JP2007068878A - Production method of tissue body and biological implant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tissue body which can remarkably increase the thickness of the tissue body to be formed and moreover, enables the massive neogenesis of blood vessels containing blood capillaries by the maturation of the tissue. <P>SOLUTION: An artificial matter which has a pyridine derivative contained layer containing a pyridine derivative at least a part of its surface and so arranged as to release the pyridine derivative is imbedded in vivo to make the tissue body grow on the perimeter of the artificial matter. At least one kind of pyridine derivative is preferable as selected from a group of nicotine, nicotinic acid, ester nicontinate and nicotinic acid amide. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a tissue body and a biological implant.

  Covering with plastic artifacts has been a fire since the unfortunate accident (breast cancer) when transplanting silicone moldings in breast augmentation.

  Excision of autologous tissue such as the large buttocks is highly invasive, and the functions of the buttocks (such as sitting stability) are impaired even if they are concealed with clothes.

  It is known to embed a stick-like artificial object in a living body and create an artificial blood vessel using this as a template.

Japanese Patent Application Laid-Open No. 2004-261260 discloses a technique for quickly forming a tissue body by applying a biological growth factor on the surface.
JP 2004-261260 A

  JP-A-2004-261260 has the following problems.

  (1) Although the period until the tissue body is formed can be shortened, the thickness of the tissue body saturates with time, and even if it is embedded for a long period of time, it is only about several hundred μm. For this reason, the obtained tissue body is insufficient in strength to be used as a graft for an aorta or a vena cava.

  (2) The inside of the obtained tissue body has insufficient blood vessel density and becomes a fibrotic tissue mainly composed of an intercellular matrix. In this case, there is also a risk that the deep tissue does not mature and becomes cancerous.

  (3) Living-derived growth factor is expressed only in the living body (for example, when platelets deform and adhere to blood vessels, when lymphocytes become infected with viruses and become apoptotic), and the expression level is also very small. Therefore, it is difficult to obtain.

  (4) Even if biologically derived growth factors are extracted from non-human animals such as bovines, there is a risk that harmful prions and viruses will be mixed in as contaminated components.

  (5) Although it is possible to synthesize by gene recombination, the activity is lower than that of living organisms (natural), and there is a risk of contamination with toxic substances derived from E. coli used for synthesis.

  (6) The growth factor itself is also unstable and easily deactivated due to factors such as pH change, heat and oxygen contact. Therefore, it is difficult to apply aseptically to the mold.

  The present invention eliminates the above-mentioned conventional problems, and can significantly increase the thickness of a tissue body to be formed, and the tissue body can be matured to produce a large amount of blood vessels including capillaries. It is an object to provide a method and a biological implant.

  The method for manufacturing a tissue body according to claim 1 is a method for manufacturing a tissue body in which an artificial object is embedded in a living body and a tissue body is formed around the artificial body, wherein at least a part of the surface of the artificial body is formed. In addition, a pyridine derivative-containing layer that contains a pyridine derivative and releases the pyridine derivative is provided.

  The method for producing a tissue body according to claim 2 is the method according to claim 1, wherein the pyridine derivative is nicotine, nicotinic acid, nicotinic acid ester, nicotinic acid amide, nicelyol all, nicochronate, nicofibrate, nicofuranose, nicomol, nicomol It is characterized in that it is at least one selected from the group consisting of morphine, nicorandil, nicothelin, nicotinyl alcohol, nifedipine, nifenazone, niflumic acid, nifroquine, nikesamide, niflupyrinol and nilvadipine and substitutions thereof.

  The method for producing a tissue body according to claim 3 is the method according to claim 1 or 2, wherein the layer containing a pyridine derivative is formed by crosslinking a layer coated with a solution containing at least a water-soluble polymer and a pyridine derivative. It is characterized by being.

    The method for producing a tissue body according to claim 4 is characterized in that, in claim 3, the water-soluble polymer is modified with a molecular group having a photosensitive group.

  [Claim 5] The method for producing a tissue body according to claim 4, wherein the molecular group having the photosensitive group is a xanthene dye, an azine dye, a thiazine dye, an oxazine dye, a quinoline dye, a pyrazolone dye, or a stilsen dye. Azo dyes, diazo dyes, anthraquinone dyes, indigo dyes, thiazole dyes, phenylmethane dyes, acridine dyes, cyanine dyes, indophenol dyes, naphthalamide dyes and perylene dyes It is characterized by being at least one selected.

  The method for producing a tissue body according to claim 6 is the method according to claim 5, wherein the molecular group having the photosensitive group is eosin, fluorescein, rose bengal, benzophenone, camphorquinone, olefin, benzalacetophenone, cinnamylideneacetyl, cinnamoyl. , Styrylpyridine, α-phenylmaleimide, phenyl azide, sulfonyl azide, carbonyl azide, o-quinonediazide, furylacryloyl, coumarin, pyrone, anthracene, benzoyl, stilbene, dithiocarbamate, xanthate, cyclopropene, 1,2,3-thiadiazole , Aza-dioxabicyclo, alkyl halide, ketone and diazo and at least one selected from the group modified with these.

  A method for producing a tissue body according to a seventh aspect is characterized in that, in any one of the third to sixth aspects, the crosslinking treatment is light irradiation.

  The method for producing a tissue body according to claim 8 is the method according to any one of claims 3 to 7, wherein the solution further contains at least one selected from the group consisting of thiol, alcohol, reducing sugar and polyphenol. It is a feature.

  The method for producing a tissue body according to claim 9 is the method according to any one of claims 3 to 8, wherein the solution is further selected from the group consisting of an amino group, an N-alkylamino group, and an N, N-dialkylamino group. And an amino compound having at least one kind of group.

    The method for producing a tissue body according to claim 10 is the method according to any one of claims 1 to 9, wherein the water-soluble polymer is gelatin, collagen, fibronectin, hyaluronic acid, keratanic acid, chondroitin, chondroitin sulfate, elastin, heparan sulfate. Laminin, thrombospondin, vitronectin, osteonectin, entactin, gazein, polyethylene glycol, polypropylene glycol, polyglycidol, polyglycidol side chain esterified ester, polyvinyl alcohol, copolymer of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, Selected from the group consisting of copolymers of hydroxyethyl methacrylate and methacrylic acid, alginic acid, polyacrylamide, polydimethylacrylamide and polyvinylpyrrolidone It is characterized in that at least one element.

  The method for manufacturing a tissue body according to claim 11 is the method according to any one of claims 1 to 10, wherein the artifact is an acrylic resin, an olefin resin, a styrene resin, a polyester resin, a polyamide resin, a vinyl chloride resin, a silicon resin, a fluorine resin. It is an object made of at least one selected from the group consisting of resin, epoxy resin, glass, titanium, platinum and stainless steel.

  A tissue body according to a twelfth aspect is manufactured by the method according to any one of the first to eleventh aspects.

  The tissue body according to claim 13 is characterized in that the tissue body according to claim 12 is further decellularized.

  The tissue body according to claim 14 is characterized in that the tissue body according to claim 12 or 13 is further freeze-dried.

  An alternative material for a living tissue according to a fifteenth aspect comprises the tissue body according to any one of the twelfth to fourteenth aspects.

  The living body implant for manufacturing a tissue body according to claim 16 is characterized in that a pyridine derivative-containing layer that contains a pyridine derivative and releases the pyridine derivative is provided on at least a part of the surface of the artificial object. Is.

  When the artificial object is implanted into the living body, a tissue body is formed so as to cover the surface of the artificial object. At this time, by providing a pyridine derivative-containing layer having releasability of the pyridine derivative on the surface of the artificial object, the pyridine derivative is continuously released from the pyridine derivative-containing layer for a long period of time, for example, two weeks or longer. In addition, the thickness of the tissue body to be formed can be dramatically increased, and the tissue can be matured to regenerate a large amount of blood vessels including capillaries.

  This pyridine derivative such as nicotine is a heat-stable synthetic substance and can be sterilized. There are no contaminating components as in biological materials, and there is no difference in activity between lots.

  Pyridine derivatives are extremely soluble in water, and when applied directly to the surface of a living implant and then embedded in the living body, the pyridine derivative diffuses quickly from the living implant and no effect is obtained. On the other hand, by embedding a pyridine derivative in a water-soluble polymer gel such as a photocrosslinkable gelatin gel, release properties are imparted to the pyridine derivative-containing layer. In the present invention, a pyridine derivative is gradually released over a long period of time, for example, 2 weeks or more, thereby obtaining a tissue body having a higher density of angiogenesis, a thickness that is, for example, several tens of times that of the prior art, and a high maturity. be able to.

  The obtained tissue body is constructed of living cells including a large amount of blood vessels, and can be used as a medical material itself.

  For example, there are many cases in which biological tissue is lost due to sports injury, traffic accident, malignant tumor, necrosis, poor limb blood flow, etc., but if the affected part affects the appearance such as face, ear, etc. Plastic surgery is performed by excising the autologous tissue from the above, and supplementing with a plastic artifact. The tissue body of the present invention can also be used in such a case.

  The tissue produced by the method of the present invention can be cut after production. It is also possible to use this tissue body for the replacement of the defective tissue. The tissue is a living tissue and has a function of growing after transplantation. Even if it is largely lost, it can be used for filling, and if it is formed into a tubular shape, it can also be used as an artificial blood vessel.

  Hereinafter, the present invention will be described in more detail.

  In the method for producing a tissue body of the present invention, an artificial body having a pyridine derivative-containing layer provided on at least a part of its surface is implanted into a living body, and a tissue body is formed around the artificial body.

  As this pyridine derivative, nicotine, nicotinic acid, nicotinic acid ester, nicotinic acid amide, nicelyol all, nicochronate, nicofibrate, nicofuranose, nicomol, nicomorphine, nicorandil, nicothelin, nicotinyl alcohol, nifedipine, nifenazone, Preference is given to at least one selected from the group consisting of niflumic acid, nifloquine, nikesamide, niflupynol and nilvadipine and their substitutions.

  Since this pyridine derivative is extremely readily soluble in water, it is formed by crosslinking a layer coated with a solution containing at least a water-soluble polymer and a pyridine derivative, that is, a gel-like product of a water-soluble polymer. It is embedded in the surface of the artificial object.

  Examples of the crosslinking treatment include a method of crosslinking a water-soluble polymer with a chemical crosslinking agent such as glutaraldehyde, ethylenediamine, hexamethylene diisocyanate, and diglycidylaniline, and a method of crosslinking by generating radicals by heat. It is preferable to crosslink by irradiation with light using a water-soluble polymer modified with a molecular group having a group.

  The concentration of the water-soluble polymer in this solution is preferably about 0.1 to 50% by weight, and the concentration of the pyridine derivative is preferably about 0.001 to 10% by weight. It is preferable to dissolve about 0.1 to 20% by weight of an amino compound described later in this solution.

  The molecular groups having this photosensitive group include xanthene dyes, azine dyes, thiazine dyes, oxazine dyes, quinoline dyes, pyrazolone dyes, stilsen dyes, azo dyes, diazo dyes, anthraquinone dyes, indigo dyes. At least one selected from the group consisting of a dye, a thiazole dye, a phenylmethane dye, an acridine dye, a cyanine dye, an indophenol dye, a naphthalamide dye, and a perylene dye is preferable. And eosin, benzophenone, camphorquinone, olefin, benzalacetophenone, cinnamylideneacetyl, cinnamoyl, styrylpyridine, α-phenylmaleimide, phenylazide, sulfonylazide, carbonylazide, o-quinonediazide, furylacryloyl , Coumarin, pyrone, anthracene, benzoyl, stilbene, dithiocarbamate, xanthate, cyclopropene, 1,2,3-thiadiazole, aza-dioxabicyclo, alkyl halides, ketones and diazo, and substances modified with these At least one selected from the group consisting of eosin is preferred.

  Examples of water-soluble polymers include gelatin, collagen, fibronectin, hyaluronic acid, keratanic acid, chondroitin, chondroitin sulfate, elastin, heparan sulfate, laminin, thrombospondin, vitronectin, osteonectin, entactin, gazein, polyethylene glycol, polypropylene glycol, Polyglycidol, side chain esterified product of polyglycidol, polyvinyl alcohol, copolymer of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, copolymer of hydroxyethyl methacrylate and methacrylic acid, alginic acid, polyacrylamide, polydimethylacrylamide and polyvinylpyrrolidone At least one selected from the group consisting of is preferable, and gelatin is particularly preferable.

  Therefore, eosinized gelatin is suitable as the water-soluble polymer modified with the above molecular group.

  In the present invention, the aqueous solution is further selected from the group consisting of thiols, alcohols, reducing sugars, polyphenols, amino groups, N-alkylamino groups, and N, N-dialkylamino groups as proton donors that are radical counters. It is preferable to contain about 5% by weight of an amino compound having at least one group, particularly aminoacrylamide, specifically, polydimethylaminopropylacrylamide. By including this amino compound, etc., it is possible to cut the intermolecular bond and generate a decomposed product. Depending on the conditions within the scope of the present invention, light can be insolubilized with light at a level used in daily life such as a fluorescent lamp.

  Next, eosinized gelatin suitable for use in the present invention will be described.

  Here, the gelatin may be a normal gelatin having a molecular weight of 5,000 to 100,000 and an amino group of about 10 to 100 per molecule.

  Eosinized gelatin is prepared by introducing eosin into the side chain of gelatin according to the following reaction.

  The number of eosin introduced into the gelatin molecule can be calculated, for example, by measuring the absorbance of an aqueous solution of eosinized gelatin at a maximum absorption wavelength of 522 nm of eosin and based on the molar extinction coefficient of eosin (ε = 94755). The number is preferably 1 to 10, particularly 2 to 5 with respect to the molecule. If the number of compounds having a photosensitive group such as eosin is small, the gelation rate is lowered, and if it is more than necessary, the flexibility inherent to gelatin may be impaired, and it becomes hardly soluble in water. End up.

  This eosinized gelatin is a viscous liquid. When this is made into an aqueous solution having a concentration of 1 to 10% by weight, for example, it is irradiated with visible light for about 0.1 to 30 minutes at a relatively low illuminance of about 300 to 30,000 lx, particularly about 300 to 15,000 lx. And can be cured into a gel. In addition, in light irradiation, it is also possible to form a higher-density gel-like hardened layer by irradiating light after drying the layer which applied the viscous liquid to the surface of the artifact beforehand.

  Artificial objects (living material) for forming the pyridine derivative-containing layer on the surface include acrylic resin, olefin resin, styrene resin, polyester resin, polyamide resin, vinyl chloride resin, silicon resin, fluorine resin, epoxy resin. It is preferably an object made of at least one selected from the group consisting of glass, titanium, platinum and SUS (stainless steel). The shape and size of the artificial object may be anything that can be embedded in the living body. If this artificial object is in the form of a thin rod, a tubular tissue body such as a blood vessel can be produced. If the artificial object is plate-like, a plate-like tissue body can be produced. Since the artificial object is to be embedded in a living body, it is preferable that the artificial object is not angular.

  The animal to be implanted with the biological implant may be a self (patient) or another animal, but a self that cannot induce an immune reaction or the like is preferable. However, in an emergency, it is possible to use a product prepared from a heterologous animal as it is (use of an immunosuppressive agent) or a product obtained by decellularization treatment. The decellularized tissue can be expected to become a mature tissue by infiltration, engraftment, and organization of host cells in the host.

  Methods for decellularization include elution of extracellular matrix by enzyme treatment such as collagenase and washing with a water-soluble organic solvent such as alcohol, but aldehyde compounds such as glutaraldehyde and formaldehyde and / or Alternatively, there is a method of treating with a water-soluble organic solvent such as methanol, ethanol or isopropyl alcohol. Specifically, a method in which the aldehyde compound is adjusted so as to have a final concentration of about 1 to 3%, and the tissue body is immersed in a fixative having a volume of about 50 times the volume of the tissue body for 2 hours or more is preferable. As a result, the structure of the tissue can be maintained by crosslinking lysine residues and the like of the protein chain.

  The tissue body after the decellularization treatment can be stably controlled in density and the like by further freeze-drying. It can be stored in water-soluble organic solvents such as alcohol, phosphate buffered saline, and physiological saline without lyophilization after decellularization treatment, but it is also lyophilized to suppress changes in physical properties during storage. It is preferable. Here, as a drying method, pores may be blocked or fiber association may occur in the shrinkage phenomenon during drying, and there is a possibility that a tissue body having useful physical properties with high reproducibility may not be obtained. Drying is preferred.

  Hereinafter, examples and comparative examples will be described. First, Comparative Example 1 will be described.

[Comparative Example 1]
In Comparative Example 1 shown in FIGS. 1 and 2, only a silicone base material having a diameter of 3 mm and a length of 30 mm is placed under the dorsum of the dorsum.

  The epidermis of the back of a rabbit that had been locally anesthetized and shaved by a normal procedure was quickly incised about 30 mm after disinfection with isodine, and a sterilized round bar was implanted under the subcutaneous tissue and sutured. The suture site was disinfected twice a day with isodine, water was freely supplied, and a proper amount of Hay Cube was fed as feed according to body weight.

  During the implantation period, there was no evidence of infection in the sutured area, and no antibiotics were required. Two weeks after implantation, a round bar was extracted by the same procedure as that at the time of implantation. The extracted round bar was uniformly coated with a tissue having a thickness of about 150 microns (see FIG. 1). FIG. 2 is a micrograph of a cross section of the tissue.

  In addition, when it was left for several months, it grew to a thickness of several hundred μm, but it changed little after that.

[Example 1]
In Example 1 shown in FIGS. 3 and 4, the same silicone substrate surface as described above was coated with a solution of 20% eosinized gelatin, 5% polydimethylaminopropylacrylamide and 0.1% nicotine, and visible light was applied for 60 seconds. Irradiated to form a nicotine-containing layer having a thickness of 100 μm. When left subcutaneously for 2 weeks in the same manner as in Comparative Example 1, a bright red and thick tissue was formed. In addition, thick blood vessels were formed on the surface.

  When the cross-sectional tissue was observed, as shown in FIG. 4, a thick tissue including a very large number of capillaries filled with blood cells was formed. The thickness of the tissue was several mm, which was several hundred times that of the comparative example, and had an internal pressure durability of several thousand mmHg or more exceeding that of a biological blood vessel.

[Comparative Example 2]
Comparative Example 2 is described in Example 3 of JP-A No. 2004-261260. In other words, a round bar made of acrylic resin with an outer diameter of 3 mm and a length of 30 mm (the round bar surface was mirror-finished and both ends were hemispherically curved so as not to physically irritate the living tissue more than necessary). A polystyrene derivative having a photopolymerizable initiator in the side chain was applied to the surface, immersed in a methyl methacrylate / benzene solution purified by a conventional method, photo-initiated graft polymerization was performed, and polymethyl methacrylate chain was grafted onto the surface. . The graft ratio was confirmed to be 0.4 in O / C ratio by X-ray photoelectron spectroscopy. Furthermore, vascular endothelial growth factor (0.5 μg / cm ² ) was immobilized on this surface. This round bar was sterilized with ethylene oxide gas by a conventional method and used for embedding. As shown in FIG. 5, the results after 2 weeks showed that the capillary was induced in the thin connective tissue tube formed as compared with Comparative Example 1, but no significant difference was observed with respect to the thickness of the tissue body. It was not possible to guide to the tissue.

2 is a photograph of Comparative Example 1. 2 is a photograph of a cross section of a tissue body of Comparative Example 1. 2 is a photograph of Example 1. 2 is a photograph of a cross section of the tissue body of Example 1. 6 is a photograph of Comparative Example 2.

Claims (16)

  A method of manufacturing a tissue body by implanting an artificial object into a living body and forming a tissue body around the artificial object, the pyridine derivative being contained in at least a part of the surface of the artificial object, A method for producing a tissue body, comprising a pyridine derivative-containing layer to be released.   The pyridine derivative according to claim 1, wherein the pyridine derivative is nicotine, nicotinic acid, nicotinic acid ester, nicotinic acid amide, nicelyl ol, nicochronate, nicofibrate, nicofuranose, nicomol, nicomorphine, nicorandil, nicothelin, nicotinyl alcohol, nifedipine , Nifenazone, niflumic acid, nifloquine, nikesamide, niflupyrinol and nilvadipine, and at least one selected from the group consisting of these substitutes,   3. The structure according to claim 1, wherein the layer containing a pyridine derivative is formed by crosslinking a layer coated with a solution containing at least a water-soluble polymer and a pyridine derivative. Method.   4. The method for producing a tissue body according to claim 3, wherein the water-soluble polymer is modified with a molecular group having a photosensitive group.   5. The molecular group having a photosensitive group according to claim 4, wherein the molecular group having xanthene dye, azine dye, thiazine dye, oxazine dye, quinoline dye, pyrazolone dye, stilsen dye, azo dye, diazo dye, anthraquinone. And at least one selected from the group consisting of a dye, an indigo dye, a thiazole dye, a phenylmethane dye, an acridine dye, a cyanine dye, an indophenol dye, a naphthalamide dye, and a perylene dye A method for producing a featured tissue body.   6. The molecular group having a photosensitive group according to claim 5, wherein eosin, fluorescein, rose bengal, benzophenone, camphorquinone, olefin, benzalacetophenone, cinnamylideneacetyl, cinnamoyl, styrylpyridine, α-phenylmaleimide, phenylazide , Sulfonyl azide, carbonyl azide, o-quinonediazide, furylacryloyl, coumarin, pyrone, anthracene, benzoyl, stilbene, dithiocarbamate, xanthate, cyclopropene, 1,2,3-thiadiazole, aza-dioxabicyclo, alkyl halide, A method for producing a tissue body, which is at least one selected from the group consisting of ketones and diazos and substances modified with these.   The method for manufacturing a tissue body according to any one of claims 3 to 6, wherein the crosslinking treatment is light irradiation.   The method for producing a tissue body according to any one of claims 3 to 7, wherein the solution further contains at least one selected from the group consisting of thiol, alcohol, reducing sugar, and polyphenol.   9. The amino acid according to claim 3, wherein the solution further comprises at least one group selected from the group consisting of an amino group, an N-alkylamino group, and an N, N-dialkylamino group. The manufacturing method of the structure | tissue characterized by including a compound.   The water-soluble polymer according to any one of claims 1 to 9, wherein the water-soluble polymer is gelatin, collagen, fibronectin, hyaluronic acid, keratanic acid, chondroitin, chondroitin sulfate, elastin, heparan sulfate, laminin, thrombospondin, vitronectin, osteonectin , Enteractin, casein, polyethylene glycol, polypropylene glycol, polyglycidol, polyglycidol side chain esterified product, polyvinyl alcohol, copolymer of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, copolymer of hydroxyethyl methacrylate and methacrylic acid, It is at least one selected from the group consisting of alginic acid, polyacrylamide, polydimethylacrylamide and polyvinylpyrrolidone. Method for producing a tissue construct and symptoms.   The artificial object according to any one of claims 1 to 10, wherein the artifact is an acrylic resin, an olefin resin, a styrene resin, a polyester resin, a polyamide resin, a vinyl chloride resin, a silicon resin, a fluorine resin, an epoxy resin, glass, titanium, platinum, and the like. A method for producing a tissue body, which is an object made of at least one selected from the group consisting of stainless steel.   A tissue body produced by the method according to any one of claims 1 to 11.   The tissue body according to claim 12, wherein the tissue body is further decellularized.   14. The tissue body according to claim 12 or 13, wherein the tissue body is further freeze-dried.   The substitute material of the biological tissue which consists of a structure | tissue of any one of Claim 12 thru | or 14.   A biological implant for producing a tissue body, wherein a pyridine derivative-containing layer that contains a pyridine derivative and releases the pyridine derivative is provided on at least a part of the surface of the artificial product.

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