed. On the other hand, for the dense layer components (collagen I, collagen III, collagen V, fibronectin), the detection sensitivity of fibronectin was decreased as shown in Fig. 4. It was confirmed that collagen I, III, and V remained in the same extent as the fresh trawling amnion with epithelium (F).

From the above results, it was shown that separation of epithelial cells and survival of basement membrane components (collagen IV, collagen VII, laminin 5) can be achieved in (Condition 16).

1-6. Most preferred trypsin processing conditions

In order to suitably use the amniotic membrane for tissue reconstruction / adhesion prevention, it is necessary that the basement membrane component remains highly, and it is preferable that the dense layer component remains as much as possible. In addition, since amniotic epithelial cells generally cause a foreign body reaction, it can be said that they are preferably removed. When trypsin is used, a treatment method that satisfies all of the following six conditions is the most preferable treatment method for amniotic membrane.

(1) When the amniotic membrane is immersed in a trypsin solution, the collagen layer is decomposed in a short time if the concentration is high, and if the concentration is low, the treatment takes time, resulting in damage to the collagen layer. Therefore, in order to leave the basement membrane component, it is preferable to use a trypsin solution having a concentration within a certain range (for example, 0.1% to 0.02%).

(2) When trypsin with the same concentration is used, the shorter the time required for treatment, the better. This is because the decomposition of the basement membrane component and the dense layer component is reduced. In the case of treatment with 0.02% trypsin, the treatment time is preferably within 1 hour, more preferably within 30 minutes.

(3) It is preferable not to shake during the trypsin treatment and to stand still. This is because the rate of penetration of the trypsin solution into the amniotic basement membrane and dense layer is slowed, and as a result, the amniotic basement membrane and dense layer are difficult to be decomposed.

(4) Epithelial detachment is preferably performed by a washing operation. This can shorten the time for trypsin treatment and realize (2). The washing step is usually performed immediately after the trypsin treatment, and any washing operation such as running water washing, shaking washing or ultrasonic washing may be used.

(5) Trypsin treatment is preferably performed only on the amnion epithelium side. Since trypsin is not in direct contact with the dense layer, it is difficult to decompose.

(6) It is preferable to pretreat the amniotic membrane to weaken the binding force between the epithelial cells and the basement membrane in advance. This method can be used to weaken the force coupling that can increase freezing and thawing. Other means may be used.

[0079] 2. Preparation of amniotic membranes after various treatments

2- 1. Cryopreservation · Preparation of amnion with epithelium (A)

Amniotic membranes were collected at the time of cesarean section in the operating room after giving sufficient informed consent with the obstetrician and gynecologist in advance for pregnant women scheduled for cesarean section without systemic complications. The operation was careful of cleanliness, and a special gown was worn after hand washing according to the surgical operation. Before delivery, a clean bat for collecting amnion and physiological saline for washing were prepared. After delivery, the placenta tissue was transferred to a vat and the amnion tissue was manually detached from the placenta. The area where the adhesion between the amniotic membrane and the placenta was strong was removed with scissors. 20ml each of the stock solution was put into a 50ml sterilized tube, and the collected amniotic membranes were labeled one by one and stored in a refrigerator at _80 ° C. As a preservation solution, 50% sterilized glycerol in DMEM (Dulbecco'S Modofied Eagle Medium: GI BCOBRL) was used.

[0080] The amniotic membrane treatment was performed in the order of (1) washing, (2) chorionic detachment, and (3) trimming. In all processes, it is preferable to operate in a clean draft. Use containers and equipment that have been sterilized. Petri dishes should be sterilized and discarded (disposable). The type was used. Blood components adhering to the collected amnion were removed while washing with physiological saline, and further washed with a sufficient amount of physiological saline (0.005% ofloxacin added). Next, the amniotic membrane was transferred to a sufficient amount of phosphate buffer (PBS), and the chorion was manually detached. When the chorion could no longer be detected visually, it was divided into sizes of about 15 X 15 cm using scissors.

4cc of the stock solution was put into a lOcc sterilized cryotube, each amnion collected was labeled and stored in a refrigerator at -80 ° C. The stock solution used was 50% sterilized dalycerol in DMEM (Dulbecco, S Modofied Eagle Medium: GIBCOBRL)

[0081] 2— 2. Cryopreservation · Preparation of epithelium-free amnion (B)

After collection, the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish. The epithelial cells were detached by the following treatment. The amniotic membrane was fixed to a frame, frozen at 80 ° C for 30 minutes, and thawed at 37 ° C for 30 minutes. Another freeze-thaw step Repeated once. Next, the epithelial side of the amniotic membrane was immersed in a trypsin solution (phosphate buffer containing 0.02% trypsin and 0.2 mM EDTA) and allowed to stand for about 15 minutes (37 ° C). Only the area in contact with the trypsin solution was cut with scissors and washed in PBS.

4cc of the stock solution was put into lOcc sterilized cryotubes, and the amnion was collected, washed and selected one by one, labeled, and stored in a refrigerator at -80 ° C. 50% sterilized glycerol in DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBR L) was used as a preservation solution.

[0082] 2— 3. Lyophilization · Amnion with epithelium (C)

After collection, the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish. The amniotic membrane was spread on a Teflon punching sheet. The whole punching sheet was transferred to a deep freezer at 80 ° C, and after confirming that the amniotic membrane was frozen, freeze drying (-50 ° C, about 1 hour) was performed using a vacuum freeze dryer. The conditions were set according to the instruction manual so that a sufficiently dry product could be obtained.

The dried amniotic membrane was peeled off from the Teflon punching sheet, transferred to a double-layer bag with polyamide nylon on the outside and polyethylene inside, and vacuum-packed using a household vacuum pack device (frame nova, magic pack). The vacuum packed amniotic membrane thus obtained was sterilized by irradiation with γ rays (about 15 kGy). The sterilized amniotic membrane was stored at room temperature in a vacuum-packed state until just before use.

[0083] 2— 4. Lyophilization · Preparation of epithelium-free amnion (D)

Epithelial cells were detached from the amnion that had been cryopreserved after collection by the same procedure as in (B). The drying process was performed in the same manner as (C). The process of γ-ray irradiation is the same as in (C), without γ-ray treatment (freeze-dried · no epithelium · γ-ray untreated amniotic membrane: D-1) and with γ-ray treatment (freeze-dried · no epithelium · γ-ray treatment Finished amniotic membrane: D-2). The preservation of the dried amniotic membrane was carried out in the same manner as (C).

[0084] 2- 5. Basement membrane damage · Cryopreservation · Preparation of epithelium-free amnion (Ε)

After collection, the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish. The process of epithelial cell detachment was performed in the order of (1) frame fixation, (2) freezing and thawing, (3) trypsin solution immersion, and (4) washing, as in (ii). However, trypsin solution immersion time is 15 minutes to 2 hours Changed to and implemented. Compared with (B), the basement membrane is severely damaged in this amniotic membrane by increasing the trypsin solution immersion time. After treatment, the amniotic membrane was preserved by a conventional method.

[0085] 2-6. Production of fresh and epithelial amniotic membrane (F)

After collecting the amniotic membrane, the following operation was performed without cryopreservation. After the treatment of the amnion chorion, put 30cc of amniotic cell culture in a 100φ Petri dish, place and label one piece of washed and washed amniotic membrane, and store in a refrigerator at 4 ° C for one day Later used for experiments. As the amniotic cell culture medium, FBS 10%, gentamicin 5 μg / ml in DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL) was used.

[0086] 2— 7. Production of fresh and fresh epithelium-free amnion (G)

After collecting the amniotic membrane, the following operation was performed without cryopreservation. The amniotic membrane was thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish. The process of epithelial cell detachment was performed in the same manner as in (B), and stored at 4 ° C as in (F).

[0087] 2-8. No cryopreservation · Freeze drying · Preparation of epithelial amniotic membrane (H)

After collecting the amniotic membrane, the following operation was performed without cryopreservation. After thoroughly washing with a sterilized phosphate buffer solution (PBS) in a petri dish, the amniotic membrane was lyophilized in the same manner as in (C) and stored in a vacuum packed state without y-ray sterilization.

[0088] 2- 9. No cryopreservation · Freeze drying · Preparation of epithelium-free amniotic membrane (I)

After collecting the amniotic membrane, the following operation was performed without cryopreservation. The epidermal cells were detached and dried by the same method as in (D). Stored in a vacuum package without y-ray sterilization

[0089] 2- 10. Fresh Fresh '10% Trypsin Treatment 'Preparation of γ-ray-untreated amniotic membrane (J)

After collecting the amniotic membrane, the following operation was performed without cryopreservation. The amniotic membrane was thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish. A solution (trypsin concentration of 10%) was prepared by dissolving 3 g of trypsin l: 250 (Sigma) in PBS to a total volume of 30 ml. 30ml trypsin solution was put into a 100φ Petri dish, and one piece of amnion that was collected and washed was put in it and left at 37 ° C for 3 hours. After thoroughly washing the amniotic membrane with pure water, 30 ml of pure water was placed in a 50 ml sterile falcon tube, and the amniotic membrane was placed therein and stored in a refrigerator at -80 ° C. [0090] 2- 11. Raw fresh · 10% trypsin treatment · Preparation of γ-ray treated amniotic membrane (K)

After collecting the amniotic membrane, the following operation was performed without cryopreservation. Trypsinization was performed in the same manner as (J). Amnion was placed in a 100 ml medium bottle with 70 ml of pure water, and sterilized by irradiating gamma rays (about 35 kGy). The sterilized amniotic membrane was taken out from the medium bottle and thoroughly washed with pure water. Then, 30 ml of pure water was placed in a 50 ml sterilized falcon tube, and the amniotic membrane was poured into the -80 ° C refrigerator.

[0091] 2- 12. Adhesion component adhesion 'Epithesis-free amniotic membrane (L)'

The amniotic membrane stored in a frozen state was thawed at room temperature, and then thoroughly washed with a sterilized phosphate buffer solution (PBS) in a petri dish. After washing, it was stored in a 0.02% EDTA solution (Nacalai tesque) for 2 hours at 37 ° C, and then the skin was mechanically repellated using a cell scraper (cell scraper, Nunc USA). After being sandwiched between a pair of sterilized plastic frames, they were fixed with clips. The whole frame was transferred to a deep freezer at 80 ° C, and it was confirmed that the amniotic membrane was frozen, and then freeze-dried using a vacuum freeze dryer (H10 ° C, approximately 1 hour).

A mixture of fibrinogen and thrombin was added dropwise so as to spread over almost the entire surface of the dried amniotic membrane (chorionic side), and then dried under reduced pressure for 1 hour at room temperature. Subsequently, the dried amniotic membrane was also removed and transferred to a two-layer bag with polyamide nylon on the outside and polyethylene strength on the inside, and vacuum-packed using a household vacuum pack device (frame nova, magic pack). The vacuum packed amniotic membrane thus obtained was sterilized by irradiation with γ rays. The amniotic membrane after sterilization treatment was stored at room temperature in a vacuum packed state until just before use.

[0092] 3. Immunostaining for basement membrane components and dense layer components

In order to work well as a material for tissue reconstruction, it is considered preferable that the basement membrane and the dense layer retain their original structures. Whether the basement membrane and the dense layer maintain the structure or not can be evaluated by examining the presence or absence of characteristic components (whether they remain or not). Therefore, whether or not the basement membrane component and the dense layer component remained in the various amniotic membranes obtained by the above treatment was examined by immunostaining (the method described in 11.). In parallel with immunostaining, HE staining (the method described in 1 1.) was performed.

[0093] The results of immunostaining and HE staining are shown in FIGS. Figures 5-10 are stained images, Figures 11 and 1 2 is a table summarizing the evaluation by stained images.

As shown in Figures 5-12, cryopreservation · amniotic membrane with epithelium (A), cryopreservation · amniotic membrane without epithelium (B), freeze-drying · amniotic membrane with epithelium (C), freeze-drying · amniotic membrane without epithelium (Dl, D- 2), fresh and fresh · Epithelium-free amniotic membrane (G), cryopreservation-freeze-dried, epithelial-containing amniotic membrane (H), cryopreservation-freeze-dried, epithelial-free amniotic membrane (I), and adhesion component adhesion · epithelium-free amnion ( In (L), it was confirmed that the basement membrane components (collagen IV, collagen VII, laminin 5) remained as much as the fresh fresh 'epithelial amniotic membrane (F) as a control. On the other hand, in the basement membrane disorder, cryopreservation, epithelium-free amniotic membrane (E), there are obvious disorders of the basement membrane components. Basement membrane components could not be detected in fresh fresh 10% trypsin-treated γ-ray-untreated amniotic membrane (J) and fresh fresh 10% trypsin-treated γ-treated amniotic membrane (Κ).

On the other hand, for the dense layer components (collagen I, collagen III, collagen V, fibronectin), the fibronectin detection sensitivity decreased in part, but it was cryopreserved 'amniotic membrane with epithelium (Α), cryopreserved · epithelium No amniotic membrane (Β), lyophilized · Epithelial amniotic membrane (C), lyophilized · No epidermis amniotic membrane (Dl, D-2), fresh · Non-epithelial amniotic membrane (G), no cryopreservation · Lyophilized, with epithelium Amniotic membrane (H), cryopreservation-freeze-dried-epithelial-free amniotic membrane (I) and adherent component adherence--epithelial-free amniotic membrane (L) is roughly the same as control fresh fresh-epithelial amniotic membrane (F) Survival was confirmed. On the other hand, fibronectin is clearly impaired in basement membrane damage, cryopreservation, epithelial-free amniotic membrane (E). In addition to the fact that fibronectin cannot be detected in fresh fresh10% trypsin-treated γ-ray-untreated amniotic membrane (J) and fresh-fresh 10% trypsin-treated γ-treated amniotic membrane (Κ), collagen V is clearly detected. Disability is recognized.

From the above results, it can be said that the structures of the basement membrane and the dense layer are highly maintained in the amniotic membranes (A to D, G to) obtained by the above treatment.

3. Tissue reconstruction using amniotic membrane

The methods of HE staining, HBME-1 staining and immunostaining in the following evaluation experiments are as follows.

(1) HE staining

The extracted tissue was embedded with OCT compound and frozen at 80 ° C to obtain a frozen specimen. The specimen was sliced using a cryostat (CM1900 Leica) in a frozen state. Then, it mounted on the slide glass and made it the frozen section. Using this frozen section, the following procedure was carried out under the same procedure and conditions as the above-mentioned HE staining for amniotic membrane.

[0095] (2) HBME-1 staining

The excised tissue was fixed by immersing in 10% formalin for 3 hours and then embedded in paraffin to form a norafin block. After slicing this block, it was mounted on a slide glass to prepare a section. Using this section, HBME-1 staining was performed according to the following procedure.

1. Deparaffinization, 2. Washing, 3. 0.3% hydrogen peroxide 5 minutes, 4. Washing, 5. HBME-1 antibody 30 minutes, 6. Washing, 7. Secondary antibody 30 minutes, 8. Washing, 9. DAB solution 5 min, 10. Wash, 11. Hematoxylin staining, 12. Dehydration, 13. Enclose

[0096] (3) Immunostaining

A frozen section of the tissue was prepared in the same procedure as for HE staining. Using this section, the following procedure was performed under the same procedure and conditions as the immunostaining for the amnion.

[0097] 3- 1. Cryopreservation · Reconstruction of rat cecal injury using epithelial amniotic membrane

Cryopreserved amniotic membrane with epithelium (A) was prepared, and its serosa reconstruction ability was evaluated in a rat cecal injury model.

Rats were anesthetized by subcutaneous injection of Nembutal and then disinfected with isodine. A midline laparotomy was performed using scissors, the cecum was removed from the body, and the serosa was physically damaged by rubbing with sandpaper. When the tissue after rubbing was extracted and stained with HE, the entire serosa layer and a part of the outer longitudinal layer were damaged. After thoroughly wiping the blood with gauze, cryopreserved in the cecum of the rat.Cover the epithelial amniotic membrane (A) with the epithelial side facing the abdominal side, and fix it by suturing so that the amniotic membrane is not displaced. I was hungry. At 1, 3, 5, 7 days, 2, 3, 4 weeks, the rats were sacrificed, the cecal tissue was removed, and the tissue sections were subjected to HE staining for histological analysis.

The analysis results are shown in Figs. Figure 13 shows the state of the cecal tissue one week after the amniotic membrane coating. Fig. 14 shows HE-stained images of cecal tissue 4 weeks after amnion coating, Fig. 15 shows HE-stained images of cecal tissue 1 day to 4 weeks after amnion coating with cryopreserved epithelium, and Fig. 16 shows cryopreservation. 'HBME-1 (mesothelial cell marker) -stained image of cecal tissue 1 day to 4 weeks after epithelial and amnion coating. As shown in these figures, the following matters became clear. Amnion in 1 day after amnion coating Was closely attached to the cecum (Figures 15 and 16). Amniotic epithelial cells disappeared in 3 days, and at the same time, rat cells were covered on the basement membrane of the amniotic membrane (Figs. 15 and 16). Since ex-SMA expressed in myoblasts was specifically detected, it was considered that these cells floated in ascites and were myoblasts. On day 5 after coating, cells infiltrated into the amniotic membrane also from the cecal muscle layer side (Figs. 15 and 16). This cell is a myoblast. One week later, myoblasts became multi-layered, and a single layer of squamous epithelial cells began to cover the myofibroblast layer (Figs. 15 and 16). It was also confirmed macroscopically that adhesion can be suppressed by covering the amniotic membrane (Fig. 13). Squamous epithelial cells were found to be mesothelial cells (serosa epithelial cells) because HBME-1 expressed specifically in the serosa was detected, and their origin was suspended in ascites. (Figure 16). Three weeks later, the biodegradation of the amniotic membrane progressed and the membranous structure became undetectable (Figs. 15 and 16). Four weeks later, mesothelial cells were covered on the entire surface of the amniotic membrane, and neovascularization was also observed (Figures 14 and 15). In addition, the serosa damaged by abrasion was regenerated and almost normal serosa was reconstructed (Figs. 14 and 15).

[0098] The above results are summarized as follows. The basement membrane side of the amniotic membrane (peritoneal side of the cecum) was covered in multiple layers by myoblasts, and a single layer of mesothelial cells was coated thereon. Myoblasts also proliferated from the chorion side of the amniotic membrane (cecal mucosa side), and the amniotic membrane was biodegraded early. The structure after 4 weeks was almost the same as that of serous tissue in the normal intestine. By applying the amniotic membrane to the damaged area, reconstruction of the damaged serosa without delaying the wound healing process was achieved.

[0099] 3- 2. Cryopreservation · Amnion without epithelium, lyophilization · Reconstruction of rat cecal injury using epithelium with epithelium

Cryopreservation · Amnion without epithelium (B) and freeze-drying · Amnion with epithelium (C) were prepared, and their serosa reconstruction ability was evaluated in a rat caecal disorder model. The experimental procedure was the same as in 3-1. For each test group, HE-stained images of rat cecal tissue sections 4 weeks after amnion coating were prepared and used for evaluation. As a result, it was observed that myoblasts and mesothelial cells were coated on the entire surface of the amniotic membrane regardless of whether amniotic membrane (C) or (C) was used. There was no difference between the histology of the group covered with amniotic membrane. The serosa, which was damaged by abrasion, was regenerated and the almost normal bowel was reconstructed. From this result, epithelial detachment It was confirmed that the amniotic membrane that had been treated and dried had the same ability to reconstruct serous membrane as the cryopreserved and epithelial amniotic membrane (A).

[0100] 3- 3. Prevention of adhesion of rat cecum by various treated amniotic membranes

Cryopreservation · Amnion with epithelium (A), Cryopreservation · Amnion without epithelium (B), dry · Amnion with epithelium (C), freeze-dried · No epithelium · γ-ray untreated amnion (D_l), freeze-dried · No epithelium · γ-ray treated amniotic membrane (D-2), basement membrane disorder · cryopreservation · epithelium-free amniotic membrane (Ε), raw fresh · epithelial amniotic membrane (F), raw fresh · epithelial-free amniotic membrane (G), no cryopreservation · frozen Dry · Epithelial amniotic membrane (Η), frozen storage · Freeze-dried · Upper skinless amniotic membrane (1), fresh and fresh · 10% trypsin treatment · γ-ray untreated amniotic membrane (J), fresh · 10% trypsin treatment · Gamma-ray treated amniotic membrane (Κ) was prepared, and its adhesion prevention ability was evaluated using a rat adhesion model. In each test group, rats were restarted after 1 week of amnion coating, and the state of cecal adhesion was observed with the naked eye. In the group using dry amniotic membrane (C, D, H, I), the cecum was wrapped with amniotic membrane, and the abdomen was closed without fixation by suture. This is because the amnion in the dry state, unlike the amniotic membrane in the cryopreserved state, has a high affinity with the tissue and the amniotic membrane does not shift after abdominal closure. As a control group, a group without amniotic membrane coating (amniotic membrane uncoated) and a cryopreserved amniotic membrane with epithelium were coated in the reverse direction (the direction in which the chorion side becomes the abdominal side) were prepared.

[0101] The degree of caecal adhesion was scored for each site to be evaluated, and the total score of all sites was used for the evaluation. In addition, adhesion formation occurs prior to serosa reconstruction. Serosa reconstruction does not always occur because no adhesion is formed, but when an adhesion is formed, a serosa similar to a normal tissue is not reconstructed. Therefore, by examining the adhesion prevention ability, it is possible to clarify the type of amniotic membrane treatment that is unsuitable for use as a serosal reconstruction application.

[0102] The degree of adhesion was scored as follows.

0: Adhesion is not recognized, 1: Adhesion is recognized, but it can be peeled off naturally, 2: Exfoliated when tension is applied, 3: Exfoliation is possible even when tension is applied.

[0103] On the other hand, the sites to be evaluated were as follows.

i: cecum-small intestine, ϋ: cecum-mesentery,: cecum-large intestine, iv: cecum-body network, V: cecum-abdominal wall

, Vi: Blind month recruitment—others.

[0104] Fig. 17 shows the evaluation results. Amnion (A) score 1.3, amniotic membrane (B) score 1, amniotic membrane (C) Score 0, amniotic membrane (D-1) score 0, amniotic membrane (D-2) score 9, amniotic membrane (E) score 1 2, amniotic membrane (F) score 9, amniotic membrane (G) score 2, amniotic membrane (H) scored 5, amniotic membrane (I) scored 3, amniotic membrane (J) scored because the rat died due to intestinal obstruction, and amnion (Κ) scored 6. In addition, the group (Μ) covered with amniotic membrane in the opposite direction had a high degree of adhesion, similar to the group not covered with amniotic membrane, with a score of 21.

From the above results, it was shown that amniotic membranes (B), (C) and (D-l) have almost the same anti-adhesion effect as cryopreserved amniotic membrane with epithelium. The adhesion score fluctuated greatly depending on the presence or absence of y-rays on lyophilized / epithelial-free amnion (D-1 and D_2). This is thought to be because the surface structure of the amniotic membrane changes due to the γ-ray treatment and the function of the anti-adhesion component disappears. The amniotic membrane (Ε) causes severe adhesions, and the difference is obvious when compared to the mild adhesions of the amniotic membrane (Β). The only difference between the amniotic membrane (Β) and the amniotic membrane (のみ) is the difference in trypsin treatment time in the process of amnion epithelial cell detachment, and it has been confirmed that the basement membrane is damaged in the amniotic membrane (Ε). Therefore, it can be said that the basement membrane component must remain intact to prevent the adhesion of the amniotic membrane. When fresh amniotic membrane is used (amniotic membrane (F), (G), (H), (1)), the score is also cryopreserved. In the amniotic membrane (F)-amniotic membrane (I), the preparation process is cryopreserved! It is thought that it was easy to form. The score of the amniotic membrane with epithelium (amniotic membrane (A), (C), (F), (H)) and the amniotic membrane without epithelium (amniotic membrane (B), (D), (G), (1)) Comparing the scores, amnion without epithelium is generally lower. From this result, it can be said that it is preferable that there is no epithelium for exhibiting the adhesion preventing ability (effect). The group (M), which had the reverse side coating, showed no adhesion-preventing ability. Thus, it can be said that only the epithelial side of the amniotic membrane has an adhesion prevention function.

Most individuals died within a week in the group coated with amniotic membrane (J) and amniotic membrane (spider). Pathological anatomy was performed using the dead individuals, and all of the organs in the abdomen were highly adhered and integrated together, and the cause of death was thought to be due to adhesive intestinal obstruction. Slightly surviving individuals were observed in rats covered with amniotic membrane (spider). Although the survival score of the surviving individuals was 6, which was not high, testicular fat and omentum had planar adhesions at the center of the amnion-covered area. Amniotic membrane (Α) to amniotic membrane (D), which are considered to have sufficient adhesion-preventing ability -In 1), adhesion may occur due to individual differences. In this case, the site of adhesion is limited to the area where the amniotic membrane and cecum are fixed by suturing or the area of the end of the amniotic membrane, and the classification of adhesion is mostly punctate and mild, and the amniotic membrane ω It was clearly different from the observed images of the amniotic membrane (κ). This suggests that the amniotic membrane ω and the amniotic membrane (κ) are less effective in preventing adhesion because the basement membrane component is highly damaged.

[0105] From the above results, it is clear that the four types of amniotic membranes (A), (Β), (C), and (D-1) have sufficient adhesion prevention functions. became. 3- Along with the results of 1 and 3-2, it can be seen that amniotic membranes (A), (B), and (C) have the ability to reconstruct serosa in addition to sufficient adhesion prevention. It was also suggested that amniotic membrane (D-1) may have serosa-rebuilding ability. Among these four types of amniotic membranes, lyophilized / epithelial amniotic membrane (C) is the most excellent. The reasons are: 1. Has anti-adhesion effect, 2. Has serous tissue reconstruction effect, 3. Easy to handle, 4. Can be applied without suturing, and 5. Gamma ray treatment (sterilization This is because sterility is guaranteed by the treatment.

[0106] 3-4. Application of cryopreserved amniotic membrane with epithelium to rat peritoneum

Cryopreserved 'amniotic membrane with epithelium (Α) was prepared and evaluated for its ability to prevent adhesion in the peritoneum.

Rats were anesthetized by subcutaneous injection of Nembutal and then disinfected with isodine. A midline laparotomy was performed using scissors, and the peritoneum was removed with scissors at a size of 1.5 x 1.5 cm. After thoroughly wiping the blood with gauze, the peritoneum removal site was cryopreserved and covered with epithelial amniotic membrane (A), sutured at the four corners so that the amnion would not slip, and then the abdomen was closed. One week later, the rats were restarted, and the anti-adhesion effect of the cryopreserved 'amniotic membrane coating was examined. As a result, in the cryopreserved / amniotic membrane-coated group, adhesion did not occur.

[0107] 3- 5. Application to the Ryukyu Region of Rats

The possibility of applying an amniotic membrane (eg, cryopreserved amniotic membrane with epithelium (A)) as an anti-adhesion agent can be examined in the following procedure using rats as a model.

First, the rat is anesthetized and HC12N is instilled under the armpit to physically injure the armpit and sclera. Due to acid damage, the rat's Ryukyu is highly adherent. One week later, the Ryukyu adhesions are physically removed and covered with amniotic membrane. Two weeks later, the rats are observed and examined for adhesion between the amniotic membrane-coated group and the control group that does not coat the amniotic membrane. In the control group, Ryukyu causes adhesion However, adhesion is expected to be suppressed in the amniotic membrane coating group.

[0108] 3-6. Adhesion component adhesion 'Application of epithelium-free amnion to the cecum

Adhesion component adhesion · The feasibility of applying epithelial-free amniotic membrane (L) as an anti-adhesion agent can be examined by the following procedure using rats as a model.

First, the rat is anesthetized, and after laparotomy, the cecum is scraped with sandpaper to physically damage the cecal serosa. If the serosa is damaged, the physical barrier of the organ disappears, and the intestine and the intestine-abdominal wall frequently adhere. This model is a clinically meaningful adhesion model. After the blood is thoroughly wiped with gauze, the cecum of the rat is covered with a dry adhesive component adhering epithelium-free amnion (L) and closed. One week later, the rat is restarted and the abdominal adhesion, extent, and serous membrane regeneration are examined between the amnion-coated group and the non-amniotic control group. In the control group, the intestines usually adhere to each other over a wide area, and the intestines are observed together. By comparing the state of the control group and the state of the amniotic membrane coating group, the effect of the adhesive component adhering 'epithelium-free amniotic membrane (L) as an adhesion inhibitor can be evaluated. In addition, histological analysis is possible by removing the tissue after a predetermined period of time and subjecting the tissue section to HE staining.

[0109] 3- 7. Adhesion component adhesion · Application of epithelium-free amnion (L) to peritoneum

Adhesion component adhesion · The possibility of application as an anti-adhesion agent for epithelial-free amniotic membrane (L) can be examined with the following procedure using rat peritoneum as the application site.

First, the rat is anesthetized and laparotomized, and the peritoneum is removed with scissors. The peritoneal defect site is abraded with sandpaper to damage the abdominal wall. This model is a model in which peritoneal intestinal adhesion occurs frequently. After thoroughly wiping the blood with gauze, the peritoneal defect is covered with dry adhering component adhering epithelium-free amnion (L) and the abdomen is closed. One week later, the rats are restarted, and the degree and extent of adhesion in the abdomen and the degree of regeneration of the peritoneum are examined between the amniotic membrane-coated group and the non-amniotic control group. In the control group, the intestine-peritoneum usually causes adhesions. By comparing the state of the control group and the state of the amniotic membrane-coated group, the effect of the adhesion component-attached / epithelial-free amniotic membrane (L) as an adhesion inhibitor can be evaluated.

[0110] 3-8. Application of Adhesive Component Adhesion 'Epithelial Amnion (L) as a Peritoneal Dissemination Prevention Agent

The following procedure can be used to examine the possibility of application of the adhesive component adhering non-epithelial amniotic membrane (L) as a peritoneal dissemination prevention sheet. First, the rat is anesthetized and laparotomized, and the peritoneum is excised with scissors, and then melanoma cells are implanted in the peritoneal defect site. Melanoma cells proliferate at the peritoneal defect site, and a peritoneal dissemination model can be created in which metastasis occurs frequently on the peritoneum and organ surfaces. Cover the peritoneum inoculated with melanoma and the surface of the visceral organ with a dry adhesive component. Cover with epithelium-free amnion (L) and close the abdomen. Rats are restarted after a certain period of time, and the number and size of spots where melanoma cells are seeded in the abdomen between the amnion-covered group and the non-amniotic control group are examined and scored. By comparing the condition of the control group and the condition of the amniotic membrane-coated group, the effect of the adhesive component adhesion / epithelium-free amnion (L) as a peritoneal dissemination prevention sheet can be evaluated.

[0111] 3- 9. Application of freeze-dried amniotic membrane with epithelium (C) to the anastomosis of the esophagus

The possibility of application of freeze-dried amnion with epithelium (C) to the anastomosis can be examined by the following procedure using Inu as a model.

First, Anu is anesthetized, and after laparotomy, the esophagus is removed and anastomosed. After the blood is thoroughly wiped with gauze, the anastomosis region is freeze-dried and wrapped with amniotic membrane with epithelium (C). The esophagus and lyophilized · Epithelial amnion (C) is sutured and fixed, and the abdomen is closed. After 4 weeks, the stomach was restarted and scored and compared to the degree of adhesion in the anastomosis area between the amniotic membrane-coated group and the non-amniotic-coated control group. The effect can be evaluated. It is also possible to observe the tissue reconstruction by removing the tissue and subjecting the tissue section to HE staining.

Industrial applicability

[0112] The tissue reconstruction material of the present invention is suitably used in a treatment aimed at reconstruction of an organ or a surface tissue disorder of an organ due to surgical invasion. The tissue reconstruction material of the present invention is expected to be applied to a wide range of fields, including the fields of gastroenterological surgery, obstetrics and gynecology, thoracic surgery, oral surgery, otolaryngology, and ophthalmic surgery. The

In the tissue reconstruction material of the present invention, it is possible to simultaneously achieve suppression of adhesion formation and tissue reconstruction. Therefore, the tissue reconstruction material of the present invention for various cases that need to promote wound healing while suppressing adhesion formation (especially cases where early wound healing is required, such as tissue reconstruction after anastomosis). Is valid.

[0113] The present invention is not limited to the description of the embodiments and examples of the invention described above. Absent. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

The contents of papers, published patent gazettes, patent gazettes, etc. specified in this specification are incorporated by reference in their entirety.

The following matters are disclosed.

(1) In the freeze-thaw treatment, the freezing temperature is about 20 ° C to about 80 ° C, and the melting temperature is about 4 ° C to about 50 ° C. Manufacturing method.

(2) The production method according to claim 16 or (1), wherein the freeze-thaw treatment is repeated twice or more.

(3) The trypsin treatment is performed using a trypsin solution having a trypsin concentration of about 0.01% (w / v) to about 0.05% (w / v). The production method described in (2).

(4) The trypsin solution contains about O.lmM to about 0.6 mM of a chelating agent selected from the group consisting of EDTA, NTA, DTPA, HEDTA, GLDA and any combination thereof. The production method according to (3).

(5) The preparation method according to any one of claims 16 and (1) to (4), wherein the trypsin treatment is performed under a condition in which a trypsin solution contacts only the amnion epithelium side.

(6) Before the step (a-1) or before the step (a-2), the following steps are performed. A production method according to any one of the above,

(A) A step of fixing the amniotic membrane to the frame in an expanded state.

Claims

The scope of the claims

[I] Tissue reconstruction material consisting essentially of amniotic membrane.

[2] The tissue reconstruction material according to claim 1, which is used for reconstruction of an organ or a surface tissue disorder of an organ due to surgical invasion.

[3] Used to reconstruct organs in the abdomen, chest, or pelvis or surface tissue of the abdomen, chest cavity, pelvic cavity, oral cavity, nasal cavity, ear cavity, throat cavity, or ocular tissue The material for tissue reconstruction according to claim 1, wherein the material is a material for reconstruction.

[4] The tissue reconstruction material according to any one of claims 1 to 3, which is in a frozen state or a dried state.

[5] The tissue reconstruction material according to any one of claims 1 to 3, which is in a lyophilized state.

6. The tissue reconstruction material according to any one of claims 1 to 5, wherein the amniotic membrane is an amniotic membrane from which an epithelial cell layer has been removed.

[7] The amniotic membrane according to any one of claims 1 to 6, wherein the basement membrane components collagen IV, collagen VII, and laminin 5 are detected with the same strength as the untreated amniotic membrane. Material for tissue reconstruction.

8. The tissue reconstruction material according to any one of claims 1 to 7, wherein the amniotic membrane is human amniotic membrane.

[9] The tissue reconstruction material according to any one of claims 1 to 8, wherein an adhesive component is attached to a surface of the amniotic membrane on the chorionic membrane side.

10. The tissue reconstructing material according to claim 9, wherein the adhesive component strength is ibrinogen and thrombin.

[II] The tissue reconstruction material according to claim 9, wherein the adhesive component is fibrinogen, thrombin, or caprotun.

12. The tissue reconstructing material according to claim 1, wherein the surface of the amniotic membrane on the side of the chorion is coated with a bioabsorbable material.

[13] A method for producing a tissue reconstruction material, including the following steps (1) to (3):

(1) a step of preparing a biomedical separated amniotic membrane,

(2) A step of freezing or drying the amniotic membrane,

(3) A step of sterilizing the amniotic membrane as an optional step.

14. The production method according to claim 13, wherein the drying process in step (2) is a freeze-drying process.

[15] The production method according to claim 13 or 14, wherein the next step (a) is performed before step (2).

(a) removing the epithelial layer from the amniotic membrane, leaving at least a portion of the basement membrane.

[16] The production method according to claim 15, wherein the step (a) includes the following steps:

(a-1) applying a freeze-thaw treatment to the amniotic membrane,

(a-2) applying a trypsin treatment to the amniotic membrane after the freeze-thaw treatment,

(a-3) A step of washing the amniotic membrane after the trypsin treatment.

[17] The production method according to any one of claims 13 to 16, wherein the next step (b) is performed before or after step (2),

(b) A step of attaching an adhesive component to the chorion side of the amniotic membrane.

[18] Use of amniotic membrane as a major component of tissue reconstruction materials.

[19] A tissue reconstruction method comprising covering a surface tissue damaged part with amniotic membrane.