Connective tissue constructs and methods of making the same

Abstract

A connective tissue construct (30) formed of collagen-containing organism-derived tissue according to one embodiment of the present disclosure has a dense surface layer (32) denser than the inside thereof on the surface of the connective tissue construct (30).

Description

Technical Field

[0001] This invention relates to connective tissue bodies formed from tissues derived from living organisms and methods for manufacturing the same. Background Technology

[0002] As a natural defense mechanism of living organisms, when a foreign object invades the subcutaneous tissue, fibroblasts in the subcutaneous layer aggregate around it and produce collagen to encapsulate the foreign object. This phenomenon is commonly known as encapsulation. In recent years, regenerative medicine has been actively researched for the artificial restoration of lost tissues or organs, and the application of encapsulation in regenerative medicine has been studied.

[0003] For example, Patent Document 1 discloses a scheme in which a connective tissue body forming substrate having two tissue forming surfaces that are opposite each other is disposed in a living organism, and a connective tissue body is formed in the tissue forming space between the two surfaces using collagen or the like generated by an encapsulation reaction.

[0004] The following scheme is shown in more detail: a connective tissue forming substrate comprises a cylindrical substrate and a core material inserted into the cylindrical substrate, wherein a tubular connective tissue body is formed in the tissue forming space therebetween. The tubular connective tissue body is envisioned for use in artificial blood vessels, etc. Furthermore, it is shown that a membrane-like connective tissue body can be formed by using a connective tissue forming substrate having two plate-like substrates facing each other. Further, the following scheme is shown: its application in heart valves, etc., is envisioned to form an artificial valve having two or more valve leaflets.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2016 / 076416

[0008] Non-patent literature

[0009] Non-patent literature 1: Journal of Biomedical Materials Research Part B 2011: 99B: 420-430. Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] However, the connective tissue formed by the method disclosed in Patent Document 1 has insufficient surface strength. For example, as reported in Non-Patent Document 1, when used in tissues within living organisms such as artificial blood vessels and artificial valves, blood can seep into the interior of the connective tissue, leading to problems such as easy thrombosis. Therefore, treatment such as coating the surface with antithrombotic substances is necessary.

[0012] Furthermore, when the resulting connective tissue is used in tissues within living organisms such as artificial esophagus, artificial lymphatic vessels, artificial diaphragms, artificial urinary catheters, and artificial urethras that come into contact with bodily fluids, digestive juices, and excretory fluids, problems arise such as tissue swelling and fragility due to water seepage. Additionally, the overall strength of thick connective tissue is insufficient to function as artificial tendons, artificial ligaments, or other locomotor organs. When artificial structures such as scaffolds are encapsulated within the tissue, issues such as tissue displacement and shape instability occur.

[0013] This invention was made in view of the above-mentioned problems. Its objective is to improve the surface strength of connective tissue bodies obtained through encapsulation reactions, for example, to improve the strength of artificial blood vessels, artificial valves, artificial esophagus, artificial lymphatic vessels, artificial diaphragms, artificial urinary catheters, artificial urethras, or the connective tissue body as a whole. Even when used in tissues within living organisms such as artificial tendons and ligaments, it can prevent blood and body fluids from seeping into the interior of the connective tissue body and forming thrombi, and prevent swelling. Furthermore, its objective is to stabilize the artificial structures encapsulated within the tissue body, preventing displacement of the encapsulated artificial structures within the tissue body, even when used in applications such as artificial tracheas, stent grafts, and covered stents.

[0014] Methods for solving problems

[0015] The inventors conducted in-depth research and discovered that the above-mentioned problems can be solved by forming a dense surface layer on the surface of connective tissue, thus completing the following invention.

[0016] [1] A connective tissue body formed from organism-derived tissue containing collagen, wherein,

[0017] The connective tissue body has a dense surface layer, which is a surface layer in which the tissue constituting any surface of the connective tissue body is denser than the portion outside the surface layer.

[0018] [2] The connective tissue body as described in [1] above, wherein the collagen comprises fibrous collagen.

[0019] [3] As described in [2] above, the connective tissue body wherein the fibrous collagen is flat and smooth on the surface of the dense surface layer.

[0020] [4] The connective tissue body as described in [2] or [3] above, wherein the fibrous collagen is arranged in a mesh-like manner in a cross-shaped manner in the portion outside the surface of the dense surface layer.

[0021] [5] The connective tissue body as described in any one of [1] to [4] above, wherein the ratio of the tissue density of the surface layer calculated along the thickness direction to the tissue density of the portion other than the surface layer is 1.1 or more.

[0022] [6] A connective tissue body as described in any one of [1] to [5] above, wherein a foreign component is embedded inside.

[0023] [7] The connective tissue body as described in [6] above, wherein the dense surface layer is formed around the heterogeneous component.

[0024] [8] The connective tissue body as described in any one of [1] to [7] above, wherein the connective tissue body is formed by an encapsulation reaction.

[0025] [9] A method for manufacturing a connective tissue body, which is a method for manufacturing a connective tissue body formed from a biological tissue containing collagen, wherein the method comprises the following steps:

[0026] The process of placing a connective tissue forming substrate having a tissue forming surface, wherein at least a portion of the tissue forming surface is made of a polymeric material, into a living organism.

[0027] A process of extracting air percutaneously and tubularly from a space within a living organism in which the aforementioned connective tissue matrix is ​​disposed;

[0028] The process of forming connective tissue on the aforementioned tissue-forming surface;

[0029] The process of removing the connective tissue forming substrate having the above-mentioned connective tissue from the biological body; and

[0030] The process of peeling off the connective tissue body from the substrate formed by the above-mentioned connective tissue body.

[0031]

[10] A connective tissue body comprising a tissue forming substrate having a tissue forming surface that separates the environment in which the biological tissue material is located from the space for forming the connective tissue body, and the tissue forming substrate having pores that connect the space and the environment and allow connective tissue to penetrate the space, wherein the connective tissue body is formed by placing the tissue forming substrate in the environment of a biological body other than a human body and by extracting air from the space in which the tissue forming substrate is disposed outside the biological body, wherein...

[0032] The aforementioned tissue formation surfaces are composed of polymeric or metallic materials;

[0033] The aforementioned connective tissue is formed from organism-derived tissue containing collagen and is used in tissues within organisms;

[0034] In the aforementioned connective tissue, the surface layer constituting the surface in contact with the tissue formation surface is a dense surface layer in which the tissue is denser than the portion outside the surface layer.

[0035] In the aforementioned dense surface layer, the ratio of the tissue density of the surface layer, calculated along the thickness direction, to the tissue density of the portion outside the surface layer is 1.1 or more.

[0036]

[11] A method for manufacturing a connective tissue body, which is a method for manufacturing a connective tissue body formed from a tissue derived from a living organism and containing collagen, and used in a tissue within a living organism, the method comprising the following steps:

[0037] A process of placing a connective tissue forming substrate having a tissue forming surface, at least a portion of which is made of a polymeric material, into a biological body other than the human body.

[0038] A process of extracting air percutaneously and tubularly from the space within the organism in which the connective tissue matrix is ​​disposed;

[0039] The process of forming connective tissue on the aforementioned tissue-forming surface;

[0040] The process of removing the connective tissue forming substrate, on which the above-mentioned connective tissue is formed, from the above-mentioned organism; and

[0041] The process of peeling off the connective tissue body from the substrate formed by the above-mentioned connective tissue body.

[0042]

[12] The method for manufacturing connective tissue as described in

[11] , wherein the connective tissue is formed by an encapsulation reaction.

[0043]

[13] A connective tissue body for regenerative medicine, comprising:

[0044] It possesses a surface layer of first fibrous collagen derived from living organisms; and

[0045] It possesses the non-surface layer portion of the second fibrous collagen derived from living organisms.

[0046] The first fibrous collagen in the aforementioned surface layer has a first microstructure and a first density.

[0047] The second fibrous collagen in the non-surface layer portion has a second microstructure different from the first microstructure and a second density lower than the first density.

[0048] The aforementioned surface layer and the aforementioned non-surface layer portions form a wall-reverse structure due to the difference between the first density of the first fibrous collagen and the second density of the second fibrous collagen, and the difference between the first microstructure of the first fibrous collagen and the second microstructure of the second fibrous collagen.

[0049]

[14] The connective tissue body as described in

[13] , wherein the surface layer and the non-surface layer portion are formed as layer boundaries solely due to the difference between the first density and the second density, and the difference between the first microstructure and the second microstructure.

[0050]

[15] Connective tissue bodies as described in

[13] , wherein,

[0051] The aforementioned first microstructure and first density endow the aforementioned surface layer with first blood impermeability.

[0052] The aforementioned second microstructure and second density impart a second blood impermeability to the aforementioned non-surface layer portion.

[0053] The first blood in the aforementioned surface layer is less prone to penetration than the second blood in the aforementioned non-surface layer portion.

[0054] The effects of the invention

[0055] According to the present invention, a connective tissue body is provided, the strength of which is improved, and even when used in tissues within a living organism such as artificial blood vessels or artificial valves, it can prevent blood from seeping into the interior and forming thrombi.

[0056] In addition, when heterogeneous components such as artificial structures are enclosed within connective tissue, the heterogeneous components can be stabilized, preventing them from shifting internally. Attached Figure Description

[0057] Figure 1 A schematic cross-sectional view showing one embodiment of connective tissue is shown.

[0058] Figure 2 A schematic cross-sectional view showing one embodiment of connective tissue is shown.

[0059] Figure 3 A schematic cross-sectional view showing one embodiment of connective tissue is shown.

[0060] Figure 4 A schematic cross-sectional view showing one embodiment of connective tissue is shown.

[0061] Figure 5 A schematic cross-sectional view showing one embodiment of connective tissue is shown.

[0062] Figure 6 A perspective view is shown representing one embodiment of a connective tissue forming substrate.

[0063] Figure 7 A perspective view is shown representing one embodiment of a connective tissue forming substrate.

[0064] Figure 8 A perspective view is shown representing one embodiment of a connective tissue forming substrate.

[0065] Figure 9 A perspective view showing one embodiment of the core material is shown.

[0066] Figure 10 A perspective view is shown representing one embodiment of a connective tissue forming substrate.

[0067] Figure 11 A perspective view is shown representing one embodiment of a connective tissue forming substrate.

[0068] Figure 12 A perspective view is shown representing one embodiment of a connective tissue forming substrate.

[0069] Figure 13 A perspective view illustrating one embodiment of a process for placing a connective tissue forming substrate within a living organism.

[0070] Figure 14 A perspective view showing one embodiment of connective tissue is shown.

[0071] Figure 15 This is a magnified photograph of the cross-section of the connective tissue from Example 1.

[0072] Figure 16 This is a magnified photograph of a cross-section of the connective tissue from Example 1 after staining.

[0073] Figure 17 The photograph is a magnified image of the portion near the inner circumferential surface of the connective tissue body after staining the cross section of the connective tissue body in Example 1.

[0074] Figure 18 The photographs are taken after staining the cross-section of the connective tissue from Example 1, and are magnified photographs of the central portion of the connective tissue in the thickness direction.

[0075] Figure 19 The photograph is a magnified image of the inner circumferential surface of the connective tissue body constituting the dense surface layer in Example 1.

[0076] Figure 20This is a magnified photograph of the interior of the dense surface layer of the connective tissue in Example 1.

[0077] Figure 21 These are magnified photographs of the portion of the connective tissue body other than the dense surface layer of Example 1.

[0078] Figure 22 The normalized brightness distribution data of Example 1 is shown.

[0079] Figure 23 The image shows connective tissue bodies from Example 1 immersed in whole blood from a dog.

[0080] Figure 24 A photograph is shown to illustrate the wall-backed structure of Embodiment 1.

[0081] Figure 25 This is a magnified photograph of a cross-section of the connective tissue from Comparative Example 1 after staining.

[0082] Figure 26 The connective tissue body in Comparative Example 1 is shown.

[0083] Figure 27 The connective tissue bodies of Comparative Example 1, when immersed in the whole blood of a dog, are shown. Detailed Implementation

[0084] [Connective tissue]

[0085] The connective tissue bodies of the present invention are formed from biologically derived tissues containing collagen. The connective tissue bodies of the present invention are typically formed via an encapsulation reaction. An encapsulation reaction is a reaction in which a foreign substance (the connective tissue body forming substrate described later) is placed in an environment containing biological tissue materials such as the subcutaneous tissue of a living organism, causing biological tissue materials such as fibroblasts to aggregate around the foreign substance and generate collagen to encapsulate it.

[0086] Connective tissue contains collagen, produced by encapsulation, as its main component. Additionally, connective tissue may also contain other biological tissue materials such as fibroblasts. Furthermore, in connective tissue, some or all of the collagen constitutes fibrous collagen.

[0087] It should be noted that the term "biological tissue material" in this specification refers to substances necessary for forming the desired biologically derived tissue. Examples include animal cells such as fibroblasts, smooth muscle cells, endothelial cells, stem cells, ES cells, and iPS cells; various proteins (collagen, elastin); sugars such as hyaluronic acid; and various physiologically active substances present in the body, such as cell growth factors and cytokines. This "biological tissue material" includes materials derived from mammals such as humans, dogs, cattle, pigs, goats, and sheep; birds; fish; other animals; or equivalent artificial materials. Biological tissue materials are preferably derived from mammals. Furthermore, "tissue derived from a living organism" refers to tissue formed from the aforementioned biological tissue materials.

[0088] In the connective tissue body of the present invention, the surface layer constituting any surface of the connective tissue body has a dense surface layer in which the tissue is denser than the portion outside the surface layer. Tissue density refers to the density of collagen in the connective tissue body. The dense surface layer, which is denser than the portion outside the surface layer, is provided on at least a portion of the surface of the connective tissue body. The surface of the connective tissue body composed of the dense surface layer (for example, the inner circumferential surface in the tubular structure described later) has high strength. It becomes a highly dense, smooth, and high-strength surface, thereby making it difficult for blood to seep in and for thrombus formation to occur. Furthermore, the resulting connective tissue body does not contain blood clots or the like.

[0089] It should be noted that the term "surface layer" is not limited to the layer constituting the outer surface of the connective tissue body; it also includes the layer within the connective tissue body that forms the interface with other components, such as heterogeneous components, present within the connective tissue body. Furthermore, in cases where there are cavities within the connective tissue body, the interface with those cavities is also included in the surface layer.

[0090] That is, as described below, when a dissimilar component is embedded inside, a dense surface layer can be provided at the interface with the dissimilar component. Furthermore, when there is a hollow portion inside, a dense surface layer can be provided at the interface with that hollow portion (e.g., the inner circumferential surface in a tubular structure).

[0091] The connective tissue body of the present invention preferably has a dense surface layer along its thickness direction, and a portion other than the surface layer that is sparser than the dense surface layer. By having a portion sparser than the dense surface layer, the thickness is increased, making it easier to process, and thus improving its flexibility so that it can easily follow the curvature of the tissue as a whole. Furthermore, the connective tissue body of the present invention preferably has a dense surface layer on both surfaces along its thickness direction, and a portion sparser than the dense surface layer between these two dense surface layers. It should be noted that, regarding the thickness direction, for example in a tubular structure, it refers to the direction consistent with its radial direction. It should also be noted that both surfaces on which the dense surface layer is formed can be the outer surface of the connective tissue body, or both can be interfaces with any of the voids and dissimilar materials, or one surface can be the outer surface and the other surface can be the aforementioned interface.

[0092] Whether the surface layer of connective tissue is a dense surface layer can be confirmed as follows: cut the connective tissue into thin slices, perform Massen trichrome staining, illuminate the stained connective tissue with a certain illuminance, and confirm by the brightness value in the obtained image.

[0093] When performing Massen's trichrome staining on connective tissue, collagen is stained blue, and denser areas are stained darker. Therefore, in a slice of connective tissue irradiated with a certain illuminance, the brightness value of denser areas is relatively lower than that of other areas, and areas with relatively lower brightness values ​​can be identified as denser areas.

[0094] That is, when the connective tissue of the present invention is stained with Massen trichrome and photographed, the brightness value of the dense surface layer in the photographic image is lower than that of other parts outside the surface layer. Therefore, it can be confirmed that it has a dense surface layer.

[0095] In this invention, the density of the surface layer of connective tissue can also be calculated by utilizing the decrease in brightness value of dense tissue in a Masson trichrome staining image.

[0096] Here, the density obtained from the Massen trichrome staining image can be defined by tissue density, which represents the density of the tissue itself (i.e., collagen). In connective tissue, capillary structures and voids (i.e., areas without tissue) are usually visible when sliced ​​at a relatively thin thickness and observed in cross-section. Tissue density is calculated after image processing to remove such voids.

[0097] In this invention, connective tissue can be sliced ​​along its thickness direction into thin sections, stained with Massen's trichrome, and the resulting cross-sections can be observed to calculate the tissue density of the surface layer. Specifically, the ratio of the tissue density of the surface layer to the tissue density of the portion outside the surface layer (hereinafter also referred to as the "tissue density ratio") can be calculated along the thickness direction of the connective tissue, thereby determining the density of the surface layer of the connective tissue.

[0098] Here, the density of the surface layer of the present invention is higher than that of the portion outside the surface layer. Therefore, the tissue density ratio of the surface layer is greater than 1, preferably 1.1 or more, and more preferably 1.2 or more. There is no particular upper limit to the tissue density ratio, for example, it is 3, and practically it is 2.5.

[0099] In this invention, when the tissue density ratio is 1.1 or higher, the collagen, especially fibrous collagen, in the surface layer is densely present, thus increasing its strength. Therefore, it can be used beneficially in vivo. Furthermore, blood does not easily penetrate, thereby reducing the likelihood of thrombosis.

[0100] It should be noted that, in this invention, the tissue density ratio of the surface layers on both surfaces may be within the above range, but the tissue density ratio of the surface layer on one surface may also be within the above range.

[0101] In the dense surface layer of the present invention, as described above, the tissue density is higher than that of other parts, thereby having a wall-repellent structure. It should be noted that the wall-repellent structure refers to a structure that can be peeled off in layers from the parts other than the dense surface layer, for example, when the end of the dense surface layer is grasped and peeled off using tweezers.

[0102] Regarding the dense surface layer, its moisture content is lower when immersed in water compared to other parts. Specifically, assuming the weight of both the dense surface layer separated by peeling and the other layers is B (g), and the weight of either the dense surface layer or the other parts after vacuum drying at 25°C for 12 hours is A (g), the moisture content of the dense surface layer, expressed as (BA) / A×100, is preferably less than 60%, more preferably less than 55%. On the other hand, the moisture content of the other parts, similarly measured, is preferably 60% or more, more preferably 70% or more. The low moisture content also indicates that the dense surface layer has a dense structure, thus preventing blood infiltration and reducing the likelihood of adverse conditions such as thrombosis. Regarding the moisture content, the moisture content of any number of points (e.g., 10 points) of both the dense surface layer and the other parts can be calculated, and the average value can be determined.

[0103] As described above, the connective tissue body of the present invention has fibrous collagen, which is preferably flat and smooth in the surface (e.g., the inner circumferential surface of the tubular structure) formed by the aforementioned dense surface layer. In the dense surface layer, the flattened fibrous collagen is smoothly disposed on its surface, and the tissue is dense, thereby presumably preventing blood from easily penetrating into the connective tissue body.

[0104] On the other hand, regarding fibrous collagen, in the portion of connective tissue other than the surface composed of a dense surface layer (e.g., the inner circumferential surface of a tubular structure), fibrous collagen is arranged in a mesh-like manner in both the dense surface layer and the sparse portion outside the dense surface layer. More specifically, it is preferable that bundles of fibrous collagen are oriented in a unidirectional direction to form a layer, and that bundles of fibrous collagen are oriented in another unidirectional direction in a manner that intersects with the collagen bundles of that layer to form another layer, and these layers are laminated into a mesh-like structure to form a laminated structure. Furthermore, this laminated structure is preferably multi-layered.

[0105] Furthermore, the bundles of fibrous collagen in the portion of connective tissue outside the surface (e.g., the inner circumferential surface of a tubular structure) formed by a dense surface layer can have a structure that is thicker in the dense surface layer and thinner in the portion outside the dense surface layer.

[0106] The connective tissue of the present invention, having a mesh-like structure similar to human collagen, is readily applicable to regenerative medicine for the artificial restoration of lost tissues or organs in the human body.

[0107] Here, regarding the unidirectional orientation of the fibrous collagen bundles, it is sufficient that the fibrous collagen is arranged in a manner that flows in approximately one direction; it is not necessary for all the fibrous collagen to be strictly parallel to each other.

[0108] The connective tissue body of the present invention can be of any shape, such as tubular, sheet-like, rod-like, or spherical. When the connective tissue body is tubular, it can be used, for example, in artificial blood vessels. When it is sheet-like, it can be used as a membranous tissue such as an artificial heart membrane, dura mater, skin, valve, cornea, diaphragm, or abdominal wall, or as a filling and repair material for tubular or pouch-like tissues such as blood vessels, esophagus, trachea, digestive tract, lymphatic vessels, urethra, ureter, and bladder. Furthermore, when it is rod-like, it can be used as a tendon or ligament.

[0109] In addition to being tubular or sheet-like, connective tissue can be shaped into any form corresponding to various organs, such as a shape corresponding to an artificial valve. Artificial valves typically have a tubular portion and multiple leaflets that bulge radially inward from the tubular portion.

[0110] When connective tissue has tubular portions (tubular structures), a dense surface layer may be formed on at least one of its two surfaces (i.e., the outer circumferential surface and the inner circumferential surface). For example, Figure 1 , 2 As shown, the connective tissue body 30 with a tubular structure preferably has a dense surface layer 32 formed at least on its inner circumferential surface 30B. Furthermore, it is also preferred that... Figure 2 The diagram shows the method of forming a dense surface layer 32 on both the inner peripheral surface 30B and the outer peripheral surface 30A.

[0111] In addition, such as Figure 1 , 2 As shown, the connective tissue body 30 with a ring structure preferably has a hollow portion 35 inside, or it can be as follows: Figure 3 The diagram shows a plurality of hollow portions 35. When a plurality of hollow portions 35 are provided, as shown... Figure 3 As shown, it is preferable to also form a dense surface layer 32 on the inner circumferential surface 30B of the tube forming the outer periphery of the hollow portion 35, and it is even more preferable to form the dense surface layer 32 on both the inner circumferential surface 30B and the outer circumferential surface 30A. By embedding a high-strength cylindrical tissue into the interior of soft tissue, it can be used in rod-shaped grafts such as tendons and ligaments.

[0112] In addition, such as Figure 4 As shown, foreign components 37, such as metals or polymers, can also be embedded inside the connective tissue body 30. When foreign components 37 are embedded, such as... Figure 4 As shown, it is preferable that a dense surface layer 32 is also formed on the inner circumferential surface 30B of the tube portion constituting the outer periphery of the hollow portion 35, and it is even more preferable that... Figure 5 The diagram shows a method in which a dense surface layer 32 is formed on both the inner peripheral surface 30B and the outer peripheral surface 30A. For example... Figure 4 , Figure 5 As shown, in the connective tissue body 30 in which the foreign component 37 is embedded, it is preferable that the surface layer surrounding the foreign component 37 is also formed in the form of a dense surface layer 32. By making the portion surrounding the boundary with the foreign component 37 a dense surface layer 32, the foreign component 37 is firmly fixed within the soft tissue while maintaining a certain degree of mobility. Therefore, the foreign component 37 can be stabilized, preventing it from shifting internally.

[0113] As a xenogeneic component 37, a scaffold is preferably used. The connective tissue body 30 embedded with the scaffold can be used in tubular grafts, and can accommodate large deformations without damaging the overall tissue, and can shrink and expand.

[0114] As a heterogeneous component 37, in addition to the support that can expand and deform, it can also be a linear, ring-shaped, or spiral structure.

[0115] Furthermore, as a heterogeneous component 37, biocompatible metals, polymers, or materials derived from living organisms can be used. Examples of metals include stainless steel, titanium, cobalt-chromium alloys, nickel-titanium alloys, and magnesium alloys. These metals can be appropriately used, for example, in scaffolds. Examples of polymers include nylon resin, PEEK resin, silicone resin, acrylic resin, fluoropolymer resin, urethane resin, vinyl resin, styrene resin, and acrylic resin. These polymers can be coated onto metal surfaces. Additionally, examples of materials derived from living organisms include inorganic materials such as hydroxyapatite, and organic materials such as cartilage, which are primarily composed of cellular components.

[0116] As described above, when connective tissue with a tubular structure is used in artificial blood vessels and artificial valves, blood flows through its inner side. If a dense surface layer is formed on the inner circumferential surface, the blood flowing through its inner side will not easily penetrate into the connective tissue through the dense surface layer, and thrombus formation will be less likely in artificial blood vessels, artificial valves, etc.

[0117] In addition, when the connective tissue is sheet-like, a dense surface layer can be formed on one surface of the sheet-like connective tissue or on both surfaces.

[0118] [Method for manufacturing connective tissue]

[0119] The connective tissue body of the present invention can be manufactured using a connective tissue forming substrate via an encapsulation reaction. Specifically, it can be manufactured subcutaneously in an organism using a manufacturing method comprising the following steps.

[0120] Step 1: The process of placing a connective tissue forming substrate having a tissue-forming surface, at least a portion of which is composed of a polymeric material, into a living organism.

[0121] Step 2: A process of percutaneously and tubularly extracting air from the space within the organism containing connective tissue matrix outside the organism.

[0122] Step 3: The process of forming connective tissue on the tissue-forming surface.

[0123] Step 4: The process of removing the connective tissue-forming substrate containing connective tissue from the biological body.

[0124] Step 5: Step of peeling off connective tissue from the substrate formed by connective tissue.

[0125] (Connective tissue forming substrate)

[0126] First, the connective tissue forming substrate used in the above manufacturing method will be explained.

[0127] The connective tissue forming substrate has tissue forming surfaces on its surface for forming connective tissue bodies. The tissue forming surfaces are preferably configured to separate spaces (tissue forming spaces) corresponding to the shape of the connective tissue body. For example, the connective tissue forming substrate may have at least a pair of tissue forming surfaces facing each other, in which connective tissue bodies are formed by an encapsulation reaction in the tissue forming space between the pair of tissue forming surfaces.

[0128] The connective tissue forming substrate can be composed of materials that have the strength to not undergo significant deformation in the presence of biological tissue materials such as those in a living organism, have chemical stability, are resistant to sterilization loads, and have no or minimal irritants to the organism.

[0129] The connective tissue forming substrate is specifically composed of polymeric materials, metallic materials, etc. Preferred examples of polymeric materials include nylon resin, PEEK resin, silicone resin, acrylic resin, fluoropolymer resin, urethane resin, ethylene resin, styrene resin, and acrylic resin, which are used as components of in-vitro medical devices. Preferred examples of metallic materials include stainless steel, titanium, cobalt-chromium alloy, nickel-titanium alloy, and magnesium alloy, which are used as components of in-vitro medical devices.

[0130] Furthermore, at least a portion of the tissue-forming surface is made of a polymeric material such as resin, preferably nylon resin, PEEK resin, silicone resin, acrylic resin, fluororesin, urethane resin, vinyl resin, styrene resin, or acrylic resin, which are used as components of in vivo contact-type medical devices. Among these resins, biological tissue materials tend to densely aggregate on their surface, easily forming a dense surface layer. Therefore, when the connective tissue forming substrate has a pair of tissue-forming surfaces facing each other, it is preferable that one tissue-forming surface is made of a polymeric material such as resin, and more preferably of a material used as a component of in vivo indwelling-type medical devices, such as nylon resin, PEEK resin, silicone resin, acrylic resin, fluororesin, urethane resin, vinyl resin, styrene resin, or acrylic resin. Additionally, it is preferable that the other tissue-forming surface is made of a metallic material.

[0131] When the tissue-forming surface is made of the aforementioned polymeric or metallic material, the entire component with the tissue-forming surface can be made of polymeric or metallic material, or only the tissue-forming surface can be made of polymeric or metallic material. For example, the tissue-forming surface can be made of polymeric material by forming a polymeric coating on the surface of a metallic component.

[0132] The shape of the connective tissue matrix can be designed according to the connective tissue being manufactured. For example, in the case of manufacturing tubular connective tissue, such as...Figure 6 As shown, the connective tissue forming substrate 10 can be made of a material having a cap member 11 made of a cylindrical body and a core material 12 inserted into the interior of the cap member 11.

[0133] Figure 6 In the connective tissue forming substrate 10 shown, the inner peripheral surface of the cover member 11 forms a tissue forming surface 11A, and the outer peripheral surface of the core material 12 forms another tissue forming surface 12A. A tissue forming space 13 is formed between the tissue forming surfaces 11A and 12A. The cover member 11 is provided with holes 17 that connect the tissue forming space 13 to the outside of the substrate. Typically, the cover member 11 is provided with multiple holes 17.

[0134] Cover bodies 14 and 15 are provided on both ends of the cover member 11, respectively blocking the two ends of the tissue forming space 13. The cover bodies 14 and 15 are annular and have locking portions 14A and 15A on their outer periphery. Multiple locking portions 14A and 15A are provided on each cover body 14 and 15. Each locking portion 14A has a groove 14C into which the end of the cover member 11 is inserted. Similarly, each locking portion 15A has a groove (not shown). In the cover bodies 14 and 15, the end of the core material 12 is inserted into the annular interior, and the end of the cover member 11 is inserted into the groove of the locking portions 14A and 15A, thereby connecting and fixing the cover member 11 and the core material 12.

[0135] The aperture 17 is not particularly limited; for example, it can be round, elliptical, or a slit. When the aperture 17 is round or elliptical, from the perspective of facilitating the penetration of connective tissue and biological tissue materials, the aperture of the round or elliptical aperture can be set to 1 mm or more, and can be set to 10 mm or less.

[0136] like Figure 6 As shown, the hole 17 is preferably a slit. By making the hole 17 a slit, it is prevented from being blocked by connective tissue, allowing connective tissue to easily invade the tissue formation space 13. The slit can have a width of 1 mm or more, allowing connective tissue and biological tissue materials to easily invade, and can have a width of, for example, 10 mm or less. In addition, in the slit, the incision length is generally preferably greater than twice the slit width, preferably less than five times, and more preferably less than three times.

[0137] Furthermore, the area ratio of the slit to the tissue-forming surface 11A of the cover member 11 can be set to 2 / 3 or less. By reducing the area ratio of the slit, it is easier to achieve a good surface condition for the connective tissue formed on the tissue-forming surface 11A. Additionally, in order to allow connective tissue and biological tissue materials to penetrate into the tissue-forming space 13 through the slit, the area ratio of the slit to the tissue-forming surface 11A of the cover member 11 is preferably 1 / 10 or more. Furthermore, when the slit is not circular or elliptical, the area ratio of the hole to the tissue-forming surface 11A of the cover member 11 is also preferably 1 / 10 or more, and more preferably 2 / 3 or less.

[0138] In addition, the slit can be like Figure 6 The slits can be arranged parallel to the central axis of the substrate along their length, or they can be arranged obliquely to the central axis of the substrate along their length. Furthermore, multiple slits can be arranged along both the circumferential and longitudinal directions.

[0139] The outer peripheral surface of the core material 12 is preferably made of the aforementioned polymeric materials, particularly nylon resin, PEEK resin, silicone resin, or acrylic resin. Therefore, in this invention, a dense surface layer can be easily formed on the inner peripheral surface of the tubular connective tissue. The core material 12, for example, includes a central material 12B and a cap tube 12C covering the outer peripheral surface of the central material 12B; the cap tube 12C can be made of the aforementioned polymeric materials.

[0140] On the other hand, the cover member 11 is made of a metallic material, preferably its tissue-forming surface 11A (i.e., the inner circumferential surface) is made of a metallic material. Compared with the aforementioned polymeric materials, the surface of a metallic material is less prone to the accumulation of biological tissue material, thereby preventing the pores 17 of the cover member 11 from being blocked by biological tissue material in the early stages. Therefore, biological tissue material can accumulate in the tissue-forming space 13 for a long period of time. However, the cover member 11 can also be formed of the aforementioned polymeric material. By being formed of a polymeric material, the aforementioned... Figure 2 As shown, a dense surface layer is formed on both the outer and inner circumferential surfaces of the tubular connective tissue.

[0141] The matrix forming connective tissue is not limited to a straight shape; it can have various shapes, including curved shapes, such as... Figure 7 , 8 As shown, a substrate 40 can be formed of connective tissue with two or more rings rolled into a spiral shape.

[0142] In connective tissue forming substrate 40, such as Figure 7 or Figure 8As shown, the cover component 41 is provided. The cover component 41 has a double or more ring-shaped spiral. The cover component 41 is, for example, composed of an upper part 41A and a lower part 41B that are divided into upper and lower parts, which are joined together to form a tubular structure. The upper part 41A and the lower part 41B are respectively provided with connecting parts 43A and 43B, which are connected and fixed to each other. The cover component 41 is provided with a plurality of holes 47, which can be arranged in multiple directions along the circumferential and length directions. The holes 47 can be arranged as follows: Figure 7 The image shown is a rectangle, but it can also be like... Figure 8 The image shown is a circular hole, but other shapes of holes are also possible.

[0143] A core material 42 (see reference) is disposed inside the cover component 41. Figure 9 ).like Figure 9 As shown, the core material 42 and the cover member 41 are respectively shaped as two or more spiral rings. The two ends 42E of the core material 42 are wider and taller than the other parts, for example, they are spherical. By having such a shape, the two ends 42E can be fitted into the recesses at both ends of the cover member 41, thereby fixing the core material 42 relative to the cover member 41. However, the two ends of the core material 42 are not limited to this configuration.

[0144] In the connective tissue forming substrate 40, a tissue forming space is formed between the inner peripheral surface of the cover member 41 and the outer peripheral surface of the core material 42. The materials of the inner peripheral surface of the cover member 41 and the outer peripheral surface of the core material 42 are as described above.

[0145] In use Figures 7-9 In the case of the connective tissue forming substrate 40 shown, the connective tissue is also formed in the tissue forming space as described above. By making the connective tissue forming substrate 40 have two or more rings, the connective tissue can be formed into a long tubular structure, and therefore it is suitable for forming connective tissue for, for example, artificial blood vessels.

[0146] Furthermore, the core material does not necessarily have to be one; two or more can be provided. Specific examples of connective tissue forming substrate 50 with two or more core materials include... Figure 10 As shown. The connective tissue forming substrate 50 includes a cover member 51 made of a cylindrical body and a plurality of core materials 52 inserted into the interior of the cover member 51. The plurality of core materials 52 can be installed on the cover bodies 54 and 55. In addition, as described above, the cover bodies 54 and 55 are provided with a plurality of locking portions 54A and 55A. For example, by inserting the end of the cover member 51 into the groove of the locking portion 54A and 55A, the cover member 51 and the core materials 52 are connected and fixed.

[0147] When multiple core materials 52 are provided, each core material 52 is rod-shaped, with a thickness of, for example, 0.2 to 3 mm, preferably 0.5 to 2.5 mm. Furthermore, the maximum cross-sectional area occupied by the core material 52 in the internal space of the cover member 51 is, for example, about 20% to 60%. It should be noted that the maximum cross-sectional area refers to the cross-sectional area of ​​the portion constituting the largest area on a section perpendicular to the axis of the connective tissue forming substrate 50.

[0148] exist Figure 10 In the cover component, multiple circular holes 57 are provided. The configuration of the holes 57 is not limited; they can be any holes as described above, or they can be slits, etc. Furthermore, Figure 10 In the middle, the circular holes 57 are arranged in an alternating pattern, but the arrangement is not limited and can be any arrangement.

[0149] Furthermore, when implanting foreign components within connective tissue, the foreign components can be placed within the tissue-forming space. For example, such as... Figure 11 As shown, in a connective tissue forming substrate having a core material 12 and a capping member 11, the heterologous component 37 can be disposed in the tissue forming space 13 between the core material 12 and the capping member 11. However, the heterologous component 37 may not be tightly fitted to the core material 12 and the capping member 11. By not tightly fitting it to the core material 12 and the capping member 11, tissue derived from a living organism can enter between the heterologous component 37 and the core material 12 or the capping member 11, such as... Figure 5 As shown, in the connective tissue body 30, a dense surface layer 32 can be easily formed integrally on the inner peripheral surface 30B and the outer peripheral surface 30A. Additionally, the heterogeneous component 37 can also be part of the connective tissue forming substrate, with its surface being the tissue forming surface.

[0150] Furthermore, in the case of forming tubular connective tissue bodies, the connective tissue forming substrate does not necessarily need to have a capping member and a core material; for example, the capping member can be omitted. An example of a connective tissue forming substrate 60 without a capping member is... Figure 12 As shown. Even without the cap component, the surface of the core material 62 becomes a tissue-forming surface, and connective tissue is formed on the surface of the core material 62 through an encapsulation reaction. Furthermore, in the connective tissue forming substrate 60 where the cap component is omitted, it is also possible to... Figure 12 As shown, a dissimilar component 37 is disposed on the outer peripheral surface of the core material 62. Preferably, the dissimilar component 37 is not in close contact with the outer peripheral surface of the core material 62, as described above. Figure 12 As shown, when the heterogeneous component 37 is arranged in the tissue forming space 13 and no cover component is provided, for example, the following can be obtained: Figure 4 Connective tissue body 30 shown.

[0151] Figure 11 , 12An example of a dissimilar component 37 as a support is shown. The support can be mounted on the outer peripheral surface of the core material 62, for example, by expanding its diameter. The dissimilar component 37 is not limited to a support, and other materials can also be used.

[0152] When fabricating sheet-like connective tissue bodies, a substrate having two plate-like components facing each other can be used as the connective tissue body forming substrate. In this case, the facing surfaces of the two plate-like components are tissue-forming surfaces, and biological tissue material is gathered in the tissue-forming space between the pair of tissue-forming surfaces to form a connective tissue body.

[0153] At this point, it is preferable that either of the pair of tissue-forming surfaces is made of a polymer material such as resin, particularly nylon resin, PEEK resin, silicone resin, or acrylic resin. By being made of these polymer materials, a dense surface layer can be easily formed on the tissue-forming surface. Furthermore, it is acceptable to form a pore such as a slit in either of the two plate-shaped components. Moreover, it is preferable that the tissue-forming surface of one plate-shaped component is made of the aforementioned polymer material, and that a pore is formed in the other plate-shaped component; more preferably, in the plate-shaped component with the pore, the tissue-forming surface is made of a metallic material.

[0154] Furthermore, in the case of manufacturing artificial valves, for example, the connective tissue forming substrate includes a core material and a cover member in which the core material is inserted. The outer peripheral surface of the core material and the inner peripheral surface of the cover member serve as tissue forming surfaces, and a tissue forming space is created between these two tissue forming surfaces. The tissue forming space is divided according to a shape corresponding to an artificial valve or artificial valve precursor (which can be appropriately processed to become an artificial valve). For example, the tissue forming space is divided according to the shape of an artificial valve or artificial valve precursor that includes a tubular portion and multiple leaflets that bulge radially inward from the tubular portion.

[0155] At this time, it is also preferable that the outer peripheral surface of the core material is made of polymer materials such as resin, especially nylon resin, PEEK resin, silicone resin, and acrylic resin. This facilitates the formation of a dense surface layer on the inner peripheral surface of the artificial valve. Additionally, the cover component may have slits or other openings, and its inner peripheral surface is preferably made of metal.

[0156] Next, the steps of the above-mentioned method for manufacturing connective tissue will be explained.

[0157] (Process 1)

[0158] In step 1, the connective tissue forming substrate is placed within a living organism. Examples of living organisms include humans, or mammals other than humans such as dogs, cattle, pigs, goats, rabbits, and sheep, as well as birds, fish, and other animals. Among these, mammals are preferred. The living organism in which the connective tissue forming substrate is placed may be, for example, subcutaneous or intraperitoneal.

[0159] As a method for placing connective tissue formation substrates within an organism, a minimal incision can be made to form an insertion port on the surface of the organism, through which the connective tissue formation substrate is implanted into the animal. The wound is then sutured. Preferred implantation sites for the connective tissue formation substrate include, for example, the abdominal cavity with a volume capable of receiving the substrate, or subcutaneously in the limbs, shoulders, back, or abdomen. Specifically, in the subcutaneous case, it can be implanted between the lower dermis and the upper fat or muscle layer; more specifically, it can be placed within a subcutaneous pouch created by adequately controlling hemostasis between the lower dermis and the upper fat or muscle layer within the organism.

[0160] Furthermore, implantation is preferably performed using minimally invasive methods and with minimal incision under adequate anesthesia. Bleeding is preferably minimized, and in the event of bleeding, thorough hemostasis is necessary. The subcutaneous pouch preferably ensures a larger space than the substrate containing the connective tissue. By creating a larger subcutaneous dissection surface than the substrate, the biological response is more stimulated, resulting in a synergistic effect with the repair and healing of the dissection surface, and promoting tissue formation within the substrate.

[0161] Specifically, an insertion port is first formed on the surface of the organism, through which the organism is dissected to create a subcutaneous sac. A connective tissue matrix is ​​then placed within the organism by inserting a tissue body through the insertion port. The insertion port is then closed by suturing or similar methods.

[0162] Furthermore, when using guide rods, it is easier to place connective tissue morphogenetic substrates within the organism while simultaneously suppressing subcutaneous bleeding. For example... Figure 13 As shown, an insertion port 18 is first formed on the surface of the organism, and the front end of a guide rod 19 with a convex curved front end is inserted into the organism through the insertion port 18. Figure 13 (a) state). Next, slide the guide rod 19 outwards, and insert the insertion tube 20 into the organism through the insertion port 18. Figure 13 (b) and (c) states). After inserting the insertion tube 20, the guide rod 19 can be pulled out. Then, the connective tissue forming substrate 10 is inserted into the insertion tube 20. Figure 13 (d) state), after being pushed in with push rod 21 ( Figure 13 In state (e), the insertion tube 20 is pulled out through the insertion port 18, thereby placing the connective tissue forming substrate 10 into the organism. After the insertion tube 20 is pulled out, the insertion port 18 is closed by suturing or the like. Figure 13(f) state). In this case, a subcutaneous pouch is also preferred to ensure a larger space than the substrate. By making the guide rod have an outer diameter larger than that of the substrate, a wide peeling surface can be formed, the biological response is more stimulated, and a synergistic effect with the repair and healing of the peeling surface is generated, promoting tissue formation inside the substrate, especially the formation of a dense collagen layer at the substrate contact surface.

[0163] (Process 2)

[0164] After step 1 is completed, air is then extracted from the space 22 within the organism where the connective tissue forming substrate 10 is disposed. The method of air extraction is not particularly limited as long as it is extracted transdermally and tubularly from outside the organism; for example, the tip of a syringe can be inserted into the space 22 from outside the organism to extract the air. The vacuum level is preferably in the range of 0.1 to 0.5 atm. Even with simple pressure applied to the skin, the substrate adheres closely to the subcutaneous tissue, bringing skin cells close to the substrate surface. However, by further creating a vacuum inside the substrate, skin cells are drawn into the interior of the substrate, promoting tissue formation within the substrate, particularly the formation of a dense collagen layer at the substrate contact surface.

[0165] (Process 3)

[0166] In step 2, after air is removed, the connective tissue forming substrate 10 is placed within the organism for a predetermined period. The placement period varies depending on the type of organism; if tissue formation lasts for more than 20 days, it can be placed for an extended period with almost no change in the shape of the formed tissue. For example, 20 to 90 days, preferably 30 to 60 days, is suitable. During this predetermined placement period, connective tissue is formed within the tissue formation space of the connective tissue forming substrate 10.

[0167] In this manufacturing method, by extracting air from space 22 in step 2, the tissue within the organism adheres tightly to the outer peripheral surface of the connective tissue forming substrate 10. Therefore, connective tissue and biological tissue materials readily aggregate in the tissue forming space, and since at least a portion of the tissue forming surface is composed of a polymer material, the connective tissue and biological tissue materials densely aggregate on the tissue forming surface made of this polymer material. Thus, in this manufacturing method, the aforementioned dense surface layer is formed on the tissue forming surface made of the polymer material.

[0168] Furthermore, the outermost surface of the connective tissue formed on the tissue-forming surface made of polymeric materials readily and smoothly contains flattened collagen. Moreover, in the portion beyond the outermost surface of this dense surface layer and further inward, the connective tissue moderately aggregates, and fibrous collagen readily forms a mesh-like structure through a cross-linking arrangement, easily creating a structure formed by the overlapping of the aforementioned laminated structures.

[0169] (Step 4)

[0170] In step 4, the connective tissue forming substrate, which has formed connective tissue in step 3, is removed from the organism. Specifically, an extraction port can be formed at the location on the surface of the organism where the connective tissue forming substrate is embedded in the organism, and the connective tissue forming substrate is removed through this extraction port.

[0171] For example, when the connective tissue forming substrate has an mounting portion formed by a threaded portion (e.g., external thread) not shown, the mounting portion can be screwed onto the threaded portion (e.g., internal thread) provided at the front end of the substrate removal tool, thereby mounting the connective tissue forming substrate at the front end of the substrate removal tool and removing the connective tissue forming substrate from the biological body.

[0172] Alternatively, the end of the connective tissue forming substrate can be clamped and fixed using pliers or similar means, thereby removing the connective tissue forming substrate from the organism.

[0173] (Step 5)

[0174] Subsequently, if necessary, after destroying the connective tissue forming substrate, the connective tissue formed in the tissue forming space is peeled off from the tissue forming surface and removed, thereby obtaining the connective tissue body. Here, in the connective tissue forming substrate such as Figure 6 When the cover member 11 with a hole 17 formed by a slit and the core material 12 are shown, the connective tissue body is as follows: Figure 14 The connective tissue body 30 is shown to be tubular. In addition, a plurality of protrusions 31 are provided on the outer peripheral surface 30A of the connective tissue body 30 corresponding to the slit, and a dense surface layer is formed on the inner peripheral surface 30B of the connective tissue body 30.

[0175] In addition, when xenografting the obtained connective tissue, it is preferable to perform immunogenic removal treatments such as decellularization, dehydration, and fixation to prevent post-transplant rejection.

[0176] It should be noted that the manufacturing method described above is not limited to the method described as an example. For instance, although a method for placing a connective tissue forming substrate in a living organism has been specifically described with reference to the accompanying drawings, the method is not limited to that specific example.

[0177] Furthermore, while the above describes a method for manufacturing connective tissue bodies within a living organism, the method is not limited to manufacturing connective tissue bodies within a living organism. For example, connective tissue bodies can also be manufactured under conditions that are the same as or similar to those in a living organism.

[0178] In this case, the connective tissue forming substrate is appropriately placed in the environment where the biological tissue material is located, and connective tissue is formed in the tissue forming space in the same way as described above. A portion of the tissue forming surface that separates the tissue forming space is made of a polymer material. This allows the biological tissue to adhere closely to the outer peripheral surface of the connective tissue forming substrate, and a dense surface layer can be easily formed on the polymer-based tissue forming surface.

[0179] [Example]

[0180] The invention is illustrated in more detail by way of examples, but the invention is not limited to these examples in any way. It should be noted that, unless otherwise stated, "%" in the following description refers to "mass %".

[0181] The method for measuring the density of the surface layer is explained in detail below.

[0182] [Density determination]

[0183] (Slicing)

[0184] The connective tissue obtained in each embodiment and comparative example was used to prepare paraffin-embedded blocks through the following steps 1) to 7).

[0185] 1) Immerse the connective tissue in 4% paraformaldehyde-phosphate buffer for 24 hours.

[0186] 2) Remove the connective tissue and immerse it in 70% ethanol.

[0187] 3) After soaking the connective tissue in 99.5% ethanol for 30 minutes, remove it and repeat this operation 5 times.

[0188] 4) Immerse the connective tissue in 100% ethanol for 30 minutes.

[0189] 5) After soaking the connective tissue in xylene for 30 minutes, remove it and repeat this operation twice.

[0190] 6) After immersing the connective tissue body in molten paraffin at 60°C for 30 minutes, remove it and repeat this operation 4 times.

[0191] 7) Add the block into the embedding mold, allowing the paraffin wax to flow in and solidify, thus creating the block.

[0192] Paraffin-embedded blocks were cut to a thickness of 3 μm to prepare sections. Connective tissue bodies were cut along the thickness direction. The sections were then deparaffinized. The deparaffinization process involved immersing the sections in xylene twice, 100% ethanol twice, 70% ethanol once, and distilled water once, with each immersion lasting 10 minutes.

[0193] (Massen trichrome staining)

[0194] Next, perform Massen trichrome staining through the steps 1) to 21) below.

[0195] 1) Immerse the slices in mordant solution (manufactured by Muto Chemical Co., Ltd., serial number "40061") for 30 minutes.

[0196] 2) Remove the slices from the mordant and rinse them with running water.

[0197] 3) Immerse the slices in an equal volume mixture of Weigert iron hematoxylin solution (manufactured by Muto Chemical Co., Ltd., number "40341") and Weigert iron hematoxylin solution 2 (manufactured by Muto Chemical Co., Ltd., number "40351")) for 10 minutes.

[0198] 4) Remove the slices from the mixed solution and rinse them with running water.

[0199] 5) Wash the slices by shaking them up and down about 5 times in 1% hydrochloric acid alcohol solution for a few seconds. It should be noted that 1% hydrochloric acid alcohol solution is a solution of hydrochloric acid diluted with 70% ethanol to a concentration of 1%.

[0200] 6) To develop the color, rinse the slices with running water for 10 minutes.

[0201] 7) Immerse in the second mordant (manufactured by Muto Chemical Co., Ltd., serial number "81411") for 30 seconds.

[0202] 8) Remove the slices from the second mordant and rinse with running water for 1 minute.

[0203] 9) Shake the solution up and down about 5 times in 1% acetic acid water for a few seconds.

[0204] 10) Immerse in 0.75% Orange G (manufactured by Muto Chemical Co., Ltd., serial number "40231") for 1 minute.

[0205] 11) Remove the slices from the orange-yellow G and wash them by shaking them up and down about 5 times in 1% acetic acid water for a few seconds.

[0206] 12) Immerse in Mason staining solution B (manufactured by Muto Chemical Co., Ltd., serial number "40251") for 20 minutes.

[0207] 13) Wash the food by shaking it up and down about 5 times in 1% acetic acid water for a few seconds.

[0208] 14) Immerse in 2.5% phosphotungstic acid solution (manufactured by Muto Chemical Co., Ltd., serial number "40181") for 30 minutes.

[0209] 15) After removing it from the phosphotungstic acid solution, rinse it by shaking it up and down about 5 times in 1% acetic acid water for a few seconds.

[0210] 16) Immerse in aniline blue solution (manufactured by Muto Chemical Co., Ltd., serial number "40201") for 30 minutes.

[0211] 17) After removing it from the aniline blue solution, wash it by shaking it up and down about 5 times in 1% acetic acid for a few seconds.

[0212] 18) Clean the container by shaking it up and down about 10 times in 100% ethanol for a few seconds.

[0213] 19) To dehydrate the slices, immerse them in 100% ethanol for 5 minutes.

[0214] 20) Immerse the slices in xylene for 5 minutes, then remove them from the xylene. Repeat this process 3 times.

[0215] 21) Add sealing agent (Entellan(R)new Millipore, 1.07961.0100, manufactured by Merck) to the slide, cover with a coverslip, and allow the sealing agent to solidify on the glass to perform sealing.

[0216] (Image observation)

[0217] The sections sealed with the sealing agent as described above were illuminated at an illuminance of 500 lx using the transmission Kohler illumination provided with the microscope (Nikon, trade name "ECLIPSE E1000"), and observed at a magnification of 10x, and 30 images were taken.

[0218] For each image, perform image analysis processing using the steps 1) to 8) below to calculate the tissue density ratio.

[0219] 1) Use the H filter in HSV space to extract the blue part (collagen part).

[0220] 2) Calculate the brightness value (256 levels) through grayscale conversion. Figure 22 The histogram based on brightness values ​​in Embodiment 1, described later, is shown in the form of a brightness histogram.

[0221] 3) Normalize the histogram using a histogram normalization function, emphasize the brightness values ​​and distribute them to obtain a normalized intensity histogram (see reference). Figure 22 Additionally, the brightness values ​​are inverted, setting the highest brightness (the lightest stained part) to 0 and the lowest brightness (the darkest stained part) to 1.

[0222] 4) Obtain an arbitrary straight line L along the thickness direction of the connective tissue (refer to...). Figure 16 Brightness data on ).

[0223] 5) Filter using a 30-point moving average zero-phase filter.

[0224] 6) Calculate the overall average value of the obtained luminance data and use this overall average value as the threshold. Then, determine whether the luminance data on a straight line L on one surface of the connective tissue (i.e., the luminance data at the end of line L) is above the threshold. If the data is above the threshold, move from one end of line L to the other end, and designate the portion of data above the threshold as the "above-threshold portion," which is then designated as the "surface layer." Subsequently, similarly move to the other end; if the data is below the threshold, designate the portion of data below the threshold as the "below-threshold portion," which is then designated as the "part outside the surface layer."

[0225] Furthermore, if the luminance data on a straight line L on one surface of the connective tissue (i.e., the luminance data at the end of the straight line L) is less than a threshold, then from one end of the straight line L to the other end, the portion of the data less than the threshold is designated as the "less than threshold portion," and this "less than threshold portion" is designated as the "surface layer." Subsequently, moving similarly to the other end, if the data is above the threshold, the portion of the data "above the threshold" is designated as the "above the threshold portion," and this "above the threshold portion" is designated as the "part outside the surface layer."

[0226] 7) Next, calculate the average brightness data (Y1) of the portion identified as the "surface layer" on line L, and the average brightness data (Y2) of the portion identified as the "other part outside the surface layer". Use Y1 / Y2 as the tissue density ratio of the surface layer of a surface.

[0227] 8) For the remaining 29 images, the tissue density ratio is calculated similarly for the surface layer of one surface of the connective tissue body. The average of the tissue density ratios calculated in the 30 images is taken as the “tissue density ratio” of the surface layer of one surface.

[0228] Furthermore, if necessary, the tissue density ratio can also be calculated similarly for the surface layer of the other surface.

[0229] [Example 1]

[0230] First, prepare Figure 6The diagram shows a connective tissue forming substrate 10 comprising a cylindrical cap member 11 and a core material 12. The core material 12 comprises a central material 12B formed of a stainless steel tube with an acrylic rod embedded inside, and a cap tube 12C formed of silicone resin. The outer diameter of the core material 12 is 16 mm. The cap member 11 has an outer diameter of 19 mm and an inner diameter of 18 mm, and is made of stainless steel, with four 1.5 × 15 mm slits formed axially and eleven circumferentially. Therefore, the tissue forming surface formed by the core material 12 is silicone resin, and the tissue forming surface formed by the cap member 11 is stainless steel.

[0231] Next, under anesthesia, an incision was made in the back skin of a 12-month-old, 8.5kg Beagle to create an insertion port on the surface of the organism. Subcutaneous tissue was then dissected and cut through this port using scissors to create a subcutaneous pouch large enough to accommodate the connective tissue formation substrate 10. The connective tissue formation substrate 10 was manually pushed into the insertion port and embedded, after which the insertion port was sutured. Subsequently, a syringe needle was inserted through the skin onto the substrate, and air was aspirated from around the embedded connective tissue formation substrate 10.

[0232] Next, after raising the Beagle in a standard feeding environment for 60 days, a second incision was performed. An access port was created on the surface of the organism, and the connective tissue forming substrate 10 containing connective tissue was removed from the organism. The connective tissue was then extracted from the connective tissue forming substrate 10, thereby obtaining... Figure 14 The cylindrical connective tissue body 30 is shown. Before being removed from the connective tissue forming substrate 10, the inner circumferential surface of the connective tissue body 30 is in contact with or opposite the tissue-forming surface (silicone resin) of the core material 12 of the connective tissue forming substrate 10. Before being removed from the connective tissue forming substrate 10, the outer circumferential surface of the connective tissue body 30 is in contact with or opposite the tissue-forming surface (stainless steel) of the cover member 11 of the connective tissue forming substrate 10.

[0233] The obtained connective tissue body 30 was cut into sections, and the cross-section was observed using an electron microscope. The resulting photographs are shown below. Figure 15 . Figure 15 In the middle, the upper part is the inner circumferential side. Furthermore, the obtained connective tissue body 30 was sectioned according to the above method, and the cross-section of the section after Massen trichrome staining was observed under a microscope. The resulting photographs are shown below. Figures 16-18 . Figure 17 This is an enlarged view of the portion near the inner circumferential surface of connective tissue body 30. Figure 18 This is an enlarged view of the central portion of the connective tissue body 30 in the thickness direction.

[0234] like Figure 16 , 17As shown, on the inner circumferential side of the cylindrical connective tissue body, there are few voids and the connective tissue is dense, which can be understood as forming a dense surface layer.

[0235] In contrast, such as Figure 16 , 18 As shown, the portion outside the dense surface layer (e.g., the central portion in the thickness direction) has more voids, which can be understood as the internal density of the connective tissue being lower than that of the surface layer.

[0236] In addition, the density of the surface layer on one surface (inner peripheral surface) was measured according to the method described in the instruction manual. The results are shown in Table 1 below, with a tissue density of 1.56. The tissue density of the surface layer on the other surface (outer peripheral surface) was 1.48.

[0237] [Table 1]

[0238]

[0239] In addition, the inner circumferential surface of the connective tissue was observed using an electron microscope, and the resulting photographs are shown below. Figure 19 The inner circumferential surface of the connective tissue was further peeled into layers, and the surface condition of the dense surface layer outside the inner circumferential surface was observed using an electron microscope. The resulting photographs are shown below. Figure 20 Further peeling was performed, and electron microscopy was used to observe the state outside the dense surface layer. The resulting photographs are shown below. Figure 21 .

[0240] like Figure 19 As can be understood, on the surface of the dense surface layer, collagen is smoothly covered in a flattened shape, and the collagen is oriented randomly rather than unidirectionally. On the other hand, regarding the portion outside the surface of the dense surface layer, such as... Figure 20 As shown, fibrous collagen bundles together and are oriented in a roughly unidirectional direction. Additionally, from... Figure 15 , 20 The photos suggest that the fibrous collagen, bundled together and oriented in a single direction, is arranged in multiple layers. Furthermore, in... Figure 15 In the photograph, the granular dots are arranged in a continuous lateral manner. This suggests that the fibrous collagen in the layer formed by bundles of unidirectionally oriented fibrous collagen is orthogonal to the fibrous collagen in other layers, and can be understood as the fibrous collagen being arranged in a mesh-like pattern. This mesh-like arrangement continues inwards, such as... Figure 21 As shown, this configuration was also observed inside connective tissue bodies with low density.

[0241] In addition, such as Figure 20As shown, in the dense surface layer, collagen density is high and collagen fibers are coarse. On the other hand, as... Figure 21 As shown, outside the dense surface layer, the bundles of fibrous collagen are thinner than the bundles of collagen in the dense surface layer.

[0242] The connective tissue obtained in Example 1 was immersed in whole blood of dogs, and removed after 1, 3, and 5 minutes. The tissue was then rinsed with physiological saline, photographed, and observed. The results are as follows: Figure 23 As shown, almost no blood clotting or thrombus formation occurred.

[0243] On the other hand, the dense surface layer of the connective tissue obtained in Example 1 was peeled off, exposing the portion outside the surface layer. This tissue was then immersed in whole blood from dogs, removed after 1, 3, and 5 minutes, rinsed with physiological saline, and then photographed for observation. The results are as follows: Figure 23 As shown, thrombi adhered to a portion of the surface at 1 minute, and thrombus formation occurred across the entire surface at 5 minutes. It should be noted that, regarding the dense surface layer, as... Figure 24 As shown, it can be easily peeled into layers using tweezers and has a wall-backed structure.

[0244] The dense surface layer was further peeled off, and its weight (B(g)) was measured. It was then vacuum-dried at 25°C for 12 hours, and its weight (A(g)) was measured. The moisture content of the dense surface layer ((BA) / A×100) was calculated from the weight change, and it averaged 56%. On the other hand, the moisture content of the portion outside the dense surface layer was similarly calculated, averaging 73%. This indicates that the dense surface layer absorbs moisture less readily than the portion outside the dense surface layer.

[0245] [Example 2]

[0246] Prepare Figure 10 The diagram shows a connective tissue forming substrate 50 comprising a cylindrical cap component 51 and four core materials 52. The core materials 52 are stainless steel round bars encased in silicone resin tubes, with an inner diameter of 1 mm and an outer diameter of 2 mm, and an outer diameter of 1 mm for the silicone resin tubes. The cap component 51 has an outer diameter of 10 mm and an inner diameter of 8 mm. 1.5 mm circular holes are formed evenly in a staggered arrangement at an opening ratio of approximately 20% (the ratio of the slit area to the tissue forming surface of the cap component), and are made of stainless steel. The four core materials 52 are arranged approximately evenly inside the cap component 51. Therefore, the tissue forming surface formed by the core materials is silicone resin, and the tissue forming surface formed by the cap component is stainless steel.

[0247] Next, under anesthesia, an incision was made in the abdominal skin of a 3-month-old domestic pig weighing 40 kg to create an insertion port on the surface of the organism. Subcutaneous tissue was then dissected and cut through this port using scissors to create a subcutaneous pouch large enough to accommodate the connective tissue formation substrate 50. The connective tissue formation substrate 50 was manually pushed in through the insertion port, embedding the substrate, and the insertion port was then sutured. Subsequently, a syringe needle was inserted through the skin onto the substrate, and air was aspirated from around the embedded connective tissue formation substrate 50.

[0248] Next, after the pigs were raised in a standard feeding environment for 60 days, a second incision was performed. An access port was created on the surface of the organism, and the connective tissue forming substrate 50 containing connective tissue was removed from the organism. The connective tissue was then extracted from the connective tissue forming substrate 50, thereby revealing... Figure 3 The cross-sectional structure of the cylindrical connective tissue body 30, which has a cylindrical hollow cavity (hollow part) inside.

[0249] Furthermore, the density of the surface layer around the hollow portion (inner circumferential surface) and the outer circumferential surface of the connective tissue body 30 was measured according to the method described in the instruction manual. The results showed that the density around the internal cavities was 1.4, and the density of the outer circumferential surface was 1.2.

[0250] [Example 3]

[0251] Preparation Figure 12 The diagram shows a connective tissue forming substrate 60, serving as a support for a dissimilar component 37, mounted on the outer periphery of a cylindrical core material 62. The core material 62 is a round rod of nylon resin with an outer diameter of 15 mm. The support is a hollow sphere expanded type cobalt-chromium alloy with an inner diameter of 16 mm. Therefore, the tissue forming surface formed by the core material 62 is nylon resin, and the tissue forming surface formed by the dissimilar component 37 is cobalt-chromium alloy.

[0252] Next, under anesthesia, an incision was made in the back skin of a 10-month-old goat weighing 35 kg to create an insertion port on the surface of the organism. Subcutaneous tissue was then dissected and cut through this port using scissors to create a subcutaneous pouch large enough to accommodate the connective tissue formation substrate. The connective tissue formation substrate was manually pushed into the insertion port and embedded. The insertion port was then sutured shut. Subsequently, a syringe needle was inserted through the skin onto the substrate, and air was aspirated from the area surrounding the embedded connective tissue formation substrate.

[0253] Next, after raising the goats in a standard feeding environment for 30 days, a second incision was performed. An access port was created on the surface of the organism, and the connective tissue-forming substrate containing connective tissue was removed from the organism. The connective tissue was then extracted from the connective tissue-forming substrate, thereby revealing...Figure 4 The cross-sectional structure of the cylindrical connective tissue body 30, which has a cylindrical hollow cavity (hollow part) inside.

[0254] In addition, the density of the surface layer of one surface (inner circumferential surface) of the connective tissue body 30 and the surface layer around the internal support were measured according to the method described in the instruction manual. As a result, the tissue density of the inner circumferential surface of the connective tissue body 30 was 1.2, and the tissue density of the surface layer around the support was 1.1.

[0255] [Example 4]

[0256] Preparation Figure 11 The connective tissue forming substrate 10 shown includes a cylindrical cap member 11 and a core material 12, with a support serving as a dissimilar component 37 disposed in the gap between the cap member 11 and the core material 12. The core material 12 includes a core material 12B formed of PEEK resin and a cap tube 12C formed of silicone resin. The outer diameter of the core material 12 is 20 mm. The cap member 11 has an outer diameter of 25 mm, an inner diameter of 23 mm, and forms a 2 mm × 20 mm slit; it is made of stainless steel. The support is made of nickel-titanium alloy and has an inner diameter of 22 mm. Therefore, the tissue forming surface formed by the core material is PEEK resin, the tissue forming surface formed by the cap member is stainless steel, and the tissue forming surface formed by the support is nickel-titanium.

[0257] Next, under anesthesia, an incision was made in the back skin of a 43kg goat that was 12 months old. An insertion port was created on the surface of the organism, and subcutaneous tissue was dissected and cut through the insertion port using scissors to create a subcutaneous pouch large enough to accommodate the connective tissue formation substrate 10. The connective tissue formation substrate 10 was manually pushed in through the insertion port and embedded, and the insertion port was then sutured. Subsequently, a syringe needle was inserted through the skin onto the substrate, and air was aspirated from around the embedded connective tissue formation substrate 10.

[0258] Next, after raising the goats in a conventional feeding environment for 60 days, a second incision was performed. An access port was created on the surface of the organism, and the connective tissue forming substrate 10 containing connective tissue was removed from the organism. The connective tissue was then extracted from the connective tissue forming substrate 10, thereby obtaining... Figure 5 The cylindrical connective tissue body 30 shown has a cross-sectional structure.

[0259] Furthermore, the density of the surface layer of one surface (inner peripheral surface), the surface layer of the other surface (outer peripheral surface), and the surface layer surrounding the internal support of the connective tissue body 30 were measured according to the method described in the instruction manual. The results showed that the tissue density of the inner peripheral surface of the connective tissue body 30 was 1.3, the tissue density of the outer peripheral surface of the connective tissue body 30 was 1.2, and the tissue density of the surface layer surrounding the support was 1.1.

[0260] [Comparative Example 1]

[0261] Except for omitting the operation of extracting air from the organism using a syringe, it was carried out in the same manner as in Example 1. In Comparative Example 1, as... Figure 25 As shown, a blood clot has formed (upper left side), the connective tissue is not fully formed, and a dense surface layer has not been formed, as... Figure 26 As shown, the liquid agglomerates form red on a portion of the white collagen surface layer. Furthermore, they lack strength and exhibit surface cracking. Figure 26 (The central part). The inner peripheral surface of Comparative Example 1 was immersed in dog whole blood, removed after 1, 3, and 5 minutes, rinsed with physiological saline, photographed, and observed. The results are as follows. Figure 27 As shown, a thrombus formed on the entire surface within 1 minute.

[0262] In this disclosure, air extraction (step 2) is advantageous for promoting contact or improving contact between the tissue-forming surface of the connective tissue-forming substrate 10 and the cells of the organism. Air extraction is advantageous for promoting the encapsulation reaction of the connective tissue-forming substrate 10 in the organism, for example, it is advantageous for promoting the initiation of the encapsulation reaction. The formation of the connective tissue 30 based on the encapsulation reaction begins from the tissue-forming surface of the connective tissue-forming substrate 10 in the organism.

[0263] In this disclosure, the formation rate (growth rate) of the connective tissue body 30 during the initial period of formation or growth (step 3) is a factor affecting the density (and microstructure) of the surface layer in the connective tissue body 30, including the surface that contacts or opposes the tissue-forming surface of the connective tissue body forming substrate 10. The polymeric material (or a combination of polymeric and metallic materials) illustrated in this disclosure is advantageous for intentionally or controllably reducing the formation rate (growth rate) of the connective tissue body during the initial period of formation or growth (step 3). Using the polymeric material (or a combination of polymeric and metallic materials) illustrated in this disclosure on the tissue-forming surface of the connective tissue body forming substrate 10 is advantageous for giving the connective tissue body 30 a dense surface layer with a higher density. It should be noted that as the formation of the connective tissue body progresses, the formation rate of the connective tissue body may not be easily affected by the material of the tissue-forming surface. The choice of material for one or more tissue-forming surfaces helps to make the dense surface layer of the connective tissue body relatively dense, while making the portion outside the dense surface layer relatively sparse, and also helps to make the microstructure of the dense surface layer different from that of the portion outside the dense surface layer. In several examples, the difference in density between the dense surface layer and the portion outside the dense surface layer, and the difference in microstructure between the dense surface layer and the portion outside the dense surface layer, which can be a continuous variation in microstructure, can be functionally observed in the form of the aforementioned wall-apart structures, for example, as distinct or blurred layer boundaries under a microscope.

[0264] The portion of the connective tissue body 30 that is sparser than the dense surface layer, excluding the surface layer, is sometimes referred to as the non-surface layer portion, the main body portion, or the basal portion of the connective tissue body. The collagen forming the dense surface layer of the connective tissue body 30 is sometimes referred to as first fibrous collagen, and the collagen forming the non-surface layer portion of the connective tissue body 30 is sometimes referred to as second fibrous collagen. The structure and density of the first fibrous collagen in the dense surface layer of the connective tissue body 30 are sometimes referred to as first microstructure and first density, respectively. The structure and density of the second fibrous collagen in the non-surface layer portion of the connective tissue body 30 are sometimes referred to as second microstructure and second density, respectively. The structure and density of the first fibrous collagen impart first blood impermeability to the dense surface layer, and the structure and density of the second fibrous collagen impart second blood impermeability to the non-surface layer portion. The first blood impermeability of the dense surface layer is higher than the second blood impermeability of the non-surface layer portion. The wall-removing structure of this disclosure is sometimes referred to as a bio-derived peelable boundary formed by a first fibrous collagen layer of the surface layer and fibrous collagen of the non-surface layer portion 2. The connective tissue body 30 of this disclosure is sometimes referred to as an implantable product containing bio-derived collagen. The connective tissue body 30 of this disclosure can be the artificial organ itself or a part thereof for regenerative medicine.

[0265] Explanation of symbols

[0266] 10 Connective tissue forming substrate

[0267] 11. Cover components

[0268] 12 core material

[0269] 11A, 12A tissue formation surfaces

[0270] 13 Organizational Formation Space

[0271] 14, 15 Cover

[0272] 17 holes

[0273] 30 Connective tissues

[0274] 30A outer periphery

[0275] 30B Inner circumferential surface

[0276] 32 Dense Surface Layer

[0277] 35 Hollow section

[0278] 37. Dissimilar components

Claims

1. A connective tissue body using a tissue body forming substrate having a tissue forming surface that separates an environment in which a biological cell and a biological tissue material are present from a space for forming a connective tissue body, and the tissue body forming substrate having a hole that communicates the space and the environment and allows a connective tissue to invade into the space, the connective tissue body being formed by disposing the tissue body forming substrate in the environment, extracting air from the space in which the tissue body forming substrate is disposed, and placing the tissue body forming substrate in the environment from which the air is extracted, thereby forming a connective tissue body containing collagen produced by the biological cell, wherein the tissue forming surface is composed of a high molecular material or a metal material, the connective tissue body has a surface layer that is in contact with the tissue forming surface and a non-surface layer portion that is not in contact with the tissue forming surface other than the surface layer, the surface layer is formed of a collagen produced by the biological cell in the environment from which the air is extracted, and the non-surface layer portion is formed of a collagen produced by the biological cell in the environment from which the air is extracted, the collagen contains fibrous collagen, the fibrous collagen is flat and smooth on the surface of the dense surface layer, the fibrous collagen is arranged in a mesh shape in a crosswise manner in a portion other than the surface of the dense surface layer, a ratio of a tissue density of the surface layer calculated in a thickness direction to a tissue density of the non-surface layer portion is 1.1 or more, a foreign component is embedded in the inside, the dense surface layer is formed around the foreign component, the connective tissue body is formed by a capsulation reaction, the method has the following steps: a step of disposing a connective tissue body forming substrate having a tissue forming surface and at least a portion of the tissue forming surface composed of a high molecular material in an environment in vitro that is similar to an inside of a living body and in which a biological cell is present; and a step of extracting air from a space in the environment in vitro that is similar to the inside of the living body and in which the biological cell is present, through the skin and through a tube, by which step contact between the biological cell in the environment and the tissue forming surface of the connective tissue body forming substrate is improved, thereby promoting production of collagen by the biological cell and promoting formation of a connective tissue body composed of a biological tissue containing the collagen on the tissue forming surface.

2. The connective tissue body according to claim 1, wherein the fibrous collagen is arranged in a mesh shape in a crosswise manner in a portion other than the surface of the dense surface layer.

3. The connective tissue body according to claim 1, wherein a ratio of a tissue density of the surface layer calculated in a thickness direction to a tissue density of the non-surface layer portion is 1.1 or more.

4. The connective tissue body according to claim 1, wherein a foreign component is embedded in the inside.

2. The connective tissue body of claim 1, wherein, 5. The connective tissue body according to claim 1, wherein the dense surface layer is formed around the foreign component.

3. The connective tissue body of claim 2, wherein, 6. The connective tissue body according to claim 1, wherein the connective tissue body is formed by a capsulation reaction.

4. The connective tissue body of claim 2 or 3, wherein, 7. The method according to claim 1, wherein the method has the following steps: a step of disposing a connective tissue body forming substrate having a tissue forming surface and at least a portion of the tissue forming surface composed of a high molecular material in an environment in vitro that is similar to an inside of a living body and in which a biological cell is present; and a step of extracting air from a space in the environment in vitro that is similar to the inside of the living body and in which the biological cell is present, through the skin and through a tube, by which step contact between the biological cell in the environment and the tissue forming surface of the connective tissue body forming substrate is improved, thereby promoting production of collagen by the biological cell and promoting formation of a connective tissue body composed of a biological tissue containing the collagen on the tissue forming surface.

5. The connective tissue bodies of claim 1 or 2, wherein, ​ 6. The connective tissue bodies of claim 1 or 2, wherein, ​ 7. The connective tissue body of claim 6, wherein, ​ 8. The connective tissue bodies of claim 1 or 2, wherein, ​ 9. A method for producing a connective tissue body, which is a method for producing a connective tissue body formed of a collagen-containing tissue derived from a living organism and having a dense surface layer, wherein, ​ ​ ​ a step of forming a connective tissue body on the tissue formation surface by placing the connective tissue body forming base material in the environment from which air has been extracted, the connective tissue body having a surface layer in contact with the tissue formation surface and a non-surface layer portion other than the surface layer and not in contact with the tissue formation surface, the surface layer being formed of collagen produced by the organism cells in the environment from which air has been extracted at a higher tissue density, the non-surface layer portion being formed of collagen produced by the organism cells in the environment from which air has been extracted at a lower tissue density, the ratio of the tissue density of the surface layer to the tissue density of the non-surface layer portion calculated in the thickness direction being 1.1 or more; a step of taking the connective tissue body forming base material on which the connective tissue body has been formed out of the environment; and a step of peeling the connective tissue body from the connective tissue body forming base material.

10. A connective tissue body formed by placing a tissue body forming base material having a tissue formation surface that separates an environment in which organism cells and organism tissue materials are present from a space for forming a connective tissue body and having a hole that communicates the space and the environment and allows a connective tissue to invade into the space in an environment in an organism other than a human body from which air has been extracted from the space of the environment in which the tissue body forming base material is disposed, and placing the tissue body forming base material in the environment from which air has been extracted, thereby forming a connective tissue body containing collagen produced by the organism cells, wherein the tissue formation surface is formed of a high molecular material or a metal material, the connective tissue body is used in a tissue in an organism, the connective tissue body has a surface layer in contact with the tissue formation surface and a non-surface layer portion other than the surface layer and not in contact with the tissue formation surface, the surface layer is formed of collagen produced by the organism cells in the environment from which air has been extracted at a higher tissue density, the non-surface layer portion is formed of collagen produced by the organism cells in the environment from which air has been extracted at a lower tissue density, and the ratio of the tissue density of the surface layer to the tissue density of the non-surface layer portion calculated in the thickness direction is 1.1 or more.

11. A method of manufacturing a connective tissue body formed of collagen derived from an organism tissue formed of collagen produced by organism cells in an environment from which air has been extracted and used in a tissue in an organism, the method comprising the steps of: disposing a connective tissue body forming base material having a tissue formation surface and at least a portion of the tissue formation surface formed of a high molecular material in an environment outside an organism and in an environment in which organism cells are present; and ​ ​ ​ ​ ​ ​ a step of drawing air percutaneously and transductively from a space in which an extracorporeal environment similar to the inside of the organism is configured with the connective tissue formation substrate, by which the contact between the organism cells and the tissue formation surface of the connective tissue formation substrate in the environment is increased, thereby promoting the production of collagen from the organism cells and promoting the formation of a connective tissue formed of organism-derived tissue containing the collagen on the tissue formation surface; a step of forming a connective tissue on the tissue formation surface by placing the connective tissue formation substrate in the environment in which air is drawn, the connective tissue having a surface layer in contact with the tissue formation surface and a non-surface layer portion not in contact with the tissue formation surface, the surface layer being formed of organism-derived tissue containing collagen produced from the organism cells in the environment in which air is drawn at a higher tissue density, the non-surface layer portion being formed of organism-derived tissue containing collagen produced from the organism cells in the environment in which air is drawn at a lower tissue density, the ratio of the tissue density of the surface layer to the tissue density of the non-surface layer portion calculated in the thickness direction being 1.1 or more; a step of taking out the connective tissue formation substrate on which the connective tissue is formed from the environment; and a step of peeling the connective tissue from the connective tissue formation substrate.

12. The method of manufacturing a connective tissue body of claim 11, wherein, The connective tissue is formed by a capsulation reaction.

13. A connective tissue which is a connective tissue for regenerative medicine, the connective tissue being formed by disposing a tissue formation substrate having a tissue formation surface that separates an environment in which organism cells and organism tissue materials are present from a space for forming a connective tissue, and the tissue formation substrate having a hole that communicates the space and the environment and allows a connective tissue to invade into the space, and drawing air from the space in which the tissue formation substrate is disposed in an environment in an organism other than a human body, and placing the tissue formation substrate in the environment in which air is drawn, thereby forming a connective tissue, the tissue formation surface being composed of a high molecular material or a metal material, the connective tissue having: a surface layer having a first fibrous collagen produced from the organism cells in the environment in which air is drawn, the surface layer being in contact with the tissue formation surface; and a non-surface layer portion having a second fibrous collagen produced from the organism cells in the environment in which air is drawn, the non-surface layer portion not being in contact with the tissue formation surface, the first fibrous collagen of the surface layer having a first microstructure and a first density, the second fibrous collagen of the non-surface layer portion having a second microstructure different from the first microstructure and a second density smaller than the first density. the surface layer having a first fibrous collagen produced from the organism cells in the environment in which air is drawn, the surface layer being in contact with the tissue formation surface; and the non-surface layer portion having a second fibrous collagen produced from the organism cells in the environment in which air is drawn, the non-surface layer portion not being in contact with the tissue formation surface, the first fibrous collagen of the surface layer having a first microstructure and a first density, the second fibrous collagen of the non-surface layer portion having a second microstructure different from the first microstructure and a second density smaller than the first density, The surface layer and the non-surface layer portion form a wall-and-back structure due to a difference between the first density of the first fibrous collagen and the second density of the second fibrous collagen, and a difference between the first microstructure of the first fibrous collagen and the second microstructure of the second fibrous collagen.

14. The connective tissue body of claim 13, wherein, The surface layer and the non-surface layer portion form a layer boundary due to only a difference between the first density and the second density, and a difference between the first microstructure and the second microstructure.

15. The connective tissue body of claim 13, wherein, the first microstructure and the first density impart a first blood-impermeability to the surface layer, the second microstructure and the second density impart a second blood-impermeability to the non-surface layer portion, the first blood-impermeability of the surface layer is higher than the second blood-impermeability of the non-surface layer portion. the first blood-impermeability of the surface layer is higher than the second blood-impermeability of the non-surface layer portion.

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