JP5643011B2 - Biological tissue, production method thereof, and apparatus for producing the same - Google Patents

Description

  The present invention relates to a living body tissue that substitutes for a defective tissue, a production method thereof, and an apparatus for producing the same.

  There have been many studies on regenerative medicine in which cells, tissues, and organs lost due to illness and accidents are revived by artificial materials and cells. Normally, the body has a self-defense function, and when a foreign object such as a thorn enters a shallow position inside the body, it tries to push it out of the body, but when a foreign object enters a deep position inside the body, It is known that fibroblasts gather and sequester in the body by forming a capsule of connective tissue composed mainly of fibroblasts and collagen and covering foreign matter. A plurality of methods for forming a tubular living body tissue using living cells in the living body using the latter self-defense reaction have been reported (see Patent Documents 1 to 3).

  In Patent Document 1 (Japanese Patent Application Laid-Open No. 2007-312821), a spiral groove is formed on the surface of a rod-like structure, and the rod-like structure is embedded in a living body, whereby a membrane-like bond is formed on the surface of the rod-like structure. It is disclosed that it forms a tissue and increases the mechanical strength of the connective tissue.

  In Patent Document 2 (Japanese Patent Laid-Open No. 2008-237896), an outer shell member is formed in a spiral shape along the outer periphery of a rod-shaped structural member, and this is embedded in a living body to form a connective tissue body on the outer edge of the rod-shaped structural member. This is disclosed. The connective tissue body enters between the outer member and the surface of the rod-shaped structural member, and the inner surface shape of the connective tissue body is formed on the same smooth surface as the surface of the rod-shaped structural member. A connective tissue body is formed to a thickness that embeds the shell member.

  Patent Document 3 (Japanese Patent Application Laid-Open No. 2010-094476) discloses that an outer member is formed on the surface of a rod-like structural member, and this is used as a connective tissue forming substrate. By embedding this base material in the living body, a film-like tissue body is formed on the surface of the base material. At that time, by using a material that is excellent in biocompatibility as a material of the outer member but is not easily invaded by the tissue body or its constituent components, the outer member adheres to the tissue body and the mechanical strength of the connective tissue body is increased. In addition, an artificial blood vessel in which the tissue body and its constituent components are not exposed on the inner surface of the outer member is obtained.

JP 2007-312821 A JP 2008-237896 A JP 2010-094476 A

  However, in the conventional biological tissue, collagen is the main constituent, and only a thin film having a thickness of about 50 to 80 μm can be formed over two months. When the film thickness is thin, the independence is poor, and when transplanting as a substitute material for vascular tissue, it is extremely difficult to perform anastomosis with an existing blood vessel. There was a possibility that the blood vessel would be blocked.

  In addition, the original blood vessels of living organisms are composed of a layered tissue consisting of multiple collagens (structural proteins) and elastins (elastic proteins), and have capillaries that provide oxygen and nutrients. It was the subject and was far from the original blood vessels. Therefore, the appearance of a living tissue having a structure similar to that of a human blood vessel has been desired.

  In view of the above, it is an object of the present invention to provide a living body-derived tissue close to the original structure of a living organism, a production method thereof, and an apparatus for producing the same.

  In general, light irradiation affects the skin, such as elastin being decomposed when ultraviolet light is applied to the skin constituting the living body, and the phenomenon that elastin proliferates by irradiating the skin with light and the wrinkles are eliminated. It is known.

  Therefore, the present inventor applied such a phenomenon to irradiate the living body with light, and obtained the knowledge that the tissue was born from the irradiated portion and the surrounding living cells, and completed the present invention. It is a thing.

  That is, the present invention is a living tissue derived from light irradiation by light irradiation means in an environment where a biological tissue material exists. By performing the light irradiation for a predetermined time, a tissue body in which a plurality of types of cells are gathered at and around the light irradiation portion is formed. This tissue body includes elastin in addition to collagen, and is composed of a tissue including a bulky portion. The tissue body is thick and has a structure close to that of an organism originally possessed. Further, as the film thickness is increased, capillaries are also formed in the tissue, and there are many cells that are the source of the blood vessels. Therefore, it is possible to produce a tissue body (biological tissue) containing elements closer to a living body.

  More specifically, in the living tissue, the light irradiation means is placed in an environment where the biological tissue material exists, and the light irradiation means has a thickness several to several tens of times that of the conventional one by performing light irradiation there. Produced by being wrapped in tissue. A tissue derived from a living body can be produced by taking out the formed tissue body from the environment where the biological tissue material exists.

  At this time, if a light source of the light irradiation means is embedded in a translucent resin structure and light is irradiated through the resin structure, a tissue body is formed around the resin structure. By removing this from the environment where the biological tissue material exists and extracting the resin structure, a tubular biological tissue can be produced. The tubular biological tissue can be used as a tubular tissue, and can also be used as a membrane tissue by crushing the tube structure as it is or by opening it in the length direction. In addition, since the shape of the biological tissue is determined by the shape of the resin structure, it is possible to construct a complicated three-dimensional structure such as a valve-like tissue.

  Further, as described above, in addition to the method of taking out the formed tissue body from the environment where the biological tissue material is present and making it as a tissue for transplantation, the light irradiation means such as an optical fiber is brought into the environment where the biological tissue material is present. Alternatively, after forming a new tissue on the irradiated portion, the light irradiation means may be taken out from the environment, and the formed living tissue body may be left in the environment to be regenerated. Using this method, it can be expected that in mammals, birds, fish, and other animals, regeneration of the defect or damaged part is promoted by injecting light irradiation means such as an optical fiber into the defect or damaged part subcutaneously.

  The light source of the light irradiation means is not limited, and examples thereof include light emitting diodes (LEDs), semiconductor lasers, general light bulbs, halogen light bulbs, fluorescent lamps, HID lamps, chemiluminescent materials, fluorescent materials, and the like. These may be used singly or in combination. However, among these, the LED is preferable because it generates a small amount of heat and can be easily controlled to be turned on and off. The number of light sources may be at least one, but a plurality of light sources may be provided in columns, in parallel, or stacked. By providing a plurality of light sources or combining with a reflecting material, a wide range of biological tissue materials can be irradiated with light.

  The wavelength of the light irradiated from the light irradiation means is not limited, and examples thereof include visible light, infrared light, and ultraviolet light. However, visible light that does not penetrate into the skin and does not damage DNA is usually used. Light is considered preferred. Further, among blue, green, yellow, and red visible light, blue to green visible light close to the high energy ultraviolet region is considered preferable. Furthermore, it is preferable that the light irradiated from the light irradiation means includes light having a wavelength of 530 nm or less. If it is in this range, it can be expected to form a tissue structure that includes elastin and capillaries, has a thick film thickness, and is closer to the organism itself.

  The light irradiation pattern of the light irradiation means may be continuous or pulsed, or the type of light wavelength, color, etc. may be changed during the process. Although the light irradiation time varies depending on the environment in which the biological tissue material exists, it has been found that a living body-derived tissue having a sufficient film thickness can be obtained in about 2 weeks when implanted under the back of a beagle dog.

  The formed living body tissue becomes a connective tissue including a membranous tissue, a valve-like tissue, or a tubular tissue. Examples of the membranous tissue include pericardium, dura mater, cornea, skin, pericardium and the like, and are planar tissues that cover the surface layer or function in a membranous form. Examples of valve-like tissues include heart valves and venous valves. Examples of the tubular tissue include blood vessels, lymphatic vessels, trachea, bile ducts, intestinal tracts, urethral tubes, ureters, oviducts, and the like.

  In the present invention, the “biological tissue material” refers to a substance necessary for forming a desired biological tissue, such as fibroblasts, smooth muscle cells, endothelial cells, stem cells, ES cells, iPS cells. Animal cells such as, various proteins (collagen, elastin), saccharides such as hyaluronic acid, and other various physiologically active substances existing in the body such as cell growth factors and cytokines.

  In the present invention, the “biological tissue material” is derived from mammals such as humans, dogs, cows, pigs, goats and sheep, birds, fish and other animals, or artificial materials equivalent thereto. included. In addition, autotransplantation, allotransplantation, and xenotransplantation may be performed for the transplant recipient, but autotransplantation or allotransplantation is preferable from the viewpoint of avoiding rejection. In the case of xenotransplantation, it is preferable to perform an immunogen removal process such as a known decellularization process in order to avoid rejection.

  In addition, “in the environment where biological tissue material is present” means in vivo (for example, limbs, mammals such as humans, dogs, cows, pigs, goats, sheep, birds, fish, and other animals). It represents the inside of an artificial environment containing a biological tissue material outside the living body of an animal). In addition, it is preferable to use a minimally invasive method under the sufficient anesthesia with respect to the spirit of animal welfare when taking the method of implantation into animals.

  By performing light irradiation with light irradiation means in an environment where a biological tissue material exists, it is possible to produce a living tissue derived from elastin, having a large film thickness, and having many cells.

It is a photograph replaced with drawing which shows sectional drawing which dye | stained the biological origin tissue concerning this invention by hematoxylin iosine dyeing | staining. It is the elements on larger scale of the intima part of FIG. It is the elements on larger scale of the middle film part of FIG. It is the enlarged view which dye | stained the membranous part of the biological tissue concerning this invention by the Elastica-Wangieson dyeing | staining. It is a photograph replaced with drawing which shows sectional drawing which dye | stained the biological origin structure | tissue concerning this invention by Masson trichrome dyeing | staining. It is a figure which shows an example of the biological origin production apparatus concerning this invention. It is a photograph replaced with drawing which shows sectional drawing which dye | stained the biological tissue of the comparative example by hematoxylin iosine dyeing | staining. It is the elements on larger scale of FIG. It is a photograph replaced with drawing which shows sectional drawing which dye | stained the biological origin structure | tissue of the comparative example by Masson trichrome dyeing | staining.

  1 to 3 are cross-sectional views of the biological tissue of the present invention stained with hematoxyliniocin. FIG. 4 is a cross-sectional view dyed by Elastica One-Geeson staining. The living body tissue of the present invention comprises a tissue having a layered structure of collagen 1 and elastin 2. The film thickness is large, and the capillaries 3 are also formed, which are close to the tissue body originally possessed by the organism.

  The living tissue of the present invention is a tissue in which the periphery of the light irradiating means 4 has a thickness several times to several tens times the conventional thickness by irradiating the light irradiating means 4 in an environment where the living tissue material exists. Wrapped with a body. By removing this from the environment where the biological tissue material exists, a biological tissue can be produced.

  As shown in FIG. 6, as an apparatus for producing a biological tissue, a biological tissue production apparatus 6 including a light irradiation unit 4 and a driving power source 5 that supplies power to the light irradiation unit 4 is illustrated. The light irradiation means 4 includes a light source 7 and a light-transmitting rod-shaped resin structure 8 that covers the periphery of the light source 7, and irradiates light from the light source 7 to the surrounding biological tissue material through the resin structure 8. To do. In addition, the light irradiation means 4 is not limited to this structure, It is good also as a structure which irradiates light directly from the light source 7, without providing the resin-made structures 8. FIG.

  A dry battery is used as the drive power source 5, but other known power sources such as a private power generation type power source utilizing blood flow may be used. As shown in FIG. 6, the outer surface of the AA battery 5 is protected by a shrink pack 5a. The dry battery 5 and the light source 7 are connected by a lead wire 10 through a lead frame 9.

  An LED is used as the light source 7 of the light irradiation means 4, but other light sources such as a semiconductor laser may be used. An LED is preferable because it generates little heat and does not cause overheating of the tissue material, and has low power and a long life. One light source 7 is provided, but a plurality of light sources 7 may be provided along the longitudinal direction of the rod-shaped resin structure 8. By providing a plurality, the influence of the light source 7 can be given to a multi-region biological tissue material. Visible light is used as the wavelength of light emitted from the light irradiation means 4, but is not limited to this, and infrared light and ultraviolet light may be used. Moreover, although blue LED is used among LED, it is not limited to this, You may use red, green, and the color (white, yellow, etc.) which mixed these.

  In the present embodiment, the reason for using the blue LED will be described. Since the blue wavelength region is difficult to penetrate into the inside through the skin, the case of irradiating from the outside of the living body and the case of irradiating it embedded in the living body I thought it was easy to compare the effects. In addition, it was initially thought that a large biological reaction is unlikely to occur because the tissue inside the skin usually does not come into contact with blue light, so there is no need to feel blue light. However, when actually performed, it was found that a thick biological tissue was formed contrary to expectation, and that collagen, elastin and neovascularization were included. It is recognized that this biological reaction has a similar effect even in green near blue.

  The resin structure 8 is a living body-derived tissue mold, is made of a light-transmitting resin, and embeds the light source 7 by sealing the periphery of the light source 7 without a gap. As a resin material, it has translucency and heat resistance, has strength (hardness) that does not greatly deform when implanted in a living body, has chemical stability, and is resistant to loads such as sterilization. A resin having no or little eluate that stimulates a living body is preferable. In view of this, silicon resin is used in the present embodiment, but other natural resins and synthetic resins may be used. In addition, a transparent structure serving as a mold of a biological tissue can be prepared separately, and the resin structure 8 can be fitted into the transparent structure. That is, the light irradiation means 4 and the structure serving as the mold of the biological tissue can be separated from each other. Accordingly, if a large number of types of transparent structures are prepared, it is possible to produce many types of biological tissue using one light irradiation means 4.

  In the present embodiment, the resin structure 8 is formed in a cylindrical bar shape having an outer diameter of 5 mm and a length of 30 mm, and an LED and a part of the lead wire 10 connected to the LED are embedded therein. The rod-shaped structure is not limited to a cylindrical shape, but may be other shapes such as a polygonal column such as a quadrangular column, but if the formed biological tissue is a tubular tissue such as a blood vessel, it is a circle. A columnar shape is preferable. Further, when an artificial blood vessel is produced as a biological tissue, the thickness of the blood vessel is determined by the outer diameter of the resin structure 8, and therefore the diameter may be changed according to the target thickness. The shape of the resin structure 8 is not limited to a rod shape, and may be other shapes such as a spherical shape, a cubic shape, a rectangular parallelepiped shape, a flat plate shape and the like according to a desired biological tissue. In addition, the mechanical strength of the living tissue may be further improved by providing irregularities and outer members on the surface of the resin structure 8.

  Next, a method for producing a biological tissue using the biological tissue producing apparatus 6 as described above will be described. First, the living body-derived tissue production apparatus 6 is placed in an environment where living tissue material exists. The environment in which the biological tissue material exists includes in an animal's living body (for example, subcutaneously or intraperitoneally embedded) or in an artificial environment such as a solution in which the biological tissue material floats outside the animal's body. . As biological tissue materials, materials derived from other mammals such as humans, dogs, cows, pigs, goats, rabbits, sheep, birds, fish, other animals, or artificial materials can be used.

  Moreover, the living body-derived tissue production apparatus 6 may place at least the resin structure 8 in an environment where the living tissue material exists, but may place the entire apparatus in an environment where the living tissue material exists. When the living body-derived tissue production apparatus 6 is implanted in an animal, it is performed with a minimum of incision under sufficient anesthesia, and the wound is sutured after implantation. In addition, when the biological tissue-producing apparatus 6 is placed in an environment where biological tissue materials are present, cell culture may be performed according to a known method in a clean environment with various culture conditions.

  The light irradiation means 4 of the biological tissue producing apparatus 6 irradiates the surrounding tissue with light through the transparent rod-shaped resin structure 8 covering the light source 7 in an environment where the biological tissue material exists. In the present embodiment, the light source 7 of the light irradiation means 4 always emits light. However, a control unit may be provided to emit light in pulses, or the power supply may be turned on and off manually from the outside by remote control operation. The intensity of the LED, the number of LEDs to emit light, and the like may be operable.

  After continuing the light irradiation for a predetermined time, the living body-derived tissue production apparatus 6 is taken out from the environment where the living tissue material exists. Then, by removing the tissue body thickly formed around the resin structure 8 from the resin structure 8, a tubular living body tissue can be produced. Note that the resin structure 8 and the tissue body can be easily separated by simply pulling out the resin structure 8. Since the peeled inner surface of the tissue body is in contact with the surface of the resin structure 8, it becomes smooth.

  The living body-derived tissue of the present invention produced as described above is composed of a tissue containing collagen 1 and elastin 2, has a large film thickness, has capillaries 3 formed therein, and is close to the tissue body originally possessed by the organism. Thus, since many collagen 1 and elastin 2 are formed, the self-supporting property of a biological tissue is high, and a tubular shape can be maintained. Therefore, when a tubular tissue body is sutured to a living body as an artificial blood vessel, an anastomosis operation can be performed with the anastomosis site opened. In addition, since many new blood vessels such as capillaries 3 are formed in the tissue body, it is expected that a neointimal membrane including endothelialization is formed early after transplantation.

  Thus, according to the present invention, formation of a tissue body can be promoted by light irradiation. In addition, in the method of promoting the formation of a tissue body by drug administration, once the drug is administered, the formation of the tissue body proceeds until all the drugs are absorbed, but in the present invention, the light irradiation is stopped. It is possible to stop the formation promotion of the tissue body at a desired growth stage.

  In addition, the living body-derived tissue of the present invention can be used not only as a tubular tissue such as an artificial blood vessel but also as a valve-like tissue and a membrane-like tissue. When using this living body-derived tissue as a membranous tissue, the tubular structure may be used as it is, or may be used by cutting it in the length direction of the tube. Furthermore, if a resin structure 8 having a three-dimensional structure is used, it is possible to form a living body-derived tissue having a complicated shape.

  In the case of xenotransplantation of the produced biological tissue, it is preferable to perform immunogen removal treatment such as decellularization treatment, dehydration treatment, and fixation treatment in order to prevent rejection after transplantation. Examples of decellularization include ultrasonic treatment, surfactant treatment, and enzyme treatment such as collagenase to elute and wash the extracellular matrix. Dehydration methods include methanol, ethanol, isopropyl alcohol, etc. There is a method of washing with a water-soluble organic solvent, and as a method of fixing, there is a method of treating with an aldehyde compound such as glutaraldehyde or formaldehyde.

  In addition, this invention is not limited to the said embodiment, Of course, many corrections and changes can be added to the said embodiment within the scope of the present invention. For example, in the above-described embodiment, the light irradiation means 4 is embedded in a living body, and a tissue body formed around the light irradiation means 4 is taken out from the living body and used as a transplanting tissue. That is, after the light irradiation means 4 is placed in an environment where a biological tissue material exists and a new tissue including new blood vessels is formed in the irradiated portion, only the light irradiation means 4 is taken out from the environment, and the formed tissue The body may be left in the environment and reproduced. According to this method, in the animal, it can be expected to promote the regeneration of the defect or damaged part by injecting and embedding the light irradiation means 4 such as an optical fiber in the defect or damaged part.

  A method for producing a living tissue using a beagle dog using a blue LED as the light source 7 will be described. First, the whole body-derived tissue production device 6 is implanted under the back of a beagle dog under a sufficient anesthesia with a minimum incision, and then the wound is sutured.

  The LED light source 7 of the light irradiating means 4 always emits blue light having a wavelength of 470 nm and passes through the resin structure 8 made of a transparent silicon resin covering the LED to irradiate the surrounding tissue with light.

  Subcutaneous LED irradiation was continued for 2 weeks, and then the biological tissue producing apparatus 6 was anesthetized and taken out from the living body of the beagle dog. The periphery of the living body-derived tissue production apparatus 6 is entirely wrapped with a tissue body, and in particular, the tissue body is thickly formed around the resin structure 8 in which the LED is embedded. A tubular living body tissue was produced by removing the tissue body from the resin structure 8 while partially cutting it as necessary.

  An enlarged photograph of the tissue structure of the portion formed around the resin structure 8 stained with hematoxylin-iocin and observed with a microscope is shown in FIGS. FIG. 4 shows an enlarged photograph observed in FIG. 4, and FIG. 5 shows an enlarged photograph observed with a microscope after staining with Masson trichrome. As a comparative example, the tissue formed in the case of non-irradiation with LED was observed by staining with hematoxyliniocin staining and is shown in FIGS. 7 and 8, and stained with Masson trichrome staining and observed. The result is shown in FIG. In addition, the comparative example was performed like the said embodiment except LED non-irradiation. In the figure, A is the circumferential direction of the tubular biological tissue, 11 is the lumen surface of the tubular biological tissue, 12 is the inner membrane part of the biological tissue, 13 is the medial part of the biological tissue, Reference numeral 14 denotes an outer membrane portion of a tissue derived from a living body, 15 denotes erythrocytes, 16 denotes inflammatory cells, and 17 denotes fibroblasts. Elastica Wangyson staining is a staining method for staining elastin 2 in blue. Masson trichrome staining is a staining method for staining collagen 1 in blue.

  As shown in FIG. 3 and FIG. 4, the tissue body of this embodiment includes inflammatory cells 16, but it can be seen that a large number of elastin 2 is formed particularly in the medial part 13 (strings or earthworms). Elastin 2 is what appears in the shape line). Further, as shown in FIG. 1, the thickness of the tissue body of this embodiment is about 700 μm, which is about 8 to 9 times thicker than the thickness of the tissue body of the comparative example of FIG. Yes. As shown in FIGS. 2 and 3, the red blood cells 15 and the capillaries 3 are also formed in the tissue body of this embodiment, particularly the intima 12 and the media 13, as shown in FIGS. Many original cells are formed. Therefore, it is possible to produce a living body-derived tissue containing elements closer to a living body. Moreover, as shown in FIG. 1, it turns out that the mucosa-like structure | tissue is formed in the outer membrane part 4 of a biological tissue. Moreover, as shown in FIG. 5, it turns out that many collagen 1 is formed in the tissue body of this embodiment.

  On the other hand, as shown in FIG. 7 and FIG. 8, the tissue body of the comparative example has a thin film thickness of about 78 μm, and although fibroblasts 17 are seen in the tissue body, the amount thereof is small, and elastin 2 is almost all. can not see. Moreover, as shown in FIG. 9, it turns out that the quantity of collagen 1 is also small in the structure | tissue of a comparative example.

  As can be seen from the above results, when a resin structure 8 made of translucent silicon resin in which a blue LED is embedded is embedded in a living body and light is emitted to propagate new cells, the surroundings of the resin structure 8 are obtained. It has been found that a biological tissue having a thickness several times that of the prior art and a large amount of collagen 1, elastin 2 and new blood vessels can be produced.

DESCRIPTION OF SYMBOLS 1 Collagen 2 Elastin 3 Capillary blood vessel 4 Light irradiation means 5 Drive power supply 6 Living body tissue production apparatus 7 Light source 8 Resin structure 9 Lead frame 10 Lead wire 11 Lumen surface 12 Inner membrane part 13 Middle membrane part 14 Outer membrane part 15 Red blood cells 16 Inflammatory cells 17 Fibroblasts

Claims (8)

In a tissue derived from a living body excluding a tissue newly formed around the light irradiation means and constituting a part of the human body by performing light irradiation with the light irradiation means in an environment where a biological tissue material exists A living tissue derived from the formation of capillaries or elastin. In a tissue derived from a living body excluding a tissue newly formed around the light irradiation means and constituting a part of the human body by performing light irradiation with the light irradiation means in an environment where a biological tissue material exists A biological tissue characterized by the formation of capillaries and elastin. The biological tissue according to claim 1 or 2, wherein the light irradiation means is a light source containing light having a wavelength of 530 nm or less. The living body tissue according to any one of claims 1 to 3, wherein the living body tissue is any of a membranous tissue, a valve-like tissue, and a tubular tissue. A living body characterized by forming a new tissue body containing capillaries or elastin around the light irradiating means by irradiating light with the light irradiating means in an environment where living tissue material excluding a human body exists The production method of the origin tissue. The structure is formed around the resin structure by embedding the light irradiation means in a resin structure having translucency and performing light irradiation through the resin structure. Item 6. A method for producing a biological tissue according to Item 5. A living body-derived tissue production apparatus comprising a light irradiation unit that performs irradiation in an environment where a biological tissue material is present, and forming a new tissue body around the light irradiation unit. The living body-derived tissue production apparatus according to claim 7, wherein the light source of the light irradiation means is embedded in a translucent resin structure.