WO2011074208A1 - 皮膚真皮又は羊膜透明化による角膜移植材料調製法 - Google Patents
皮膚真皮又は羊膜透明化による角膜移植材料調製法 Download PDFInfo
- Publication number
- WO2011074208A1 WO2011074208A1 PCT/JP2010/007119 JP2010007119W WO2011074208A1 WO 2011074208 A1 WO2011074208 A1 WO 2011074208A1 JP 2010007119 W JP2010007119 W JP 2010007119W WO 2011074208 A1 WO2011074208 A1 WO 2011074208A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tissue
- transparent
- crosslinking
- amniotic membrane
- transparency
- Prior art date
Links
Images
Classifications
-
- 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
- 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
- A61L27/362—Skin, e.g. dermal papillae
-
- 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
- A61L27/3641—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 site of application in the body
-
- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
-
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Definitions
- the present invention relates to a method for making skin dermis or amniotic membrane transparent. More specifically, the present invention relates to a method of irreversibly clearing the skin dermis or amniotic tissue, the transparent skin dermis or transparent amniotic membrane obtained by the method, and its application to a corneal transplant material.
- corneal epithelial transplantation that accounts for 10% of transplantation is only cultured mucosal epithelial sheet transplantation developed by the inventors (Patent Document 1 and Non-Patent Document 1).
- corneal parenchyma transplantation which is said to be 40% of transplantation, there is no cell therapy applied clinically.
- Non-Patent Document 2 the use of glass and synthetic polymers (PMMA, PHEMA, etc.) has already been attempted for more than 200 years (Non-Patent Document 2).
- PMMA glass and synthetic polymers
- Non-patent Document 3 the use of glass and synthetic polymers
- an artificial cornea that can be used as a standard treatment has not yet been developed.
- Non-Patent Document 4 the development of an artificial cornea that has gelled collagen, which is the main component of the cornea, has been reported, many problems to be overcome in clinical application such as vulnerability to sutures remain (Non-Patent Document 4). ).
- the inventors have established a technique for making the isolated sclera (white eye) transparent and using it as a material for transplanting the corneal stroma (Patent Document 2).
- this technique may cause rejection because the scleral tissue used is derived from another family, and its supply is limited.
- the transparency of the sclera obtained is only about 60% even when crosslinked, there is a problem that further processing such as laser treatment is required in order to obtain the transparency of 70 to 80% necessary for clinical use. .
- Non-patent Document 5 A phenomenon has been reported in which the skin becomes transparent due to dehydration by chemicals and natural drying (Non-patent Document 5).
- the examination of the transparent conditions for the purpose of clinical application and the development of the technology for maintaining the transparency under the humid conditions (physiological conditions) indispensable as a transplant material have not been performed.
- skin tissue and oral mucosal tissue can be easily and non-invasively extracted as compared with other tissues, they are extremely useful as a tissue / cell source in regenerative medicine.
- keratoplasty if an alternative material can be developed using skin tissue or oral mucosa tissue, it can be a promising solution to the problems of current transplantation treatment.
- amniotic membrane has features such as anti-adhesion, anti-inflammatory, vascular invasion, antibacterial and anti-rejection effects, and is used in the ophthalmic field for the purpose of protecting the ocular surface and wound healing. Yes.
- the amniotic membrane is low in transparency, when it is applied to the eye, a significant reduction in visual acuity occurs. If the transparency of the amniotic membrane can be improved, it is extremely useful as an ophthalmic transplant material for protecting the ocular surface while maintaining visual acuity.
- the object of the present invention is to use a tissue that is abundant in the living body and can be isolated relatively non-invasively, such as skin tissue, or amniotic membrane that is already used in the ophthalmic field, and is highly transparent.
- the purpose is to develop a new corneal transplant material that is less likely to cause a reaction, and to solve the problems of donor shortage and rejection in corneal transplantation.
- the inventors paid attention to the skin dermis rich in collagen fibers like the cornea and sclera, and succeeded in making the skin dermis tissue transparent by a special method. Furthermore, the present inventors have found a method for maintaining transparency of the dermal tissue that has been clarified even under wet conditions (physiological conditions). Also, focusing on amniotic membrane, which has excellent effects such as adhesion prevention and suppression of vascular invasion, and is unlikely to cause rejection, this amniotic membrane is made transparent by a special method, and its transparency is maintained under wet conditions (physiological conditions). As a result, the inventors have found a method for maintaining the temperature and completed the present invention.
- the present invention is to prepare a transparent tissue characterized by obtaining a tissue that remains transparent even under moist physiological conditions by dehydrating the isolated skin dermis or amniotic membrane and performing a crosslinking treatment. Regarding the method.
- Dehydration can be performed, for example, by natural drying.
- a method for dehydrating the skin dermis or amniotic tissue by natural drying for example, 0 to 40 ° C., preferably 2 to 10 ° C., more preferably 4 ° C. under the condition that at least a part of the skin dermis or amniotic membrane is in contact with the outside air.
- At -10 ° C. at least 5 minutes, preferably 1 hour or more, more preferably 12 hours or more in the case of skin dermis, at least 5 minutes or more, preferably 30 minutes or more, more preferably 12 hours or more in the case of amniotic membrane.
- the method of leaving still can be mentioned.
- the skin dermis or amniotic membrane may be dehydrated by treatment with a hygroscopic compound.
- the crosslinking treatment can be performed by chemical crosslinking using a crosslinking agent, or physical crosslinking including thermal crosslinking or ultraviolet crosslinking.
- crosslinking agent examples include water-soluble carbodiimide-based crosslinking agents including 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide.
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
- NHS N-hydroxysuccinimide
- N-hydroxysulfosuccinimide examples include water-soluble carbodiimide-based crosslinking agents including 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide.
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
- NHS N-hydroxysuccinimide
- succinimide-based crosslinking agent Including succinimide-based crosslinking agent; isocyanate-based crosslinking agent such as hexamethylene diisocyanate; polyepoxy-based crosslinking agent such as ethylene glycol diethyl ether; ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, glycerol polyglycidyl ether 1 or a combination of two or more selected from glycidyl ether-based cross-linking agents including: glutaraldehyde; paraformaldehyde; and transglutaminase Rukoto can.
- isocyanate-based crosslinking agent such as hexamethylene diisocyanate
- polyepoxy-based crosslinking agent such as ethylene glycol diethyl ether
- ethylene glycol diglycidyl ether polyethylene glycol diglycidyl ether, polyglyce
- crosslinking treatment may be performed once, or may be performed twice or more if necessary.
- Clear tissue derived from skin dermis is cross-linked between fibers and the distance between fibers is kept shorter than physiological conditions (the distance between fibers of skin dermis tissue is extremely shorter than physiological conditions and there is almost no gap. It is in a state).
- the clear tissue derived from the amniotic membrane is characterized by containing fine and uniform collagen fibers having a fiber diameter of 100 ⁇ m or less, and the interfiber distance is shorter than the physiological conditions even under wet conditions.
- the fibers are densely packed by low temperature drying, pressurization, or the like, and this state is maintained by the cross-linking introduced between the fibers, so that the transparency is maintained.
- the transparent tissue derived from the skin dermis is characterized by having a transmittance in the visible region of at least 40% or more. With this level of transparency, the transmittance of 70-80 or higher required in clinical practice can be achieved by thinning or laser treatment.
- the transparent tissue derived from amniotic membrane has a transmittance in the visible region of at least 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more. If it has such transparency, it can be used as it is as an ophthalmic transplant material such as a cornea protective material without any special treatment.
- the transparent tissue of the present invention is characterized in that transparency is maintained even under humid physiological conditions. Therefore, the transparent tissue of the present invention can be used as a corneal transplant material such as a corneal parenchyma substitute. When higher transparency is required, the transparency can be improved by thinning or laser treatment as described above.
- the transparent tissue of the present invention can also be used as a carrier for cultured mucosal epithelial cell sheets and cultured endothelial sheets.
- a transparent tissue can be easily prepared using a skin tissue that is abundant in a living body and can be isolated relatively noninvasively, or an amnion usually discarded after childbirth.
- the skin dermis can also be prepared using the patient's own skin tissue (skin dermis).
- Amniotic membrane has an anti-adhesion effect, an anti-inflammatory effect, an anti-vascular invasion effect, and an antibacterial effect, and is unlikely to cause rejection. Therefore, the transparent tissue of the present invention has no risk of rejection and side effects unlike an artificial material using an inorganic / polymer material, and is useful as a corneal transplant material (particularly, a corneal parenchyma substitute).
- the amniotic membrane when the amniotic membrane is stored in a wet state, it is usually frozen at -80 ° C.
- the lyophilized transparent amniotic membrane of the present invention can basically be stored at room temperature. Therefore, there is an advantage that it is easy to handle in the medical field.
- FIG. 1 is a diagram illustrating the transparency of naturally-dried rabbit skin dermis tissue.
- A is a skin dermis tissue that was naturally dried on a slide glass on the right side (Dry), and a skin dermis tissue that was immersed in PBS for the same period on the left side (Wet).
- B is a graph showing the transmittance of each tissue sample shown in A (the horizontal axis is the wavelength, and the vertical axis is the transmittance).
- FIG. 2 is a diagram illustrating the maintenance of clearness of a dried and clarified cutaneous dermal tissue by chemical crosslinking using EDC / NHS under wet conditions.
- B is a graph showing the transmittance of a dried and transparent skin dermal tissue subjected to crosslinking under wet conditions and a dried and transparent skin dermal tissue not subjected to crosslinking (the horizontal axis represents wavelength and the vertical axis represents transmittance).
- FIG. 3 is a diagram representing the maintenance of transparency by various concentrations of EDC / NHS (2: 1).
- A is a photograph of a dried and transparent skin dermal tissue that has been cross-linked under wet conditions.
- B is a graph showing the cross-linked dry-transparent cutaneous dermal tissue (Cross-linked) transmittance under wet conditions.
- the transmissivity of the cutaneous dermal tissue (Wet) before drying treatment and the dry and transparent skin dermal tissue (Dry) before crosslinking were also shown.
- FIG. 4 is a graph showing the transmittance of a cross-linked dry and transparent skin dermis tissue under wet conditions when the cross-linking treatment with various concentrations of EDC / NHS (2: 1) is performed 1 to 3 times. is there.
- FIG. 5 is a scanning electron microscopic image of the skin dermis tissue dried at a critical point (A, C, E are samples subjected to crosslinking treatment, B, D, F are samples not subjected to crosslinking treatment. A, B). (Cross section / low magnification (100 times), C and D are cross section / high magnification (5000 times), and E and F are plane / high magnification (5000 times)).
- FIG. 6 is a diagram schematically showing the mechanism of skin dermatological transparency caused by presumed crosslinking.
- FIG. 6 is a diagram schematically showing the mechanism of skin dermatological transparency caused by presumed crosslinking.
- A is a graph showing the transmittance at 400 to 700 nm of human amniotic membrane (Dry) after low-temperature drying and untreated wet human amniotic membrane (Native-Wet) (the horizontal axis represents wavelength, and the vertical axis represents transmittance).
- B is a graph showing the transmittance at 400 nm, 550 nm, and 700 nm of human amniotic membrane (Dry) after low temperature drying and untreated wet human amniotic membrane (Native-Wet) (the horizontal axis represents wavelength and the vertical axis represents transmission). rate).
- A shows a photograph of each sample under wet conditions (from left, non-treatment, 0% EDC / 0% NHS cross-linking treatment, 0.1% EDC / 0.05% NHS cross-linking treatment, 1.0% EDC / 0.5% NHS cross-linking treatment).
- B is a graph showing the transmittance of each sample in a wet state (the horizontal axis is wavelength, the vertical axis is transmittance: from the bottom of the graph, native, 0% EDC / 0% NHS cross-linking treatment, 0. 1% EDC / 0.05% NHS crosslinking treatment, 1.0% EDC / 0.5% NHS crosslinking treatment).
- FIG. 9 represents the transparency of porcine amniotic membrane crosslinked with EDC / NHS (2: 1).
- A is a photograph of porcine amniotic membrane (in PBS) before cross-linking treatment.
- B is a photograph of a pig amniotic membrane subjected to crosslinking under wet conditions (from left, EDC concentrations 0%, 0.01%, 0.1%, 1.0%, 10.0%).
- FIG. 10 is a photomicrograph of A: tissue cut surface, B: cultured epithelial surface, and C: cultured epithelial surface (enlarged) of rabbit corneal epithelial cells cultured on the rabbit transparent skin dermis.
- the present invention relates to a method for transparentizing skin dermis and amniotic membrane, a transparent tissue obtained by the method, and application of the transparent tissue to a corneal transplant material.
- the skin is the tissue that covers the entire surface of the body, borders the outside world, and protects internal organs such as internal organs from external stimuli and impacts.
- the skin is divided into three layers, the epidermis, the dermis, and the subcutaneous tissue in order from the surface.
- the skin dermis tissue is a tissue that supports the epidermis from the inside and contains a lot of moisture, and consists of collagen, elastin, and a matrix that fills in between them. It is a layer of connective tissue.
- the dermis is divided into a papillary layer and a reticular layer and is composed of cells such as fibroblasts, histocytes / macrophages, mast cells, and plasma cells.
- Skin is excellent as a source of transplant material in that it is abundant in vivo with an average of 1.6 m 2 in adults and can be isolated relatively non-invasively.
- the skin dermis tissue has collagen as a main component like the cornea and has sufficient strength to maintain the shape of the eyeball. Therefore, the transparent tissue is a transplant material that is very useful in the treatment of patients with corneal injury. Can be.
- the amniotic membrane is a part of the placenta, a thin membrane that wraps the fetus and retains the amniotic fluid.
- the amniotic membrane is composed of an epithelial cell layer, a basement membrane layer, and a dense layer. In the case of human amniotic membrane, the size is about 20 cm ⁇ 30 cm, and the thickness is 50 to 80 ⁇ m.
- Amniotic membrane has a thick basement membrane made of collagen, laminin, and nidogen, does not contain vascular components, and has the feature that rejection reaction does not easily occur even when used for transplantation. Amniotic membranes also have the characteristics of being very thin, transparent, flexible and strong. Furthermore, since amniotic membrane also has an anti-inflammatory action, an adhesion prevention effect, an antibacterial effect, a basement membrane retention function, and the like, it has long been used as a wound dressing material in surgical fields and the like and an adhesion prevention material after surgery.
- amniotic membrane can be stored at room temperature by freeze-drying, and can easily regain flexibility by adding water again.
- amniotic membrane is currently treated as waste after giving birth, so its use has no ethical problems and is extremely promising as a material for regenerative treatment.
- the transparent amniotic membrane of the present invention which has been made transparent, is very useful in the treatment of patients with corneal injury.
- the origin of the skin dermis and amniotic membrane used in the present invention is not particularly limited as long as it is a mammal having skin tissue or amniotic membrane close to humans, but when used as a transplant material, it may be the same species as the individual receiving the transplant. preferable.
- “Homogeneous” means that the animal (donor) from which the skin dermis or amniotic membrane is removed and the animal (recipient) to which the material obtained therefrom is transplanted belong to the same animal species. For example, if the recipient who is transplanted with the material produced using the method of the invention is a human, it is preferred that the donor is also a human.
- the donor and recipient By making the donor and recipient the same species, it is possible to prevent rejection caused by transplanting a heterogeneous biological tissue.
- the donor and the recipient are the same species in terms of preventing cross-infection of pathogens between different animals.
- the transplant material it is most preferable that the skin dermis and amniotic membrane are those of the patient who needs the transplant.
- Transparency means that the visible light transmittance is at least partially increased.
- the finally achieved transmission is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in the visible region for clear tissue from skin dermis. %, Preferably at least 80%.
- the transmittance finally achieved refers to the transmittance finally achieved including further processing such as thinning and laser processing after dehydration and crosslinking treatment.
- a clear tissue derived from amniotic membrane it is at least 55%, preferably at least 70%, more preferably at least 80%, and further preferably at least 90%.
- the fiber diameter is smaller (less than 100 ⁇ m) and uniform compared to other tissues, and the transparency is lost even under wet conditions by treatment with temporary dehydration.
- the transparency is irreversibly maintained.
- the structure at the time of dehydration is effectively maintained, and the transparency under wet conditions is further improved.
- dehydration means that the water content of the skin dermis or amniotic membrane extracted from a living body is at least partially reduced.
- the water content achieved in the dehydration step in the present invention is preferably 30%, 20%, 15%, 10%, 5% or 2%, more preferably 10% or less.
- Dehydration can be performed using natural drying or a hygroscopic compound, as will be described later.
- Crosslinking is mainly a bond between polymer compounds (or inside a polymer compound, a polymer compound and a low molecular compound).
- the crosslinks are formed in the skin dermis or amniotic tissue, in particular between the collagen fibers constituting it.
- the method of crosslinking is not particularly limited, and any of chemical crosslinking, physical crosslinking, and photocrosslinking may be used. The cross-linking method and cross-linking conditions will be described in detail in “2.2 Cross-linking step”.
- the transparent tissue according to the present invention can be prepared by dehydrating the isolated skin dermis or amnion and subjecting it to a crosslinking treatment.
- dehydration can be performed, for example, by natural drying, freeze drying, drying under reduced pressure, or treatment with a hygroscopic compound.
- the dehydration step is performed by natural drying.
- the drying time depends on the thickness of the skin dermis and amniotic membrane. For example, in the case of human amniotic membrane, it becomes transparent in about 2 hours at a normal thickness (50 to 80 ⁇ m). In the case of skin dermis, if it has a thickness of 200 ⁇ m, it may become transparent in about 2 hours.
- the crosslinking treatment is repeated, the time required for the second and subsequent drying is further shortened, and transparency may be obtained even for about 30 minutes to 1 hour. Basically, the transparency improves as it is kept at a low temperature for a long time, but it may become transparent in about 5 minutes depending on the setting of conditions.
- the dehydration step is performed at 0-40 ° C., preferably 2-10 ° C., more preferably 4-10 ° C. under conditions where at least a portion of the skin dermis or amniotic membrane is in contact with the outside air. Then, it is carried out by standing for at least 5 minutes, preferably 1 hour, more preferably 12 hours or more, and for amniotic membrane at least 5 minutes, preferably 30 minutes, more preferably 6 hours or more. In this embodiment, the skin dermis or amniotic membrane is naturally dried by contact with the outside air. These embodiments are very advantageous in that they do not require special equipment and reagents and can be carried out easily.
- the dehydration step is allowed to stand at 2 to 10 ° C. for at least 8 hours, preferably 16 hours, with the skin dermis or amniotic membrane sandwiched between two planar supports.
- the skin dermis or amniotic membrane is sandwiched between two planar supports facing each other and naturally dried.
- the support is not particularly limited, but is preferably made of a transparent material such as glass because the dry state can be observed.
- the support is, for example, a cover glass and a slide glass.
- the dehydration step is performed by treating the skin dermis or amniotic membrane with a hygroscopic compound.
- the “hygroscopic compound” refers to any compound having an affinity for water and having a high degree of adsorbing water contained in the substance in contact with the air.
- the hygroscopic compound is, for example, alcohol.
- examples of hygroscopic compounds that can be used include glycerol, diethylene glycol, polyethylene glycol, sorbitol, and sucrose.
- the hygroscopic compound is glycerol.
- Glycerol treatment is performed by immersing the skin dermis and amniotic membrane in a glycerol-containing solution.
- the glycerol concentration of the glycerol solution is preferably 40 to 100% (v / v), more preferably 60 to 100% (v / v), and most preferably 100%.
- Glycerol is preferably of the grade recommended for use on the human body.
- the glycerol solution solvent include water, physiological saline, and phosphate buffered physiological saline (PBS). PBS is preferred.
- the immersion time is not particularly limited as long as the tissue is sufficiently transparent, but is preferably at least 3 minutes, more preferably at least 5 minutes, and even more preferably at least 10 minutes.
- dispase treatment In the case of skin dermis, it is desirable to perform dispase treatment to remove tissues other than skin dermal tissue such as conjunctiva before glycerol treatment.
- the dispase treatment can be performed, for example, by immersing the skin dermis tissue in a 2.4 U / mL dispase solution at 4 ° C. for 45 hours. Dispase is commercially available from Invitrogen Corporation.
- the tissue that has been clarified by a dehydration step such as natural drying or glycerol treatment as described above easily becomes opaque again when hydrated in an aqueous solution.
- a crosslinking treatment By performing the crosslinking treatment, the dehydrated skin dermis or amniotic membrane can be kept transparent even when immersed in an aqueous solution, and therefore can be used for transplantation.
- the crosslinking can be carried out by chemical crosslinking using a crosslinking agent or a condensing agent, physical crosslinking using ⁇ rays, ultraviolet rays, thermal dehydration, electron beam, or the like.
- Chemical cross-linking can be performed using a cross-linking agent or a condensing agent that can cross-link proteins or sugars in animal tissues.
- the crosslinking conditions are appropriately set according to the amount of the crosslinking agent and the sample used. For example, it can be performed by immersing dehydrated skin dermis or amniotic membrane in an aqueous crosslinking agent solution for an appropriate time.
- EDC and NHS were used.
- concentration ratio of EDC and NHS is preferably about 2: 1 (weight ratio), and the concentration of EDC is 0.01 to 20% by weight, particularly 0.1 to 10% by weight. Is preferred.
- EDC and NHS are preferably of the grades recommended for use on the human body.
- crosslinking treatment when carried out in an aqueous solution, since it becomes a competitive reaction between swelling and crosslinking, it may be difficult to obtain an effect when the crosslinking reaction is slow.
- UV crosslinking is considered to be the cross-linking of proteins, sugars, and the like in tissues by the energy of irradiated light.
- photocrosslinking by ultraviolet irradiation can be carried out by allowing to stand for about 5 to 14 days under a UV lamp light usually used in a clean bench or the like.
- riboflavin treatment can be performed, for example, by immersing the tissue in 0.1 wt% riboflavin / PBS at 4 ° C. for 2 to 3 hours.
- the preferred degree of crosslinking cannot be shown as a specific value, but the fiber gap is extremely narrow and stabilized so that light scattering within the skin dermis or amniotic membrane is difficult to occur, and the transparency of the tissue can be maintained. May be introduced. This is because if the crosslinking is insufficient, the transparency cannot be maintained, and if the crosslinking is excessive, the transparency is impaired.
- the cross-linking step may be repeated two or more times.
- uniform crosslinking can be performed by repeating the process of dehydration and crosslinking a plurality of times. Moreover, by repeating such a process, the amount of the crosslinking agent required for obtaining transparency can be reduced.
- a combination of EDC / NHS was used.
- the concentration ratio of EDC and NHS is preferably about 2: 1 (weight ratio), and the concentration of EDC is 0.01 to 10% by weight, particularly 0.1 to 1% by weight. It is preferable to repeat twice or more.
- the transparent tissue derived from the skin dermis obtained by drying and cross-linking treatment has a transmittance of about 40% in the visible region. This is considered to be mainly caused by irregular reflection due to surface irregularities such as pores. Further, as an element other than the irregular reflection on the surface, irregular reflection in the tissue can be considered, which is proportional to the thickness of the tissue. Therefore, by applying a surface processing treatment with an excimer laser or the like, or thinning, the transparent tissue can have a transmittance of 70 to 80 or more which is required in clinical practice.
- a clear tissue derived from amniotic membrane obtained by drying and cross-linking treatment has a transmittance in the visible region of 70% to 80% or more, and can be applied clinically as it is as a protective agent in the ophthalmic region.
- the transmittance can be further increased by smoothing the surface with an excimer laser or the like.
- Transparent tissue 3.1 Transparent tissue derived from skin dermis (1) Transparency
- the transparent tissue derived from skin dermis obtained by the above-described method maintains its transparency even under physiological conditions, that is, wet conditions. .
- the transparent skin dermis tissue has a transmittance of at least about 40% in the visible region. If it has such a degree of transparency, it can be made to have a transmittance of 70 to 80 or more required in clinical practice by thinning or laser treatment.
- the visible region specifically refers to a wavelength region of about 350 to 750 nm.
- Collagen fiber In the transparent dermal tissue of the present invention, cross-linking is performed between collagen fibers. Thereby, irreversible transparency is achieved. In other words, collagen fibers are densely parked in the slowly dried skin dermis, and light scattering is less likely to occur, but this is stabilized by cross-linking and changes in the structure of dense collagen fibers even under humid conditions. Sustained transparency is achieved without occurring. In addition, a material having a high elastic modulus is achieved by such structural conversion and crosslinking.
- the transparent tissue derived from amniotic membrane obtained by the above-described method maintains its transparency even under physiological conditions, that is, wet conditions.
- the fibers in the amniotic membrane are thin and uniform in diameter compared to other tissues, so that treatment with temporary dehydration is irreversible without loss of transparency even under wet conditions. This transparency is maintained. Further, by performing a crosslinking treatment after dehydration, the structure at the time of dehydration is effectively maintained, and the transparency under wet conditions is further improved.
- the transparency of the above-mentioned transparent amniotic membrane is about 70% in the case of dehydration-uncrosslinked treatment, and about 80% in the case of being accompanied by the crosslinking treatment.
- the visible region specifically refers to a wavelength region of about 350 to 750 nm.
- the transparency of the conventionally known transparent sclera is 10% or less before cross-linking and about 60% after cross-linking, so that the transparency required in clinical practice (70 to 80%) is achieved. Therefore, it was necessary to perform further processing such as thinning and laser processing.
- the transparent amniotic membrane of the present invention has high transparency as described above, and can be clinically used without further processing. Furthermore, since the transparency required for clinical practice can be obtained without carrying out the cross-linking treatment accompanied by the risk of toxicity, early practical application is expected.
- Collagen fibers in the amniotic membrane contain V-type collagen that plays a role in controlling the fine fiber diameter, and are finely and uniformly controlled (about 50 nm) from other tissues.
- V-type collagen that plays a role in controlling the fine fiber diameter, and are finely and uniformly controlled (about 50 nm) from other tissues.
- improvement in transparency is achieved.
- collagen fibers are densely packed in the slowly dried amniotic tissue, and light scattering is less likely to occur, but even when rehydrated, the fiber structure at the time of dehydration is reflected in the structure at the time of hydration, Diffuse reflection of light is suppressed.
- This phenomenon is specific to amniotic membrane, and in other tissues such as the sclera where the fine fiber diameter is large and non-uniform and the light transmission is low, even when dehydration is performed, transparency is improved upon rehydration. Absent.
- the specific structure of the amniotic membrane as described above affected the high initial light transmittance and the irreversibility of the fiber structure after rehydration, which led to the improvement of the characteristic transparency obtained in the present invention.
- the cross-linking treatment after dehydration maintains the dense collagen fiber structure at the time of drying more stably, and higher transparency than that at the time of non-cross-linking is obtained.
- a material having a high elastic modulus is achieved by such structural conversion and cross-linking between and within the collagen fibers.
- Corneal transplant material The transparent tissue of the present invention maintains its transparency stably even under physiological conditions.
- the skin dermis tissue has collagen as a main component like the corneal stroma and has sufficient strength to maintain the shape of the eyeball.
- amniotic membrane has an anti-inflammatory effect and an anti-adhesion effect, and is originally widely used in the ophthalmic field for the purpose of protecting the ocular surface and wound healing. Therefore, the transparent tissue of the present invention is useful as a corneal transplant material, particularly as a substitute for corneal parenchyma.
- the skin is abundant in the living body and can be isolated relatively noninvasively. Therefore, by using the patient's own skin that requires transplantation, a safe corneal transplant without risk of rejection can be achieved.
- the transparent tissue of the present invention is preferably stored by freezing or refrigeration when not used immediately after preparation.
- the transparent tissue of the present invention is applied to an eyeball, it is necessary to prepare it in advance so as to have the same curvature as the eyeball.
- Carrier of cultured cell sheet The transparent tissue of the present invention has appropriate strength and elasticity. Therefore, by culturing cultured mucosal epithelial cells, corneal epithelium or corneal endothelial cells using this as a support, these cultures are cultured. It can also be used as a carrier for cell sheets produced using cells.
- the transparency method according to the present invention can be applied to other living tissues and the like.
- it can be used for the oral mucosa.
- amniotic membranes are also used as medical contact lenses. It is considered that the transparent tissue of the present invention, particularly the transparent tissue derived from amniotic membrane having high transparency, can also be used as a material for contact lenses.
- Therapeutic material for pets The application of the transparent tissue of the present invention is not limited to humans, and can be used for all mammals such as cats, dogs and other domestic animals.
- the dried skin dermis tissue sample was observed for transparency with the naked eye and its transmittance was measured. As a control, the transmittance was also measured in the same manner for skin dermis tissue immersed in PBS for the same period.
- the dried skin dermal tissue sample had a transmittance of at least 40% in the visible region, while the skin dermal tissue immersed in PBS was almost opaque (FIG. 1B).
- Clarification maintenance material and method by chemical cross-linking A skin dermis tissue sample dried naturally according to the method of Example 1 was distilled using distilled water containing a cross-linking agent EDC / NHS (10 wt% / 5 wt%) or no cross-linking agent. Treated with water at room temperature (about 23 ° C.) for 2 hours and washed with PBS.
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (Pierce)
- NHS N-hydroxysuccinimide (Wako Pure Chemical Industries)
- the cross-linked dry clarified skin tissue sample was immersed in PBS and then its transmittance was measured.
- the cross-linked dried clear skin tissue sample was immersed in PBS, and then its transmittance was measured.
- the cross-linking step (EDC concentration: 0.0 to 10%) was carried out 1, 2, and 3 times, and the cross-linked dry and transparent skin tissue sample was immersed in PBS, and the transmittance was measured.
- Transparent skin dermis tissue sample (thickness 200 ⁇ m) prepared using the crosslinking agent EDC / NHS (10 wt% / 5 wt%) according to the method of Example 1 and skin dermis tissue (thickness) without any clearing treatment 200 ⁇ m) was dried at the critical point.
- the critical point drying is usually performed in the same manner as in scanning microscope observation. That is, the tissue was washed twice in distilled water for 10 minutes at 3 ° C.
- results and Discussion The results are shown in FIG. A, C, and E are skin dermis tissue samples that have been subjected to clearing and crosslinking treatment and then dried at a critical point, and B, D, and F are skin dermis tissue samples that have been subjected to critical point drying without being subjected to transparency and crosslinking treatment. What appears to be fibrous seems to be collagen fibers.
- the cross-linked sample has a smooth surface with densely packed collagen fibers compared to the sample not subjected to the cross-linking treatment.
- Cutaneous dermal tissue is removed from an autologous corneal transplant experiment rabbit and sliced to a thickness of 200 ⁇ m.
- the skin is pasted on the same sphere as the curvature of the rabbit eye, and slowly dried at low temperature to become transparent.
- crosslinking using EDC / NHS (10 wt% / 5 wt%) is repeated twice and washed with PBS.
- the above-mentioned transparent skin dermal tissue is transplanted into the cortex of the rabbit. Observe the transplanted eye to observe epithelial regeneration and transparency of the transplanted dermal tissue.
- the obtained human amniotic membrane was cut into 5 mm ⁇ 15 mm as a test sample, placed on one slide glass, covered with a cover glass with a silicon spacer in between so as to be maintained at 200 ⁇ m, and between the sample and the glass. Saline was quickly added to fill the gap.
- the amniotic membrane sample dried according to the above method does not contain distilled water containing a crosslinking agent EDC / NHS (0.1 wt% / 0.05 wt%, 1.0 wt% / 0.5 wt%) or a crosslinking agent.
- EDC / NHS 0.1 wt% / 0.05 wt%, 1.0 wt% / 0.5 wt%
- the mixture was treated with distilled water at room temperature (about 23 ° C.) for 2 hours and washed with physiological saline. Since EDC / NHS does not remain in the sample after cross-linking, there is no risk that toxic substances derived from the cross-linking agent will be released due to its decomposition when the cross-linked amniotic membrane is clinically applied.
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (Pierce)
- NHS N-hydroxysuccinimide (Wako Pure Chemical Industries)
- the cross-linked amniotic membrane sample (n 3) was measured for transmittance at 400 to 700 nm in a wet state.
- the transmittance (550 nm) was measured.
- the transmittances of the amniotic membrane (Wet) before the drying treatment and the dried transparent amnion (Dry) before the crosslinking treatment are also shown.
- FIG. 10 shows micrographs of the tissue cut surface (FIG. 10A) and the cultured epithelial surface (FIGS. 10B and C). Rabbit corneal epithelial cells adhered to the rabbit's transparent skin dermis and reached confluence. Microvilli of corneal epithelial cells were observed on the cell surface. Rabbit clarified dermis has sufficient cell adhesion and was shown to be usable as a carrier for corneal epithelial cell sheets.
- the transparent tissue of the present invention is abundant in the living body, and can be easily made of skin tissue that can be isolated relatively noninvasively or amniotic membrane that is normally treated as waste and has no ethical problems in its use. Can be prepared.
- the transparent tissue of the present invention has high transparency even in a wet state, has no risk of rejection and side effects, and can be stored easily at room temperature. Moreover, it can be suitably used not only for humans but also for pets such as cats and dogs. Therefore, the transparent tissue of the present invention is useful as a corneal transplant material (particularly, a corneal parenchyma substitute), a cultured cell sheet carrier, a medical contact lens, and a pet treatment material.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
Description
また、癒着防止、血管浸入抑制等の優れた効果を有し、拒絶反応が生じにくい羊膜に注目し、この羊膜を特殊な方法により透明化し、その透明性を湿潤条件下(生理的条件下)においても維持させる方法を見出し、本発明を完成させた。
上記した方法のほか、吸湿性化合物で処理することにより皮膚真皮又は羊膜を脱水してもよい。
皮膚真皮
皮膚は、身体の全表面を覆って、外界との境をなし、内臓などの内部諸器官を外部の刺激や衝撃から保護する組織である。皮膚は表面から順番に表皮、真皮、皮下組織の3つの層に分かれており、皮膚真皮組織は表皮を内側から支える組織で、水分を多く含み、コラーゲンとエラスチン、これらの間を埋める基質からなる結合組織の層である。また、真皮は乳頭層と網状層に分かれ、線維芽細胞、組織球・マクロファージ、肥満細胞、形質細胞などの細胞から構成される。
羊膜は胎盤の一部で、胎児を包み、羊水を保持する薄い膜である。羊膜は、上皮細胞層、基底膜層、緻密層の三層からなり、ヒト羊膜の場合、大きさは約20cm×30cm、厚さは50~80μmである。
本発明において「透明化」とは、可視光の透過率が少なくとも部分的に上昇することを意味する。好ましくは、最終的に達成される透過率は、皮膚真皮由来の透明化組織の場合は、可視領域で少なくとも40%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、又は少なくとも90%であり、好ましくは少なくとも80%である。ここで、最終的に達成される透過率とは、脱水、架橋処理後の、菲薄化やレーザー処理等のさらなる処理をも含んで最終的に達成される透過率をいう。
本発明において「脱水」とは、生体から摘出された皮膚真皮や羊膜の水分量が少なくとも部分的に低下することを意味する。本発明における脱水工程で達成される水分含有量は、好ましくは30%、20%、15%、10%、5%又は2%であり、より好ましくは10%以下である。脱水は、後述するように、自然乾燥あるいは吸湿性化合物を用いて実施できる。
「架橋」は、主に高分子化合物同士(あるいは高分子化合物内部、高分子化合物と低分子化合物)の結合である。本発明の場合、架橋は皮膚真皮又は羊膜組織内、とくにこれを構成するコラーゲン線維間に形成される。架橋の方法は特に限定されず、化学的架橋、物理的架橋、光架橋のいずれであってもよい。架橋方法や架橋条件については、「2.2 架橋工程」において詳述する。
本発明にかかる透明化組織は、単離された皮膚真皮又は羊膜を脱水し、架橋処理を施すことにより調製することができる。
本発明において、脱水は、例えば、自然乾燥、凍結乾燥、減圧乾燥、吸湿性化合物での処理などにより行うことができる。
本発明の一実施形態では、脱水工程は、自然乾燥により行う。乾燥時間は、皮膚真皮や羊膜の厚さ等に依存する。例えば、ヒト羊膜の場合は、通常の厚み(50~80μm)であれば、2時間程で透明になる。皮膚真皮の場合も、200μmの厚みであれば、2時間程で透明になる場合もある。また架橋処理を繰り返す場合は、2回目以降の乾燥に要する時間はさらに短くなり、30分~1時間程度でも透明性が得られる場合もある。基本的には、低温で長時間おく程透明性は向上するが、条件の設定によっては5分程度で透明になることもある。
本発明の別の実施形態では、脱水工程を、皮膚真皮又は羊膜を吸湿性化合物で処理することにより行う。本明細書中、「吸湿性化合物」とは、水との親和性を有し、接触している物質又は大気に含まれる水を吸着する度合が大きい、いずれかの化合物を指す。吸湿性化合物とは、例えば、アルコールである。用い得る吸湿性化合物としては、グリセロール、ジエチレングリコール、ポリエチレングリコール、ソルビトール、スクロースなどが挙げられる。
本発明において、架橋は、架橋剤や縮合剤を用いた化学的架橋、γ線、紫外線、熱脱水、電子線等を用いた物理的架橋などにより実施できる。
化学的架橋は、動物組織中のタンパク質又は糖を架橋し得る架橋剤や縮合剤を用いて実施できる。例えば、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)を含む水溶性カルボジイミド系架橋剤;N-ヒドロキシスクシンイミド(NHS)やN-ヒドロキシスルホスクシンイミドを含むスクシンイミド系架橋剤;ヘキサメチレンジイソシアネート等のイソシアネート系架橋剤;エチレングリコールジエチルエーテル等のポリエポキシ系架橋剤;エチレングリコールジグリシジルエーテル、ポリエチレングリコールジグリシジルエーテル、ポリグリセロールポリグリシジルエーテル、グリセロールポリグリシジルエーテルを含むグリシジルエーテル系架橋剤;グルタールアルデヒド;パラホルムアルデヒド;及び、トランスグルタミナーゼから選ばれる1又は2以上の架橋剤を用いて実施できる。
紫外線架橋とは、照射した光のエネルギーにより組織中のタンパク質、糖などが架橋されることであると考えられる。紫外線照射による光架橋は、具体的には、クリーンベンチ内などで通常用いられるUVランプ光の下、5~14日間程度静置することにより行うことができる。この場合、架橋効率を向上させるために、脱水前に皮膚真皮又は羊膜をリボフラビンで処理するのが望ましい。リボフラビン処理は、例えば、4℃にて2~3時間、0.1重量%リボフラビン/PBS中に組織を浸漬することにより行うことができる。
ブタの皮膚真皮組織を加圧すると透明になるという現象が報告されている。加圧は脱水を伴って線維間距離を短縮させうる。それゆえ、加圧工程を行ってから架橋工程を行い、透明化組織を得ることもできる。
乾燥、架橋処理によって得られる皮膚真皮由来の透明化組織は、可視領域における透過率が40%程度である。これは、毛穴等の表面の凹凸により、乱反射が起きていることが主な原因と考えられる。また、表面の乱反射以外の要素として、組織内の乱反射が考えられ、これは組織の厚みに比例する。それゆえ、エキシマレーザー等で表面加工処理を施したり、菲薄化することにより、透明化組織は臨床で必要とされる70~80以上の透過率のものにすることができる。
架橋を用いて透明化した皮膚真皮や羊膜は固くなる傾向があるため、スライドガラスなどの平面上で作製した場合、眼球の曲率にフィットしないことがある。したがって、眼球への適用を考慮した場合、あらかじめ眼球と同様の曲率を有する面(球体表面、コンタクトレンズのモールド等)上で、乾燥処理及び架橋処理を行うことが好ましい。後述する実施例では、上記した方法で皮膚真皮を家兎眼の曲率と同じ球体上に成形して適用している。
3.1 皮膚真皮由来の透明化組織
(1)透明性
上記した方法によって得られた皮膚真皮由来の透明化組織は、生理条件下、すなわち湿潤条件においてもその透明性が維持される。つまり、皮膚真皮組織の透明化は、生理条件下での乾燥<=>湿潤によって透明性が失われることなく、またその透明度の大きな変化も生じない、不可逆的な透明化である。
本発明の透明化皮膚真皮組織においては、コラーゲン線維間に架橋が施されている。これにより、不可逆的な透明性が達成される。すなわち、ゆっくりと乾燥された皮膚真皮組織内ではコラーゲン線維が密にパーキングされ、光の散乱が生じにくくなるが、これが架橋によって安定化され、湿潤条件下においても密なコラーゲン線維の構造に変化を生じることなく持続的な透明性が達成される。また、この様な構造的変換と架橋により高い弾性率を有する素材が達成される。
(1)透明性
上記した方法によって得られた羊膜由来の透明化組織は、生理条件下、すなわち湿潤条件においてもその透明性が維持される。つまり、羊膜中の線維は他組織と比較して細線維径が細く均一であるため、一時的な脱水を伴う処理を行うことにより、湿潤条件下でもその透明性が失われることなく、不可逆的にその透明性が維持される。また、脱水後に架橋処理を行うことにより脱水時の構造が効果的に保持され、湿潤条件下での透明性がさらに向上する。
羊膜中のコラーゲン線維には細線維径を制御する役割を担うV型コラーゲンが含まれており、他の組織より細く(50nm程度)均一に制御されている。本発明の透明化羊膜においては、脱水処理を行うことでコラーゲン線維間の距離を著しく短くし、その後再水和させても、透明性の向上が達成される。すなわち、ゆっくりと乾燥された羊膜組織内ではコラーゲン線維が密にパッキングされ、光の散乱が生じにくくなるが、再水和させてとしても脱水時の線維構造が水和時の構造に反映され、光の乱反射が抑制される。この現象は羊膜特異的であり、強膜の様に細線維径が大きく不均一で光透過性の低い他の組織では、脱水処理を行ったとしても再水和時に透明性の向上は得られない。上記の様な羊膜の特異的な構造的は、高い初期光透過率や再水和後の線維構造の不可逆性に影響し、本発明で得られる特徴的な透明性の向上につながった。また強膜の場合と同様に脱水後の架橋処理により、乾燥時の密なコラーゲン線維構造がより安定的に維持され、未架橋時よりも高い透明性が得られる。また、この様な構造的変換とコラーゲン線維内及び線維間の架橋により高い弾性率を有する素材が達成される。
本発明の透明化組織は、生理的条件下においても安定的に透明性が維持される。
本発明の透明化組織は適当な強度と弾力性を有する、それゆえ、これを支持体として、培養粘膜上皮細胞や、角膜上皮あるいは角膜内皮細胞を培養することにより、これら培養細胞を用いて作製される細胞シートのキャリアとしても利用できる。
アメリカでは羊膜を医療用のコンタクトレンズとして使用することも行われている。本発明の透明化組織、特に透明度の高い羊膜に由来する透明化組織はコンタクトレンズの素材としても利用することが可能と考えられる。
本発明の透明化組織の適用はヒトに限定されず、ネコやイヌをはじめとするペットや家畜などあらゆる哺乳動物に利用可能である。
材料及び方法
白色家兎より摘出した皮膚真皮組織(1.5cm×1.5cm程度)を凍結標本作製用包埋液(OCT compound)にいれ、-80℃にて凍結した。得られた凍結ブロックを厚み200μmで薄切し、PBS及び蒸留水で洗浄した。この皮膚真皮組織試料を1枚のスライドガラス上に置き、余分な水分を除去した後、4℃で1日乾燥させた。
結果を図1に示す。スライドガラス上で低温でゆっくりと乾燥させた皮膚真皮組織試料は徐々に透明化し、1日程度で組織全体が透明になった(図1A)。これは蒸留水(PBS)に浸漬すると再び白濁してもとの状態に戻る可逆的な現象であった。
材料及び方法
実施例1の方法にしたがって自然乾燥した皮膚真皮組織試料を、架橋剤EDC/NHS(10wt%/5wt%)を含む蒸留水もしくは架橋剤を含まない蒸留水で常温(約23℃)にて2時間処理し、PBSで洗浄した。
EDC:1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(ピアス社製)
NHS:N-ヒドロキシスクシンイミド(和光純薬工業製)
架橋した乾燥透明化皮膚組織試料はPBSに浸漬した後、その透過率を測定した。
結果を図2に示す。自然乾燥による皮膚真皮組織の透明化は、PBSに浸漬すると再び白濁してもとの状態に戻る可逆的な現象であるが、架橋処理後の乾燥透明化皮膚真皮組織試料は、PBSでも白濁することなく、透明性が維持されることが確認された(図2A及びB)。
材料及び方法
1.架橋剤の濃度
実施例1の方法にしたがって自然乾燥した皮膚真皮組織試料を、架橋剤EDC/NHSを含む蒸留水もしくは架橋剤を含まない蒸留水で常温(約23℃)にて2時間処理し、PBSで洗浄した。架橋剤は、EDC/NHS(2:1)の比率を維持し、EDCの濃度を0.0~20%まで変化させた。
上記の架橋工程(EDC濃度:0.0~10%)を1、2、3回実施し、架橋した乾燥透明化皮膚組織試料はPBSに浸漬した後、その透過率を測定した。
結果を図3及び図4に示す。10wt% EDCまでは架橋剤の濃度の上昇にしたがい、架橋皮膚真皮組織試料の透明度は増加したが、20wt% EDCでは架橋処理を施さない乾燥皮膚真皮組織と同程度の透明度であった(図3A)。一方、それぞれのサンプルに関して透明処理前(Wet)、及び透明処理後(Dry)のデータと図3Bで比較する。どのサンプルにおいても乾燥処理により5%程度から40%程度にまで透明性の上昇が得られ、架橋処理することにより、透明性が維持されている。この実験においては特に0.1、1、又は10%のEDCを使用した際に乾燥時よりも透明性が高くなる現象が観察された。乾燥サンプルの場合は空気と凹凸が激しい皮膚表面間での乱反射が大きいのに対して、架橋後のサンプルに関してはPBSを使用しているため、空気と比較して界面での屈折率の差が少なくなり、乱反射が抑えられたためと考えている。
架橋剤の濃度が低い場合は架橋を繰り返すことで透過率を高めることができた(図4)。
上記のとおり、皮膚真皮組織は自然乾燥処理による脱水によって透明化し、さらに架橋処理を施すことで透明化は湿潤状態でも維持されることが確認された。そこで、その機構を検討するために、走査型電子顕微鏡による観察を行った。
実施例1の方法にしたがっ架橋剤EDC/NHS(10wt%/5wt%)を用いて調製した透明化皮膚真皮組織試料(厚さ200μm)と透明化処理を一切しない皮膚真皮組織(厚さ200μm)を臨界点乾燥した。臨界点乾燥は、通常、走査型顕微鏡観察において行われるのと同様に行った。すなわち、組織を蒸留水中で3℃にて10分間×2回洗浄し、エタノール系列を用いて置換し(50%エタノール:10分間、60%エタノール:10分間、100%エタノール:10分間、無水エタノールに浸漬;エタノール:和光純薬工業製)、その後t-ブチルアルコール(和光純薬工業製)にて置換した。続いて臨界点乾燥機(日立社製)を用いて超臨界二酸化炭素による臨界点乾燥を行った。
架橋処理後の試料は、オスミウムコーティング処理後、走査型電子顕微鏡にて観察を行った。
結果を図5に示す。A,C,Eが透明化及び架橋処理を施したのち臨界点乾燥した皮膚真皮組織試料、B,D,Fが透明化及び架橋処理を施さずに臨界点乾燥した皮膚真皮組織試料である。線維状に見えるものはコラーゲン線維であると思われる。架橋処理した試料は、架橋処理を施さない試料と比較して、コラーゲン線維が密に充填され滑らかな表面となっている。
家兎より皮膚真皮組織を摘出し、200μmの厚さに薄切する。家兎眼の曲率と同じ球体上に皮膚を張り付け、低温でゆっくりと乾燥させ透明化する。次いで、実施例2の方法にしたがい、EDC/NHS(10wt%/5wt%)を用いた架橋を2回繰り返し、PBSで洗浄する。
材料及び方法
ヒト羊膜(東北大学病院産婦人科より供与)は、PBS中、-80℃にて凍結保存した。この凍結羊膜を4~8℃(冷蔵庫内)で解凍し、生理食塩水を用いてオーバーナイトで洗浄し残留する血液を除去した。
次いで、1時間室温に置いた後、水分を加えることなく、乾燥状態のサンプルの透過率を測定した。
図7に示されるように、羊膜の400~700nmでの透過率は、簡単な低温乾燥によって顕著に向上することが確認された。また、透過率は短波長側でより高い値を示した(図7B)。
材料及び方法
臨床においては、透明性が湿潤状態においても維持されることが必要である。そこで、上記の方法にしたがって乾燥させた羊膜サンプルを、架橋剤EDC/NHS(0.1wt%/0.05wt%、1.0wt%/0.5wt%)を含む蒸留水もしくは架橋剤を含まない蒸留水で室温(約23℃)にて2時間処理し、生理食塩水で洗浄した。EDC/NHSは架橋後のサンプルに残留しないため、架橋化羊膜を臨床適用した際に、その分解によって架橋剤由来の有毒な物質が放出される恐れがない。
EDC:1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(ピアス社製)
NHS:N-ヒドロキシスクシンイミド(和光純薬工業製)
架橋後の羊膜サンプル(n=3)は、湿潤状態にて400~700nmでの透過率を測定した。
低温乾燥による強膜の透明化は可逆的であり、水溶液中で膨潤し、乾燥処理前と同様にまで透明性が低下する。しかし図8に示されるように、低温乾燥による羊膜の透明化は、不可逆的であり、水溶液中で膨潤させても高い光透過率が維持され(550nmで70.1%)、乾燥処理前のレベル(native)にまで透明性が低下しない。また架橋処理後の羊膜の透過率は湿潤状態においてさらに高い透過率(550nmで80%)を有する。
材料及び方法
1.架橋剤の濃度
実施例1の方法にしたがって低温乾燥したブタ羊膜サンプルを、架橋剤EDC/NHSを含む蒸留水もしくは架橋剤を含まない蒸留水で常温(約23℃)にて2時間処理し、PBSで洗浄した。架橋剤は、EDC/NHS(2:1)の比率を維持し、EDCの濃度を0.0~10.0%まで変化させた。
図9に示すように、0.01~1.0wt% EDCまでは、架橋剤の濃度にかかわらず90%以上の透過率が得られたが、10wt% EDCでは架橋処理を施さない乾燥羊膜よりも低い透過率であった(図9C)。適切な架橋剤濃度(0.01~1.0wt% EDC)においては、湿潤状態において、極めて高い透明性を有する羊膜が得られることが確認された。
材料及び方法
実施例1及び2の方法にしたがって調製した家兎透明化皮膚真皮上にて家兎角膜上皮細胞を培養し、細胞の接着性を評価した。
図10に組織切断面(図10A)、培養上皮表面(図10B及びC)の顕微鏡写真を示す。家兎角膜上皮細胞は家兎透明化皮膚真皮上に接着し、コンフルエントにまで達した。細胞表面上には角膜上皮細胞が有する微絨毛が観察された。家兎透明化真皮は十分な細胞接着性を有しており、角膜上皮細胞シートのキャリアとして利用可能であることが示された。
Claims (15)
- 単離された皮膚真皮又は羊膜を脱水し、架橋処理を施すことにより、湿潤な生理条件下でも透明性が持続する組織を得ることを特徴とする、透明化組織の調製方法。
- 自然乾燥により皮膚真皮又は羊膜を脱水することを特徴とする、請求項1に記載の方法。
- 皮膚真皮又は羊膜の少なくとも一部分が外気と接触する条件下で、0~40℃にて少なくとも5分以上静置することにより皮膚真皮組織を脱水することを特徴とする、請求項1又は2に記載の方法。
- 吸湿性化合物で処理することにより皮膚真皮又は羊膜を脱水することを特徴とする、請求項1に記載の方法。
- 架橋剤を用いた化学的架橋、あるいは熱架橋や紫外線架橋を含む物理的架橋を行うことを特徴とする、請求項1~4のいずれか1項に記載の方法。
- 架橋剤が、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)を含む水溶性カルボジイミド系架橋剤;N-ヒドロキシスクシンイミド(NHS)やN-ヒドロキシスルホスクシンイミドを含むスクシンイミド系架橋剤;ヘキサメチレンジイソシアネート等のイソシアネート系架橋剤;エチレングリコールジエチルエーテル等のポリエポキシ系架橋剤;エチレングリコールジグリシジルエーテル、ポリエチレングリコールジグリシジルエーテル、ポリグリセロールポリグリシジルエーテル、グリセロールポリグリシジルエーテルを含むグリシジルエーテル系架橋剤;グルタールアルデヒド;パラホルムアルデヒド;及び、トランスグルタミナーゼから選ばれる1又は2以上の組合せである、請求項5に記載の方法。
- EDC及び/又はNHSを用いて化学的架橋を行うことを特徴とする、請求項6に記載の方法。
- 請求項1~7のいずれか1項に記載の方法により調製された、透明化組織。
- 線維間が架橋され、線維間距離が生理条件よりも短く維持されていることを特徴とする、単離された透明化組織。
- 可視領域における透過率が少なくとも40%以上であることを特徴とする、請求項9に記載の透明化組織。
- 羊膜由来の透明化組織であって、線維径100μm以下の細く均一なコラーゲン線維を含み、湿潤条件下でもその線維間距離が生理条件よりも短いことを特徴とする、請求項9に記載の透明化組織。
- 可視領域における透過率が少なくとも60%以上であることを特徴とする、請求項11に記載の透明化組織。
- 湿潤な生理条件下においても透明性が維持されることを特徴とする、請求項9~12のいずれか1項に記載の透明化組織。
- 請求項9~13のいずれか1項に記載の透明化組織を利用した角膜移植材料。
- 角膜実質組織代替物である、請求項14に記載の角膜移植材料。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2009-287890 | 2009-12-18 | ||
JP2009287890A JP2013048642A (ja) | 2009-12-18 | 2009-12-18 | 皮膚真皮透明化による角膜移植材料調製法 |
JP2010-051310 | 2010-03-09 | ||
JP2010051310A JP2013048643A (ja) | 2010-03-09 | 2010-03-09 | 透明化羊膜の作製方法及び透明化羊膜 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011074208A1 true WO2011074208A1 (ja) | 2011-06-23 |
Family
ID=44166982
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2010/007119 WO2011074208A1 (ja) | 2009-12-18 | 2010-12-07 | 皮膚真皮又は羊膜透明化による角膜移植材料調製法 |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2011074208A1 (ja) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5946046B2 (ja) * | 2012-12-27 | 2016-07-05 | 新田ゼラチン株式会社 | ヒト角膜内皮細胞シート |
JP2017514879A (ja) * | 2014-05-07 | 2017-06-08 | オシリス セラピューティクス,インコーポレイテッド | 免疫適合性のある羊膜生成物 |
EP3305339A4 (en) * | 2015-06-03 | 2019-01-23 | Sewon Cellontech Co., Ltd | METHOD FOR PRODUCING A COLLAGEN FILM BY THE ULTRAVIOLETTEM LIGHT, COLLAGEN FILM MANUFACTURED THEREOF AND BIOMATERIAL PRODUCED BY USING THE COLLAGEN FILM |
JP2019504708A (ja) * | 2016-02-11 | 2019-02-21 | ライフセル コーポレーションLifeCell Corporation | 酵素分解に対するコラーゲン含有組織製品の安定化方法 |
US10576104B2 (en) | 2010-02-18 | 2020-03-03 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible amniotic membrane products |
JP2021523811A (ja) * | 2018-05-17 | 2021-09-09 | キョンブク ナショナル ユニバーシティ インダストリー−アカデミック コーオペレーション ファウンデーション | 部分硬化されたコンタクトレンズ型羊膜ドレッシング及びその製造方法 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09225018A (ja) * | 1995-12-21 | 1997-09-02 | Yoshihiko Shimizu | ヒト由来の天然コラーゲン膜からなる医用材料 |
WO2004078225A1 (ja) * | 2003-02-26 | 2004-09-16 | Amniotec Inc. | 羊膜由来医用材料、及びその作製方法 |
JP2006507851A (ja) * | 2002-03-26 | 2006-03-09 | アンスロジェネシス コーポレーション | コラーゲンバイオ繊維、ならびにその調製方法および使用 |
WO2007013331A1 (ja) * | 2005-07-25 | 2007-02-01 | Arblast Co., Ltd. | シート状組成物 |
WO2009084507A1 (ja) * | 2007-12-28 | 2009-07-09 | Osaka University | 積層コラーゲンゲルの作製方法及び積層コラーゲンゲル |
JP2009285155A (ja) * | 2008-05-29 | 2009-12-10 | Tohoku Univ | 強膜透明化による角膜移植材料調製方法 |
-
2010
- 2010-12-07 WO PCT/JP2010/007119 patent/WO2011074208A1/ja active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09225018A (ja) * | 1995-12-21 | 1997-09-02 | Yoshihiko Shimizu | ヒト由来の天然コラーゲン膜からなる医用材料 |
JP2006507851A (ja) * | 2002-03-26 | 2006-03-09 | アンスロジェネシス コーポレーション | コラーゲンバイオ繊維、ならびにその調製方法および使用 |
WO2004078225A1 (ja) * | 2003-02-26 | 2004-09-16 | Amniotec Inc. | 羊膜由来医用材料、及びその作製方法 |
WO2007013331A1 (ja) * | 2005-07-25 | 2007-02-01 | Arblast Co., Ltd. | シート状組成物 |
WO2009084507A1 (ja) * | 2007-12-28 | 2009-07-09 | Osaka University | 積層コラーゲンゲルの作製方法及び積層コラーゲンゲル |
JP2009285155A (ja) * | 2008-05-29 | 2009-12-10 | Tohoku Univ | 強膜透明化による角膜移植材料調製方法 |
Non-Patent Citations (1)
Title |
---|
YUMIKO ABE: "Saisei Iryo no Shorai to Sanfujinka 10. Yomaku o Mochiita Saisei Iryo", OBSTETRICS & GYNECOLOGY, vol. 76, no. 10, October 2009 (2009-10-01), pages 1233 - 1237 * |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11207353B2 (en) | 2010-02-18 | 2021-12-28 | Osiris Therapeutics, Inc. | Immunocompatible amniotic membrane products |
US11590172B2 (en) | 2010-02-18 | 2023-02-28 | Osiris Therapeutics, Inc. | Immunocompatible chorionic membrane products |
US11590173B2 (en) | 2010-02-18 | 2023-02-28 | Osiris Therapeutics, Inc. | Methods of manufacture of therapeutic products comprising vitalized placental dispersions |
US11510947B2 (en) | 2010-02-18 | 2022-11-29 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible amniotic membrane products |
US10576104B2 (en) | 2010-02-18 | 2020-03-03 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible amniotic membrane products |
US10646519B2 (en) | 2010-02-18 | 2020-05-12 | Osiris Therapeutics, Inc. | Methods of manufacture of therapeutic products comprising vitalized placental dispersions |
US11638725B2 (en) | 2010-02-18 | 2023-05-02 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible chorionic membrane products |
US11986498B2 (en) | 2010-02-18 | 2024-05-21 | Osiris Therapeutics, Inc. | Therapeutic products comprising vitalized placental dispersions |
JP5946046B2 (ja) * | 2012-12-27 | 2016-07-05 | 新田ゼラチン株式会社 | ヒト角膜内皮細胞シート |
JP2017514879A (ja) * | 2014-05-07 | 2017-06-08 | オシリス セラピューティクス,インコーポレイテッド | 免疫適合性のある羊膜生成物 |
EP3305339A4 (en) * | 2015-06-03 | 2019-01-23 | Sewon Cellontech Co., Ltd | METHOD FOR PRODUCING A COLLAGEN FILM BY THE ULTRAVIOLETTEM LIGHT, COLLAGEN FILM MANUFACTURED THEREOF AND BIOMATERIAL PRODUCED BY USING THE COLLAGEN FILM |
US11179505B2 (en) | 2016-02-11 | 2021-11-23 | Lifecell Corporation | Methods for stabilizing collagen-containing tissue products against enzymatic degradation |
JP2019504708A (ja) * | 2016-02-11 | 2019-02-21 | ライフセル コーポレーションLifeCell Corporation | 酵素分解に対するコラーゲン含有組織製品の安定化方法 |
JP2021523811A (ja) * | 2018-05-17 | 2021-09-09 | キョンブク ナショナル ユニバーシティ インダストリー−アカデミック コーオペレーション ファウンデーション | 部分硬化されたコンタクトレンズ型羊膜ドレッシング及びその製造方法 |
JP7141666B2 (ja) | 2018-05-17 | 2022-09-26 | キョンブク ナショナル ユニバーシティ インダストリー-アカデミック コーオペレーション ファウンデーション | 部分硬化されたコンタクトレンズ型羊膜ドレッシング及びその製造方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR101132625B1 (ko) | 콘택트 렌즈 형태의 양막 드레싱의 제조 방법 | |
Grolik et al. | Hydrogel membranes based on genipin-cross-linked chitosan blends for corneal epithelium tissue engineering | |
JP5661722B2 (ja) | 眼用デバイス及びその製造方法 | |
Lai et al. | Ocular biocompatibility of carbodiimide cross-linked hyaluronic acid hydrogels for cell sheet delivery carriers | |
US9474791B2 (en) | Sterile autologous, allogenic or xenogenic implant and the method of its production | |
US9314531B2 (en) | Wound healing compositions comprising biocompatible cellulose hydrogel membranes and methods of use thereof | |
US20100028407A1 (en) | Layered bio-adhesive compositions and uses thereof | |
US20050287223A1 (en) | Use of amniotic membrane as biocompatible devices | |
US9441080B2 (en) | Cellulose hydrogel compositions and contact lenses for corneal applications | |
WO2016178586A2 (en) | Collagen compositions and preparation and uses thereof | |
US20100198348A1 (en) | Biomaterials with modified optical character and methods for preparing and using same | |
WO2011074208A1 (ja) | 皮膚真皮又は羊膜透明化による角膜移植材料調製法 | |
CN115845141B (zh) | 一种干态羊膜的制备方法及应用 | |
Sarvari et al. | A comprehensive review on methods for promotion of mechanical features and biodegradation rate in amniotic membrane scaffolds | |
JP7141666B2 (ja) | 部分硬化されたコンタクトレンズ型羊膜ドレッシング及びその製造方法 | |
JP5388090B2 (ja) | 強膜透明化による角膜移植材料調製方法 | |
CN110420352A (zh) | 一种生物型眼组织修复材料及其制备方法 | |
US20210268033A1 (en) | Flowable birth tissue composition and related methods | |
RU2714943C1 (ru) | Искусственная роговица, представляющая собой мембрану гетерогенной жесткости на основе коллагена, и способ ее получения и применения | |
Hong et al. | Ultra-stiff compressed collagen for corneal perforation patch graft realized by in situ photochemical crosslinking | |
JP2013048642A (ja) | 皮膚真皮透明化による角膜移植材料調製法 | |
Li et al. | Properties of the acellular porcine cornea crosslinked with UVA/riboflavin as scaffolds for Boston Keratoprosthesis | |
CN107050515B (zh) | 一种角膜基质、制备方法与应用 | |
Widiyanti et al. | Collagen-chitosan-glycerol-HPMC composite as cornea artificial candidate | |
JP2013048643A (ja) | 透明化羊膜の作製方法及び透明化羊膜 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10837244 Country of ref document: EP Kind code of ref document: A1 |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10837244 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 10837244 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: JP |