JP2008126005A - Safe preparation of cell tissue-hydroxyapatite complex - Google Patents

Abstract

Hydroxyapatite, which approximates the composition and crystal structure of bone constituents and is expected to have excellent biocompatibility to cells or cell aggregates, is efficiently and firmly produced at a very high production rate and is strongly Provided is a method for producing a hydroxyapatite composite that can be attached to a living tissue, a cell obtained by the method, or a similar living tissue.
A cell or a cell population includes a calcium solution containing calcium ions and substantially free of phosphate ions, and a phosphate solution containing phosphate ions and substantially free of calcium ions. A method for producing a hydroxyapatite complex, comprising the step of alternately dipping and generating and fixing hydroxyapatite on at least the surface of the cell or cell population.
[Selection figure] None

Description

The present invention is a method for producing a cell / hydroxyapatite complex that can be used for various biological tissues such as artificial bones, medical materials, and the like, using cells that are bone constituents, and obtained by the production method. The present invention relates to a cell / hydroxyapatite composite and a biocompatible material.

  In recent years, regenerative treatment using genetic engineering, tissue tissue engineering, regenerative medicine, etc. has attracted attention as a new treatment for severe organ failure and intractable diseases, and the most important and most difficult research theme of advanced medicine in the 21st century As one of them, many researchers worldwide are working vigorously.

  Estimated regenerative (tissue engineering) medical-related market is around 48 trillion yen in the world according to the materials of the New Energy and Industrial Technology Development Organization, about 5 trillion yen in Japan. The world's expectations are high as the next-generation new industry in this field.

  The present inventors have also worked on the development of a regenerative treatment in the musculoskeletal tissue and circulatory tissue regions, and have reported a combined method of cell transplantation and growth factors, and a tissue transplant regeneration method by tissue engineering. However, in order to perform regenerative medicine by such cell transplantation or tissue transplantation, it is urgently and most important to secure an autologous cell source. Many cells of musculoskeletal tissues have high self-repairing ability, and the presence of cells having functions as stem cells has been reported.

Living bones, teeth, and the like are matrices in which hydroxyapatite (hereinafter sometimes abbreviated as hydroxyapatite), which is an inorganic substance, and collagen, which is a protein, are complexed and arranged three-dimensionally. It is known that a biocompatible ceramic material or the like can be used for repair when a bone or tooth is damaged. For example, a product name “Bioglass” (manufactured by Nippon Electric Glass Co. Ltd., Otsu. Siga. Japan, component; Na ₂ O—CaO—SiO ₂ —P ₂ O ₅ ), which is mainly used as a periodontal filler. Apatite and wollastonite (used as hydroxyapatite sintered body (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), artificial pancreas, iliac spacer, etc., mainly used as a bone filler) CaO-SiO ₂₎ and crystallized glass containing (trade name "Cerabone a-W", NipponElectric glass Co. Ltd., Otsu. Siga.Japan , Ltd.) and the like are known. In order to use these ceramic materials as substitutes for bone, attempts have been made to form them on the surface of a material having high strength such as metal. In addition, a method of forming a hydroxyapatite layer on the surface of various organic polymer materials that are highly flexible and durable and expected to be applied to artificial biological tissues other than bone, so-called biomimetic reaction A method called is being developed. In this biomimetic reaction, glass particles mainly composed of CaO and SiO ₂ are immersed in an aqueous solution (pseudo body fluid) having an ionic concentration equal to that of human body fluid, and then an organic polymer material is immersed in the organic polymer material. After a large number of apatite nuclei are formed on the surface of this, only this organic polymer material is immersed and reacted in an aqueous solution having an ion concentration 1.5 times that of the simulated body fluid. According to this biomimetic reaction, it has been reported that an apatite nucleus grows naturally on an organic polymer material, and a dense and homogeneous bone-like hydroxyapatite layer is formed in an arbitrary thickness (non-patent document). Literature 1 = J. Biomed. Mater. Res. Vol. 29, p349-357 (1995)). However, this biomimetic reaction has a slow hydroxyapatite production rate, and even if it is reacted for a long period of 2 weeks or longer, it cannot produce hydroxyapatite to the extent that it can be used for an artificial bone on an organic polymer material. This is the actual situation.

  In addition, as a new method for synthesizing hydroxyapatite, there is an alternating dipping method in which a material is immersed in a calcium solution and then immersed in a phosphoric acid solution, and this process is made one cycle, and this process is repeated to sequentially form a hydroxyapatite layer. Non-patent document 2 = Chem. Lett., Pp. 711-712, 1998; Non-patent document 3 = J Biomed Mater Res Part B: Appl. Biometer 66B: 429-438, 2003; = J Biomed Mater Res Part B: Appl Biometer 75B: 378-386, 2005).

Main constituent layers such as bones and teeth in the living tissue are composed of an inorganic solid substance approximated to hydroxyapatite (hereinafter sometimes abbreviated as hydroxyapatite). It is known that a biocompatible ceramic material or the like can be used for repair when a bone or tooth is damaged. For example, the product name “Bioglass” (manufactured by Nippon Electric Glass Co. Ltd., Otsu. Siga. Japan, component; Na ₂ O—CaO—SiO ₂ —P ₂ O ₅ ), which is mainly used as a periodontal filler. Apatite and wollastonite (used as hydroxyapatite sintered body (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), artificial pancreas, iliac spacer, etc., mainly used as a bone filler) CaO-SiO ₂₎ and crystallized glass containing (trade name "Cerabone a-W", Nippon Electric glass Co.Ltd., Otsu.Siga.Japan , Ltd.) and the like are known.

In order to use these ceramic materials as substitutes for bone, attempts have been made to form them on the surface of a material having high strength such as metal. In addition, a method of forming a hydroxyapatite layer on the surface of various organic polymer materials that are flexible and durable, and that can be easily processed, with the expectation of application to artificial biological tissues other than bone, so-called biomimetic. A method called reaction has been developed.
In this biomimetic reaction, glass particles mainly composed of CaO and SiO ₂ are immersed in an aqueous solution (pseudo body fluid) having an ionic concentration equal to that of human body fluid, and then an organic polymer material is immersed in the organic polymer material. After a large number of apatite nuclei are formed on the surface of this, only this organic polymer material is immersed and reacted in an aqueous solution having an ion concentration 1.5 times that of the simulated body fluid. According to this biomimetic reaction, it has been reported that apatite nuclei grow naturally on organic polymer materials, and a dense and homogeneous bone-like hydroxyapatite layer is formed to an arbitrary thickness (non-patent document). Literature 5 = J. Biomed. Mater. Res. Vol. 29, p349-357 (1995)).
However, this biomimetic reaction has a slow hydroxyapatite production rate, and even if it is reacted for a long period of 2 weeks or longer, it cannot produce hydroxyapatite to the extent that it can be used for an artificial bone on an organic polymer material. This is the actual situation.
By the way, for example, the above-mentioned ceramic material is required to have a biocompatibility that can be combined with living bones, and researches centering on composition and form are being conducted. Recently, it has been found that improvement in biocompatibility is important not only in the composition and form of the ceramic material but also in its crystal structure. For example, the crystal structure of human bone is known to show a specific diffraction peak by X-ray diffraction (Non-patent Document 6 = Biomaterials, 11, p568-572, 1990).
However, hydroxyapatite having a crystal structure close to this has not been known. For example, hydroxyapatite obtained by the above-described biomimetic reaction, which is reported to form a bone-like hydroxyapatite layer, does not have such a crystal structure.

  Patent Document 1 (Japanese Patent Laid-Open No. 2002-219167) discloses a method of preparing a polymer-apatite complex by mixing a naturally-derived polymer with a solution of calcium and phosphoric acid. However, the polymer used here is separated from the biomaterial and does not use the cell itself.

Patent Document 2 (Japanese Patent Laid-Open No. 2000-327314) describes the production of a hydroxyapatite composite by an alternating dipping method for polymers. However, this document does not disclose biomaterials such as cells themselves.
J. et al. Biomed. Mater. Res. vol. 29, p349-357 (1995) Chem. Lett. , Pp. 711-712, 1998 J Biomed Mater Res Part B: Appl. Biometer 66B: 429-438, 2003; J Biomed Mater Res Part B: Appl Biometer 75B: 378-386, 2005 J. et al. Biomed. Mater. Res. vol. 29, p349-357, 1995 Biomaterials, 11, p568-572, 1990 JP 2002-219167 A JP 2000-327314 A

  The object of the present invention is to efficiently produce hydroxyapatite, which is close to the composition and crystal structure of bone constituents and is expected to have excellent biocompatibility, in cells or cell aggregates such as polymer materials, at an extremely high production rate. And a method for producing a hydroxyapatite complex that can be firmly attached to a cell or a similar biological tissue, to a cell or a similar biological tissue obtained by the method, and to provide a cell or a similar biological tissue .

  The present invention has solved the above problems by successfully complexing with hydroxyapatite without killing a cell such as a mesenchymal stem cell or a tissue composed of the cell. In particular, in the present invention, in order to solve the above-mentioned problems, the point devised as compared with the conventional complex is that the complex with hydroxyapatite is achieved while the cells are alive. There is no example in which living cells and their tissues were combined with hydroxyapatite by alternate immersion.

  In particular, there has never been a case where a three-dimensional tissue in which mesenchymal stem cells are uniformly distributed in an undifferentiated state is combined with hydroxyapatite. In the present invention, it is possible to safely prepare in vitro a complex of hydroxyapatite with a cell such as a mesenchymal stem cell required for bone / cartilage regeneration therapy or a tissue composed of the cell, A therapeutic effect is expected. The economic effect of the product is also expected to be large.

  Cells such as mesenchymal stem cells or their cell-derived self-assembled three-dimensional artificial tissues can be combined with hydroxyapatite, and the cells remain alive after the combination, making them extremely safe. It is the greatest feature of the present invention that it is a technique. Moreover, since the amount of hydroxyapatite can be easily controlled by changing the alternate dipping conditions, the degree of compounding can be controlled according to the place and conditions of use. Further, since hydroxyapatite formed by the present invention is low crystalline and bioabsorbable, it is expected to induce bone formation and be decomposed and absorbed after implantation in the living body. The present invention is an epoch-making technique capable of safely complexing cell tissues with hydroxyapatite.

  In one embodiment, a mesenchymal stem cell-derived self-organized three-dimensional artificial tissue prepared by adding L-ascorbic acid 2-phosphate and culturing is prepared using calcium chloride / Tris buffer (pH = 7.4). ), For example, for 10 seconds, and excess calcium ions are removed by immersing in, for example, 10 seconds in a Tris buffer (pH = 7.4). After that, it is immersed in a disodium hydrogen phosphate / Tris buffer (pH = 7.4) for 10 seconds, for example, and is then immersed in a Tris buffer (pH = 7.4) for 10 seconds to remove excess phosphate ions. .

  In one embodiment, by repeating this cycle, hydroxyapatite is formed on the surface of the mesenchymal stem cell-derived self-organized three-dimensional artificial tissue. The amount of hydroxyapatite formed can be controlled by the number of cycles and the solution concentration.

Accordingly, the present invention provides the following.
(1) A complex of cells and hydroxyapatite.
(2) The composite according to Item 1, wherein the hydroxyapatite is attached to the surface of the cell.
(3) The composite according to item 1, wherein the hydroxyapatite is directly bonded to the surface of the cell.
(4) The complex according to item 1, wherein the hydroxyapatite is present in a state where microcrystals are formed on the cell membrane surface of the cell.
(5) The complex according to item 1, wherein the cell has a three-dimensional tissue structure.
(6) The complex according to item 1, wherein the cell is biologically integrated in the third dimension.
(7) The complex according to Item 1, wherein the cell has an integration capability with surroundings.
(8) The complex according to item 4, wherein the biological binding ability includes the ability to adhere to surrounding cells and / or extracellular matrix.
(9) The complex according to item 1, comprising an extracellular matrix derived from the cell.
(10) The complex according to item 9, wherein the extracellular matrix includes at least one selected from the group consisting of collagen I, collagen III, vitronectin and fibronectin.
(11) The complex according to item 10, comprising 10 to 90% of collagen I as collagen.
(12) The complex according to item 10, comprising 10 to 90% of collagen II as collagen.
(13) The complex according to item 10, comprising 10 to 90% of collagen III as collagen.
(14) The complex according to item 1, comprising a proteoglycan derived from the cell.
(15) The complex according to item 14, wherein the proteoglycan contains at least 0.1 to 90% of hyaluronic acid.
(16) The complex according to item 14, wherein the proteoglycan contains at least 0.1 to 90% of fibronectin.
(17) The complex according to item 10, wherein the extracellular matrix is disposed between the cell and the hydroxyapatite.
(18) The complex according to item 10, wherein the extracellular matrix is dispersed in the complex.
(19) The complex according to item 10, wherein the extracellular matrix is uniformly dispersed in the complex.
(20) The composite according to item 1, wherein the hydroxyapatite is present as a crystal.
(21) The composite according to item 20, wherein the crystal has a form selected from a hexagonal crystal, a plate shape, and a rod shape.
(22) The complex according to item 1, wherein the cell is a stem cell.
(23) The complex according to item 1, wherein the cell is a tissue stem cell.
(24) The complex according to item 1, wherein the cell is a cell expressing collagen.
(25) The complex according to item 1, wherein the cell is a cell that expresses type I collagen and type III collagen.
(26) The complex according to item 1, wherein the cell is a mesenchymal stem cell.
(27) The complex according to item 1, wherein the cell is a living cell.
(28) The complex according to item 1, wherein the cell retains at least 50% of the amount of DNA compared to the amount of DNA before complex formation or is detected by a live cell detection method.
(29) The complex according to item 28, wherein the live cell detection method is an MTT assay or a WST-1 assay.
(30) A cell or a cell population is divided into a calcium solution containing calcium ions and substantially free of phosphate ions, and a phosphate solution containing phosphate ions and substantially free of calcium ions. A method for producing a hydroxyapatite complex, comprising a step of alternately dipping to produce and fix hydroxyapatite on at least the surface of the cell or cell population.
(31) The production method according to item 30, wherein the cell or cell population is an artificial tissue.
(32) A production method according to item 30, wherein the cell or cell population contains stem cells.
(33) The production method according to item 30, wherein the concentration substantially free of calcium ions is a concentration at which formation of hydroxyapatite does not proceed.
(34) The manufacturing method according to Item 30, wherein the concentration substantially free of phosphate ions is a concentration at which formation of hydroxyapatite does not proceed.
(35) After the step of generating and fixing the hydroxyapatite, the method includes confirming the degree of hydroxyapatite deposition on the cell surface in the complex, and repeating the step of generating and fixing based on the result. Item 30. The method according to Item 30.
(36) The production method according to item 30, wherein the ion concentration is 10 to 200 mM.
(37) The manufacturing method of the item 30 whose density | concentration of the said ion is 10-50 mM.
(38) The manufacturing method according to Item 30, wherein the immersion time is 1 minute or less.
(39) A hydroxyapatite composite obtained by the production method according to item 30.
(40) A biocompatible material substantially consisting of the composite according to item 1 or 39.

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  Conventional cell transplantation and commercially available apatite have insufficient bone and cartilage induction, and crystalline apatite is not decomposed in the living body and remains for a long time. In the present invention, not only can cell transplantation and bone induction by hydroxyapatite be achieved simultaneously, but also hydroxyapatite is bioabsorbable, and thus has a great feature that it is rapidly decomposed and absorbed after bone induction. In addition, since organic substances are only cells and extracellular matrix components produced by the cells, they are extremely excellent in safety.

  The fact that hydroxyapatite microcrystals can be formed on the cell membrane surface in a state where the cells are alive is also one of the features achieved for the first time in the present invention, and the prior art forms hydroxyapatite microcrystals on the cell membrane surface. I couldn't. When cells adhere to a substance, they do not adhere to the entire cell membrane, but adhere to the substance at a point via a membrane protein such as an integrin. Therefore, when cells adhere to a substance, the cell membrane does not come into contact with the substance. Therefore, it can be said that the state in which cells are adhered to the hydroxyapatite surface and the state in which hydroxyapatite is formed on the cell membrane surface are completely different, and this can be observed objectively. The technique of the present invention can be said to be the first example in which hydroxyapatite microcrystals are directly formed in a state where cells are alive on the cell membrane surface.

  The conventional cell / hydroxyapatite complex is not a complex in the true sense, but rather is in a state to be called a mixture, and has a difficulty in bone / cartilage induction. For example, although it has been reported that bone is induced by the effect of hydroxyapatite, there are few that can obtain an osteoinductive effect that is actually used in treatment. In the treatment, it is important to induce both bone and cartilage as found in cancellous bone, but there is no report on a material that exhibits such an effect.

  The difference between cancellous bone and cortical bone is that when the bone is classified according to its properties and functions, it is composed of a portion with a hollow structure like cancellous (cancellous bone) and a hard dense portion (cortical bone) surrounding it. . The cancellous bone refers to the cancellous bone contained in the spinal vertebral body, ribs, near the ends of the long bones, and the like. The function of cancellous bone is mainly called metabolic bone because it contributes to bone metabolism. The cancellous bone has a metabolic rate 5 to 8 times faster than the cortical bone, and nearly 20% of the bone is replaced with new bone in one year. Cortical bone refers to bone that thinly covers cancellous bone, or bone that is thick bamboo tube like the central part of long bone. Cortical bone serves to maintain mechanical strength. New bone replacement is characterized by a slow metabolic rate of 4% per year. When the present invention was used, it was found that a bone having a Shanghai cottonbone-like morphology was formed in the defect part in CT and tissue by a transplantation experiment of HA-3DBT.

  From the above, a great contribution to the industry is expected as a new regenerative medical material for bone and cartilage.

  1) A mesenchymal stem cell-derived self-organized three-dimensional artificial tissue hydroxyapatite complex could be prepared by the alternate immersion method.

  2) Formation of hydroxyapatite was confirmed by X-ray diffraction measurement.

  3) Formation of hydroxyapatite on the extracellular matrix and the cell surface was confirmed by electron microscope observation.

  4) The survival of the cells after conjugation was confirmed by WST-1 staining.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the above field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

(Regenerative medicine)
As used herein, “regeneration” refers to a phenomenon in which a remaining tissue grows and is restored when a part of the tissue of an individual is lost. Depending on the tissue species between animal species or in the same individual, the degree and mode of regeneration varies. Many human tissues have limited regenerative ability, and complete regeneration cannot be expected if they are largely lost. In a large injury, a tissue with strong proliferative power different from the lost tissue proliferates, and incomplete regeneration may occur in which the tissue is regenerated incompletely and the function cannot be recovered. In this case, a structure made of a bioabsorbable material is used to prevent the invasion of a tissue having a strong proliferation ability into the tissue defect portion, thereby securing a space in which the original tissue can grow, and further, a cell growth factor. Regenerative medicine is performed to replenish the natural tissue and enhance the regenerative capacity of the original tissue. Examples of this include regenerative medicine of cartilage, bone, heart and peripheral nerves. It has previously been thought that cartilage, nerve cells and myocardium are incapable or remarkably incapable of regeneration. In recent years, the existence of tissue stem cells (somatic stem cells) having the ability to differentiate into these tissues and the ability to self-proliferate has been reported, and expectations for regenerative medicine using tissue stem cells are increasing. Embryonic stem cells (ES cells) are cells that have the ability to differentiate into all tissues. Regeneration of complex organs such as kidneys and livers using these cells has been attempted but has not been realized.

A “cell” as used herein is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism, surrounded by a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention. The number of “cells” used in the present invention can be counted through an optical microscope. When counting through an optical microscope, counting is performed by counting the number of nuclei. The tissue is used as a tissue slice, and hematoxylin-eosin (HE) staining is performed, whereby the extracellular matrix (for example, elastin or collagen) and the nucleus derived from the cells are dyed with a dye. This tissue section can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 μm × 200 μm) can be estimated and counted. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). The source of cells can be, for example, a single cell culture, or such as cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line Examples include but are not limited to cell mixtures. As the cells, for example, primary cultured cells can be used, but subcultured cells can also be used. Preferably, cells that have passed through passage 3 to 8 are used. In the present specification, the cell density can be represented by the number of cells per unit area (for example, cm ² ).

  In this specification, the state in which a cell is “alive” can be evaluated by, for example, the amount of DNA. For example, a cell is evaluated to be “alive” if it retains at least 50% of the amount of DNA compared to the amount of DNA prior to complex formation of the present invention. Alternatively, for example, when the amount of DNA is maintained at least 50% compared to the amount of DNA before complex formation of the present invention, or when detected by a live cell detection method such as MTT assay or WST-1 assay, the cell Evaluates to be “live”.

Here, in the MTT assay, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazole bromide (MTT) is reduced by intracellular dehydrogenase produced by living cells to produce formazan. Thus, formazan can be measured by measuring the absorbance at 570 nm. The WST-1 assay is an improved version of the MTT assay,
2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt (WST-1) is 1-methyl-5-methylphenylsulphate When PMS) is used as an electron carrier, it is reduced by intracellular dehydrogenase to produce water-soluble formazan, and the absorbance at 450 nm of formazan can be measured.

These assays can be performed, for example, using a TaKaRa kit (Premix WST-1 25 ml). “Premix WST-1” is a ready-to-use Premix solution containing sterile WST-1 diluted with a phosphate buffer and an electron binding reagent. Regardless of the cell concentration, 10 μl of Premix WST-1 may be added in the case of 100 μl / well, and 20 μl of Premix WST-1 may be added in the case of 200 μl / well (10: 1). When cells are cultured at 100 μl / well, this product corresponds to 2500 times (25 microtiter plates) per 1 bottle. This reagent is a reagent for quantifying cell proliferation ability and cell viability by color measurement. It is based on the conversion of tetrazolium salt (WST-1) to formazan dye by mitochondrial dehydrogenase in living cells, and can be measured by non-RI instead of [ ³ H] -thymidine incorporation measurement. The tetrazolium salt added to the medium is present in the mitochondrial respiratory chain and converted into a formazan dye by “succinate-tetrazolium reductase” (EC 1.3.99.1), which is active only in living cells. Converted. As the number of viable cells increases, the overall activity of mitochondrial dehydrogenase in the sample increases, and this increase in enzyme activity leads to increased production of formazan dye, so that formazan dye and metabolic activity in the medium are increased. It shows a linear correlation with the number of cells with a gap.
This measurement method does not require washing or uptake of cells, and has the advantage that the same microtiter plate can be used from the start of microculture to data analysis using an ELISA reader.
In addition, in cell proliferation and drug sensitivity measurement methods, cell proliferation and viability can be quantified with a spectrophotometer using non-RI.

Different tetrazolium salts such as MTT, XTT, and MTS are used to measure cell growth and viability. Tetrazolium salts are present in the mitochondrial respiratory chain and are broken down into formazan dyes by “succinate-tetrazolium reductase” (EC 1.3.99.1), which is active only in living cells. ⁹⁾ Increasing the number of viable cells will increase the overall activity of mitochondrial dehydrogenase in the sample. Since this increase in enzyme activity leads to an increase in the production of formazan dye, there is a linear correlation between formazan dye and the number of metabolically active cells in the medium. The formazan dye produced by metabolically active cells can be quantified by measuring the absorbance of the dye solution with an ELISA reader. Thereby, it is possible to see cell proliferation ability and cell viability.

  A formazan dye is formed by the above reductase (Chemical Formula 1). By quantifying this formazan dye, living cells (= cells having metabolic activity) can be measured. There is a linear relationship between the absorbance of the formazan dye solution and the number of viable cells.

The protocol for WST-1 is as follows.
A. Necessary equipment and incubator (37 ℃)
・ Centrifuge ・ Microtiter plate (ELISA) reader with filter corresponding to wavelength of 420-480 nm (filter with control wavelength of 600 nm or more is desirable)
・ Microscope ・ Hematocytometer ・ Multi-channel pipetter (10, 50, 100 μl)
・ Sterilized pipette tips ・ Microtiter plate (tissue culture grade, 96 holes, flat bottom)

B. Operating procedure Cultivate cells in a microtiter plate under humidification and normal pressure (eg, 37 ° C., 5% CO ₂ ) so that the final volume of the medium is 100 μl / well ^* .
* Culture time and cell concentration depend on the individual experimental conditions and the cell line used. In most experiments, a cell concentration of 0.1 to 5 × 10 ⁴ cells / well and a culture time of 24 to 96 hours are appropriate.
2. After completion of cell culture, 10 μl of Premix WST-1 is added per well.
3. Incubate for 0.5 to 4 hours under humidification and normal pressure (for example, 37 ° C., 5% CO ₂ ).
4). Measure the absorbance of the sample against a background control (see “C”) using a microtiter plate (ELISA) reader. The wavelength for measuring the absorbance of the formazan product is between 420-480 nm, depending on the available filters of the ELISA reader (maximum absorbance is about 440 nm). The reference wavelength is preferably 600 nm or more.
C. Background control (blank)
Add 100 μl of the medium used in “B-1” and 10 μl of Premix WST-1 to one well. This is used as a background control (cell-free medium + Premix WST-1 absorbance) at the blank position of the ELISA reader. Even when Premix WST-1 is added to cell-free medium, there is a slight spontaneous absorbance. The absorbance of this background depends on the medium, incubation time, and exposure to light. Typical background absorbance after 2 hours is between 0.1 and 0.2.

In the WST-1 assay, note the following points.
-The optimal incubation time after the addition of Premix WST-1 varies depending on individual experiments (for example, the type and concentration of cells used). Therefore, in the experiment, the optimum incubation time is determined by repeatedly measuring the absorbance at different times (for example, 0.5, 1, 2, 4 hours) after adding Premix WST-1.
• If high sensitivity is desired, cells should be incubated for a longer time in the presence of Premix WST-1.

  As used herein, “stem cell” refers to a cell having a self-renewal ability and having pluripotency (ie, “pluripotency”). Stem cells are usually able to regenerate the tissue when the tissue is damaged. As used herein, stem cells can be embryonic stem (ES) cells or tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), but are not limited thereto. In addition, as long as it has the above-mentioned ability, an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.) can also be a stem cell. Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981, and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Unlike embryonic stem cells, tissue stem cells are cells in which the direction of differentiation is limited, are present at specific positions in the tissue, and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. As used herein, stem cells may preferably be embryonic stem cells, although tissue stem cells can also be used depending on the circumstances.

  When classified according to the site of origin, tissue stem cells are classified into, for example, the skin system, digestive system, myeloid system, and nervous system. Examples of skin tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive tissue stem cells include pancreatic (common) stem cells and liver stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells, mesenchymal stem cells (for example, fat-derived, cartilage-derived, bone-derived, bone marrow-derived). Examples of nervous system tissue stem cells include neural stem cells and retinal stem cells.

  In the present specification, “somatic cells” refers to all cells that are cells other than germ cells such as eggs and sperm, and that do not deliver the DNA directly to the next generation. Somatic cells usually have limited or disappeared pluripotency. The somatic cells used in the present specification may be naturally occurring or genetically modified.

  Cells can be classified into stem cells derived from ectoderm, mesoderm and endoderm by origin. Cells derived from ectoderm are mainly present in the brain and include neural stem cells. Cells derived from mesoderm are mainly present in bone marrow, and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like. Endoderm-derived cells are mainly present in organs, and include liver stem cells, pancreatic stem cells, and the like. As used herein, somatic cells may be derived from any mesenchyme. Preferably, a mesenchymal cell can be used as the somatic cell.

  As the cells constituting the artificial tissue, complex, and three-dimensional structure provided in the present invention, for example, differentiated cells or stem cells derived from the above-described ectoderm, mesoderm, and endoderm can be used. Examples of such cells include mesenchymal cells. In certain embodiments, such cells can be adipocytes, fibroblasts, synoviocytes, and the like. As such cells, differentiated cells can be used as they are, or stem cells can be used as they are, but cells differentiated from stem cells in a desired direction can be used.

  As used herein, “mesenchymal stem cell” refers to a stem cell found in mesenchyme. In this specification, it may be abbreviated as MSC. Here, the mesenchyme is formed by a population of free cells with stellate or irregular projections that fill the gaps between epithelial tissues, which are observed at each stage of development of multicellular animals, and the accompanying cytoplasm An organization. Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, muscle cells, stromal cells, tendon cells, and adipocytes. Mesenchymal stem cells are used to culture or proliferate bone marrow cells collected from patients, differentiate them into chondrocytes or osteoblasts, or as reconstruction materials for bone, cartilage, joints, etc. of alveolar bone, arthropathy, etc. The demand is high. Therefore, the artificial tissue, complex or three-dimensional structure containing the mesenchymal stem cell or differentiated mesenchymal stem cell of the present invention is particularly useful when a structure is required in these applications.

  As used herein, “isolated” means that the substances naturally associated with it in a normal environment are at least reduced, preferably substantially free. Thus, an isolated cell, tissue, etc. refers to a cell that is substantially free of other materials (eg, other cells, proteins, nucleic acids, etc.) that accompany it in its natural environment. When referring to a tissue, an isolated tissue is a substance other than the tissue (for example, in the case of an artificial tissue or complex, the material, scaffold, sheet, coating, etc. used in producing the artificial tissue). Refers to an organization that is substantially free of. In the present specification, isolated preferably means that the scaffold is not contained (that is, scaffold free). Accordingly, it is understood that in an isolated state, components used in producing the artificial tissue or complex provided in the present invention such as a medium may be contained. When referring to a nucleic acid or polypeptide, “isolated” means, for example, substantially free of cellular material or culture medium when made by recombinant DNA technology, and precursor when chemically synthesized. Refers to a nucleic acid or polypeptide that is substantially free of somatic or other chemicals. An isolated nucleic acid preferably includes sequences that are naturally flanking in the organism from which the nucleic acid is derived (ie, sequences located at the 5 'and 3' ends of the nucleic acid). Absent.

  In this specification, “scaffold-free (scaffold-free, no base material; scaffold-free)” is substantially free of materials conventionally used when producing artificial tissue (base material = scaffold). That means. Examples of such scaffold materials include, but are not limited to, chemical polymer compounds, ceramics, or biologics such as polysaccharides, collagen, gelatin, and hyaluronic acid. A scaffold refers to a material that is substantially solid and contains strength that can support cells or tissues.

  As used herein, “established” or “established” cells maintain certain properties (eg, pluripotency) and the cells continue to grow stably under culture conditions. State. Thus, established stem cells maintain pluripotency.

  As used herein, “non-embryonic” means not derived directly from an early embryo. Therefore, cells derived from body parts other than early embryos fall under this category, but cells obtained by modifying embryonic stem cells (eg, genetic modification, fusion, etc.) are also within the scope of non-embryonic cells. It is in.

  As used herein, “differentiated cells” refers to cells with specialized functions and morphology (eg, muscle cells, nerve cells, etc.), and unlike stem cells, there is no or almost no pluripotency. . Examples of differentiated cells include epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells, Examples include smooth muscle cells, fat cells, bone cells, chondrocytes.

  As used herein, “tissue” refers to a cell population having the same function and form in a cell organism. In multicellular organisms, the cells that make up it usually differentiate, function becomes specialized, and division of labor occurs. Therefore, it cannot be a mere assembly of cells, and an organization as an organic cell group or social cell group having a certain function and structure is formed. Examples of tissues include, but are not limited to, integument tissue, connective tissue, muscle tissue, nerve tissue, and the like. The tissue of the present invention may be any organ or tissue derived from an organism. In a preferred embodiment of the present invention, the tissue targeted by the present invention includes bone, cartilage, tendon, ligament, meniscus, intervertebral disc, periosteum, blood vessel, blood vessel-like tissue, heart, heart valve, pericardium, dura mater, etc. Examples include but are not limited to organizations.

  As used herein, “artificial tissue” refers to a tissue different from the natural state. In this specification, the artificial tissue is typically prepared by cell culture. A tissue that has been extracted from a living organism as it is is not called an artificial tissue in this specification. Therefore, the artificial tissue can include a substance derived from a living body and a substance not derived from a living body. The artificial tissue provided in the present invention is usually composed of cells and / or biological materials, but may contain other materials. More preferably, the artificial tissue provided in the present invention is substantially composed only of cells and / or biological materials. Such a biological substance is preferably a substance derived from cells constituting the tissue (for example, an extracellular matrix).

  As used herein, the term “transplantable artificial tissue” can be transplanted in actual clinical practice among artificial tissues, and can also serve as a tissue at a site transplanted at least for a certain period after transplantation. An artificial tissue. An implantable artificial tissue usually has sufficient biocompatibility, sufficient biofixation, and the like.

  The sufficient strength of the implantable artificial tissue varies depending on the portion intended for transplantation, but those skilled in the art can appropriately set the strength. This strength is sufficient for self-supporting and can be set according to the environment to be transplanted. Such strength can be measured by measuring the stress and strain characteristics described below, or by performing a creep characteristic indentation test. Strength can also be assessed by observing the maximum load.

  The sufficient size of the artificial tissue that can be transplanted in the present invention varies depending on the portion intended for transplantation, but those skilled in the art can appropriately set the size. This size can be set according to the environment to be transplanted.

However, when implanted, it is preferable to have at least a certain size, and such a size is usually at least 1 cm ² in terms of area, preferably at least 2 cm ² , more preferably at least 3 cm ² . is there. More preferably at least 4 cm ² , at least 5 cm ² , at least 6 cm ² , at least 7 cm ² , at least 8 cm ² , at least 9 cm ² , at least 10 cm ² , at least 15 cm ² Alternatively, it can be at least 20 cm ² , but is not limited thereto, and the area can be 1 cm ² or less or 20 cm ² or more, depending on the application. It is understood that the essence of the present invention is that any size (area, volume) can be produced, and the size is not limited.

  Sufficient biocompatibility in an implantable artificial tissue varies depending on the portion intended for transplantation, but those skilled in the art can appropriately set the degree of biocompatibility. Usually, the desired biocompatibility includes, but is not limited to, performing biological binding with surrounding tissues without causing inflammation or the like and without causing an immune reaction. In some cases, for example, in the cornea and the like, it is difficult to cause an immune reaction. Therefore, even if there is a possibility of causing an immune reaction in other organs, it can be considered biocompatible for the purpose of the present invention. Examples of the biocompatibility parameter include, but are not limited to, the presence / absence of an extracellular matrix, the presence / absence of an immune response, and the degree of inflammation. Such biocompatibility can be determined by checking the compatibility at the transplant site after transplantation (for example, confirming that the transplanted artificial tissue has not been destroyed) (human transplant organ rejection) Histopathological diagnosis criteria for differential diagnosis and biopsy specimen handling (diagrams) Kidney transplantation, liver transplantation and heart transplantation. See Japanese Society of Transplantation, Japanese Pathology Society, Kanehara Publishing Co., Ltd. (1998)). According to this document, Grade 0, 1A, 1B, 2, 3A, 3B, and 4 are classified into Grade 0 (no accurate rejection), and biopsy specimens have no evidence of acute rejection, cardiomyocyte damage, or the like. State. Grade 1A (focal, mild accurate rejection) is a state in which large lymphocytes infiltrate locally, around blood vessels or in the stroma, but without cardiomyocyte injury. This finding is observed in one or more biopsy specimens. Grade 1B (diffuse, mild accurate rejection) is a state in which large lymphocytes are more diffusely infiltrated around the blood vessel, stroma or both, but there is no cardiomyocyte injury. Grade 2 (focal, moderate rate rejection) is a state in which an inflammatory cell infiltrate clearly demarcated from the surrounding area is found in only one place. Inflammatory cells consist of large, activated lymphocytes that may be eosinophils. Cardiomyocyte injury with alteration of myocardial architecture is observed in the lesion. Grade 3A (multifocal, moderate accurate rejection) is a state in which inflammatory cell infiltrates composed of large activated lymphocytes are formed in a multiplicity, and may also cause eosinophils. Two or more of these multiple inflammatory cell infiltrates are accompanied by cardiomyocyte injury. Sometimes accompanied by coarse inflammatory cell infiltration into the endocardium. This invasive lesion is found in one or more biopsy specimens. Grade 3B (multifocal, borderline severe-acute-rejection) is a condition in which the inflammatory cell infiltrate seen in 3A becomes more confluent or diffuse and is found in more biopsy specimens. There is cardiomyocyte injury, with inflammatory cell infiltration involving large lymphocytes and eosinophils, and sometimes neutrophils. There is no bleeding. In Grade 4 (severe account rejection), various inflammatory cell infiltrations including activated lymphocytes, eosinophils, and neutrophils are diffusely observed. Cardiomyocyte injury and cardiomyocyte necrosis are always present. Edema, bleeding, and vasculitis are also usually observed. Inflammatory cell infiltration into the endocardium, which is different from the “Quilty” effect, is usually observed. Edema and hemorrhage can be more prominent than cell infiltration if treated with immunosuppressants for a significant period of time.

  Sufficient biofixability in a transplantable artificial tissue varies depending on the portion intended for transplantation, but those skilled in the art can appropriately set the degree of biofixability. Examples of the biocompatibility parameter include, but are not limited to, biological connectivity between the transplanted artificial tissue and the transplanted site. Such biostability can be determined by the presence of biological binding at the site of transplantation after transplantation. In the present specification, preferable bio-fixation property includes that the transplanted artificial tissue is arranged so as to exhibit the same function as the transplanted site.

  As used herein, “self-supporting” refers to a property that a prosthetic tissue is not substantially destroyed when at least one point of the tissue (eg, prosthetic tissue) is fixed in space. In this specification, the self-supporting property is obtained by picking up a tissue (for example, artificial tissue) with tweezers having a tip having a thickness of 0.5 to 3.0 mm (preferably, a tip having a thickness of 1 to 2 mm). Pinch the tissue with tweezers having a 1 mm thick tip; where the tweezers are preferably pre-curved) is observed by not being substantially destroyed. Such tweezers are commercially available, and are available from, for example, Natsume Seisakusho. The pick-up force employed here is usually comparable to the force applied by medical personnel when handling tissue. Therefore, the self-supporting ability can be expressed by the feature that it is not destroyed when picked up by hand. As such tweezers, for example, a bent taperless tweezers (for example, number A-11 (tip is 1.0 mm thick) sold by Natsume Seisakusho), A-12-2 (tip is 0.5 mm) However, the present invention is not limited to these, but it is easier to pick up the artificial tissue, but it is not limited to bend.

  In this specification, “organ” and “organ” are used interchangeably, and a function of a living individual is localized and operated in a specific part of the individual, and the part is morphologically A structure with independence. In general, in a multicellular organism (eg, animal, plant), an organ is composed of several tissues having a specific spatial arrangement, and the tissue is composed of many cells. Such organs or organs include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, brain, joint, bone, cartilage, extremity, retina, etc. It is not limited to them. Such organs or organs also include epidermis, pancreatic parenchymal system, pancreatic duct system, liver system, blood system, myocardial system, skeletal muscle system, osteoblast system, skeletal myoblast system, nervous system, vascular endothelial system, pigment system And organs such as, but not limited to, smooth muscle system, fat system, bone system, and cartilage system.

  In one embodiment, organs targeted by the present invention include, but are not limited to, intervertebral disc, cartilage, joint, bone, meniscus, synovium, ligament, tendon and the like.

  In this specification, “replace” refers to replacing a lesion part (a part of a living body) with a cell that is originally present in the lesion part by a cell supplied by an artificial tissue or a complex provided in the present invention. It means being done. Examples of diseases for which replacement is appropriate include, but are not limited to, ruptured sites. Replacing can be said to “fill” in another expression. By assessing such substitutions, the in vivo effects of the complexes of the invention can be assessed.

  In this specification, “a sufficient time for biological binding” varies depending on a combination of a certain portion and an artificial tissue or a complex, but those skilled in the art can easily determine appropriately according to the combination. be able to. Examples of such time include, but are not limited to, one week, two weeks, one month, two months, three months, six months, and one year after surgery. In the present invention, since the artificial tissue preferably contains substantially only cells and a substance derived therefrom, a substance to be removed after surgery is not particularly necessary. It does not matter. Accordingly, in this case, it can be said that the longer is preferable, but it can be said that the reinforcement is substantially completed when it is substantially extremely long.

  As used herein, “immune reaction” refers to a reaction due to impaired immune tolerance between a graft and a host. For example, hyperacute rejection (within several minutes after transplantation) (immunization with an antibody such as β-Gal) Reaction), acute rejection (reaction due to cellular immunity about 7 to 21 days after transplantation), chronic rejection (rejection due to cellular immunity after 3 months) and the like.

  In the present specification, whether or not to induce an immune response is determined based on the pathology of the species, number, etc. of cells (immune system) infiltration into a transplanted tissue by staining including HE staining, immunostaining, and microscopic examination of tissue sections. It can be determined by histological examination.

  As used herein, “calcification” refers to the deposition of calcareous substances in a living organism.

  In the present specification, whether or not to “calcify” in vivo can be determined by measuring alizarin red staining or calcium concentration, removing the transplanted tissue, dissolving the tissue section by acid treatment, etc. Can be measured and quantified with a trace element quantification device such as atomic absorbance.

  As used herein, “in vivo” or “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo.

  In the present specification, “ex vivo” refers to a series of operations ex vivo when a target cell for gene transfer is extracted from a subject, a therapeutic gene is introduced in vitro, and then returned to the same subject again.

  In the present specification, the “substance derived from a cell” refers to all substances originating from a cell. In addition to substances constituting a cell, a substance secreted by the cell. Examples include, but are not limited to, metabolized substances. Examples of substances derived from typical cells include, but are not limited to, extracellular matrix, hormones, cytokines and the like. Substances derived from cells usually do not have a detrimental effect on the cell and the host of the cell, so such substances are usually adversely affected even if included in artificial tissues, three-dimensional structures, etc. I don't have it.

  In the present specification, “extracellular matrix” (ECM) is also called “extracellular matrix” and refers to a substance existing between somatic cells regardless of whether they are epithelial cells or non-epithelial cells. The extracellular matrix is usually produced by cells and is therefore one of the biological materials. The extracellular matrix is responsible not only for tissue support, but also for the construction of the internal environment necessary for the survival of all somatic cells. The extracellular matrix is generally produced from connective tissue cells, but some are also secreted from the cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells. The fiber component is roughly classified into a fiber component and a matrix filling the space, and the fiber component includes collagen fiber and elastic fiber. The basic component of the substrate is glycosaminoglycan (acid mucopolysaccharide), most of which binds to non-collagenous protein to form a polymer of proteoglycan (acid mucopolysaccharide-protein complex). In addition, laminin of the basement membrane, microfibril around the elastic fiber, fiber, and glycoprotein such as fibronectin on the cell surface are included in the substrate. The basic structure is the same in specially differentiated tissues, for example, hyaline cartilage produces chondrocytes that contain a large amount of proteoglycan, characteristically by chondroblasts, and bone produces bone matrix that causes calcification by osteoblasts. . Here, as a typical extracellular matrix, for example, collagen I, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycans (for example, decorin, biglycan, fibromodulin, lumican, Hyaluronic acid, aggrecan, and the like), but are not limited thereto, and various substances can be used in the present invention as long as they are extracellular matrices responsible for cell adhesion. As collagen, 10 to 90% of collagen I may be contained. Alternatively, 10 to 90% of collagen II may be included as collagen. Furthermore, 10 to 90% of collagen III may be included as collagen. It is understood that these collagen amounts can be appropriately selected according to the purpose of the present invention. Representative examples include, for example, 60% collagen type I, 5% collagen type II, 30% collagen type III, and 5% collagen type V.

  In a preferred embodiment, the complex of the invention comprises a proteoglycan derived from a cell. The proteoglycan may contain at least 0.1 to 90% hyaluronic acid. Alternatively, the proteoglycan may contain at least 0.1-90% fibronectin.

  In one embodiment of the present invention, the artificial tissue, three-dimensional structure, complex, etc. provided in the present invention is an extracellular matrix (eg, elastin, collagen (eg, type I, type III, type IV, etc.) , Laminin, etc.) may advantageously be similar to the composition of the extracellular matrix at the site of the organ intended for transplantation. In the present invention, the extracellular matrix includes cell adhesion molecules. As used herein, “cell adhesion molecule” or “adhesion molecule” is used interchangeably and refers to the proximity of two or more cells (cell adhesion) or adhesion between a substrate and a cell. A molecule that mediates Generally, a molecule (cell-cell adhesion molecule) relating to cell-cell adhesion (cell-cell adhesion) and a molecule (cell-substrate adhesion molecule) involved in adhesion between cells and extracellular matrix (cell-substrate adhesion). Divided. The artificial tissue or three-dimensional structure provided in the present invention usually contains such a cell adhesion molecule. Therefore, in the present specification, the cell adhesion molecule includes a protein on the substrate side during cell-substrate adhesion, but in this specification, a protein on the cell side (for example, integrin etc.) is also included. Even so, as long as it mediates cell adhesion, it falls within the concept of cell adhesion molecules or cell adhesion molecules herein.

  Of particular interest in the present invention is that the artificial tissue or complex provided in the present invention comprises a cell and an extracellular matrix that the cell itself purifies (autologous). Therefore, complex containing collagen I, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycans (for example, decorin, biglycan, fibrojuline, lumican, hyaluronic acid, aggrecan, etc.) It has a characteristic composition. No artificial tissue containing such cell-derived components has been conventionally provided. Artificial tissues having such a composition are substantially impossible when artificial materials are used, and the composition itself (in particular, collagen I, collagen III, etc.) is native. It can be said that it is a combination.

  More preferably, the extracellular matrix comprises all of collagen (type I, type III, etc.), vitronectin and fibronectin. In particular, it has been understood that artificial tissues formulated with vitronectin and / or fibronectin have not been provided so far, and in that respect already, the artificial tissues or complexes provided in the present invention are novel. The

  In the present specification, “arranged” of an extracellular matrix, hydroxyapatite or the like refers to the presence of an extracellular matrix in the artificial tissue when referring to the artificial tissue provided in the present invention. It is understood that such an arrangement can be observed by staining the target extracellular matrix by immunostaining or the like and visualizing it.

  In this specification, extracellular matrix, hydroxyapatite, etc. “attach” to the surface of a cell means that it does not leave another substance, and that when different substances come into contact with each other, they are attached to each other by intermolecular forces. Say.

In the present specification, the phrase “extracellular matrix, hydroxyapatite and the like are“ dispersed ”” means that they exist without being localized. Such extracellular matrix dispersion is usually about 1:10 to 10: 1, typically about 1: 3 to 3: 1 when comparing the distribution density in any two 1 cm ² sections. Dispersion that falls within a ratio within a range, preferably, dispersion that falls within a ratio within a range of about 1: 2 to 2: 1 when comparing distribution densities in any two 1 cm ² sections. More preferably, it is advantageous to be in a substantially equivalent ratio in any section, but is not limited thereto. Due to the presence of the extracellular matrix on the surface without being localized and dispersed, the artificial tissue provided in the present invention has a biological binding ability evenly with respect to the surroundings, and therefore in the course after transplantation. The effect is excellent.

  Regarding cell-cell adhesion, cadherin, many molecules belonging to the immunoglobulin superfamily (NCAM, L1, ICAM, fasciclin II, III, etc.), selectins, etc. are known, and each binds the cell membrane by a unique molecular reaction. Is also known. Therefore, in one embodiment, the artificial tissue, the three-dimensional structure, etc. provided in the present invention have the same composition as the cadherin, a molecule belonging to the immunoglobulin superfamily, etc. It is preferable that the composition is of the order.

  Since a variety of molecules are involved in cell adhesion and have different functions, those skilled in the art can appropriately apply artificial tissues and three-dimensional structures provided in the present invention according to the purpose. The molecules to be included can be selected. Techniques relating to cell adhesion are well known in addition to those described above, and are described, for example, in extracellular matrix-clinical application-medical review.

  Whether a certain molecule is a cell adhesion molecule is determined by biochemical quantification (SDS-PAG method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, immunohistological examination), PCR method, It can be determined by determining that it is positive in an assay such as a hybridization method. Examples of such cell adhesion molecules include collagen, integrin, fibronectin, laminin, vitronectin, fibrinogen, immunoglobulin superfamily (eg, CD2, CD4, CD8, ICM1, ICAM2, VCAM1), selectin, cadherin and the like. It is not limited. Many of such cell adhesion molecules transmit an auxiliary signal of cell activation by cell-cell interaction into the cell simultaneously with adhesion to the cell. Therefore, the adhesion factor used in the tissue piece of the present invention is preferably one that transmits such an auxiliary signal for cell activation into the cell. This is because the cell activation can promote the proliferation of cells gathered therein and / or cells in the tissue or organ after being applied to a damaged site in a tissue or organ as a tissue piece. Whether such auxiliary signals can be transmitted into cells is determined by biochemical quantification (SDS-PAG method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, immunohistochemistry). It can be determined by determining that it is positive in an assay such as a PCR method and a hybridization method.

  As a cell adhesion molecule, for example, cadherin is widely known as a cell adhesion molecule widely used in tissue-adherent cell systems, and cadherin can be used in a preferred embodiment of the present invention. On the other hand, in non-adherent blood / immune system cells, examples of cell adhesion molecules include immunoglobulin superfamily molecules (LFA-3, CD2, CD4, CD8, ICAM-1, ICAM2, VCAM1, etc.); integrin family Molecules (LFA-1, Mac-1, gpIIbIIIa, p150, 95, VLA1, VLA2, VLA3, VLA4, VLA5, VLA6, etc.); selectin family molecules (L-selectin, E-selectin, P-selectin, etc.) But not limited to them. Thus, such molecules may be particularly useful for treating tissues or organs of the blood / immune system.

  Cell adhesion molecules require adhesion to non-adherent cells in order to work in a specific tissue. In that case, it is considered that adhesion between cells is gradually strengthened by primary adhesion by a selectin molecule that is constantly expressed and subsequent secondary adhesion by an integrin molecule that is activated. Therefore, as the cell adhesion molecule used in the present invention, such a factor that mediates primary adhesion, a factor that mediates secondary adhesion, or both can be used together.

  As used herein, the term “actin-regulating substance” refers to a substance having a function of changing the form or state of actin by directly or indirectly interacting with intracellular actin. It is understood that the actin depolymerization promoting factor and the actin polymerization promoting factor are classified according to the action on actin. Examples of such substances include Slingshot, cofilin, CAP (cyclase-related protein), AIP1 (actin-interacting-protein 1), ADF (actin depolymerizing factor), destrin, depactin, and actolin. , Cytochalasin and NGF (never grow factor), for example, RhoA, mDi, profilin, Rac1, IRSp53, WAVE2, ROCK, LIM kinase, cofilin, cdc42, N-WASP, Arp2 / 3, Drf3, Mena, LPA (lysophosphatic acid), insulin, PDGFa PDGFb, there may be mentioned chemokines and TGFβ is understood that the invention is not limited to them. Such actin-modulating substances include substances identified by the following assays. In this specification, the interaction with actin is evaluated by visualizing actin with an actin staining reagent (Molecular Probes, Texas Red-X phalloidin), etc., and then microscopically observing actin aggregation and cell extension. This is determined by confirming the phenomenon of aggregation, reconstitution and / or improvement of cell spreading rate. These determinations can be made quantitatively or qualitatively. Such an actin-regulating substance is used in the present invention to promote separation of artificial tissue or promote stratification. When the actin-regulating substance used in the present invention is derived from a living body, its origin may be anything, and examples thereof include mammalian species such as humans, mice and cows.

  Factors involved in actin polymerization as described above are, for example, Rho-related actin polymerization control, and include the following (for example, “I understand the cytoskeleton and movement” (edited by Hiroaki Miki) see Yodosha )

Actin polymerization (see Takenaka T et al. J. Cell Sci., 114: 1801-1809, 2001)
RhoA->mDi->Profilin-> Actin polymerization RhoA-> ROCK / Rho-> LIM kinase-> Phosphorylation (inhibition) of cofilin-> Actin polymerization Rac1->IRSp53->WAVE2-> Profilin, Arp2 / 3-> Actin polymerization cdc42->N-WASP-> Profi Phosphorus, Arp2 / 3⇒Actin polymerization cdc42 → Drf3 → IRSp53 → Mena⇒Actin polymerization (In the above, → indicates a signal transduction pathway such as phosphorylation.) In the present invention, any factor involved in such a pathway is selected. Can be used.

Actin depolymerization Slingshot → Cofilin dephosphorylation (activation) ⇒ Actin depolymerization Actin depolymerization is controlled by the balance of cofilin phosphorylation by LIM kinase activity and dephosphorylation by Slingshot. As other factors that activate cofilin, CAP (cycle-associated protein) and AIPI (actin-interacting-protein 1) have been identified, and it is understood that any of them can be used. .

  LPA (lysophosphatidic acid) having any chain length can be used.

  Any chemokine can be used, and preferred examples include interleukin 8, MIP-1, and SDF-1.

  Arbitrary things can be used as TGFβ, but preferably TGF-β1 and TGF-β3 can be used. Since TGFβ1 and TGFβ3 also have an extracellular matrix production promoting action, they can be used with care in the present invention.

  As used herein, “tissue strength” refers to a parameter indicating the function of a tissue or organ, and is the physical strength of the tissue or organ. Tissue strength can generally be determined by measuring tensile strength (eg, breaking strength, stiffness, Young's modulus, etc.). Such general tensile tests are well known. By analyzing data obtained by a general tensile test, various data such as breaking strength, rigidity, Young's modulus, etc. can be obtained, and such values are also used as an index of tissue strength in this specification. be able to. In the present specification, it is usually required to have a tissue strength that can be clinically applied.

  Here, the tensile strength of the artificial tissue, the three-dimensional structure and the like provided in the present invention can be measured by measuring stress / strain characteristics. Briefly, tensile strength is applied by applying a load to the sample, for example, 1ch is strained and 2ch is input to each AD converter (eg ELK-5000) to measure the stress and strain characteristics. Can be determined. Tensile strength can also be achieved by testing creep properties. The creep characteristic indentation test is a test for examining how the material expands with time under a certain load. An indentation test of a minute material, a thin material, or the like is performed by using a triangular pyramid indenter having a tip radius of about 0.1 to 1 μm, for example. First, an indenter is pushed into the test piece to give an addition. Then, when the test piece is pushed into the test piece by about several tens nm to several μm, the indenter is returned and unloaded. What is obtained by such a test method is called a load unloading curve. Hardness, Young's modulus, and the like can be obtained from the load applied and the behavior of the indentation depth obtained from this curve.

  The artificial tissue provided in the present invention may have a low tensile strength. The tensile strength increases as the matrix ratio in the cell / extracellular matrix ratio increases, and decreases as the cell ratio increases. One feature of the present invention is that the strength can be freely adjusted as necessary. It is characterized by being able to be relatively strong and weak relative to the tissue to be transplanted. Therefore, it is understood that the purpose can be set according to such an arbitrary place.

  As used herein, “differentiation” refers to a process of development of a state of a part of an organism such as a cell, tissue or organ, which forms a characteristic tissue or organ. “Differentiation” is mainly used in developmental biology, developmental biology and the like. Until a fertilized egg consisting of one cell divides and becomes an adult, an organism forms various tissues and organs. In the early stages of development, such as before division or when division is not sufficient, each cell or group of cells does not show any morphological or functional characteristics and is difficult to distinguish. Such a state is called “undifferentiated”. “Differentiation” also occurs at the organ level, and the cells that make up the organ develop into a variety of distinctive cells or groups of cells. This is also called differentiation within the organ in organ formation. Therefore, the artificial tissue and the three-dimensional structure provided in the present invention may use a tissue containing differentiated cells.

  When producing the artificial tissue provided in the present invention, if differentiation is required, it may be differentiated before the organization is started or may be differentiated after the organization.

In the present specification, “differentiation factor” is also referred to as “differentiation promoting factor”, and any factor (for example, chemical substance, temperature, etc.) known to promote differentiation into differentiated cells can be used. It may be a factor. Examples of such factors include various environmental factors. Examples of such factors include temperature, humidity, pH, salt concentration, nutrition, metal, gas, organic solvent, pressure, and chemical substances. (For example, steroids, antibiotics, etc.) or any combination thereof, but not limited thereto. Representative differentiation factors include, but are not limited to, cell bioactive substances. Representative examples of such factors include DNA demethylating agents (such as 5-azacytidine), histone deacetylating agents (such as trichostatin), nuclear receptor ligands (eg, retinoic acid (ATRA), vitamins) D _3, T3, etc.), cell growth factors (activin, IGF-1, FGF, PDGFa , PDGFb, TGF-β, such as BMP2 / 4), cytokines (LIF, IL-2, IL -6 etc.), hexamethylene bis Examples include, but are not limited to, acetamide, dimethylacetamide, dibutyl cAMP, dimethylol sulfoxide, iododeoxyuridine, hydroxylurea, cytosine arabinoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyuridine, polybrene, selenium and the like. .

  Specific differentiation factors include the following. These differentiation factors can be used alone or in combination.

A) cornea: epidermal growth factor (EGF);
B) Skin (keratinocytes): TGF-β, FGF-7 (KGF: keratinocyte growth factor), EGF
C) Vascular endothelium: VEGF, FGF, angiopoietin
D) Kidney: LIF, BMP, FGF, GDNF
E) Heart: HGF, LIF, VEGF
F) Liver: HGF, TGF-β, IL-6, EGF, VEGF
G) Umbilical cord endothelium: VEGF
H) Intestinal epithelium: EGF, IGF-I, HGF, KGF, TGF-β, IL-11
I) Nerve: nerve growth factor (NGF), BDNF (brain-derived neurotrophic factor), GDNF (glial cell-derived neurotrophic factor), neurotrophin, IL-6, TGF-β, TNF
J) Glial cells: TGF-β, TNF-α, EGF, LIF, IL-6
K) Peripheral nerve cells: bFGF, LIF, TGF-β, IL-6, VEGF,
L) Lung (alveolar epithelium): TGF-β, IL-13, IL-1β, KGF, HGF
M) Placenta: growth hormone (GH), IGF, prolactin, LIF, IL-1, activin A, EGF
N) Pancreatic epithelium: growth hormone, prolactin O) Pancreatic Langerhans islet cells: TGF-β, IGF, PDGFa, PDGFb, EGF, TGF-β, TRH (thyroropin)
P) Synovial cells: FGF, TGF-β (particularly TGF-β1, TGF-β3)
Q) Osteoblast: BMP (particularly BMP-2, BMP-4, BMP-7), FGF
R) Chondroblasts: FGF, TGF-β (especially TGF-β1, TGF-β3), BMP (especially BMP-2, BMP-4, BMP-7), TNF-α, IGF
S) Retinal cells: FGF, CNTF (Ciliary neurotrophic factor)
T) Adipocytes: insulin, IGF, LIF
U) Muscle cells: LIF, TNF-α, FGF.

  As used herein, “bone differentiation” refers to differentiation of arbitrary cells into bone. Such bone differentiation is known to be promoted in the presence of dexamethasone, β-glycerophosphate and ascorbic acid diphosphate. An osteogenic factor (BMP, (especially BMP-2, BMP-4, BMP-7)) may be added. This is because bone formation is promoted.

  As used herein, “cartilage differentiation” refers to differentiation of arbitrary cells into cartilage. Such cartilage differentiation is known to be promoted in the presence of pyruvate, dexamethasone, ascorbyl diphosphate, insulin, transferrin and selenite. Bone morphogenetic factors (BMP (particularly BMP-2, BMP-4, BMP-7)), TGF-beta (particularly TGF-beta1, TGF-beta3), FGF, TNF-alpha, and the like may be added. This is because cartilage formation is promoted.

  As used herein, “adipose differentiation” refers to differentiation of any cell into fat. Such adipose differentiation is known to be promoted in the presence of insulin, IGF, LIF and ascorbyl diphosphate.

  As used herein, “graft”, “graft”, and “tissue graft” are used interchangeably and are the same or different groups of tissues or cells that are to be inserted into a specific part of the body and inserted into the body. It will be a part of it later. Therefore, the artificial tissue and three-dimensional structure provided in the present invention can be used as a graft. Examples of grafts include organs or parts of organs, blood vessels, blood vessel-like tissues, skin pieces, hearts, heart valves, pericardium, dura mater, articular membranes, bone pieces, cartilage pieces, corneal bone pieces, teeth, etc. But not limited to. Thus, a graft includes everything that is used to fill a defect in a part and make up for the defect. Examples of the graft include, but are not limited to, an autograft, an allograft (allograft), and a xenograft depending on the type of donor.

  In this specification, an autograft (tissue, cell, organ, etc.) or autograft (tissue, cell, organ, etc.) refers to an individual, and a graft (tissue, cell, organ, etc.) derived from the individual. ). In this specification, the term “autograft (tissue, cell, organ, etc.)” refers to a graft (tissue, cell, organ, etc.) from another genetically identical individual (eg, monozygotic twin) in a broad sense. May also be included. As used herein, such expression with self is used interchangeably when derived from a subject. Thus, as used herein, the expression not derived from a subject has the same meaning as not being self (ie, non-self).

  In the present specification, “allograft (allogeneic allograft) (tissue, cell, organ, etc.)” refers to a graft (tissue, cell, Organs). Because of genetic differences, allogeneic grafts (tissues, cells, organs, etc.) can elicit an immune response in the transplanted individual (recipient). Examples of such grafts (tissues, cells, organs, etc.) include, but are not limited to, parent-derived grafts (tissues, cells, organs, etc.). The prosthetic tissue provided in the present invention should be noted in the sense that it can also be used in allografts and has demonstrated good therapeutic results.

  As used herein, “xenograft (tissue, cell, organ, etc.)” refers to a graft (tissue, cell, organ, etc.) transplanted from a xenogeneic individual. Thus, for example, when a human is a recipient, a graft (tissue, cell, organ, etc.) from a pig is referred to as a xenograft (tissue, cell, organ, etc.).

  As used herein, a “recipient” (recipient) refers to an individual that receives a graft (tissue, cell, organ, etc.) or a transplant (tissue, cell, organ, etc.), and is also referred to as a “host”. In contrast, an individual that provides a graft (tissue, cell, organ, etc.) or transplant (tissue, cell, organ, etc.) is called a “donor” (donor).

  Any artificial tissue derived from any cell can be used by using the artificial tissue formation technology provided in the present invention. This is because the artificial tissue (eg, membranous tissue, organ, etc.) formed by the method of the present invention maintains the tissue damage rate that is not damaged for therapeutic purposes (ie, keeps it low), while maintaining the target function. It is because it can exhibit. Therefore, there has been a situation in which the tissue or organ itself as it is has been used as a transplant. In such a situation, the fact that it has become possible to use such a three-dimensional artificial tissue by forming a three-dimensionally connected tissue from cells is achieved by the conventional technology. This is one of the special effects of the present invention that could not be achieved.

  As used herein, “subject (also subject)” refers to an organism to which the treatment of the present invention is applied, and is also referred to as a “patient”. The patient or subject may preferably be a human.

  The cells used as necessary in the artificial tissue, three-dimensional structure, and tissue graft provided in the present invention may be derived from the same line (derived from the self (self)) or from the same type (derived from another individual (other family)). ) Or from different species. Self-derived cells are preferred because rejection is considered, but they may be allogeneic if rejection is not a problem. Moreover, what causes rejection reaction can also be utilized by performing the treatment which eliminates rejection reaction as needed. Procedures for avoiding rejection are well known in the art. For example, the New Surgery System, Vol. 12, Organ Transplantation (From Heart Transplantation / Lung Transplantation Technical and Ethical Development to Implementation) (Revised 3rd Edition) ), Listed in Nakayama Shoten. Examples of such methods include methods such as the use of immunosuppressants and steroids. Immunosuppressants that prevent rejection are currently "cyclosporine" (Sandimune / Neoral), "Tacrolimus" (Prograf), "Azathioprine" (Imlan), "Steroid Hormone" (Predonin, Methylpredonin), "T cells There are “antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as a preventive immunosuppressive therapy is a combination of three drugs “cyclosporine, azathioprine, steroid hormone”. The immunosuppressive agent is desirably administered at the same time as the medicament of the present invention, but it is not always necessary. Therefore, as long as the immunosuppressive effect is achieved, the immunosuppressive agent can be administered before or after the regeneration / treatment method of the present invention.

  The cells used in the present invention may be cells derived from any organism (for example, vertebrates and invertebrates). Preferably, cells derived from vertebrates are used, and more preferably cells derived from mammals (for example, primates, rodents, etc.) are used. More preferably, cells derived from primates are used. In the most preferred embodiment, human-derived cells are used. Usually, it is preferable to use cells of the same species as the host.

  Examples of the combination of the subject targeted by the present invention and the artificial tissue include joint damage or degeneration, cartilage damage or degeneration, osteonecrosis, meniscal damage or degeneration, intervertebral disc degeneration, ligament damage or degeneration, fracture, bone defect Examples include, but are not limited to, transplantation of joints, cartilage, and bone to a patient having the disease, transplantation of the cornea of the present invention to a patient having a damaged cornea, and the like.

  The tissue targeted by the present invention may be any organ or organ of an organism, and the tissue targeted by the present invention may be derived from any type of organism. Examples of organisms targeted by the present invention include vertebrates and invertebrates. Preferably, the organism targeted by the present invention is a mammal (eg, primate, rodent, etc.). More preferably, the organism targeted by the present invention is a primate. Most preferably, the present invention is directed to humans.

  As used herein, “flexible” artificial tissue refers to resistance to physical stimulation (eg, pressure) from the external environment. The artificial tissue having flexibility is preferable when the site to be transplanted moves or deforms autonomously or under the influence of others.

  In this specification, the artificial tissue having “stretchability” refers to a property having resistance to stretchable stimuli (for example, pulsation) from the external environment. A stretchable artificial tissue is preferred when the site to be transplanted is accompanied by a stretch stimulus. Examples of such a site accompanied by elastic stimulation include, but are not limited to, muscles, joints, cartilage, and tendons.

  As used herein, the term “part” refers to any part, tissue, cell, organ in the body. Such portions, tissues, cells, organs include, but are not limited to, portions that can be treated with skeletal myoblasts, fibroblasts, synoviocytes, stem cells. As long as it is a specific marker, any parameters such as nucleic acid molecule (mRNA expression), protein, extracellular matrix, specific phenotype, and cell shape can be used. Therefore, even if the marker is not specifically described in the present specification, any marker can be used in the present invention as long as it can indicate that it is derived from the portion. An artificial tissue may be determined. Representative examples of such parts include, for example, parts of the heart other than the myocardium, parts containing mesenchymal stem cells or cells derived therefrom, tissues, organs, myoblasts (for example, skeletal myoblasts), fibroblasts, etc. Examples include, but are not limited to, cells and synovial cells.

  When looking at cartilage tissue or the like, the following markers can be used as indices.

  Sox9 is (human: accession number NM_000346) and is a marker specific for chondrocytes. This marker can be confirmed mainly by observing the presence of mRNA (Kulyk WM, Franklin JL, Hoffman LM. Sox9 expression durning chondrogenesis, in the 15th anniversary of the development of the United States of America. ): 327-32.).

  Col 2A1 is (human: accession number NM_001844) and is a marker specific for chondrocytes. This marker can be confirmed mainly by observing the presence of mRNA (Klyk WM, Franklin JL, Hoffman LM. Sox9 expression durning chondrogenesis in micromolecules of the United States of America. ): 327-32.).

  Aggrecan is (human: accession number NM_001135) and is a marker specific for chondrocytes. This marker can be confirmed mainly by observing the presence of mRNA (Klyk WM, Franklin JL, Hoffman LM. Sox9 expression durning chondrogenesis in micromolecules of the United States of America. ): 327-32.).

  Bone sialoprotein (Bone sialoprotein) is (human: accession number NM_004967) and is a marker specific to osteoblasts. This marker can be confirmed mainly by observing the presence of mRNA (Haase HR, Ivanovski S, Waters MJ, Bartold PM. Growth hormonal regulation of human biomolecules in the bloodstream. Res. 2003 Aug; 38 (4): 366-74.).

  Osteocalcin is (human: accession number NM199173) and is a marker specific to osteoblasts. This marker can be confirmed mainly by observing the presence of mRNA (Haase HR, Ivanovski S, Waters MJ, Bartold PM. Growth hormonal regulation of human biomolecules in the bloodstream. Res. 2003 Aug; 38 (4): 366-74.).

  GDF5 is (human: accession number NM_000557) and is a marker specific to ligament cells. This marker can be confirmed mainly by observing the presence of mRNA (Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, Dube JL, DiBlasio-Smith E, Jon J, Nov J Wozney JM, Rosen V. Ectopic Induction of tendon and ligation in rats by Je gro s., J. et al. 30.).

  Six1 is (human: accession number NM — 005982) and is a marker specific to ligament cells (Dreyer SD, Naruse T, Morello R, Zabel B, Winterpatch A, Johnson RL, Lee B, Obberg KC Lb. expression duration joint and tendon formation: localization and evaluation of potential downstream targets. Gene Expr Patterns. 2004 Jul; 4 (4): 397-405. This marker can be confirmed mainly by observing the presence of mRNA.

  Scleraxis is (human: accession number BK000280) and is a marker specific to ligament cells (Brent AE, Schweitzer R, Tabin CJ. A somic component of tendon 3 cells. 113 (2): 235-48.). This marker can be confirmed mainly by observing the presence of mRNA.

  In other embodiments, other markers specific for other tissues can be utilized. Such markers include, for example, Oct-3 / 4, SSEA-1, Rex-1, Otx2, etc. for embryonic stem cells; VE-cadherin, Flk-1, Tie-1, for endothelial cells PECAM1, vWF, c-kit, CD34, Thy1, Sca-1, etc .; for skeletal muscle, in addition to those described above, skeletal muscle α-actin and the like; for neurons, Nestin, Glu receptor, NMDA receptor, GFAP, neurite Examples of hematopoietic cell lines include c-kit, CD34, Thy1, Sca-1, GATA-1, GATA-2, and FOG.

  As used herein, the term “derived from” means that a certain cell has been separated, isolated or extracted from the cell mass, tissue, organ, etc. from which the cell originally resided. Or the cell was derived from a stem cell.

  As used herein, the term “three-dimensional structure” includes cells in which the matrix is three-dimensionally oriented and the cells are arranged three-dimensionally and retain the connection and orientation between the cells. An object that spreads in a three-dimensional direction. The composite of the present invention is usually a three-dimensional structure.

  As used herein, “biological integration” refers to the relationship between biological entities, where there is some biological interaction between two biological entities. Say something. Examples of such interactions include interactions via biomolecules (eg, extracellular matrix), interactions via information transmission, and electrical interactions (electrical coupling such as synchronization of electrical signals). But not limited to them. Biological bonds include biological bonds within an artificial tissue and biological bonds that a certain artificial tissue has with the surroundings (eg, surrounding tissues after transplantation, surrounding cells, etc.). When confirming an interaction, an appropriate assay method is used depending on the characteristics of the interaction. For example, when confirming a physical interaction via a biomolecule, the strength (for example, a tensile test) of an artificial tissue, a three-dimensional structure, or the like is confirmed. In the case of information transmission, whether or not signal transmission is performed is confirmed through gene expression or the like. Alternatively, in the case of an electrical interaction, it can be confirmed by measuring a potential state in an artificial tissue, a three-dimensional structure, etc., and checking whether the potential is propagated with a constant wave. In the present invention, normal biological bonds have biological bonds in all three dimensions. Preferably, it may be advantageous to have biological bonds approximately equally in all three dimensions, but in another embodiment, it has biological bonds approximately equally in two dimensions, Artificial tissues, three-dimensional structures, etc. that have a slightly weak biological bond in the three directions can also be used. Alternatively, in the case of biological binding via an extracellular matrix, the extracellular matrix can be stained and the degree of staining can be observed. As a method for observing biological binding in vivo, there is a binding experiment using cartilage. In this experiment, the surface of the cartilage was excised and digested with chondroitinase ABC (Hunziker EB et al., J Bone Joint Surg Am. 1996 May; 78 (5): 721-33). After transplanting to the excised surface and culturing for about 7 days, the subsequent binding is observed histologically. It is understood that the ability to adhere to surrounding cells and / or extracellular matrix can be measured by experiments using such cartilage.

  The artificial tissue, three-dimensional structure and the like provided in the present invention can be provided as pharmaceuticals, or can be provided by known preparation methods as animal drugs, quasi drugs, marine drugs, cosmetics, and the like.

  Accordingly, the animal targeted by the present invention may be any organism (eg, animal (eg, vertebrate, invertebrate)) as long as it has an organ or an organ. Preferably, it is a vertebrate (for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably mammals (for example, single holes, marsupials, Rodents, crustaceans, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents , Rabbit eyes, etc.). Exemplary subjects include, but are not limited to, animals such as cows, pigs, horses, chickens, cats, dogs and the like. More preferably, a primate (for example, chimpanzee, Japanese monkey, human) is used. Most preferably humans can be targeted. This is because there is a limit in transplantation treatment.

  When the present invention is used as a medicament, the medicament of the present invention may further contain a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. Examples include, but are not limited to, agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants.

  The amount of the pharmaceutical (artificial tissue, pharmaceutical compound used together) used in the treatment method of the present invention is the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, It can be easily determined by those skilled in the art in view of the form or type of tissue. The frequency with which the treatment method of the present invention is applied to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  In the present specification, “administering” means that the artificial tissue, three-dimensional structure or the like provided in the present invention or a medicament containing the same is administered alone or in combination with other therapeutic agents. . For the artificial tissue provided in the present invention, the following introduction method, introduction form, and introduction amount to a treatment site (for example, a damaged heart) can be used. That is, the method for administering the artificial tissue and the three-dimensional structure provided in the present invention includes, for example, direct application to a damaged part of myocardial tissue that has suffered ischemic damage due to myocardial infarction, angina, etc., and suturing after application And insertion methods. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes presentation where the combined drugs are administered together as a therapeutic mixture, and the combined drugs are separate but at the same time (eg, artificial tissue or the like is provided by direct surgery, other drugs are intravenously injected) Also included are procedures administered (if given by). “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

  In this specification, “reinforcement” refers to improving the function of an intended part of a living body.

  In the present specification, the “instruction” refers to a method of administering or diagnosing the pharmaceutical of the present invention, such as a reagent handling, usage method, preparation method, artificial tissue preparation method, contraction method, etc. It is described for the person who performs the diagnosis and the person who diagnoses (which may be the patient). This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.) in the country where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page (web site) or e-mail provided on the Internet) It can also be provided in such a form.

  As used herein, “extracellular matrix production promoting factor” refers to any factor that promotes the production of extracellular matrix in cells. In the present invention, when an extracellular matrix production promoting factor is added to a cell sheet, the cell sheet is provided with an environment in which exfoliation from the culture container is promoted, and such a sheet biologically binds in a three-dimensional direction. Here, biological binding includes binding between cells in a tissue and an extracellular matrix and binding between extracellular matrices. Here, three-dimensionalization is further promoted by self-shrinkage. Such factors typically include factors that promote extracellular matrix secretion (eg, TGF-β1, TGF-β3, etc.). In the present invention, typical extracellular matrix production promoting factors include TGF-β1, TGF-β3, ascorbic acid, ascorbic acid diphosphate, a derivative thereof, or a salt thereof. Preferably, the extracellular matrix production promoting factor is the component (s) that promote secretion of the extracellular matrix so that it is similar to the compositional component and / or amount of the extracellular matrix that is intended for application. It is preferable that When such an extracellular matrix production-promoting factor includes a plurality of components, such a plurality of components is configured to be similar to the composition component and / or the amount of the extracellular matrix of the portion intended for application. obtain.

  As used herein, “ascorbic acid or a derivative thereof” includes ascorbic acid and analogs thereof (eg, ascorbic acid diphosphate) and salts thereof (eg, sodium salt, magnesium salt, potassium salt, etc.). . Ascorbic acid is preferably L-form, but is not limited thereto.

(Preparation of artificial tissue with factors that promote extracellular matrix production)
The method for producing the tissue of the present invention comprises: A) providing a cell; B) containing the desired artificial tissue size containing a cell culture medium containing an extracellular matrix production promoting factor. Placing in a container having a sufficient bottom area; and C) culturing the cells in the container for a time sufficient to form an artificial tissue having a size of the desired size. it can.

  The cell used here may be any cell. Methods for providing cells are well known in the art. For example, a method of extracting a tissue and separating the cell from the tissue, a method of separating a cell from a body fluid containing blood cells, or a cell line Examples include, but are not limited to, methods prepared by artificial culture. The cells used in the present specification can be any stem cells or differentiated cells, and in particular, myoblasts, mesenchymal stem cells, adipocytes, synovial cells, bone marrow cells, and the like can be used. As the mesenchymal stem cells used in the present specification, for example, adipose tissue-derived stem cells, bone marrow-derived stem cells, and the like can be used.

  In the artificial tissue production method used in the present invention, a cell culture medium containing an extracellular matrix production promoting factor is used. Examples of such an extracellular matrix production promoting factor include ascorbic acid or a derivative thereof, and examples thereof include, but are not limited to, ascorbic acid diphosphate and L-ascorbic acid.

  The cell culture medium used in the present invention may be any medium as long as the target cell grows. For example, DMEM, MEM, F12, DME, RPMI1640, MCDB104, 199, MCDB153, L15, A SkBM, Basal medium or the like to which glucose, FBS (fetal bovine serum) or human serum, and antibiotics (penicillin, streptomycin, etc.) are appropriately added can be used.

As long as the container used in the method of the present invention has a bottom area sufficient to accommodate a desired artificial tissue size, a container that is usually used in the art can be used. A container having a large bottom area (for example, 1 cm ² or more), such as a mold container, may be used. Any material may be used for the container, and glass, plastic (for example, polystyrene, polycarbonate, etc.), silicone, and the like may be used, but are not limited thereto.

  In a preferred embodiment, the artificial tissue production method used in the present invention may further include a step of separating the produced artificial tissue. In this specification, “separate” refers to separation of the artificial tissue from the container after the artificial tissue provided in the present invention is formed in the container. Separation can be achieved by, for example, physical means (for example, pipetting of a medium), chemical means (addition of a substance), and the like. In the present invention, the prosthetic tissue can be separated by applying a stimulus by physical means or chemical means around the artificial tissue without actively invading the artificial tissue such as proteolytic enzyme treatment. In this respect, it should be noted that there is an effect of high performance as a graft, such as simplicity that could not be achieved in the past and the resulting damage to the artificial tissue.

  In a preferred embodiment, the present invention further comprises the step of separating the artificially organized cells. Here, in a more preferred embodiment, the separating step can apply a stimulus that causes the artificial tissue to contract. Includes physical stimulation (eg pipetting). It is a preferable feature in the present invention that such physical stimulation does not directly act on the produced artificial tissue. This is because damage to the artificial tissue can be suppressed by not directly acting on the artificial tissue. Alternatively, the separating step includes chemical means such as addition of an actin modulating substance. Such actin-regulating substances include chemical substances selected from the group consisting of actin depolymerization promoting factors and actin polymerization promoting factors. For example, actin depolymerization promoting factors include Slingshot, cofilin, CAP (cyclase-related protein), AIP1 (actin-interacting-protein 1), ADF (actin depolymerizing factor), destrin, depactin, actofolin, cytochalasin v and eGF growth factor) and the like. On the other hand, actin polymerization promoting factors are RhoA, mDi, profilin, Rac1, IRSp53, WAVE2, ROCK, LIM kinase, cofilin, cdc42, N-WASP, Arp2 / 3, Drf3, Mena, LPA (lysophosphatic acid), insulin, PDGFa, PDGFb, chemokine, TGFβ and the like can be mentioned.

  Without wishing to be bound by theory, these actin modulators cause contraction or relaxation of the cytoskeleton of the act-myosin system, thereby regulating the contraction or stretching of the cell itself, resulting in It is considered that the action of promoting or delaying the separation of the artificial tissue itself from the bottom surface of the container is achieved.

  In another embodiment, one of the features is that the artificial tissue or complex provided in the present invention is produced from cells cultured in a monolayer. Despite the monolayer culture, as a result, it has become possible to construct artificial tissues having various thicknesses. For example, when a conventional temperature-responsive sheet is used, a plurality of sheets are used. It can be said that this was an effect that could not have been predicted from the fact that a thick structure could not be constructed without overlaying. The present invention has achieved for the first time a three-dimensional method that does not require feeder cells and enables the layering of ten or more cells. As a cell transplantation method that does not use a scaffold, cell sheet engineering technology using a temperature-sensitive culture dish according to Non-Patent Document 3 is representative, and international evaluation has been obtained as an original technology. However, when this cell sheet technology is used, a single sheet is often fragile, and in order to obtain a strength that can withstand surgical operations such as transplantation, it has been necessary to devise operations such as overlapping sheets. .

  Unlike the cell sheet technology, the cell / matrix complex developed in this study does not require a temperature-sensitive culture dish, and it is easy to layer cells / matrix. There is no technology in the world that can produce a complex layered in 10 layers or more without using so-called Feeder cells such as rodent stromal cells in about 3 weeks. In addition, by adjusting the matrix production conditions of synovial cells, it is also possible to create a complex that retains strength that allows surgical operations such as grasping and moving the complex without the need for special instruments. This method may be an innovative and innovative method for surely and safely performing cell transplantation even in the world.

  In a preferred embodiment, the extracellular matrix production promoting factor used in the artificial tissue production method used in the present invention includes ascorbic acid 2-phosphate (Hata R, Senoo H. J Cell Physiol. 1989; 138 (1)). : See 8-16). In the present invention, the production of extracellular matrix is promoted by adding a certain amount or more of ascorbic acid diphosphate. As a result, the resulting artificial tissue or complex is cured and easily peeled off. Thereafter, self-contraction is performed by applying a stimulus for peeling. It is not described in the report of Hata et al. That the tissue is hardened and acquires the property of being easily detached after culturing with the addition of ascorbic acid. Without being bound by theory, one notable difference is that Hata et al. Differ significantly in the cell density used. Moreover, Hata et al. Did not even suggest a curing effect, and such a curing and shrinkage effect and an effect of being easily peeled were found for the first time by the present invention and are provided in the present invention. It can be said that the artificial tissue is at least completely different from the conventionally produced artificial tissue in that it is produced through procedures such as hardening, shrinkage, and peeling.

  It is a surprising effect that when the culture was peeled, it contracted and three-dimensionalization, stratification, etc. were promoted. Such a thing has not been reported so far.

  In a preferred embodiment, the ascorbic acid diphosphate used in the present invention is usually present at at least 0.01 mM, preferably at least 0.05 mM, more preferably at least 0.1 mM. More preferably it is present at a concentration of at least 0.2 mM. More preferably, it is present at a concentration of 0.5 mM, even more preferably at a concentration of 1.0 mM. Here, any concentration can be used as long as it is 0.1 mM or more, but there may be an aspect in which it may be desirable that the concentration is preferably 10 mM or less.

  In a preferred embodiment, the extracellular matrix production promoter used in the present invention includes ascorbic acid diphosphate or a salt thereof and L-ascorbine or a salt thereof.

  In a preferred embodiment, the artificial tissue production method used in the present invention further includes a step of detaching and self-contracting the artificial tissue following the culturing step. Peeling can be promoted by applying a physical stimulus (for example, applying a physical stimulus (shear stress application, pipetting, deformation of the container, etc.) with a stick or the like to the corner of the container). Self-shrinking occurs naturally after such debonding when a physical stimulus is applied. In the case of chemical stimulation, self-contraction and peeling occur in parallel. Self-contraction promotes biological coupling, particularly in the third dimension (when referring to tissue on the sheet, the direction perpendicular to the two-dimensional direction). Since it is manufactured in this manner, it can be said that the artificial tissue provided in the present invention takes the form of a three-dimensional structure.

  In the artificial tissue production method used in the present invention, the sufficient time varies depending on the intended use of the artificial tissue, but preferably means at least 3 days (3-7 days as an exemplary period) Can be mentioned).

  In another embodiment, the artificial tissue production method used in the present invention may further comprise the step of differentiating the artificial tissue. This is because, by differentiating, it is possible to adopt a form closer to the desired tissue. Examples of such differentiation include, but are not limited to, cartilage differentiation and bone differentiation. In a preferred embodiment, bone differentiation can be performed in a medium containing dexamethasone, beta glycerophosphate and ascorbic acid diphosphate. More preferably, bone morphogenetic proteins (BMPs) are added. This is because such BMP2, BMP-4, and BMP-7 further promote bone formation.

  In another embodiment, the artificial tissue production method used in the present invention is a step of differentiating an artificial tissue, and examples of differentiation include a form in which cartilage differentiation is performed. In a preferred embodiment, cartilage differentiation can be performed in a medium containing pyruvate, dexamethasone, ascorbyl diphosphate, insulin, transferrin and selenite. More preferably, bone morphogenetic proteins (BMP-2, BMP-4, BMP-7, TGF-β1, TGF-β3) are added. This is because such BMP further promotes the formation of cartilage.

It should be noted that in the present invention, a tissue capable of differentiating into various differentiated cells such as bone and cartilage can be produced. Differentiation into cartilage tissue has been difficult with other scaffold-free artificial tissues. When a certain size is required, conventionally, it has been necessary to co-culture with the scaffold, to make it three-dimensional, and to contain a cartilage differentiation medium. Conventionally, it was difficult to differentiate cartilage in a scaffold-free manner. In the present invention, for the first time, cartilage differentiation in an artificial tissue has become possible. This is an unproven result and is one of the characteristic effects of the present invention. In cell therapy aiming at tissue regeneration, it has been difficult to effectively and safely perform treatment using a sufficiently large tissue without a scaffold. In this sense, the present invention can be said to achieve a remarkable effect. In particular, the significance is deep in that it has become possible to freely manipulate differentiated cells, such as cartilage, which was impossible in the past. Conventionally, for example, cells could be collected in a pellet form, and the cell mass could be differentiated to make about 2 mm ³ , but if it exceeds this size, use a scaffold. I have to.

  The differentiation step included in the artificial tissue preparation method provided in the present invention can be performed before or after the provision of the cells.

  The cells used in the present invention can be those in primary culture, but are not limited thereto, and subcultured cells (for example, three or more generations) can also be used. Preferably, when cells that have been passaged are used, it is preferable to use cells that have been passaged 4 or more, more preferably cells that have been passaged 5 or more, and more preferably cells that have been passaged 6 or more. It is advantageous to use. This is because the upper limit of the cell density increases as the number of passages increases to a certain extent, so it is considered that a denser artificial tissue can be produced, but this is not a limitation, but rather a certain range of passage numbers. (E.g. 3rd to 8th generations) seems appropriate.

In the present invention, the cells are preferably provided at a cell density of 5.0 × 10 ⁴ / cm ² or more, but not limited thereto. This is because a sufficiently strong artificial tissue can be provided by sufficiently increasing the cell density. However, it is understood that the lower limit can be lower. It is understood that those skilled in the art can define such a lower limit based on the description herein.

  In one embodiment, as cells that can be used in the present invention, for example, myoblasts, synovial cells, adipocytes, mesenchymal stem cells (eg, derived from adipose tissue or bone marrow) can be used. It is not limited to. Such cells are applicable to, for example, the heart, bone, cartilage, tendon, ligament, joint, and meniscus.

(Artificial tissues and composites)
The artificial tissue or complex provided in the present invention can be implemented by any method described in the present invention or the method described in WO2005 / 012512. In this specification, the artificial tissue provided in the present invention is an implantable artificial tissue. In the past, attempts have been made to produce artificial tissues by cell culture, but all have become artificial tissues suitable for transplantation due to their size, strength, physical damage when detached from the culture vessel, etc. There wasn't. The present invention provides a tissue culture method that is not problematic in terms of size, strength, and the like, and is not particularly difficult when exfoliated, by culturing cells in the presence of the above-described extracellular matrix production promoting factor. sponsored. By providing such a tissue culture method, an implantable artificial tissue is provided for the first time.

  It will be understood that the complex provided in the present invention comprising a cell and a component derived from the cell preferably consists essentially of the cell and a component derived from the cell. Here, the composite of the present invention is provided for reinforcing, repairing or regenerating a part of a living body.

  As used herein, “complex” refers to a complex of a cell and another component due to some interaction. Therefore, it is understood that the composite of the present invention often has an artificial tissue-like appearance, and in this sense, what is referred to as an artificial tissue overlaps.

  The present invention provides a scaffold-free artificial tissue or complex. By providing such a scaffold-free artificial tissue, a therapeutic method and a therapeutic agent excellent in the course after transplantation are provided.

  The present invention also solves the problem caused by contamination of the scaffold itself, which is one of the long-term regulatory problems in biologics, due to the fact that it is a scaffold-free artificial tissue. In addition, despite the absence of the scaffold, the therapeutic effect is better than the conventional one.

  In addition, when a scaffold is used, the orientation of transplanted cells in the scaffold, problems with intercellular adhesion, changes in the scaffold itself (inflammation), and engraftment of the scaffold to the recipient tissue Can solve problems such as sex.

  The artificial tissues and complexes provided in the present invention can also be distinguished from conventional cell therapies in that they are self-assembled and have biological connections within them.

  The prosthetic tissue and composites provided in the present invention should also be noted for their versatility because they can easily form a three-dimensional form and can be easily designed to the desired form.

  Since the artificial tissue and complex provided in the present invention have a biological bond with the surrounding tissue, cells and the post-transplant environment, post-surgery colonization is good, cells are well supplied, etc. Excellent effect is achieved. As an effect of the present invention, there is also a point that complicated treatment can be performed by forming a composite tissue with other artificial tissue or the like because of such good biological connectivity.

  Another advantage of the present invention is that differentiation can be induced after being provided as an artificial tissue and a complex. Alternatively, before being provided as an artificial tissue and / or complex, differentiation can be induced to form such an artificial tissue and / or complex.

  Another effect of the present invention is that, from the viewpoint of cell transplantation, compared to conventional transplantation of only cells, transplantation using a sheet, etc., there is an effect such as comprehensive cell supply by coating, which has better replaceability. Is in the point to be.

  According to the present invention, an implantable artificial tissue having a biological binding ability is provided. Such a tissue has the above-mentioned features and effects, so that it has become possible to treat a site where transplantation with an artificial product has not been considered. The artificial tissue provided in the present invention has biological bonds within the tissue and between other tissues and actually functions in transplantation therapy. Such an artificial tissue is not provided in the prior art, but is provided for the first time. The artificial tissue provided in the present invention has the ability to biologically bind to surrounding tissues, cells and the like after transplantation (preferably by an extracellular matrix), and thus has an excellent postoperative course. Conventionally, there is no artificial tissue having such a biological binding ability. Therefore, the present invention has a therapeutic effect that cannot be achieved by the conventional artificial tissue.

  Furthermore, the present invention has enabled medical procedures that provide a therapeutic effect by filling and replacing diseased portions and / or covering diseased sites.

  In addition, the present invention can be used in combination with other artificial tissues (for example, artificial bones made of hydroxyapatite, microfibrous collagen medical materials, etc.) and biologically combined with other artificial tissues. The improvement of the establishment of artificial tissue that could not be achieved was obtained.

  In the artificial tissue provided in the present invention, an extracellular matrix such as fibronectin or vitronectin or a cell adhesion factor is distributed throughout the tissue, whereas in cell sheet engineering, the cell adhesion factor is localized on the adhesion surface of the cultured cells to the petri dish. Yes. The main difference is that in cell sheet engineering, the main body of the sheet is a cell, and the sheet is a mass of cells with an adhesive factor glue attached to the bottom, but our artificial tissue literally wraps the cells in the extracellular matrix. However, it can be said that the “organization” is significantly different from the conventional one.

  As a cell transplantation method that does not use a scaffold (base) material, a cell sheet engineering technique using a temperature sensitive culture dish described in Non-Patent Document 3 is representative, and an international evaluation has been obtained as an original technique. Yes. However, when this cell sheet technology is used, a single sheet is often fragile, and in order to obtain a strength that can withstand surgical operations such as transplantation, it has been necessary to devise operations such as overlapping sheets. . The present invention has solved these problems.

  Unlike the cell sheet technology, the cell / matrix complex developed in this study does not require a temperature-sensitive culture dish, and it is easy to layer cells / matrix. There is no technology in the world that can produce a complex layered in 10 layers or more without using so-called Feeder cells such as rodent stromal cells in about 3 weeks. In addition, by adjusting the matrix production conditions of synovial cells, it is also possible to create a complex that retains strength that allows surgical operations such as grasping and moving the complex without the need for special instruments. The present invention can be said to have an effect from the viewpoint of being an innovative and innovative method for reliably and safely performing cell transplantation even in the world.

  In a preferred embodiment, the artificial tissue provided in the present invention has an integration capability with the surroundings. Here, the surroundings typically refer to the environment to be transplanted, and examples thereof include tissues and cells. The biological binding ability of surrounding tissues, cells, etc. can be confirmed by, for example, micrographs, physical tests, staining of biological markers, and the like. Conventional artificial tissues have little affinity with tissues in the transplanted environment, and it has not been assumed that biological binding ability can be exhibited. Rather, the conventional artificial tissue relies on the regenerative ability of the living body and plays a role in bridging the self-cells and the like to accumulate and regenerate, and therefore was not intended for permanent use. Therefore, the artificial tissue provided in the present invention should be construed as being able to constitute a transplant treatment in a true sense. Accordingly, the biological binding ability referred to in the present invention preferably includes the ability to adhere to surrounding cells and / or extracellular matrix. Such adhesive ability can be measured by in vitro culture assays with tissue pieces (eg, cartilage pieces).

  `` Disease '' targeted by the present invention refers to any disease accompanied by degeneration, necrosis, damage, etc., for example, osteoarthritis, osteochondral damage, refractory fracture, osteonecrosis, cartilage damage, half-moon damage, It can be ligament injury, tendon injury, cartilage degeneration, meniscal degeneration, disc degeneration, ligament degeneration or tendon degeneration, any heart disease with tissue damage. Examples of such heart diseases include heart failure, refractory heart failure, myocardial infarction, cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, and the like. The combination therapy of the present invention can also be applied to regenerate damage to organs other than the heart as long as the purpose is to regenerate tissue damage. In a specific embodiment, the disease targeted by the method of the present invention is refractory heart failure.

  As used herein, “prophylaxis” or “prevention” refers to a certain disease or disorder that prevents or reduces such a condition before it is triggered. Treatment that occurs in a condition or delays the occurrence of the condition.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means that it is reduced. As used herein, “curative treatment” refers to treatment involving the eradication of the root or cause of a pathological process. Therefore, when a radical treatment is given, in principle, the disease will not recur.

  As used herein, “prognosis” is also referred to as prognosis treatment, and refers to diagnosing or treating a post-treatment condition for a certain disease or disorder.

  In a preferred embodiment, the artificial tissue or complex provided in the present invention is biologically bound in three dimensions. Here, biological binding has been described elsewhere herein and includes, but is not limited to, physical binding, electrical coupling, etc., by an extracellular matrix. In particular, in a preferred embodiment, it is important that the extracellular matrix in the tissue is biologically bound. Since no artificial tissue or composite comprising such a biologically bound hydroxyapatite has been provided so far, the artificial tissue or composite provided in the present invention of this embodiment is It can be said that the structure is also novel. Furthermore, in a preferred embodiment having the ability to biologically bind to the surroundings, an artificial tissue or composite that has not been heretofore provided in that it can form a part of a living body even after transplantation. It can be said to be a body.

In one preferred embodiment, the artificial tissue or complex provided in the present invention can be said to differ from a conventional hydroxyapatite complex in that it contains cells with attached hydroxyapatite. In particular, the high density should be noted in that the cell density can include, for example, up to 5 × 10 ⁶ / cm ² .

  Preferably, the artificial tissue or complex provided in the present invention is substantially composed of a cell or a substance derived from the cell. Biocompatibility and biofixability can be improved by being composed only of cells and substances derived from the cells (for example, extracellular matrix). As used herein, “substantially composed” includes a cell and a substance derived from the cell, as long as no other harmful effects (here, mainly adverse effects on transplantation) are given. , Is defined as including other substances and should be understood as such herein. Substances that do not have such harmful effects are known to those skilled in the art or can be identified by performing simple tests. Representative examples include, but are not limited to, any additive component approved by the Ministry of Health, Labor and Welfare, FDA, and the like, components associated with cell culture, and the like. Here, the substance derived from a cell typically includes an extracellular matrix. In particular, the artificial tissue or complex provided in the present invention preferably contains cells and an extracellular matrix in an appropriate ratio. Such a suitable ratio is, for example, a ratio of cells to extracellular matrix in the range of 1: 3 to 20: 1, and the tissue strength is regulated by the ratio of cells to extracellular matrix. It can be used by adjusting the ratio of cells and extracellular matrix according to the application and the mechanical environment at the transplant destination. Preferred ratios will vary depending on the treatment intended, but such fluctuations will be apparent to those skilled in the art and can be estimated by examining the ratio of cells and extracellular matrix in the organ of interest. .

  Preferably, an artificial tissue substantially composed of a cell and an extracellular matrix derived from the cell has not been known so far. Therefore, it can be said that the present invention provides a completely new artificial tissue.

  Preferably, the extracellular matrix constituting the present invention includes collagen I, collagen III, vitronectin and fibronectin. Preferably, such various extracellular matrices are preferably included in all listed ones, and are preferably mixed together. Alternatively, they are preferably interspersed with extracellular matrix throughout. Such a distribution has a particular effect in that it improves compatibility and affinity with the environment when transplanted. In the present invention, intercellular matrix that promotes cell adhesion to a substrate, cell spreading, and cell chemotaxis by containing collagen (I, III), vitronectin, fibronectin and the like in particular. It is known to have the feature that adhesion is also promoted. However, an artificial tissue in which collagen (I, III), vitronectin, fibronectin and the like are integrated and contained has not been provided so far. Although not wishing to be bound by theory, collagen (types I and III), vitronectin, fibronectin and the like are thought to play a role in exerting biological binding ability with the surroundings. Accordingly, in a preferred embodiment, it is advantageous that vitronectin is dispersed and disposed on the surface in the artificial tissue or complex provided in the present invention. This is because adhesion, establishment, and stability after transplantation are considered to be significantly different.

  Fibronectin is also preferably placed in the artificial tissue or complex provided in the present invention. Fibronectin is known to have a role in regulating cell adhesion, cell shape control, and cell migration. However, an artificial tissue in which fibronectin is expressed has not been provided so far. Without wishing to be bound by theory, fibronectin is also thought to play a role in exerting biological binding ability with the surroundings. Therefore, in a preferred embodiment, it is advantageous that fibronectin is also dispersed and placed on the surface in the artificial tissue provided in the present invention. This is because adhesion, establishment, and stability after transplantation are considered to be significantly different.

  In a preferred embodiment, it will be understood that placing the extracellular matrix used in the present invention in an artificial tissue or complex is easily accomplished by the artificial tissue production method provided in the present invention, It is understood that the manufacturing method is not limited thereto.

  In a more preferred embodiment, the extracellular matrix used in the present invention is advantageously distributed. It is understood that the arrangement of the extracellular matrix in such a dispersed form is impossible with conventional artificial tissues and is provided for the first time by the present invention.

In a preferred embodiment, the extracellular matrix dispersed in the artificial tissue or complex is within the range of about 1: 3 to 3: 1 when comparing distribution densities in any two 1 cm ² sections. It is preferable to be within the ratio. For the measurement of the distribution density, any method known in the art can be used, and examples thereof include immunostaining.

In a preferred embodiment, the extracellular matrix used in the present invention is about 1: 2 to 2: 1, more preferably about 1.5: when comparing the distribution density in any two 1 cm ² sections. The ratio falls within the range of 1 to 1.5: 1. Advantageously, the distribution of the extracellular matrix arrangement is evenly distributed. Therefore, it may be preferably dispersed substantially uniformly, but is not limited thereto. As used herein, “homogeneous” refers to a state in which an extracellular matrix is evenly dispersed in a complex.

  In one embodiment, the extracellular matrix disposed in the present invention can include collagen I, collagen III, vitronectin, fibronectin and the like.

  In another embodiment, the artificial tissue or complex provided in the present invention can use heterologous cells, allogeneic cells, syngeneic cells or autologous cells. In the present invention, even when allogeneic cells were used, it was found that harmful side reactions such as immune rejection hardly occur particularly when mesenchymal cells are used. Therefore, the present invention is not only used for ex vivo treatment but also for a treatment method in which an artificial tissue is produced and used by using another person's cells without using an immune rejection inhibitor or the like. .

  In one preferred embodiment, the cells included in the artificial tissue or complex provided in the present invention may include both stem cells and differentiated cells. In a preferred embodiment, the cell contained in the three-dimensional structure of the present invention is a mesenchymal cell. Although not bound by theory, mesenchymal cells are preferable because they are excellent in compatibility with organs such as the heart, and may have the ability to differentiate into various tissues or organs. It is.

  Such mesenchymal cells may be mesenchymal stem cells or mesenchymal differentiated cells.

  Examples of mesenchymal cells used in the present invention include, but are not limited to, bone marrow, adipocytes, synoviocytes, myoblasts, skeletal muscle cells, and the like. As the mesenchymal stem cells used in the present specification, for example, adipose tissue-derived stem cells, bone marrow-derived stem cells, and the like can be used.

  In a preferred embodiment, the cells used in the present invention are advantageously cells derived from the subject to which the artificial tissue or complex is applied. In such cases, the cells used herein are also referred to as autologous cells, but by using autologous cells, immune rejection can be prevented or reduced.

  Alternatively, in another embodiment, the cells used in the present invention may be cells not derived from the subject to which the artificial tissue or complex is applied. In this case, it is preferable that measures for preventing immune rejection are taken.

  The artificial tissue or complex provided in the present invention may be provided as a medicament. Alternatively, the artificial tissue or complex provided in the present invention may be prepared by a doctor or the like at a medical site, or after a doctor prepares a cell, the cell is cultured by a third party to obtain a three-dimensional structure. It may be prepared as a body and used for surgery. In this case, cell culture can be carried out by a person skilled in the art of cell culture without being a doctor. Accordingly, those skilled in the art can determine the culture conditions according to the type of cells and the intended transplant site after reading the disclosure in this specification.

  In another embodiment, the artificial tissue or complex provided in the present invention is preferably isolated. In this case, the isolation means that it is separated from the scaffold, support, culture solution, etc. used for the culture. Due to the substantial absence of substances such as scaffolds, the artificial tissue provided in the present invention can suppress adverse reactions such as immune rejection and inflammatory reactions after transplantation.

Bottom area of the artificial tissue provided in the present invention may be, for ^example, a 1 cm 2 to 20 cm ^2, is not limited thereto and may be a 1 cm ² or less, say even 20 cm ² or more. It is understood that the essence of the present invention is that any size (area, volume) can be produced, and the size is not limited.

  In a preferred embodiment, the artificial tissue provided in the present invention is thick. The wall thickness refers to a thickness that is usually strong enough to cover a site to be transplanted. Such an area is, for example, at least about 50 μm or more, more preferably at least about 100 μm or more, at least about 200 μm or more, at least about 300 μm or more, more preferably at least about 400 μm or more, Or even more preferably at least about 500 μm or about 1 mm. It is understood that in some cases, thicknesses of 3 mm or more and 5 mm or more can be produced. Alternatively, such a thickness may be less than 1 mm. It is understood that the essence of the present invention is that any thickness of tissue or composite can be made and is not limited to size.

  The present invention provides a scaffold-free artificial tissue or complex. By providing such a scaffold-free artificial tissue, a therapeutic method and a therapeutic agent excellent in the course after transplantation are provided.

  The present invention also solves the problem caused by contamination of the scaffold itself, which is one of the long-term regulatory problems in biologics, due to the fact that it is a scaffold-free artificial tissue. In addition, despite the absence of the scaffold, the therapeutic effect is better than the conventional one.

  In addition, when a scaffold is used, the orientation of transplanted cells in the scaffold, problems with intercellular adhesion, changes in the scaffold itself (inflammation), and engraftment of the scaffold to the recipient tissue Can solve problems such as sex.

  The artificial tissues and complexes provided in the present invention can also be distinguished from conventional cell therapies in that they are self-assembled and have biological connections within them.

  The prosthetic tissue and composites provided in the present invention should also be noted for their versatility because they can easily form a three-dimensional form and can be easily designed to the desired form.

  The artificial tissue and complex provided in the present invention have a biological bond with the surrounding tissue, cells, and the post-transplant environment, so that postoperative colonization is good, cells are reliably supplied, etc. Excellent effect is achieved. As an effect of the present invention, there is also a point that complicated treatment can be performed by forming a composite tissue with other artificial tissue or the like because of such good biological connectivity.

  Another advantage of the present invention is that differentiation can be induced after being provided as an artificial tissue and a complex. Alternatively, before being provided as an artificial tissue and / or complex, differentiation can be induced to form such an artificial tissue and / or complex.

  Another effect of the present invention is that, from the viewpoint of cell transplantation, compared to conventional transplantation of only cells, transplantation using a sheet, etc., there is an effect such as comprehensive cell supply by coating, which has better replaceability. Is in the point to be.

  According to the present invention, an implantable artificial tissue having a biological binding ability is provided. Such a tissue has the above-mentioned features and effects, so that it has become possible to treat a site where transplantation with an artificial product has not been considered. The artificial tissue provided in the present invention has a biological connection within the tissue and between the environment and actually functions in transplantation therapy. Such an artificial tissue is not provided in the prior art, but is provided for the first time. The artificial tissue provided in the present invention has the ability to biologically bind to surrounding tissues, cells and the like after transplantation (preferably by an extracellular matrix), and thus has an excellent postoperative course. Conventionally, there is no artificial tissue having such a biological binding ability. Therefore, the present invention has a therapeutic effect that cannot be achieved by the conventional artificial tissue.

  Furthermore, the present invention has enabled medical procedures that provide a therapeutic effect by filling and replacing diseased portions and / or covering diseased sites.

  In addition, the present invention can be used together with other artificial tissues (for example, artificial bones made of hydroxyapatite, microfibrous collagen medical materials, etc.), and by combining biologically with other artificial tissues, An improvement in the establishment of artificial tissue that could not be achieved was obtained. Such an organization is called a composite organization.

  In the artificial tissue or complex provided in the present invention, an extracellular matrix such as fibronectin or vitronectin or a cell adhesion factor is distributed throughout the tissue. On the other hand, in cell sheet engineering, the cell adhesion factor is localized on the adhesion surface of the cultured cells to the petri dish. Exist. The main difference is that in cell sheet engineering, the main body of the sheet is a cell, and the sheet is a mass of cells with an adhesive factor glue attached to the bottom, but our artificial tissue literally wraps the cells in the extracellular matrix. However, it can be said that the “organization” is significantly different from the conventional one.

  As a cell transplantation method that does not use a scaffold (base) material, a cell sheet engineering technique using a temperature-sensitive culture dish according to Non-Patent Document 3 is representative, and international evaluation has been obtained as an original technique. However, when this cell sheet technology is used, a single sheet is often fragile, and in order to obtain a strength that can withstand surgical operations such as transplantation, it has been necessary to devise operations such as overlapping sheets. . The present invention has solved these problems.

  Unlike the cell sheet technology, the cell / matrix complex developed in this study does not require a temperature-sensitive culture dish, and it is easy to layer cells / matrix. There is no technology in the world that can produce a complex layered in 10 layers or more without using so-called Feeder cells such as rodent stromal cells in about 3 weeks. In addition, by adjusting the matrix production conditions of the cells, it is possible to create a complex that retains the strength that enables surgical operations such as grasping and moving the complex without the need for special instruments. The invention can be said to have an effect from the viewpoint of being an original and epoch-making method for reliably and safely carrying out cell transplantation from a global perspective.

  In another embodiment, the artificial tissue or composite provided in the present invention is flexible. The flexibility makes it particularly suitable for the reinforcement of motor organs. Examples of such organs include, but are not limited to, the heart, blood vessels, and muscles.

  In another embodiment, the artificial tissue or composite provided in the present invention has elasticity. By having elasticity, it can be applied to expanding and contracting organs such as the heart and muscles. Such stretchability is a property that has not been achieved by a cell sheet or the like prepared by a conventional method. Preferably, the artificial tissue or composite provided in the present invention has a strength capable of withstanding the pulsating motion of the heart. The strength that can withstand such pulsation is, for example, that it is at least about 50% or more, preferably at least about 75% or more, more preferably at least about 100% or more of the strength of the natural myocardium. But not limited to them.

  In a preferred embodiment, the artificial tissue or complex provided in the present invention has biological bonds in all three dimensions. Some artificial tissues prepared by conventional methods have some biological binding in the two-dimensional direction, but tissues that are in the three-dimensional direction have not been prepared. Thus, the prosthetic tissue provided in the present invention thus has the property of being substantially implantable in any application by having all biological connections in three dimensions.

  Examples of biological bonds serving as an index in the artificial tissue or complex provided in the present invention include, but are not limited to, extracellular matrix mutual coupling, electrical coupling, and intercellular communication. The interaction of the extracellular matrix can be observed by appropriately staining the adhesion between cells with a microscope. Electrical coupling can be observed by measuring the potential.

  In a preferred embodiment, the artificial tissue provided in the present invention has a tissue strength that can be clinically applied. The tissue strength that can be clinically applied varies depending on the site where the application is intended. Such strength can be determined by those skilled in the art by referring to the disclosure of the present specification and taking into account well-known techniques in the art. The artificial tissue provided in the present invention may have a low tensile strength. Such tensile strength increases as the matrix concentration in the cell / extracellular matrix ratio increases and decreases as the cell ratio increases. One feature of the present invention is that the strength can be freely adjusted as necessary. It is characterized by being able to be relatively strong and weak relative to the tissue to be transplanted. Therefore, it is understood that the purpose can be set according to such an arbitrary place.

  In another embodiment, the strength of the artificial tissue or composite provided in the present invention is preferably sufficient to be self-supporting. Conventional artificial tissues did not have self-supporting properties after production. Therefore, when the conventional artificial tissue is moved, at least a part thereof is damaged. However, when the technique of the present invention was used, an artificial tissue having self-supporting property was provided. Accordingly, the present invention provides an artificial tissue that could not be provided by the prior art. Such self-supporting property is obtained when the tissue is picked up with tweezers having a tip of 0.5 to 3 mm (preferably a tip having a thickness of 1 to 2 mm, more preferably a tip having a thickness of 1 mm). It is preferred that it is not substantially destroyed. Here, it can be confirmed by visual observation that it is not substantially destroyed. However, after picking up under the above conditions, for example, it is confirmed by checking that water does not leak when a water leak test is performed. Can do. Alternatively, the self-supporting property as described above can be confirmed not by tweezers but by being broken when picked up by hand.

  In certain embodiments, the portion intended for clinical application includes, but is not limited to, bone, joint, cartilage, meniscus, tendon, ligament, kidney, liver, synovium, heart and the like. The origin of the cells contained in the artificial tissue provided in the present invention is characterized in that it is not affected by the clinically applied use.

  Further, when the tissue is cartilage, the artificial tissue remains without being artificially fixed after the artificial tissue is transplanted into the defect in the joint tissue (for example, after a few minutes). Can be used to test adhesion.

  In another aspect, the present invention provides a cell culture composition for producing artificial tissue from cells. The cell culture composition includes components for maintaining or growing cells (for example, a commercially available medium); and an extracellular matrix production promoting factor. The explanation regarding such an extracellular matrix production promoting factor was described in detail in the above-mentioned artificial tissue production method. Therefore, this extracellular matrix production promoting factor is ascorbic acid or a derivative thereof (for example, TGF-β1, TGF-β3, ascorbic acid 1-phosphate or a salt thereof, ascorbic acid 2-phosphate or a salt thereof, L-ascorbic acid or Its salts). Here, ascorbic acid diphosphate or a salt thereof contained in the culture composition of the present invention is present at least 0.1 mM, or in the case of a concentrated culture composition, it is at least 0.1 mM at the time of preparation. include. Alternatively, ascorbic acids appear to have little effect if they are 0.1 mM or higher. It can be said that 0.1 mM is sufficient. In the case of TGF-β1 and TGF-β3, an amount of 1 ng / ml or more, typically 10 ng / ml may be sufficient.

  Alternatively, the present invention can provide a composition for artificial tissue production comprising such an extracellular matrix production promoting factor.

  The extracellular matrix production promoting factor used in the artificial tissue production method used in the present invention includes ascorbic acid 2-phosphate (see HataR, SenooH. JCell Physiol. 1989; 138 (1): 8-16). In the present invention, the production of extracellular matrix is promoted by adding a certain amount or more of ascorbic acid diphosphate. As a result, the resulting artificial tissue or complex is cured and easily peeled off. Thereafter, self-contraction is performed by applying a stimulus for peeling. Moreover, it has not been described in the report of Hata et al. That the tissue is hardened and acquires the property of being easily detached after being cultured with such ascorbic acid added. Without being bound by theory, one notable difference is that Hata et al. Differ significantly in the cell density used. Moreover, Hata et al. Did not even suggest a curing effect, and such a curing and shrinkage effect and an effect of being easily peeled were found for the first time by the present invention and are provided in the present invention. It can be said that the artificial tissue is at least completely different from the conventionally produced artificial tissue in that it is produced through procedures such as hardening, shrinkage, and peeling.

  In a preferred embodiment, the ascorbic acid diphosphate used in the present invention is usually present at at least 0.01 mM, preferably at least 0.05 mM, more preferably at least 0.1 mM. More preferably it is present at a concentration of at least 0.2 mM, more preferably at a concentration of at least 0.5 mM. More preferably, the minimum concentration can be 1.0 mM.

Although it does not specifically limit about a cell density, In one embodiment, a cell is arrange | positioned by 5 * 10 < ⁴ > cell-5 * 10 < ⁶ > cell per cm < ² >. This condition can be applied to, for example, myoblasts. In this case, the extracellular matrix production promoting factor is preferably provided as at least 0.1 mM as ascorbic acids. This is because a thick artificial tissue can be created. In this case, when the concentration is increased, a dense artificial tissue is formed in the extracellular matrix. When the amount is small, the amount of extracellular matrix decreases, but the self-supporting property is maintained.

(Hydroxyapatite and hydroxyapatite complex formation method)
Living bones, teeth, and the like are matrices in which hydroxyapatite, which is an inorganic substance (hereinafter sometimes abbreviated as hydroxyapatite), and collagen, which is a protein, are complexed at a molecular level and arranged three-dimensionally. It is known that a biocompatible ceramic material or the like can be used for repair when a bone or tooth is damaged. For example, the product name “Bioglass” (manufactured by Nippon Electric Glass Co. Ltd., Otsu. Siga. Japan, component mainly used as periodontal filler; Na ₂ O—CaO—SiO ₂ —P ₂ O ₅ ), Apatite and wollastonite, mainly used as bone filler, sintered hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), artificial pancreas, iliac spacer, etc. (CaO-SiO ₂₎ crystallized glass containing a (trade name "Cerabone a-W", Nippon Electric glass Co.Ltd., Otsu.Siga.Japan , Ltd.) and the like are known. In order to use these ceramic materials as substitutes for bone, attempts have been made to form them on the surface of a material having high strength such as metal. In addition, a method of forming a hydroxyapatite layer on the surface of various organic polymer materials that are highly flexible and durable and expected to be applied to artificial biological tissues other than bone, so-called biomimetic reaction A method called is being developed. In this biomimetic reaction, glass particles mainly composed of CaO and SiO ₂ are immersed in an aqueous solution (pseudo body fluid) having an ionic concentration equal to that of human body fluid, and then an organic polymer material is immersed in the organic polymer material. After a large number of apatite nuclei are formed on the surface of this, only this organic polymer material is immersed and reacted in an aqueous solution having an ion concentration 1.5 times that of the simulated body fluid. According to this biomimetic reaction, it has been reported that apatite nuclei grow naturally on organic polymer materials, and a dense and homogeneous bone-like hydroxyapatite layer is formed to an arbitrary thickness (J. et al. Biomed.Matter.Res.vol.29, p349-357 (1995)). However, this biomimetic reaction has a slow hydroxyapatite production rate, and even if it is reacted for a long period of 2 weeks or longer, it cannot produce hydroxyapatite to the extent that it can be used for an artificial bone on an organic polymer material. This is the actual situation.

  As a method for synthesizing hydroxyapatite used in the present invention, the material is immersed in a calcium solution and then immersed in a phosphoric acid solution. This process is set as one cycle, and this process is repeated to alternately form a hydroxyapatite layer. Some of the inventors have developed an immersion method.

  In the method for producing a hydroxyapatite complex of the present invention, cells or cell aggregates having at least a surface hydrophilized are alternately immersed in a specific calcium solution and a specific phosphoric acid solution, so that the cells or cell aggregates are immersed. A step of generating and fixing hydroxyapatite on at least the surface of the substrate.

  The calcium solution used in the method of the present invention is an aqueous solution containing calcium ions and substantially free of phosphate ions. When phosphate ions are present, the production rate of hydroxyapatite may be reduced. Therefore, the calcium solution is usually an aqueous solution containing calcium ions and no phosphate ions. Examples of the calcium solution include a calcium chloride aqueous solution, a calcium acetate aqueous solution, a calcium chloride tris buffer solution, a calcium acetate tris buffer solution, and a mixture thereof. In the calcium solution, the calcium ion concentration is preferably 0.01 to 10 mol / liter, particularly preferably 0.1 to 1 mol / liter, considering the production rate and production efficiency of hydroxyapatite. A preferable concentration for cell survival is 10 to 200 mM, preferably 10 to 100 mM, and more preferably 10 to 50 mM. The pH of the calcium solution is not particularly limited, but when a Tris buffer solution is used, it is preferably pH 6 to 10, particularly preferably pH 7.4.

  The phosphate solution used in the present invention is an aqueous solution containing phosphate ions and substantially free of calcium ions. When calcium ions are present, the production rate of hydroxyapatite may be reduced. Therefore, the phosphoric acid solution is usually an aqueous solution containing phosphate ions and not containing calcium ions at all. Examples of the phosphoric acid solution include a sodium hydrogen phosphate aqueous solution, a sodium ammonium dihydrogen phosphate aqueous solution, a sodium hydrogen phosphate tris buffer solution, a sodium trihydrogen phosphate tris buffer solution, or a mixture thereof. In the phosphoric acid solution, the phosphate ion concentration is preferably 0.01 to 10 mol / liter, particularly preferably 0.1 to 1 mol / liter, considering the production rate and production efficiency of hydroxyapatite. A preferable concentration for cell survival is 10 to 200 mM, preferably 10 to 100 mM, and more preferably 10 to 50 mM. The pH of the phosphoric acid solution is not particularly limited, but when a Tris buffer solution is used, it is preferably pH 6 to 10, particularly preferably pH 7.4.

The combination of the calcium solution and the phosphoric acid solution used in the present invention is not particularly limited. For example, a combination of a calcium chloride aqueous solution and a sodium hydrogen phosphate aqueous solution, a combination of a calcium acetate aqueous solution and a sodium ammonium dihydrogen phosphate aqueous solution, etc. Can be mentioned. In the calcium solution and the phosphoric acid solution, other ions may be present within a range in which the desired object of the present invention is not impaired. However, when magnesium ions (Mg ²⁺ ) of 2.5 mM or more are present. Is not preferable because tricalcium phosphate (TCP) may be formed.

  In the production method of the present invention, as a method of immersing cells or cell aggregates in a calcium solution and a phosphate solution, (1) after immersing the cells or cell aggregates in a calcium solution, cells are added to the phosphate solution. Or a method of immersing the cell aggregate in one cycle or more, (2) an operation of immersing the cell or cell aggregate in the calcium solution after immersing the cell or cell aggregate in the phosphoric acid solution. The method of performing 1 time or more as 1 cycle, etc. are mentioned. At this time, the amount of hydroxyapatite produced can be increased by repeating the above operations. The number of repetitions of the operation is usually 1 to 200 times, preferably 5 to 100 times. In the case of repeating the above operation, in the method (1), it is not always necessary to end the cell or cell aggregate by immersing the cell or cell aggregate in the phosphate solution, but the cell or cell aggregate is immersed in the calcium solution. It may be terminated. Similarly, in the method (2), it is not always necessary to end the cell or cell aggregate by immersing the cell or cell aggregate in the calcium solution. The method is ended by immersing the cell or cell aggregate in the phosphate solution. May be. At this time, in alternately immersing the cells or cell aggregates in the calcium solution and the phosphate solution, before each immersion, the calcium ions or phosphate ions remaining on the cell or cell aggregate surface are washed with water or the like. After the removal, it is preferable to immerse in the next solution.

  The immersion time for immersing the cells or cell aggregates in the calcium solution can be appropriately selected in consideration of the generation rate and generation efficiency of hydroxyapatite. Usually, the total immersion time is 10 minutes to 7 days, preferably 30 minutes to 3 days, particularly preferably 1 hour to 24 hours. On the other hand, the immersion time for immersing the cells or cell aggregates in the phosphoric acid solution can be appropriately selected in consideration of the generation rate and generation efficiency of hydroxyapatite. Usually, the total immersion time is 10 minutes to 7 days, preferably 30 minutes to 3 days, particularly preferably 1 hour to 24 hours. The immersion time for each time when the operation of immersing cells or cell aggregates in the calcium solution and the phosphoric acid solution is repeated can be appropriately selected in consideration of the preferable total immersion time. The solution temperature of each solution when immersing cells or cell aggregates in each solution can be appropriately selected in consideration of the production rate and production efficiency of hydroxyapatite, and is usually 0 to 90 ° C., preferably 4 ~ 80 ° C.

  The hydroxyapatite composite of the present invention can be obtained by the production method described above. The hydroxyapatite of this composite contains crystalline hydroxyapatite, and the crystal form can form various forms of apatite such as flakes and plates. In particular, it is possible to obtain a hydroxyapatite having a novel structure in which a constant crystal plane in a dense layer shape, which has not been obtained conventionally, is maintained. In addition, the hydroxyapatite layer in this composite is firmly attached to the substrate, and it is considered that the polymer chain and hydroxyapatite are complexed at the molecular level. In the composite of the present invention, the thickness of the hydroxyapatite layer can be appropriately selected depending on the type and shape of cells or cell aggregates, the use of the composite, and the like. For example, the thickness of the hydroxyapatite layer in the composite is suitably about 0.0001 to 5 mm. The shape of the complex can be changed to various shapes by appropriately selecting the shape of the cell or cell aggregate, or by processing the complex into a desired shape. Therefore, the composite of the present invention can be made into various biocompatible materials including artificial bones by appropriately selecting the type and form of cells or cell aggregates and processing the composite into a desired shape. be able to. The composite of the present invention can be subjected to a known sintering step, surface treatment step and the like depending on the application.

  In the preferable method for producing hydroxyapatite according to the present invention, cells or cell aggregates having at least a hydrophilic surface are used as cells or cell aggregates, and hydroxyapatite is generated and fixed by an alternate immersion method. Since the hydroxyapatite adheres firmly to the body and the hydroxyapatite exhibits crystallinity, a composite having a composition and structure similar to bone can be obtained quickly and easily. The composite obtained by this method is useful as various biocompatible materials including artificial bones.

A preferred hydroxyapatite has a composition substantially Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ , and the crystal structure measured by X-ray diffraction is a specific diffraction peak in the crystal structure of human bone And have diffraction peaks at least at 31 to 32 degrees and 26 degrees, respectively. As an apparatus used for the X-ray diffraction, a trade name “Geigerflex 2013” (manufactured by Rigaku Co., Tokyo, Japan) or the like can be used. The X-ray diffraction conditions include X-rays: CuKα / 30 kV / 15 mA, scan speed: 2 degrees / min.

In a preferred hydroxyapatite, the composition substantially indicates Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ may contain inevitable components at the time of production or the like, and the dissolved carbonate ions at the time of production. This means that carbonate apatite and / or apatite partially lacking Ca may be included. Furthermore, the hydroxyapatite crystal structure of the present invention may have other peaks in addition to the specific diffraction peak in X-ray diffraction as long as it approximates that of human bone.

  In order to produce a preferred hydroxyapatite of the present invention, for example, cells or cell aggregates are immersed in a specific first aqueous solution and a specific second aqueous solution, and at least on the surface of the cells or cell aggregates. The step (A) for producing the hydroxyapatite of the present invention and the step (B) for recovering the hydroxyapatite of the present invention from cells or cell aggregates can be obtained by a method of performing as essential steps.

  The specific first aqueous solution used in the step (A) used in the method of the present invention is an aqueous solution containing calcium ions and substantially free of phosphate ions. When phosphate ions are present, the production rate of the hydroxyapatite of the present invention may be reduced. Therefore, the first aqueous solution usually contains calcium ions and does not contain phosphate ions at all. . Examples of the first aqueous solution include a calcium chloride aqueous solution, a calcium acetate aqueous solution, a calcium chloride tris buffer solution, a calcium acetate tris buffer solution, and a mixture thereof.

  In the first aqueous solution used in the method of the present invention, the calcium ion concentration is preferably 0.01 to 10 mol / liter, particularly preferably 0.8, in consideration of the production rate and production efficiency of the hydroxyapatite of the present invention. 1 to 1 mol / liter. The pH of the first aqueous solution is not particularly limited, but when a Tris buffer solution is used, it is preferably pH 6 to 10, particularly preferably pH 7.4.

  The specific second solution used in the step (A) used in the method of the present invention is an aqueous solution containing phosphate ions and substantially free of calcium ions. When calcium acid ions are present, the production rate of the hydroxyapatite of the present invention may be reduced. Therefore, the second aqueous solution usually contains phosphate ions and does not contain any calcium ions. . Examples of the second aqueous solution include a sodium hydrogen phosphate aqueous solution, a sodium ammonium dihydrogen phosphate aqueous solution, a tris buffer solution of sodium hydrogen phosphate, a tris buffer solution of sodium ammonium dihydrogen phosphate, or a mixture thereof.

  In the second aqueous solution used in the method of the present invention, the phosphate ion concentration is preferably 0.01 to 10 mol / liter, particularly preferably 0 in consideration of the production rate and production efficiency of the hydroxyapatite of the present invention. .1 to 1 mol / liter. The pH of the second aqueous solution is not particularly limited, but when a Tris buffer solution is used, it is preferably pH 6 to 10, particularly preferably pH 7.4.

  The combination of the first aqueous solution and the second aqueous solution used in the method of the present invention is not particularly limited. For example, a combination of a calcium chloride aqueous solution and a sodium hydrogen phosphate aqueous solution, a calcium acetate aqueous solution and a sodium ammonium dihydrogen phosphate A combination with an aqueous solution can be mentioned.

In the first aqueous solution and the second aqueous solution used in the method of the present invention, other ions may be present as long as the desired purpose of the present invention is not impaired. The presence of (Mg ²⁺ ) is not preferable because tricalcium phosphate (TCP) may be formed.

  As the cell or cell aggregate used in the step (A) used in the method of the present invention, any cell described in this specification can be used.

  Examples of various metals used in the method of the present invention include stainless steel, titanium, platinum, tantalum, cobalt, chromium, molybdenum, or alloys of these two or more metals; sol-gel method products using titanium such as titania gel. It is done.

  In the step (A), as a method of immersing cells or cell aggregates in the first aqueous solution and the second aqueous solution, (1) after immersing cells or cell aggregates in the first aqueous solution, A method of immersing cells or cell aggregates in the aqueous solution 2 at least once, (2) after immersing cells or cell aggregates in the second aqueous solution, cells or cell aggregates in the first aqueous solution; The method etc. which perform the operation to immerse 1 time or more are mentioned. At this time, the production amount of the hydroxyapatite of the present invention can be increased by repeating the above operations. The number of repetitions of the operation is usually 2 to 20 times, preferably 5 to 10 times. In the case of repeating the above operation, in the method (1), it is not always necessary to finally end the cells or cell aggregates by immersing the cells or cell aggregates in the second aqueous solution. It may be dipped and terminated. Similarly, in the method (2), it is not always necessary to end the process by immersing cells or cell aggregates in the first aqueous solution, but by immersing the cells or cell aggregates in the second aqueous solution. It may be terminated.

  The soaking time for immersing cells or cell aggregates in the first aqueous solution can be appropriately selected in consideration of the production rate and production efficiency of the hydroxyapatite of the present invention. Usually, the total immersion time is 10 minutes to 7 days, preferably 30 minutes to 3 days, particularly preferably 1 hour to 24 hours. On the other hand, the immersion time for immersing cells or cell aggregates in the second aqueous solution can also be appropriately selected in consideration of the generation rate and generation efficiency of the hydroxyapatite of the present invention. Usually, the total immersion time is 10 minutes to 7 days, preferably 30 minutes to 3 days, particularly preferably 1 hour to 24 hours. The immersion time for each time when the operation of immersing cells or cell aggregates in the first aqueous solution and the second aqueous solution is repeated can be appropriately selected in consideration of the preferable total immersion time. For example, each dipping time can be 10 seconds to 1 minute. This can be repeated 10 to 100 times, for example, 10 cycles, 20 cycles, 30 cycles.

  The solution temperature of each aqueous solution when cells or cell aggregates are immersed in each aqueous solution used in the method of the present invention can be appropriately selected in consideration of the production rate and production efficiency of the hydroxyapatite of the present invention. 0 to 90 ° C, preferably 4 to 80 ° C.

  By the above step (A), the hydroxyapatite of the present invention can be adhered and formed even inside the cell or cell aggregate depending on at least the surface of the cell or cell aggregate, the type and form of the cell or cell aggregate, and the like. it can. Usually, the hydroxyapatite of the present invention is formed in a layered manner on the surface of a cell or cell aggregate. The complex of the hydroxyapatite of the present invention obtained by this step (A) and cells or cell aggregates can be used for the complex of the present invention, artificial living tissue, and medical material described later.

  In order to produce the preferred hydroxyapatite of the present invention, the step (B) of recovering the hydroxyapatite of the present invention from cells or cell aggregates is then performed.

  The obtained hydroxyapatite of the present invention can be used for production of various medical materials as it is or after being molded into a desired shape, or by performing various known sintering treatments and surface treatments.

  A preferred complex of the present invention comprises the hydroxyapatite of the present invention at least on the surface of the cell or cell aggregate, and depending on the type and form of the cell or cell aggregate, even inside the cell or cell aggregate. . The hydroxyapatite of the present invention formed on cells or cell aggregates is usually layered, but the thickness of the layer is appropriately selected depending on the type and shape of the cells or cell aggregates, the use of the complex, etc. can do. For example, the thickness of the hydroxyapatite layer of the present invention in the composite is suitably about 0.0001 to 5 mm. The shape of the complex can be changed to various shapes by appropriately selecting the shape of the cell or cell aggregate, or by processing the complex into a desired shape.

  Accordingly, the composite of the present invention can be made into various artificial biological tissues including artificial bones by appropriately selecting the type and form of cells or cell aggregates and processing the composite into a desired shape. Can do. For example, when the complex cells or cell aggregates are organic polymer polymers such as hydrogel materials, which are flexible among the aforementioned organic polymer polymers, they are highly elastic and maintain a cartilage-like shape. And a composite having excellent biocompatibility due to the hydroxyapatite of the present invention. Since such a composite has good adhesion to a flesh tissue in a living body, is rich in elasticity, and has excellent durability against torsion and the like, it has been difficult to carry out with various conventional ceramic materials. Application to artificial living tissue is possible.

  In order to manufacture the preferable composite_body | complex of this invention, it can manufacture by the process (A) mentioned above, for example. The types of cells or cell aggregates are preferably the above-mentioned specific examples, and can be appropriately selected according to the application. Moreover, after a process (A), it can process into a desired shape, a well-known sintering process, a surface treatment process, etc. can be performed.

  The preferred hydroxyapatite of the present invention has both a composition close to that of bone and a crystal structure, and is therefore useful as various biological tissues including artificial bones.

(Best Mode for Carrying Out the Invention)
The best mode of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Complex)
In one aspect, the present invention provides a complex of cells and hydroxyapatite.

  In a preferred embodiment, the hydroxyapatite contained in the complex of the present invention is attached to the surface of the cell. This attachment may be in any form, but preferably the hydroxyapatite is directly bonded to the surface of the cell. More preferably, the hydroxyapatite is present in a state where microcrystals are formed on the cell membrane surface of the cell.

  In one preferred embodiment, the cells contained in the complex of the present invention have a three-dimensional tissue structure. Preferably, the cell is biologically integrated in the third dimension. More preferably, the cell has an integration capability with the surroundings. Such biological binding ability may include the ability to adhere to surrounding cells and / or extracellular matrix. These bindings can be measured by any method described herein. Having these bonds can be functionally present in constructing a three-dimensional structure such as bone and is suitable for transplantation.

  In one preferred embodiment, it comprises an extracellular matrix derived from the cells included in the present invention. The extracellular matrix contained here may contain collagen I, collagen III, vitronectin, fibronectin and the like. Preferably, the collagen may contain 10 to 90% collagen I and 90 to 10% collagen III. The extracellular matrix contained here may contain 10 to 90% of collagen I. The extracellular matrix contained here may contain 10 to 90% collagen II. The extracellular matrix contained here may contain 10 to 90% of collagen III. In one preferred embodiment, a proteoglycan derived from a cell included in the present invention is included. The proteoglycans contained here may include hyaluronic acid, fibronectin and the like. The proteoglycan contained here may contain at least 0.1 to 90% of hyaluronic acid. The proteoglycan contained here may contain at least 0.1 to 90% of fibronectin.

  In one embodiment, an extracellular matrix is disposed between the cells and the hydroxyapatite. Here, in a preferred embodiment, the extracellular matrix is dispersed and arranged in the complex.

In one preferred embodiment, the hydroxyapatite is present as crystals. The reason why it is preferable to exist as a crystal is, for example, the reason that it is preferable to exist as a crystal includes, for example, the reasons of mechanical strength, elastic modulus, cell adhesion, protein adsorption, and orientation of collagen fibers. Can be mentioned. The crystal state is, for example, hexagonal, plate-like, rod-like, particle-like, rod-like particle, etc., and the degree of crystallinity is preferably 60% or less like the degree of crystallinity of bone. In one embodiment, the present invention The cells used in this complex are stem cells. By using stem cells, it can be applied to various regenerative treatments. Preferably, the cell is a tissue stem cell. Tissue stem cells can be collected from various tissues, have few ethical problems, and have a certain fate. Therefore, tissue stem cells are particularly preferable for use in applications where the fate is determined. The cells used here are cells that express collagen. Preferably, the cell is a cell that expresses type I collagen and type III collagen. Such cells can be suitably used as a complex used for bone grafting.

  In a preferred embodiment, the cell is a mesenchymal stem cell. Such stem cells can be collected from, for example, bone marrow and fat.

  In a preferred embodiment, the cell is a living cell. Including living cells in a complex has not been achieved by conventional techniques and has been provided for the first time in the present invention. In this sense, the present invention can be said to be groundbreaking. Such a living state can be measured by confirming that it retains at least 50% of the amount of DNA compared to the amount of DNA before complex formation, or by being detected by a live cell detection method. . The live cell detection method can be an MTT assay or a WST-1 assay.

(Hydroxyapatite production method)
In one aspect, the present invention provides a calcium solution containing calcium ions and substantially free of phosphate ions, and a phosphate solution containing phosphate ions and substantially free of calcium ions. A method for producing a hydroxyapatite complex comprising the step of alternately immersing cells or cell populations to produce and fix hydroxyapatite on at least the surface of the cells or cell populations.

  In one embodiment, the cell or cell population is an artificial tissue. It has not been possible for an artificial tissue to form a complex with hydroxyapatite. In the present invention, the first artificial tissue is provided. Such cells or cell populations preferably include stem cells.

  In one embodiment, the concentration substantially free of calcium ions is a concentration at which the formation of hydroxyapatite does not proceed. Such a concentration is a concentration of about 0.001 to 0.1 mM, but varies depending on the situation and environment, and such a value can be appropriately determined by those skilled in the art.

  In one embodiment, the concentration substantially free of phosphate ions is a concentration at which the formation of hydroxyapatite does not proceed. Such a concentration is a concentration of about 0.001 to 0.1 mM, but varies depending on the situation and environment, and such a value can be appropriately determined by those skilled in the art.

  In one embodiment, after the step of producing and fixing hydroxyapatite, the method includes the step of confirming the degree of hydroxyapatite deposition on the cell surface in the complex and repeating the step of producing and fixing based on the result. To do. One of the features in a preferred embodiment of the present invention is that the alternate soaking time is extremely short (eg, typically 10 minutes or less, eg 1 minute or less, illustratively 10 seconds), and calcium and phosphoric acid. By reducing the solution concentration to about 10 mM, the effect on the cells is made as low as possible. Therefore, the immersion time may be longer (eg, 30 minutes, 1 hour, etc.) as long as it does not affect the cells, and less than 10 seconds as long as hydroxyapatite is deposited. It is understood that it may be. Accordingly, the immersion time may be a unit of several hours such as 1 hour or 30 minutes or a unit of several tens of minutes, preferably 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, hydroxyapatite. As long as deposition is confirmed, it may be as short as 10 seconds or less. In addition, since hydroxyapatite is sufficiently deposited even for 10 seconds, the present invention should have one of the effects in that it has been found that a composite can be easily and efficiently produced by such a method. is there.

  In a preferred embodiment, the concentration of phosphate ion and / or calcium ion to be used is 10 to 200 mM, preferably 10 to 100 mM, more preferably 10 to 50 mM as a preferable concentration for cell survival.

  In one aspect, the present invention provides a hydroxyapatite composite obtained by the production method of the present invention.

  In one other aspect, the present invention provides a biocompatible material consisting essentially of the composite of the present invention. Such materials can be used for transplantation surgery.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited only to the embodiments, but only by the claims.

  In the following examples, experiments were conducted after all the target patients were notified in advance and received an outlet. Animal handling complied with the standards stipulated by Osaka University. Reagents used in the following examples were obtained from Wako Pure Chemicals, Sigma-Aldrich Japan and the like.

(Preparation Example 1: Synoviocytes)
In this preparation example, various synovial cells were used to produce an artificial tissue, that is, a cell aggregate. This will be described below.

<Preparation of cells>
Swine (LWD ternary hybrid, 2 to 3 months old used when collecting cells) Synovial cells are collected from knee joint, treated with collagenase, then 10% FBS-DMEM medium (available from HyClone, DMEM from GIBCO Culture and subculture were performed using the obtained one). Regarding the number of passages, synovial cells have been reported to have pluripotency even at 10 passages. In this example, up to 10 passages were used, but more passages were used depending on the purpose. It is understood that cells are also used. When transplanting into the human body, it is autotransplantation, but it is necessary to shorten the culture period in order to reduce the risk of infection and the like while ensuring a sufficient number of cells.

  In consideration of these, various passage cells were used. Actually conducted cells were tested in primary culture cells, 1 passage, 2 passages, 3 passages, 4 passages, 5 passages, 6 passages, 8 passages, 10 passages. Using these, an artificial tissue was used.

<Production of artificial tissue>
On a 35 mm dish, 60 mm dish or 100 mm dish (BD Biosciences, cell culture dish / multiwell cell culture plate), 4.0 × 10 ⁶ synovial cells were seeded in 2 ml of 10% FBS-DMEM medium and cultured. At that time, ascorbic acid was added. The dish, ascorbic acid and cell concentrations are as follows:
Dish: BD Biosciences, cell culture dish / multiwell cell culture plate Ascorbic acid diphosphate: 0 mM, 0.1 mM, 0.5 mM, 1 mM, 2 mM, and 5 mM
Number of cells: 5 × 10 ⁴ cells / cm ² , 1 × 10 ⁵ cells / cm ² , 2.5 × 10 ⁵ cells / cm ² , 4.0 × 10 ⁵ cells / cm ² , 5 × 10 ⁵ cells / cm ² , 7.5 × 10 ⁵ cells / cm ² , 1 × 10 ⁶ cells / cm ² , 5 × 10 ⁶ cells / cm ² , and 1 × 10 ⁷ cells / cm ²
The medium was changed twice / week until the planned culture period. When the culture period was reached, the cell sheet and the dish were separated while pipetting with a 100 μl pipetteman around the dish. Once separated, the cell sheet was made as flat as possible by gently shaking the dish. Thereafter, 1 ml of a medium was added, the cell sheet was completely suspended, and the cell sheet contracted by being left for 2 hours, and an artificial tissue was prepared by three-dimensionalization.

(Hematoxylin and eosin (HE) staining)
In order to observe the fixing and fate of the support in the cells, HE staining was performed. The procedure is as follows. Deparaffinization (for example, with pure ethanol) and water washing were performed as necessary, and the sample was soaked with omni hematoxylin for 10 minutes. Thereafter, it was washed with running water and colored with ammonia water for 30 seconds. Thereafter, it is washed with running water for 5 minutes, dyed with a 10-fold diluted solution of eosin hydrochloride for 2 minutes, dehydrated, clarified and sealed.

(Various extracellular matrix)
1) Prepare 5 μm thick sections from frozen stock.

2) Fix this section in acetone for 5-10 minutes at -20 ° C (paraffin blocks need to be paraffin removed and rehydrated)
3) Endogenous peroxide activity is blocked in 0.3% H ₂ O ₂ in methanol for 20 minutes at room temperature (1 ml 30% H ₂ O ₂ +99 ml methanol).
4) Wash with PBS (3 x 5 minutes).

  5) Incubate with monoclonal primary antibody (mouse or rabbit antibody against various extracellular matrix) (1 μl antibody + 200 μl PBS / slide) overnight at 4 ° C. in a wet chamber.

  6) Wash with PBS the next day (3 x 5 minutes).

  7) anti-mouse and anti-rabbit no. 1 Biotinylated bond is applied for 30 minutes-1 hour at room temperature (3 drops directly on slide).

  8) Wash with PBS (3 x 5 minutes).

  9) Streptavidin HRP no. 2 soak for LSAB and soak for 1015 minutes.

  10) Wash with PBS (3 x 5 minutes).

11) Drop DAB (5ml DAB + 5μl H2O2)
12) Observe the brownish color with a microscope.

  13) Immerse in water for about 5 minutes.

  14) Soak HE for 30 seconds-1 minute.

  15) Wash several times.

  16) Wash once with ion exchange water.

  17) Wash with 80% ethanol for 1 minute.

  18) Wash with 90% ethanol for 1 minute.

19) Wash with 100% ethanol for 1 minute. (3 times)
20) Wash with xylene three times for 1 minute. Then apply a coverslip.

  21) Look at color development.

  As a result, when ascorbic acid 2-phosphate is added as an extracellular matrix production promoting factor, cell stratification is only slightly observed. On the other hand, it was observed that the stratification progressed and the three-dimensionalization was promoted, as shown on the left, by exfoliating the sheet-like cells from the bottom of the culture and causing them to self-shrink. Even when synovial cells were used, large tissues without pores were produced. This tissue was thick and rich in extracellular matrix. It can be seen that when 0.1 mM or more was added, formation of the extracellular matrix was promoted. It can be seen that the tissue becomes strong enough to allow detachment after 3 days of culture. As the number of culture days increases, the extracellular matrix density fluctuates and increases.

  These were artificial tissues that were peeled from the bottom of the culture dish and self-contracted. The artificial tissue is produced in a sheet form, and when left after being peeled from the dish, self-shrinkage occurs and three-dimensionalization proceeds. The tissue findings show that the cells are scattered in multiple layers and stratified.

  Next, various markers including the extracellular matrix were stained.

  It can be seen that various extracellular matrices (collagen type I, type II, type III, type IV, fibronectin, vitronectin, etc.) were present. In the immunostaining, collagens I and III were strongly stained, but collagen II staining was limited to a part. From a strong viewpoint, collagen is stained at a site slightly away from the nucleus, confirming that it is an extracellular matrix. On the other hand, fibronectin and vitronectin, which are considered to be important as cell adhesion factors, are stained in a region close to the nucleus unlike collagen, and can be confirmed to exist around the cell.

  Also, for the production of artificial tissue, passage 3 to 8 seems to be preferred, but it seems that any passage can be used.

  As a comparison, an example of staining with normal tissue and collagen sponge (CMI, Amgen, USA) is shown. For example, a normal tissue (normal synovial tissue, tendon tissue, cartilage tissue, skin and meniscal tissue) can be compared with a staining example of a commercially available collagen sponge used as a comparative example. Fibronectin, vitronectin, negative controls, and HE staining can be used. Conventional artificial tissue is not stained with fibronectin and vitronectin. Therefore, the artificial tissue that can be used in the present invention is different from the conventional artificial tissue, and the existing collagen scaffold does not include the fibronectin and vitronectin adhesion factors. The originality of the organization becomes clear. No extracellular matrix is stained. Comparing the normal tissue and the artificial tissue of this example, it is confirmed that the artificial tissue is close to the natural state.

  In addition, when the artificial tissue of the present invention was brought into contact with a filter paper for removing water, it was difficult to adhere to the filter paper and peel it off by hand.

  Furthermore, the amount of hydroxyproline revealed that collagen production was significantly promoted when 0.1 mM or more of ascorbic acid diphosphate was added. The production amount was almost proportional to the culture period.

(Confirmation of biological integration)
It is known that biological binding cannot be ensured after transplantation using conventional collagen gels. In this example, a conventional collagen gel (trade name 3% type I collagen, Koken Co., Ltd., Tokyo, Japan) was used to embed synovial cells (10 ⁵ cells / ml) therein, and cartilage When transplanted into the defect, the bond between the collagen gel and the adjacent cartilage was insufficient and a crack was observed.

  On the other hand, when the artificial tissue produced according to the present invention is introduced into the pig body in vivo, it can be seen that a biological bond is established histologically.

(Preparation Example 2: Fat-derived tissue)
Next, an artificial tissue was prepared using cells derived from adipose tissue.

  A) Cells were collected as follows.

  1) A sample was collected from a fat pad of the knee joint.

  2) This specimen was washed with PBS.

  3) This specimen was minced as finely as possible.

  4) 10 ml of collagenase (0.1%) was added and shaken in a 37 ° C. water bath for 1 hour.

  5) The same amount of DMEM (supplemented with 10% FBS) was added and passed through a 70 μl filter (available from Millipore, etc.).

6) Cells passed through the filter and the residue remaining in the filter were placed in 5 ml of DMEM supplemented with 10% FBS and cultured in a 25 cm ² flask (available from Falcon et al.).

  7) Cells (including mesenchymal stem cells) attached to the bottom of the flask were taken out and subjected to the following artificial tissue preparation.

B) Production method of artificial tissue Next, an artificial tissue was produced using the cells derived from fat. Ascorbic acid diphosphate was used under the conditions of 0 mM (none), 0.1 mM, 0.5 mM, 1.0 mM, and 5.0 mM. The production was performed according to the method for producing the synovial cells (described in Example 1). As initial seeding conditions, 5 × 10 ⁴ cells / cm ² were used. The culture period was 14 days. Artificial tissues were also created from cells derived from adipose tissue, and fibronectin and vitronectin were abundant as in synovial cell-derived artificial tissues. Collagen I and III were also abundantly expressed.

Ascorbic acid diphosphate 0 mM: Tangential coefficient (Young's modulus) 0.28
Ascorbic acid diphosphate 1.0 mM: Tangential coefficient (Young's modulus) 1.33
(Preparation Example 3: Production of artificial tissue from human adipocytes)
By local anesthesia, a small incision is made in a portion to be collected (for example, around the knee joint) of a human patient, and a few dozen mg of fat cells are collected. The collected adipocytes can be processed in the same manner as synovial cells to produce a three-dimensional artificial tissue.

(Example 1: Preparation of cell hydroxyapatite complex by alternate dipping method)
In this example, a cell hydroxyapatite complex was prepared by an alternate immersion method. The protocol is shown below. The cells or cell populations used in the preparation examples were used.

1. 3DST was immersed in 5 mL of a solution of 1.200 mM calcium chloride / 0.05 M Tris buffer (pH = 7.4) for 10 seconds.

2. The excessively adsorbed calcium ions were removed by immersing in 5 mL of a 0.05 M Tris buffer solution (pH = 7.4) for washing for 10 seconds.

3. 3DST was immersed in 5 mL of a solution of 120 mM mM disodium hydrogen phosphate / 0.05 M Tris buffer (pH = 7.4) for 10 seconds.

4). The excessively adsorbed phosphate ions were removed by immersing in 5 mL of a 0.05 M Tris buffer solution (pH = 7.4) for washing for 10 seconds.

  The above four steps were set as one cycle, and the number of times was repeated as appropriate.

  A schematic diagram is shown in FIG. 1A. The actual cycle pattern is shown in FIG. 1B.

  FIG. 2 shows a photograph of a three-dimensional structure (also referred to as 3DST) after repeating alternate dipping for 5 × 20 cycles using the above-mentioned calcium and phosphate solutions.

  FIG. 3 shows that 3DST obtained by alternately immersing 5 · 10 · 20 cycles using calcium and phosphoric acid solutions at the above concentrations was fixed by immersing in 10% formalin solution for 10 minutes, and using a freeze dryer. After performing lyophilization for one day, analysis is performed using an infrared absorption spectrum measuring instrument, and multiple results are shown. Expression of absorption spectra of 560, 600, 960, and 1070 cm −1 characteristic of apatite was confirmed.

  FIG. 4 shows that 3DST obtained by alternately immersing 5 · 10 · 20 cycles using calcium and phosphoric acid solutions at the above concentrations was fixed by immersing in 10% formalin solution for 10 minutes, and then using a freeze dryer. The results of measurement using an X-ray diffractometer after lyophilization for a day are shown. 26, 32, and 39 (degree) peaks specific to the crystal structure of apatite were confirmed.

  FIG. 5 shows that 3DST obtained by alternately immersing for 10 and 20 cycles using the above-described calcium and phosphoric acid solutions was fixed by immersing in 10% formalin solution for 10 minutes, and 50%, 60%, 70%, After immersing in 80%, 90%, 100% ethanol for 10 minutes each, immersing in 100% tert-butanol for 10 minutes, freezing overnight in a freezer, and lyophilizing for 3 hours using a freeze dryer, The result of having observed the state of 3DST combined with apatite with a scanning electron microscope is shown. The upper left 0 cycle is 450 times, the 20 upper right is 3000 times, and the lower right is 35000 times.

(Example 2: Confirmation of cell viability)
In Example 2, it was confirmed whether or not the cells were alive in the complex prepared in Example 1. The result is shown in FIG.

As shown in FIG. 6, human synovial cells adhered to a cover glass at a cell concentration of 3 × 10 ⁴ cells / cm ² for 24 hours were 10, 50, 100, 200 mM calcium chloride / 0.05 M Tris. Cells are viable by LIVE / DEAD staining by alternately dipping for 10-20 cycles using buffer (pH = 7.4) and disodium hydrogen phosphate / 0.05 M Tris buffer (pH = 7.4). -The death was confirmed (the 3DST was not used, and the toxicity of the calcium and phosphate solution concentrations on the cells was evaluated by directly immersing the cells alternately). From this result, it was found that a calcium / phosphate solution of 50 mM or less does not show cytotoxicity. At 100 and 200 mM, the presence of dead cells was confirmed because the cells stained red.

  LIVE / DEAD staining: After 10-20 cycles of alternate soaking using calcium / phosphate solution of each concentration, cover glass is placed in a 35φ culture dish and contains 1 μM calcein AM and 2 μM ethidium homodimer-1. 2 mL of phosphate buffered saline (PBS) was added to the dish and incubated in an incubator for 45 minutes. Thereafter, the solution was removed, and 1 mL of PBS was added for rinsing, followed by immobilization by immersion in a 10% formalin solution for 10 minutes. After rinsing with 1 mL of PBS, one drop of the gel mount agent was dropped on the cover glass, and the gel was solidified in the dark and covered with a new cover glass to prepare a sample. After the gel was sufficiently solidified, it was observed using a fluorescence microscope. When cells were alive, calcein reacted and emitted green fluorescence, but when cells died, ethidium homodimer-1 reacted and emitted red fluorescence.

(Example 3: Effect of solution concentration and number of alternate soaking by evaluation of 3DST DNA amount after alternate soaking)
In this example, the effects of the solution concentration and the number of alternate soaking times by the evaluation of the 3DST DNA amount after the alternate soaking were confirmed. The result is shown in FIG.

  As shown in FIG. 7, 10, 50, 100, 200 mM calcium chloride / 0.05 M Tris buffer (pH = 7.4) and disodium hydrogen phosphate / 0.05 M Tris buffer (pH = 7.4) was used, and 3DST was alternately immersed for 10.20 cycles, and the amount of DNA was quantified using the QIAGEN DNeasy Tissue Kit. A decrease in the amount of DNA means cell death. This result shows that there is no cytotoxicity at the concentration up to 50 mM as in the case of LIVE / DEAD staining as shown in FIG. Decreased and confirmed to be cytotoxic.

  From these results, it was found that, by controlling the alternate immersion concentration, it can typically be complexed with hydroxyapatite in the state where cells are alive in the range of 10 to 50 mM.

(Example 4: Confirmation of crystal on composite)
Next, the crystal on the composite was confirmed in this example. The result is shown in FIG.

  FIG. 8 shows the immobilization of 3DST, which was alternately immersed for 20 cycles using 10 mM calcium and phosphate solution, by immersing in 10% formalin solution for 10 minutes, 50%, 60%, 70%, 80% Soaked in 90%, 100% ethanol for 10 minutes each, immersed in 100% tert-butanol for 10 minutes, frozen in a freezer overnight, lyophilized for 3 hours using a freeze dryer, It is the result of having observed the mode of 3DST composited with apatite with an electron microscope. The upper left 0 cycle is 350 times, the upper right 10 mM-20 cycle is 950 times, and the lower left is 1000 times. At 10 mM-20 cycle, apatite crystals were observed on the cell membrane surface and on the ECM fiber surface where the cells were produced. The apatite crystal is about 100 to 500 nm in size.

(Example 5: Confirmation of cell viability)
In this example, the viability of the cells in the complex of the present invention was confirmed by other staining methods. FIG. 9A shows the result.

  As shown in FIG. 9A, 3DST that was alternately immersed for 20 cycles using 10 mM calcium and phosphate solution was placed on a 60φ culture dish, and 2 mL of phenol red-free medium containing 10% WST-1 reagent was added. It is the dyeing | staining photograph after adding and incubating for 1 hour using an incubator. When the cells are alive, WST-1 is changed to formazan and develops a yellow color. The fact that the entire 3DST is stained yellow means that the cells are alive. FIG. 9B shows the coloring mechanism.

  As described above, the complex produced in the present invention is composed only of cells and ECM components produced by the cells in addition to hydroxyapatite, does not contain a synthetic compound, and is excellent in safety.

  Moreover, since it adheres to a living tissue, it is excellent for transplantation.

  Typically, it can be prepared simply by adding ascorbic acid 2-phosphate (phosphorylated vitamin C), and anyone can easily create it.

  In a preferred embodiment, the complex of the present invention comprises mesenchymal stem cells (MSCs), so that highly treated human synovial cells (passages 4-7) are treated with 0.1 mM ascorbic acid 2-phosphate (phosphorylated vitamins). It can be prepared simply by adding C). Thus, it is expected as a new treatment method having a higher bone regeneration effect in place of the conventional treatment methods for bone / cartilage damage (autologous bone transplantation / apatite implantation / mesenchymal stem cell transplantation).

(Example 6: Animal experiment using rabbits)
In this example, it was confirmed whether or not bone repair progressed mainly in the deep layer in the group in which the cell defect-hydroxyapatite complex was filled in the bone defect at the time of 6 weeks after the operation. 3DBT and HA-3DBT were transplanted into mature Japanese white rabbits, and histological evaluation, safranin O staining, and mechanical tests were performed.

(Rabbit surgery protocol)
(anesthesia)
After injecting Nembutal 50 mg / kg into the 1-2-year-old adult rabbit (Japanese white rabbit) with physiological saline over 5-10 minutes from the ear vein, Selactal 5-7 mg / kg is injected intramuscularly in the buttocks I do. Antibiotic (cefamedin 0.2 g) is administered to prevent postoperative infection.

(Surgery)
After positioning and cleansing the surgical field with sterile drape, the parapatellar approach approached into the knee joint. The front surface of the femoral condyle was exposed by rolling the patella outward. A cylindrical osteochondral defect of φ4 mm × 4 mm is prepared using Mosaic Plastic DP and a dental laboratory drill. After filling the bone and cartilage defect with the cell tissue alone or the cartilage layer corresponding to the cell tissue body and the subchondral bone level corresponding to the cell tissue body-hydroxyapatite complex, the hemostasis was confirmed and the joint was confirmed. The operation was completed by suturing the sac, subcutaneous and epidermis in layers. The state of the experiment is shown in FIG. As shown in FIG. 10, 3 DBT (3DBT group; n = 3) or HA-3DBT alone (HaP-3DBT group; n = 3) was transplanted into an osteochondral defect of φ4 mm × 4 mm prepared in the knee joint, and 6 weeks Later, tissue findings and micro CT were used for evaluation. As in the above example, 3DST prepared 4.0 × 10 ⁵ / cm ² rabbit synovial cells in a monolayer under the addition of ascorbic acid, suspended this, induced self-contraction, and created 3DST. .

(Evaluation method)
Post-operative evaluation was performed by histological examination, radiography, and micro CT.

(DAPI staining)
Since DAPI [4 ′, 6-diamino-2-phenylindole] stains the nuclei of living organisms, it is used in combination with protozoan identification tests.

DAPI dispensing ・ DAPI is weighed in microtubes after purchase.
・ Stand the microtube on a precision top balance and tare it.
・ Take a small amount with a spatula, put it in a microtube, and record the amount.
・ Store in the manner specified by the manufacturer (shading, refrigeration or freezing).

Dissolve in a ratio of 1 mL of methanol (2 mg / mL) to 2 mg of DAPI preservation solution / DAPI.
・ After adjustment, shield from light and refrigerate.

Add 10 to 50 μL of DAPI stock solution to 50 mL of DAPI staining solution / PBS and mix (0.4 to 2.0 μg / mL).
・ After adjustment, shield from light and refrigerate and use as soon as possible.
DAPI stain ・ Place appropriate amount on sample and stain for 2-5 minutes.
• Wash with PBS.

  The morphology of the nucleus stained as described above is observed with a fluorescence microscope, and the evaluation is performed based on the degree of aggregation and the color development amount.

(result)
At 6 weeks after surgery, bone repair was significantly enhanced mainly in the deep layer in the group in which the bone defect part was filled with the cell tissue-hydroxyapatite complex. No repair reaction was observed. FIG. 11 shows the formed three-dimensional composite. The right side is a complex, and the left side is a three-dimensional tissue unit. FIG. 11 shows that hydroxyapatite (HaP) crystals were successfully deposited on the surface (HaP-3DBT) while cells survived by alternately immersing 3DBT in Ca ⁺ solution and PO 4 ⁻ solution. .

  Specific results are shown in FIGS.

  FIG. 12 shows the result of transplanting a three-dimensional tissue alone. Upper left: Micro CT, Lower left: Tissue image (1x), Right: Tissue image magnification (200x). No bone formation was observed at the 3DBT transplant. a shows a corresponding part.

  FIG. 13 shows the result of transplanting the three-dimensional tissue complex. Upper left: Micro CT, Lower left: Tissue image (1x), Right: Tissue image magnification (200x). No bone formation was observed at the 3DBT transplant. In the HA-3DBT transplantation group, good bone formation was observed at the center of the deep layer of 3DBT transplanted into the bone defect. a shows a corresponding part.

(result)
According to the present example, it can be said that a bone showing a form of Shanghai cotton bone in CT and tissue was formed in the defect part by the transplantation experiment of HA-3DBT.

  Here, the difference between cancellous bone and cortical bone is that if the bone is classified according to its properties and functions, it can be divided into a portion with a hollow structure like cancellous (cancellous bone) and a hard and dense portion surrounding it (cortical bone). is made of. The cancellous bone refers to the cancellous bone contained in the spinal vertebral body, ribs, near the ends of the long bones, and the like. The function of cancellous bone is mainly called metabolic bone because it contributes to bone metabolism. The cancellous bone has a metabolic rate 5 to 8 times faster than the cortical bone, and nearly 20% of the bone is replaced with new bone in one year. Cortical bone refers to bone that thinly covers cancellous bone, or bone that is thick bamboo tube like the central part of long bone. Cortical bone serves to maintain mechanical strength. New bone replacement is characterized by a slow metabolic rate of 4% per year.

  From the above, it is considered possible to interpret that a bone showing a Shanghai cottonbone-like morphology in CT and tissue was formed in the defect by HA-3DBT transplantation experiment.

(Example 7: Determination of collagen composition)
Next, in this example, the collagen composition of the complex of the present invention was determined.

(Collagen composition ratio analysis method)
1. Pretreatment of 3DBT and extraction of collagen with pepsin Wetly weigh 10 mg of 3DBT under 200 mM of 5 mM EDTA / distilled aqueous solution.
II. Centrifugation (10,000 × g, 15 minutes)
III. Discard the supernatant, add the EDTA solution again and sonicate.
IV. Repeat II-III until the supernatant is clear.
V. The precipitate was extracted with 50 ml of 2M urea · 5 mM EDTA / 0.05M Tris-HCl (pH 7.5). Repeat 3 times a day at 4 ° C.
VI. The precipitate was extracted with 50 ml of 4M guanidine hydrochloride / 0.05M Tris-HCl (pH 7.5). Repeat 3 times a day at 4 ° C.
VII. The precipitate is stirred with 20 μg pepsin / 50 mL 0.5 M acetic acid solution and repeated 3 times a day at 4 ° C.
VIII. Add 5M NaOH, quickly neutralize to pH 8-8.5 and leave at room temperature for one day.

(2 Fractionation by salting out)
Collagen is a technique of separating and extracting each type of collagen by utilizing the fact that the solubility of each type varies depending on various conditions of NaCl concentration and pH.
I. Separation of type I / III and type IV / V / VI collagen i. 1-VIII is dialyzed against a 0.7 M NaCl / 0.5 M acetic acid solution.
ii. Ultracentrifugation (100,000 × g, 1 hour).
iii. Preserve the precipitate (including type I and type III collagen).
iv. The supernatant is dialyzed against a 1.2M NaCl / 0.5M acetic acid solution.
v. Ultracentrifugation (100,000 × g, 1 hour).
vi. Preserve the precipitate (including type IV and type V collagen)
vii. Save the supernatant (including type VI collagen)
II. Fractionation of type I and type III collagen i. The precipitate stored in 2-I-iii is dissolved in 1.0 M NaCl / 0.05 M Tris-HCl (pH 7.5).
ii. Ultracentrifugation (100,000 × g, 1 hour).
iii. NaCl is added to the supernatant to make 4.5 M, and it is left overnight at 4 ° C. iv. Ultracentrifugation (100,000 × g, 1 hour).
v. Wash with 4.5 M NaCl / 0.05 M Tris-HCl (pH 7.5) and leave at 4 ° C. overnight.
vi. Ultracentrifugation (100,000 × g, 1 hour).
vii. The precipitate was extracted with 2.0M NaCl / 0.05M Tris-HCl (pH 7.5). Repeat three times.
viii. Ultracentrifugation (100,000 × g, 1 hour).
ix. The supernatant is stored as a type I collagen solution.
x. The precipitate is extracted with 1.7M NaCl / 0.05M Tris-HCl (pH 7.5). Repeat three times. The extract is stored as a type I collagen solution.
xi. Ultracentrifugation (100,000 × g, 1 hour).
xii. The supernatant is stored as a type I collagen solution.
xiii. The precipitate was extracted with 1.0 M NaCl / 0.05 M Tris-HCl (pH 7.5). Repeat three times.
xiv. Ultracentrifugation (100,000 × g, 1 hour).
xv. The supernatant is stored as a type III collagen solution.
III. Fractionation of type IV and type V collagen i. The precipitate stored in 2-I-vi is dissolved in 1.0 M NaCl / 0.05 M Tris-HCl (pH 7.5).
ii. Ultracentrifugation (100,000 × g, 1 hour).
iii. NaCl is added to the supernatant to make 4.5M, and it is left overnight at 4 ° C.
iv. Ultracentrifugation (100,000 × g, 1 hour)
v. Dissolve the precipitate in 0.5M acetic acid.
vi. Ultracentrifugation (100,000 × g, 1 hour)
vii. Add NaCl to the supernatant to 1.2M and leave at 4 ° C. overnight.
viii. Ultracentrifugation (100,000 × g, 1 hour)
ix. The precipitate is dissolved in 0.1 M NaCl / 0.05 M Tris-HCl (pH 7.5).
x. Ultracentrifugation (100,000 × g, 1 hour)
xi. Dialyze against 0.02M NaCl / 0.05M Tris-HCl (pH 7.5).
xii. Ultracentrifugation (100,000 × g, 1 hour)
xiii. The supernatant is stored as a type IV collagen solution.
xiv. The precipitate is dissolved in 0.5 M acetic acid solution and stored as a type V collagen solution.
IV. Fractionation of type VI collagen i. Add NaCl to the supernatant stored in 2-I-vii to 2.0M.
ii. Ultracentrifugation (100,000 × g, 1 hour)
iii. The precipitate is dissolved in 0.5 M acetic acid solution and stored as a type VI collagen solution.

(Separation of I / III type collagen peptide chain by SDS-PAGE method and composition ratio calculation)
Type I collagen is composed of two α1 chains and one α2 chain, and type III collagen is composed of three α1 chains. Type I collagen α1-chain-α1 (I) and type III collagen α1-chain-α1 (III) Are shown to exhibit the same molecular weight by electrophoresis by urea-free SDS-PAGE method. The type I / III collagen solution obtained in 3 above is run by SDS-PAGE, the gel is stained with Coomassie brilliant blue, the concentration of α1 chain band is quantified, and the ratio of type I / type III collagen is determined. Can do.
I. Preparation i. Electrophoresis gel: 5% acrylamide gel (no urea)
ii. Concentration gel: 3% acrylamide gel (no urea)
iii. Electrophoresis buffer: SDS-Tris-Glycine iv. Sample for electrophoresis: 0.1 M dithiothreitol / type I / III type collagen solution 14 μL + 3 × concentrated SDS buffer 7 μL
II. Electrophoresis until marker dye reaches bottom of gel III. The gel is stained with Coomassie Brilliant Blue, and the color development of the band is quantified by computer processing.
IV. Type I: type 3 collagen composition ratio = α1 (I) ÷ 2: α1 (III) ÷ 3.

(Calculation of collagen composition ratio by hydroxyproline quantification method (Wossner method))
Since 10% of all amino acids of collagen is hydroxyproline, this is a method of measuring hydroxyproline in each type of collagen solution to obtain a collagen composition ratio. Since the ratio of type I to type III is determined as 3, this method uses the collagen solution obtained in 2 other than type III as a sample to determine the ratio to type I.
I. The I / III / IV collagen solution is dialyzed against Tris-HCl (pH 7.5), and the V / VI collagen is dialyzed against a 0.5 M acetic acid solution to remove NaCl.
II. Each sample is reacted in a sealed tube with 6N HCl at 110 ° C. for 24 hours to hydrolyze the collagen.
III. Make the necessary reagents listed below.
i. Standard solution of hydroxyproline / 0.001N HCl (0.1 mg / ml). Standard hydroxyproline solutions of various concentrations are prepared by diluting this 10 times, 100 times, 1000 times, 10000 times, and 100000 times.
ii. Acetic acid citrate buffer (pH 6.0) 50 g sodium citrate (monohydrate), 12 ml glacial acetic acid, 120 g sodium acetate (trihydrate), 34 g sodium hydroxide were dissolved in pure water to make 1 L in total, pH 6.0 Adjusted to. Store cold.
iii. Chloramine T solution A solution obtained by dissolving 1.4 g of chloramine T in 20 ml of pure water and adding 30 ml of methyl cellosolve and 50 ml of acetic acid citrate buffer. Cannot be saved.
iv. p-dimethylaminobenzaldehyde solution 20 g of p-dimethylaminobenzaldehyde was dissolved in methyl cellosolve to make a total of 100 ml. Store cold.
IV. Add 1 ml of chloramine T solution to 2 ml of sample solution and standard solution made in III-i.
V. Leave at room temperature for 20 minutes.
VI. Add 1 ml perchloric acid solution (27 ml of 70% perchloric acid + pure water, 100 ml total).
VII. Leave at room temperature for 5 minutes.
VIII. Add 1 ml p-dimethylaminobenzaldehyde solution IX. Heat at 60 ° C. for 20 minutes and cool with water.
X. Measure absorbance at 560 nm. From the hydroxyproline concentration-absorbance standard curve obtained from the absorbance of III-i, the hydroxyproline concentration of each collagen solution is determined. The ratio is defined as the collagen composition ratio of each type.

(result)
By these assays, it can be confirmed whether or not a complex having a desired collagen composition is obtained. For example, it can be confirmed that collagen type I is 60%, collagen type II is 5%, collagen type III is 30%, and collagen type V is 5%.

(Example 8: Induction of bone differentiation)
In this example, it was confirmed whether or not the complex of the present invention functions even when bone differentiation is induced.

  First, an artificial tissue is prepared from synovial cells induced by osteogenesis by culturing from an osteogenesis induction medium (10% FBS-DMEM + 0.1 μM dexamethasone, 10 mM β-glycerophosphate, 0.2 mM ascorbic acid diphosphate) from the beginning. did.

  In addition, it was confirmed that a three-dimensional artificial tissue was prepared without inducing bone differentiation, and then the culture medium was changed to a bone differentiation induction medium and further cultured, whereby calcified bone was generated in the artificial tissue.

  The artificial tissue that does not induce differentiation in appearance is white, while the artificial tissue that has formed bone is white, compared to being transparent. Alizarin Red is strongly stained, and alkaline phosphatase (ALP) staining is also more strongly stained than the control. Thus, it can be confirmed that the artificial tissue by synovial cells has bone differentiation ability.

(Example 9: Induction of cartilage differentiation)
Next, whether or not cartilage differentiation induction can be used in the method for producing a complex of the present invention was confirmed.

(Culture conditions)
Cell density: 4 × 10 ⁴ / cm ²
Conditions: CO ₂ 5%, air 95%, 37 ° C
Using this medium, culture was performed using the following cartilage differentiation induction medium to prepare an artificial tissue.

Cartilage differentiation induction medium: DMEM (GIBCO), FBS (HyClone) 10%, ITS + Premix (insulin, transferrin, selenite) (BD Biosciences) 6.25 μg / ml, dexamethasone (Sigma) 10 ⁻⁷ M, ascorbic acid (WAKO) 50 μg / ml, pyruvate (SIGMA) 100 μg / ml.

  Examples of the results include those obtained by culturing an artificial tissue in a normal medium, cartilage differentiation induction medium, cartilage differentiation induction medium + BMP-2, and cartilage differentiation induction medium + TGF-b1. In any case, the color is stained blue by Alcian blue staining, and enhancement of cartilage-like matrix production can be confirmed, but the effect is remarkable in the BMP-2 addition group.

  When the expression of cartilage-related genes in the artificial tissue (Aggrecan, Col II, Sox9) was tested, the expression of the Sox9 gene, which is a cartilage differentiation marker, increased when the artificial tissue was changed from a normal medium to a cartilage differentiation-inducing medium. When cultured in induction medium + BMP-2, Collagen II gene expression is also enhanced, and more powerful cartilage differentiation can be confirmed. When the cartilage differentiation reaction when the same differentiation-inducing stimulus is applied is compared between the synovial cells cultured in a single layer and the synovial cells that are three-dimensionally artificially synthesized, the stimulation of the cartilage differentiation-inducing medium or cartilage differentiation-inducing medium + BMP-2 is When added, it is confirmed that the expression of the cartilage differentiation marker gene is more clearly expressed in the artificial tissue, and the three-dimensional artificial tissue is confirmed to have a strong cartilage differentiation ability.

(Example 10: cartilage repair)
Next, it is checked whether cartilage repair is possible. Here, allogeneic artificial tissue is used.

In order to confirm the presence or absence of the adhesive ability of the artificial tissue, the same kind of artificial tissue was transplanted on pork cartilage pieces. The artificial tissue was prepared under the conditions of 4.0 × 10 ⁶ cells / 35 mm dish, 1 mM ascorbic acid, and culture period of 7 to 14 days. A wound with a diameter of 6 mm was created on the cartilage piece, the upper layer was excised with a scalpel, chondroitinase ABC (1 U / 1 ml) was added, and the mixture was treated for 5 minutes. The artificial tissue was cast and transplanted to a diameter of 6 mm, and cultured for 7 days. The artificial tissue adheres closely to the cartilage fragment adhesion surface, and fibronectin collects on the adhesion surface.

  Next, pig cartilage transplantation was performed. Similar to the above, a wound with a diameter of 6 mm was created in the femoral endophysis of a living body, the upper layer was excised with a scalpel, chondroitinase ABC (1 U / 1 ml) was added, and after treatment for 5 minutes, the homogenous artificial tissue was treated. Molded and transplanted to a diameter of 6 mm and raised for 10 days. It becomes clear that the artificial tissue and the cartilage tissue are not directly bonded via cells, but the artificial tissue matrix and the cartilage matrix are directly bonded. It can be seen that fibronectin and vitronectin are accumulated on the adhesive surface. That is, it is suggested that these adhesion factors are involved in the adhesion between the artificial tissue and the transplanted tissue. Therefore, it can be seen that the present invention is also characteristic in that natural healing is more significant than conventional artificial tissues or cells.

  Furthermore, the tissue findings after about one month confirm that the prosthetic tissue was firmly attached without causing inflammation because the cartilage damage was biologically bound. Observation of union with surrounding tissues reveals that biological bonds were made. The surface layer portion of the artificial tissue is mainly composed of fibroblast-like cells, but the deep layer portion is mainly composed of cartilage-like cells. This indicates that the transplanted artificial tissue differentiates into cartilage-like tissue over time in the deep layer. No significant rejection was observed at any time, and there was no fear of rejection during allogeneic transplantation.

  Therefore, it can be seen that treatment can be performed without side effects by transplantation using an allogeneic artificial tissue.

(Example 11: Half-moon repair)
Next, it is confirmed whether the complex of the present invention can be used in half-moon repair.

Similar to Example 6 above, the same type of artificial tissue was prepared under the conditions of 4.0 × 10 ⁶ cells / 35 mm dish, 1 mM ascorbic acid diphosphate, and culture period of 7 to 14 days. After creating a defect with a diameter of 5.5 mm in a half moon, an artificial tissue was transplanted. As a protection until the artificial tissue was engrafted, the defective part was covered with a collagen sheet (Nipro Co., Ltd.) and reared for one month. The protocol is shown below.

(anesthesia)
A 15-17 week-old pig (LWD ternary breed) was intramuscularly injected with ketalal 20 mg / kg + ceractal 10 mg / kg from the back neck. Then, after securing an infusion route from the ear vein, the airway was secured by endotracheal intubation, and anesthesia was maintained by continuous infusion of dipriban 0.5 mg / kg / hr. Antibiotic (cefamedin 1 g) was administered to prevent postoperative infection.

(Surgery)
After taking the position and cleansing the surgical field with sterile drape, it entered the knee joint with the paramedial patella approach. After identifying the medial joint capsule, cut off at the center. The assistant flexes and rotates the lower leg, and further pulls forward to expand the anterior corner of the inner half-moon. A cylindrical-shaped defect of φ6.5 mm was created in the same part by mosaic plastic DP (Smith & Nephew). The defect was filled with artificial tissue. In order to protect the artificial tissue until engraftment, a collagen sheet (Nipro Co., Ltd.) was wrapped around the half moon and fixed by sewing. After confirming hemostasis, repair the dissected medial collateral ligament and suture the joint capsule, subcutaneous, and epidermis. The cast was fixed at the knee flexion position, and the operation was completed.

(Evaluation method)
It was performed by macroscopic findings and histological examination.

(result)
The repair was significantly improved in the group filled with the artificial tissue by macroscopic and histological findings 4 weeks after the operation.

  In the histology, notably, eosin positivity was observed in the artificial tissue at 4 weeks after transplantation, formation of a half-moon-like matrix was observed, and biological association with the adjacent half-moon tissue was completed. It was.

(Example 12: Repair of porcine tendon / ligament tissue)
In this embodiment, a repair operation for tendon / ligament tissue is performed in a pig. The injured state of the tendon / ligament tissue is confirmed, and at that time, a part of the synovial cells is collected, the synovial cells are cultured, and an artificial tissue is prepared using the protocol as described in Example 1.

  Next, at the time of surgery, the area around the damaged site of the tendon / ligament tissue is excised and refreshed, and then the artificial tissue prepared as described above is placed. At that time, since the artificial tissue has an adhesive factor, it is fused without the need for suturing. The protocol is shown below.

(anesthesia)
A 15-17 week-old pig (LWD ternary breed) was intramuscularly injected with ketalal 20 mg / kg + ceractal 10 mg / kg from the back neck. Then, after securing an infusion route from the ear vein, the airway was secured by endotracheal intubation, and anesthesia was maintained by continuous infusion of dipriban 0.5 mg / kg / hr. Antibiotic (cefamedin 1 g) was administered to prevent postoperative infection.

(Surgery)
After taking the position and cleansing the surgical field with sterile drape, it entered the knee joint with the paramedial patella approach. The medial collateral ligament was identified by advancing the exfoliation of the medial joint capsule, and a defect of φ3.5mm was created one mosaic finger distal from the femoral attachment of the medial collateral ligament by mosaic plastic DP (Smith & Nephew) . The defect is filled with artificial tissue and then covered with a joint capsule. Sustained joint capsule, subcutaneous and epidermis confirmed hemostasis. The cast was fixed at the knee flexion position, and the operation was completed.
(Evaluation method)
Histological examination was performed based on the method of Frank et al. (J. Orthod. Res. 13; 923-9, 1995).
(result)
The repair is significantly improved in the group filled with artificial tissue by macroscopic and histological findings 6 weeks after surgery.

Example 13: Porcine bone repair
In this example, a bone repair experiment is performed in a pig. Using a protocol as described in Example 1, synovial cell complexes are made.

  The artificial tissue is then applied to the bone. It is applied to the affected area mainly by covering the bone cortex like the periosteal tissue. As a result, it can be seen that the artificial tissue using synovial cells is effective in bone repair. The protocol is shown below.

(anesthesia)
A 15-17 week-old pig (LWD ternary breed) is intramuscularly injected with ketal 20 mg / kg + seractal 10 mg / kg from the back neck. Then, after securing an infusion route from the ear vein, the airway is secured by endotracheal intubation, and anesthesia is maintained by continuous infusion of Diprivan 0.5 mg / kg / hr. Antibiotic (cefamedin 1 g) is administered to prevent postoperative infection.

(Surgery)
After taking the position and cleansing the surgical field with sterile drape, a longitudinal incision was made on the second metatarsal and invaded. The periosteum of the second metatarsal was excised as much as possible with a scalpel to expose the metatarsal surface. A 1.5 cm wide x 3 cm vertical fenestration was performed on the metatarsal surface with a bone flea, and then the bone surface of the fenestration portion was covered with an expanded artificial tissue. Check the adhesion of the artificial tissue and suture the subcutaneous and epidermis. Fix the lower leg cast and finish the operation.

(Evaluation method)
X-ray photography. Micro CT. Histological examination (Result)
In the evaluation 4 weeks after the operation, a significant increase in bone formation in the fenestration part is confirmed in the group coated with the artificial tissue.

  As mentioned above, although this invention was illustrated using the preferable embodiment and Example of this invention, it is understood that the scope of this invention should be interpreted only by a claim. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention has the utility of providing a fundamental treatment method, technique, medicine, and medical device for diseases that have been difficult to treat. In particular, it provides breakthrough treatment and prevention in the sense of promoting the recovery to a state close to natural by complexing hydroxyapatite with cells in a natural form, and a drug used for that purpose, It will be appreciated that the present invention is useful in providing cells, tissues, compositions, systems, kits and the like.

  The need for joint tissue repair / regeneration centering on bone and cartilage targeted by the present invention is the target of bone regeneration, and the number of fractured patients is several hundred thousand per year. The number of potential patients with osteoarthritis is said to be 30 million, the potential market is huge, and its usefulness in the surrounding industries is high. Regenerative medicine research targeting joint tissues centering on bone and cartilage has started intense competition in the world, but the artificial tissue of the present invention is a safety created from cells collected from living bodies such as patients. In addition, it is an original material and highly useful in terms of side effects.

FIG. 1A is a schematic view of the alternate dipping method of the present invention. FIG. 1B shows the actual cycle pattern of the alternate immersion method of the present invention. FIG. 2 shows a photograph (3DST derived from human synovial cells before and after the alternate dipping method) of a three-dimensional structure (also referred to as 3DST) after repeating alternate dipping for 5 × 20 cycles using the calcium and phosphate solutions having the above concentrations. Show. FIG. 3 shows that 3DST obtained by alternately immersing 5 · 10 · 20 cycles using calcium and phosphoric acid solutions at the above concentrations was fixed by immersing in 10% formalin solution for 10 minutes, and using a freeze dryer. The result (IR spectrum measurement) which analyzed using the infrared absorption spectrum measuring machine after performing freeze-drying for one day is shown. Expression of absorption spectra of 560, 600, 960, and 1070 cm −1 characteristic of apatite was confirmed. FIG. 4 shows that 3DST obtained by alternately immersing 5 · 10 · 20 cycles using calcium and phosphoric acid solutions at the above concentrations was fixed by immersing in 10% formalin solution for 10 minutes, and then using a freeze dryer. The result (X-ray diffraction measurement) which measured using the X-ray-diffraction measuring machine after performing freeze-drying for the day is shown. 26, 32, and 39 (degree) peaks specific to the crystal structure of apatite were confirmed. FIG. 5 shows that 3DST obtained by alternately immersing for 10 and 20 cycles using the above-described calcium and phosphoric acid solutions was fixed by immersing in 10% formalin solution for 10 minutes, and 50%, 60%, 70%, After immersing in 80%, 90%, 100% ethanol for 10 minutes each, immersing in 100% tert-butanol for 10 minutes, freezing overnight in a freezer, and lyophilizing for 3 hours using a freeze dryer, The result (scanning electron micrograph) of observing the state of apatite composite 3DST with a scanning electron microscope is shown. The upper left 0 cycle is 450 times, the 20 upper right is 3000 times, and the lower right is 35000 times. As shown in FIG. 6, human synovial cells adhered to a cover glass at a cell concentration of 3 × 10 ⁴ cells / cm ² for 24 hours were 10, 50, 100, 200 mM calcium chloride / 0.05 M Tris buffer. (PH = 7.4) and disodium hydrogen phosphate / 0.05 M Tris buffer solution (pH = 7.4) are used for 10-20 cycles, so that cells survive or die by LIVE / DEAD staining. (3DST was not used, and the toxicity of the calcium and phosphate solution concentration on the cells was evaluated by directly immersing the cells alternately.) Impact). From this result, it was found that a calcium / phosphate solution of 50 mM or less does not show cytotoxicity. At 100 and 200 mM, the presence of dead cells was confirmed because the cells stained red. FIG. 7 shows 10, 50, 100, 200 mM calcium chloride / 0.05 M Tris buffer (pH = 7.4) and disodium hydrogen phosphate / 0.05 M Tris buffer (pH = 7.4). The results of quantifying the amount of DNA using QIAGEN DNeasy Tissue Kit (influence of the solution concentration and the number of alternate soaking by evaluating the amount of 3DST DNA after alternate soaking) Show. A decrease in the amount of DNA means cell death. This result shows that there is no cytotoxicity at the concentration up to 50 mM as in the case of LIVE / DEAD staining as shown in FIG. Decreased and confirmed to be cytotoxic. FIG. 8 shows the immobilization of 3DST, which was alternately immersed for 20 cycles using 10 mM calcium and phosphate solution, by immersing in 10% formalin solution for 10 minutes, 50%, 60%, 70%, 80% Soaked in 90%, 100% ethanol for 10 minutes each, immersed in 100% tert-butanol for 10 minutes, frozen in a freezer overnight, lyophilized for 3 hours using a freeze dryer, It is the result (scanning electron micrograph after 20 cycles at 10 mM) of observing the state of apatite composite 3DST with an electron microscope. The upper left 0 cycle is 350 times, the upper right 10 mM-20 cycle is 950 times, and the lower left is 1000 times. At 10 mM-20 cycle, apatite crystals were observed on the cell membrane surface and on the ECM fiber surface where the cells were produced. The apatite crystal is about 100 to 500 nm in size. As shown in FIG. 9A, 3DST that was alternately immersed for 20 cycles using 10 mM calcium and phosphate solution was placed on a 60φ culture dish, and 2 mL of phenol red-free medium containing 10% WST-1 reagent was added. It is the dyeing | staining photograph after adding and incubating for 1 hour using an incubator (The cell survival confirmation by WST-1 dyeing | staining after 20 cycles at 10 mM). When the cells are alive, WST-1 is changed to formazan and develops a yellow color. The fact that the entire 3DST is stained yellow means that the cells are alive. FIG. 9B shows the coloring mechanism. FIG. 9B shows the color development mechanism (confirmation of cell survival of HAp-3DST) used in FIG. 9A. FIG. 10 shows the state of the experiment of Example 7. After filling the bone and cartilage defect with the cell tissue alone or the cartilage layer corresponding to the cell tissue body and the subchondral bone level corresponding to the cell tissue body-hydroxyapatite complex, the hemostasis was confirmed and the joint was confirmed. The operation was completed by suturing the sac, subcutaneous and epidermis in layers. FIG. 11 shows the formed three-dimensional composite. The right side is a complex, and the left side is a three-dimensional tissue unit. FIG. 12 shows the result of transplanting a three-dimensional tissue alone. Upper left: Micro CT, Lower left: Tissue image (1x), Right: Tissue image magnification (200x). No bone formation was observed at the 3DBT transplant. a shows a corresponding part. FIG. 13 shows the result of transplanting the three-dimensional tissue complex. Upper left: Micro CT, Lower left: Tissue image (1x), Right: Tissue image magnification (200x). No bone formation was observed at the 3DBT transplant. In the HA-3DBT transplantation group, good bone formation was observed at the center of the deep layer of 3DBT transplanted into the bone defect. a shows a corresponding part.

Claims (40)

A complex of cells and hydroxyapatite. The composite according to claim 1, wherein the hydroxyapatite is attached to a surface of the cell. The composite according to claim 1, wherein the hydroxyapatite is directly bound to the surface of the cell. The complex according to claim 1, wherein the hydroxyapatite is present in a state in which microcrystals are formed on a cell membrane surface of the cell. The complex according to claim 1, wherein the cell has a three-dimensional tissue structure. The complex of claim 1, wherein the cells are biologically integrated in a third dimension. The complex according to claim 1, wherein the cells have an integration capability with surroundings. The complex according to claim 4, wherein the biological binding ability includes adhesion ability with surrounding cells and / or extracellular matrix. The complex of claim 1, comprising an extracellular matrix derived from the cells. 10. The complex according to claim 9, wherein the extracellular matrix comprises at least one selected from the group consisting of collagen I, collagen III, vitronectin and fibronectin. The complex according to claim 10, comprising 10 to 90% of collagen I as collagen. The complex according to claim 10, comprising 10 to 90% of collagen II as collagen. The complex according to claim 10, comprising 10 to 90% of collagen III as collagen. The complex according to claim 1, comprising a proteoglycan derived from the cell. 15. The complex according to claim 14, wherein the proteoglycan contains at least 0.1 to 90% hyaluronic acid. 15. The complex of claim 14, wherein the proteoglycan contains at least 0.1-90% fibronectin. The composite according to claim 10, wherein the extracellular matrix is disposed between the cells and the hydroxyapatite. The complex according to claim 10, wherein the extracellular matrix is dispersed in the complex. The complex according to claim 10, wherein the extracellular matrix is uniformly dispersed in the complex. The composite according to claim 1, wherein the hydroxyapatite is present as crystals. 21. The composite according to claim 20, wherein the crystal has a form selected from hexagonal, plate-like and rod-like. The complex according to claim 1, wherein the cell is a stem cell. The complex according to claim 1, wherein the cell is a tissue stem cell. The complex according to claim 1, wherein the cell is a cell expressing collagen. The complex according to claim 1, wherein the cells are cells expressing type I collagen and type III collagen. The complex according to claim 1, wherein the cell is a mesenchymal stem cell. The complex of claim 1, wherein the cell is a living cell. The complex according to claim 1, wherein the cell retains at least 50% of the amount of DNA compared to the amount of DNA before complex formation or is detected by a live cell detection method. 29. The complex of claim 28, wherein the live cell detection method is an MTT assay or a WST-1 assay. Cells or cell populations are alternately immersed in a calcium solution containing calcium ions and substantially free of phosphate ions and a phosphate solution containing phosphate ions and substantially free of calcium ions And producing and fixing hydroxyapatite on at least the surface of the cell or cell population. The production method according to claim 30, wherein the cell or the cell population is an artificial tissue. The production method according to claim 30, wherein the cell or cell population comprises a stem cell. The manufacturing method according to claim 30, wherein the concentration substantially free of calcium ions is a concentration at which formation of hydroxyapatite does not proceed. The manufacturing method according to claim 30, wherein the concentration substantially free of phosphate ions is a concentration at which formation of hydroxyapatite does not proceed. The method includes the step of confirming the degree of deposition of hydroxyapatite on the cell surface in the complex after the step of generating and fixing the hydroxyapatite and repeating the step of generating and fixing based on the result. The manufacturing method as described in. The manufacturing method according to claim 30, wherein the concentration of the ions is 10 to 200 mM. The manufacturing method of Claim 30 whose density | concentration of the said ion is 10-50 mM. The manufacturing method according to claim 30, wherein the immersion time is 1 minute or less. A hydroxyapatite composite obtained by the production method according to claim 30. 40. A biocompatible material consisting essentially of the composite of claim 1 or 39.