WO2006129673A1 - Matériel pour la reconstruction de tissus et son utilisation - Google Patents

Matériel pour la reconstruction de tissus et son utilisation Download PDF

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Publication number
WO2006129673A1
WO2006129673A1 PCT/JP2006/310806 JP2006310806W WO2006129673A1 WO 2006129673 A1 WO2006129673 A1 WO 2006129673A1 JP 2006310806 W JP2006310806 W JP 2006310806W WO 2006129673 A1 WO2006129673 A1 WO 2006129673A1
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WIPO (PCT)
Prior art keywords
amniotic membrane
tissue
membrane
amniotic
tissue reconstruction
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PCT/JP2006/310806
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English (en)
Japanese (ja)
Inventor
Yoshiaki Kuriu
Yuen Nakase
Akeo Hagiwara
Hisakazu Yamagishi
Eiji Kurihara
Junji Hamuro
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Arblast Co., Ltd.
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Publication of WO2006129673A1 publication Critical patent/WO2006129673A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/005—Ingredients of undetermined constitution or reaction products thereof

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • serosal epithelial cells which are representative of mechanical and chemical stimuli caused by surgical operation of the intestinal tract and peritoneum during open surgery.
  • serosal epithelial cells can easily fall off and damage organs.
  • the serosa 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Sepra Finorem Genzym
  • Surmenore Johnson and Johnson
  • Gelfoam Apjon
  • Interseed Chondron
  • Xelan Shionogi
  • Veriblast ZLB Behring
  • Tissir Baxter
  • Bolhir Ye Blood Research Institute
  • Octopus Octopus
  • 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.
  • anti-adhesive agents such as polysaccharides, chondroitin sulfate, cellulose, collagen, gelatin, or substances that are reconstituted by mixing these ingredients! / Speak. Disclosure of the invention
  • 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.
  • 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.
  • Sepurafilm is generally fragile and easily breaks, so it requires skill.
  • 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.
  • 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.
  • 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.
  • amniotic membrane 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a tissue reconstruction material is constructed using human amniotic membrane! Speak.
  • 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.
  • the surface of the amniotic membrane is the amniotic membrane coated with a bioabsorbable material. The material for tissue reconstruction is built!
  • 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.
  • the drying process in step (2) is a freeze-drying process.
  • a step (step (a)) of removing the epithelial layer from the amniotic membrane leaving at least a part of the basement membrane is performed before the step (2).
  • 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.
  • 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.
  • 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.
  • FIG. 1 is a diagram for explaining a method for fixing an amniotic membrane.
  • A A pair of frames sandwich the amniotic membrane,
  • 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.
  • 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.
  • FIG. 10 HE stained image and immunostained image of raw fresh, 10% trypsinized, and ⁇ -treated amniotic membrane ( ⁇ ). The signal of each antibody is green in the immunostained image. Cell nuclei are displayed in red.
  • ⁇ 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.
  • ⁇ 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.
  • Figure 13 shows the state of the cecal tissue one week after the amniotic coating.
  • the cecum and abdominal wall, testicular fat, small intestine and omentum are highly adhered.
  • Fresh fresh ⁇ 10% trypsin treatment ⁇ ⁇ -ray treated amniotic membrane has adhesions between the cecum, omentum and testicular fat.
  • no adhesion was observed in the cryopreserved / epithelial-coated group.
  • 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.
  • mesothelial cells are present in the same manner as in the normal intestinal tract, and the serosa is reconstructed.
  • 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.
  • 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 is a table summarizing the adhesion score and the presence or absence of serosal reconstruction of amniotic membrane after various treatments.
  • 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.
  • 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).
  • tissue 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.
  • 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).
  • the first aspect of the present invention relates to a tissue reconstruction material.
  • amniotic membrane is used as a main constituent.
  • the tissue reconstruction material of the present invention is substantially composed solely of amniotic membrane.
  • 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.
  • 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.
  • 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.
  • 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.
  • the tissue reconstruction material of the present invention may be constructed using amniotic membrane with the epithelial layer remaining.
  • sufficient sterilization treatment such as ⁇ -ray treatment can be performed at the production stage, and safety can be improved.
  • 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.
  • a frozen state having an epithelial layer 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.
  • 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.
  • 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”.
  • 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.
  • the component is detected.
  • 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.
  • the tissue reconstruction material of the present invention can also be constituted using the reconstructed amniotic membrane.
  • 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.
  • the tissue reconstruction material of the present invention is formed into a very thin sheet.
  • the tissue reconstruction material of the present invention is prepared to a thickness of 10 m to 500 m, for example.
  • 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 ⁇ m to 30 ⁇ m) is removed, or a bioabsorbable material
  • the thickness may be about 100 ⁇ m to 500 ⁇ m.
  • 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.
  • 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.
  • 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”.
  • the adhesive component for example, fibrinogen and thrombin can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • fibrinogen and thrombin derived from the blood of a patient (recipient) who receives the transplantation of the tissue reconstruction material of the present invention are particularly preferable to use. This is because there is no risk of inducing immune rejection caused by these adhesive components.
  • fibrinogen and thrombin origins of fibrinogen and thrombin are not necessarily the same.
  • human blood-derived fibrinogen and sushi blood-derived thrombin can be used in combination.
  • the amount of fibrinogen and thrombin attached is not particularly limited.
  • the adhesion amount of thrombin can be set in the range of 0.5 ⁇ mg to 10 mg per 1 cm 2 of amniotic membrane.
  • 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).
  • 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.
  • aprotinin is not particularly limited.
  • aprochons derived from the spleen such as ushi, horse, hidge, pig, monkey and chimpanzee can be used.
  • the amount of adhesion is not particularly limited.
  • 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).
  • aprotun 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.
  • fibrin clots are used as adhesives. In such applications, it is common to use aprotune together.
  • tissue reconstruction material of the present invention can provide sufficient adhesion to a living body without using aprochun.
  • 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.
  • 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.
  • a bioabsorbable material used for such a purpose it is preferable to employ a material that is decomposed and absorbed earlier than amniotic membrane.
  • 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”!
  • 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.
  • the tissue reconstruction material of the present invention is provided packaged so as to be practically free of contact with oxygen.
  • 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.
  • 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).
  • Step (1) Step of preparing amniotic membrane
  • the amniotic membrane used in this step is preferably human amniotic membrane.
  • human amnion can also collect force such as human fetal membrane and placenta obtained as a postpartum at delivery.
  • 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.
  • a method for preparing such human amniotic membrane a known method such as the method described in JP-A-5-56987 can be employed.
  • 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.
  • 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.
  • DMEM Denbecco's modified Eagle's medium
  • 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.
  • the amnion after collection may be stored refrigerated rather than frozen and used for subsequent processing.
  • the amniotic membrane is frozen or dried.
  • This treatment results in an amniotic membrane that has excellent storage stability and is easy to handle.
  • the amniotic membrane that was cryopreserved after collection exhibited better anti-adhesion action than the amniotic membrane that was cryopreserved after collection.
  • 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.
  • the amniotic membrane is very highly preserved and convenient.
  • freeze-drying treatment is preferable from the viewpoint of maintaining the structure of the basement membrane component of the amniotic membrane.
  • the boiling point generally ranges from about -20 ° C (107 Pa, 0.8 Torr) to about -50 ° C (4 Pa, 0.
  • freeze-drying process 1 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.
  • 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
  • step (2) “step of removing epithelial layer from amniotic membrane leaving at least part of basement membrane (step (a))” is performed.
  • the epithelial layer is removed, but at this time, at least a part of the epithelial layer remains without removing the basement membrane.
  • 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.
  • 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.
  • pretreatment treatment with EDTA or the like
  • pretreatment 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.
  • 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.
  • 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, ⁇ 2, and ⁇ 5), collagen VII, and laminin 5).
  • 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).
  • the amniotic membrane 10 is fixed using the frame 3 and the plate-like member 4.
  • the amniotic membrane 10 is spread on the plate-like member 4.
  • the epithelial side of the amniotic membrane 10 is turned up.
  • 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.
  • 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).
  • 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.
  • 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.
  • a melting temperature of about 4 ° C to about 50 ° C can be employed. Preferably the melting temperature is about 37 ° C.
  • freeze-thaw treatment it is preferable to repeat the freeze-thaw treatment.
  • 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.
  • 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.
  • 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.
  • a trypsin solution having a trypsin concentration of about 0.01% (w / v) to about 0.05% (w / v) can be used.
  • 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.
  • 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.
  • trypsins are commercially available such as those derived from ushi, porcine, and human.
  • Trypsin-EDTA Invitrogen
  • trypsin 1: 250 Sigma
  • a chelating agent is usually added to the trypsin solution, but the chelating agent is not essential.
  • 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.
  • 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.
  • 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.
  • 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.
  • the trypsin treatment can be performed in a plurality of times. [0042] (Washing: Step a-3)
  • 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.
  • the liquid used for washing include physiological saline, phosphate buffer, pure water, and DMEM.
  • the washed amniotic membrane may be refrigerated or frozen until use.
  • 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)).
  • glyceride for example, 50% glycerol-containing DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL)
  • step (b) before or after step (2), (b) a step of attaching an adhesive component to the chorion side of the amniotic membrane is performed.
  • step (b) is usually performed after step (a).
  • fibrinogen and thrombin can be used as the adhesive component.
  • fibrinogen and thrombin can be used as the adhesive component.
  • amniotic membrane it is preferable to dry the amniotic membrane prior to the adhering operation of the adhesive component.
  • an adhesive component such as fibrinogen can be better adhered.
  • 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.
  • 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.
  • 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.
  • fibrinogen and thrombin are simultaneously attached to the amniotic membrane surface.
  • a fibrinogen solution is prepared. Specifically, fibrinogen is dissolved in a solvent (solvent) such as ethanol (for example, 94% ethanol) to a desired concentration.
  • a solvent such as ethanol (for example, 94% ethanol)
  • alcohols such as absolute ethanol, isopropanol, and methanol, and acetone can be used as the solvent.
  • a thrombin solution separately by the same procedure.
  • ethanol for example, 99.5% ethanol
  • alcohols such as absolute ethanol, isopropanol and methanol, acetone, and the like can be used.
  • 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.
  • 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.
  • fibrinogen and thrombin adhere to the amniotic membrane in a state where they do not act in advance.
  • 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.
  • 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.
  • 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.
  • 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.
  • human-derived fibrinogen can be purchased from Paxter.
  • human-derived thrombin can be purchased from Baxter.
  • 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.
  • a commercially available aprotune may be used.
  • aprochun derived from Ushi can be purchased from Bayer Yakuhin.
  • step of attaching caprotun 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.
  • 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.
  • 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 include polydaractin 910, gelatin, collagen, polylactic acid and the like.
  • Amnion can be sterilized by EOG (ethylene oxide gas), UV (ultraviolet light), ⁇ -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.
  • 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.
  • tissue reconstruction material of the present invention can be broadly classified into the following three types based on the application method and application purpose.
  • 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.
  • 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.
  • Sepura film is 1 2 It is the same usage pattern.
  • Sepurafilm cannot be used in the following usage modes 1 ⁇ 3, 2 ⁇ 2.
  • 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.
  • the surface of the injured organ is covered with a tissue reconstruction material to reconstruct the tissue and prevent adhesion.
  • 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
  • dry amniotic membranes are preferable because they are easy to handle, but they are frozen in areas where the carrier needs flexibility such as the heart.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a tissue reconstruction material constructed with cryopreserved 'amniotic membrane with epithelium' from the viewpoint of strength.
  • 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.
  • 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.
  • peritoneal dissemination 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.
  • 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.
  • 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.
  • the application site is covered with a tissue reconstruction material so as to wrap the tissue.
  • 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).
  • 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.
  • tissue reconstructing material constructed with lyophilized / epithelial amniotic membrane.
  • the intestine is wrapped in a tube shape with a tissue reconstruction material at the stage where adhesions are physically separated after laparotomy.
  • the tissue reconstruction material is applied so that the basement membrane side of the amniotic membrane becomes the abdominal cavity side.
  • 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.
  • tissue reconstruction material constructed of amniotic membrane with an adhesive component attached 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.
  • 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.
  • 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.
  • 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.
  • tissue reconstruction material constructed with freeze-dried / epithelial amniotic membrane.
  • the application site is covered with a tissue reconstruction material so as to wrap the tissue.
  • 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.
  • the form and application method of the tissue reconstruction material used are the same as in 1-3.
  • Glaucoma is a disease in which the optic nerve is damaged, the visual field narrows, and visual acuity is reduced.
  • 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.
  • amniotic membrane is considered effective for this problem.
  • tissue reconstruction material is inserted under the conjunctiva.
  • 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.
  • Ryukyu adhesion is a disease in which scarring occurs from the eyelid conjunctiva to the eyeball, causing adhesion between the eyelid and the eyeball.
  • 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.
  • the scarred conjunctival tissue is peeled, the sclera is exposed, and the tissue is reconstructed.
  • 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.
  • tissue reconstruction material it is preferable to use a material for tissue reconstruction that is constructed with an amnion without an adhesive skin.
  • 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.
  • 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.
  • the pterygium tissue is peeled off and the sclera is exposed, and then covered with a tissue reconstruction material.
  • 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.
  • tissue reconstruction material constructed with an amnion with no epithelium.
  • 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.
  • 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.
  • PBS phosphate buffer
  • 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.
  • 50% sterilized glyceride in DMEM Dulbecco'S Modofied Eagle Medium: GIBCOBRL was used.
  • 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.
  • 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.
  • 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 wash 30 minutes 9. Enclosed.
  • the encapsulated sample was observed under a fluorescence microscope (Leica DMIRB).
  • Collagen I Collagen I
  • Collagen III Collagen III
  • Collagen IV Collagen IV
  • Collagen V Collagen V
  • Collagen VII Collagen VII
  • Chemicon MAB1345 Laminin-5 (Chemicon MAB19562, Fibronectin (bronectin): LSL LB-1021.
  • 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.
  • 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.
  • the encapsulated sample was observed under an optical microscope (Olympus BX50).
  • 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
  • condition 5) was treated for 12 hours. It was possible to detach the cells.
  • amniotic epithelial cells can be detached by treatment with trypsin 0.2% to 0.01%.
  • 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. .
  • (Condition 2) to (Condition 4) are preferred. This is because the basement membrane component collagen IV remains.
  • 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.
  • the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish.
  • PBS sterile phosphate buffer
  • 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.
  • immunostaining and HE staining were performed according to the procedure described in 11.1.
  • the treatment time was preferably 1 hour or less, more preferably 30 minutes or less in order to keep the basement membrane component remaining.
  • 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.
  • 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.
  • 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).
  • 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.
  • HE staining and immunostaining were performed according to the procedure described in 1-1.
  • 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.
  • trypsin a treatment method that satisfies all of the following six conditions is the most preferable treatment method for amniotic membrane.
  • the treatment time is preferably within 1 hour, more preferably within 30 minutes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 50% sterilized glycerol in DMEM Dulbecco'S Modofied Eagle Medium: GI BCOBRL was used.
  • 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.
  • PBS phosphate buffer
  • the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish.
  • PBS sterile phosphate buffer
  • 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.
  • 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.
  • trypsin solution phosphate buffer containing 0.02% trypsin and 0.2 mM EDTA
  • the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish.
  • PBS sterile phosphate buffer
  • 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.
  • freeze drying -50 ° C, about 1 hour
  • 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 ⁇ rays (about 15 kGy).
  • the sterilized amniotic membrane was stored at room temperature in a vacuum-packed state until just before use.
  • 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 ⁇ -ray irradiation is the same as in (C), without ⁇ -ray treatment (freeze-dried ⁇ no epithelium ⁇ ⁇ -ray untreated amniotic membrane: D-1) and with ⁇ -ray treatment (freeze-dried ⁇ no epithelium ⁇ ⁇ -ray treatment Finished amniotic membrane: D-2).
  • the preservation of the dried amniotic membrane was carried out in the same manner as (C).
  • the amniotic membrane cryopreserved was thoroughly washed with a sterile phosphate buffer (PBS) in a petri dish.
  • PBS sterile phosphate buffer
  • 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).
  • trypsin solution immersion time is 15 minutes to 2 hours Changed to and implemented.
  • the basement membrane is severely damaged in this amniotic membrane by increasing the trypsin solution immersion time.
  • the amniotic membrane was preserved by a conventional method.
  • the following operation was performed without cryopreservation. After the treatment of the amnion chorion, put 30cc of amniotic cell culture in a 100 ⁇ 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 ⁇ g / ml in DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL) was used.
  • the amniotic membrane was thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish.
  • PBS sterilized phosphate buffer
  • the process of epithelial cell detachment was performed in the same manner as in (B), and stored at 4 ° C as in (F).
  • the amniotic membrane 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.
  • PBS sterilized phosphate buffer solution
  • the amniotic membrane was thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish.
  • a solution trypsin concentration of 10%
  • PBS sterilized phosphate buffer
  • a solution trypsin concentration of 10%
  • trypsin solution was put into a 100 ⁇ Petri dish, and one piece of amnion that was collected and washed was put in it and left at 37 ° C for 3 hours.
  • 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).
  • PBS sterilized phosphate buffer solution
  • 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 ⁇ rays. The amniotic membrane after sterilization treatment was stored at room temperature in a vacuum packed state until just before use.
  • 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.
  • FIGS. Figures 5-10 are stained images
  • Figures 11 and 1 2 is a table summarizing the evaluation by stained images.
  • 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-free amniotic membrane I
  • 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.
  • the fibronectin detection sensitivity decreased in part, but it was cryopreserved 'amniotic membrane with epithelium ( ⁇ ), cryopreserved ⁇ epithelium No amniotic membrane ( ⁇ ), 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.
  • fibronectin is clearly impaired in basement membrane damage, cryopreservation, epithelial-free amniotic membrane (E).
  • E epithelial-free amniotic membrane
  • collagen V is clearly detected. Disability is recognized.
  • 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.
  • 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.
  • 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.
  • 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.
  • A epithelial amniotic membrane
  • FIG. 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
  • Fig. 16 shows cryopreservation.
  • 'HBME-1 mesothelial cell marker
  • amniotic membrane uncoated 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.
  • 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.
  • cecum-small intestine ⁇ : cecum-mesentery,: cecum-large intestine, iv: cecum-body network, V: cecum-abdominal wall
  • Fig. 17 shows the evaluation results.
  • the group ( ⁇ ) 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.
  • 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 ⁇ -ray treatment and the function of the anti-adhesion component disappears.
  • the amniotic membrane ( ⁇ ) causes severe adhesions, and the difference is obvious when compared to the mild adhesions of the amniotic membrane ( ⁇ ).
  • amniotic membrane ( ⁇ ) and the amniotic membrane ( ⁇ ⁇ ) are the differences 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 ( ⁇ ). Therefore, it can be said that the basement membrane component must remain intact to prevent the adhesion of the amniotic membrane.
  • amniotic membrane (F), (G), (H), (1) the score is also cryopreserved.
  • the preparation process is cryopreserved! It is thought that it was easy to form.
  • 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.
  • 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 ⁇ It was clearly different from the observed images of the amniotic membrane ( ⁇ ). This suggests that the amniotic membrane ⁇ and the amniotic membrane ( ⁇ ) are less effective in preventing adhesion because the basement membrane component is highly damaged.
  • amniotic membranes (A), ( ⁇ ), (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.
  • Cryopreserved 'amniotic membrane with epithelium ( ⁇ ) 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.
  • epithelial amniotic membrane A
  • amniotic membrane eg, cryopreserved amniotic membrane with epithelium (A)
  • A epithelium
  • 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.
  • 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.
  • 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.
  • the cecum of the rat is covered with a dry adhesive component adhering epithelium-free amnion (L) and closed.
  • L epithelium-free amnion
  • the intestines In the control group, the intestines usually adhere to each other over a wide area, and the intestines are observed together.
  • the effect of the adhesive component adhering 'epithelium-free amniotic membrane (L) as an adhesion inhibitor can be evaluated.
  • histological analysis is possible by removing the tissue after a predetermined period of time and subjecting the tissue section to HE staining.
  • 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.
  • 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.
  • the peritoneal defect is covered with dry adhering component adhering epithelium-free amnion (L) and the abdomen is closed.
  • 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.
  • the intestine-peritoneum usually causes adhesions.
  • the effect of the adhesion component-attached / epithelial-free amniotic membrane (L) as an adhesion inhibitor can be evaluated.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the freezing temperature is about 20 ° C to about 80 ° C
  • the melting temperature is about 4 ° C to about 50 ° C. Manufacturing method.
  • 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) is performed using a trypsin solution having a trypsin concentration of about 0.01% (w / v) to about 0.05% (w / v).
  • 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.
  • a chelating agent selected from the group consisting of EDTA, NTA, DTPA, HEDTA, GLDA and any combination thereof.

Abstract

L’invention concerne un matériel ayant de nombreux champs d'application et qui peut faciliter la reconstruction d'un tissu endommagé tout en empêchant son adhésion (c.-à-d., un matériel pour la reconstruction de tissus) ; une méthode pour le produire ; une méthode pour l'utiliser ; et ainsi de suite. Un matériel de reconstruction de tissus est produit au moyen de l'amnios.
PCT/JP2006/310806 2005-05-30 2006-05-30 Matériel pour la reconstruction de tissus et son utilisation WO2006129673A1 (fr)

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JPWO2008102847A1 (ja) * 2007-02-23 2010-05-27 国立大学法人富山大学 医用代用膜、その使用及び生体内部の膜組織の修復方法
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