Fibrinogen gel as biomaterial for hemostasis, tissue adhesion / closure, cell scaffold material, and so forth

Abstract

As one embodiment of the present invention, the present application discloses a dry gel of fibrinogen that is useful as a biomaterial for hemostasis, tissue adhesion / closure, a cell scaffold material, and the like.

Description

Fibrinogen gel as a biomaterial for hemostasis, tissue adhesion and closure, and cell scaffolding

[0001] The present invention relates to a fibrinogen gel or the like that is useful as a biomaterial for hemostasis, tissue adhesion / closure, cell scaffolding material, etc., and is useful, for example, in the field of medicine.

[0002] Fibrin glue has been widely used in medical settings for hemostasis and tissue adhesion / closure (e.g., Patent Documents 1 to 5). Fibrin glue preparations are broadly divided into liquid and sheet formulations. Liquid fibrin glue involves reacting fibrinogen and thrombin solutions on the surface of a wound to produce fibrin, which then polymerizes and crosslinks to form a three-dimensional fibrin network microscopically and a gel macroscopically, which then covers the wound to achieve the intended purpose. Even liquid fibrin glues are provided as compositions of dried fibrinogen and dried thrombin to improve storage stability. Each composition must be dissolved over time before use to obtain two liquids: fibrinogen and thrombin (Patent Documents 1 and 2). To address this issue, fibrin glues composed of fibrinogen and thrombin solutions that can be stored in liquid form have also been devised (Patent Documents 3 and 4). When fibrinogen and thrombin solutions are applied to a treatment site, it takes time for the two solutions to react and form a fibrin gel. This can lead to dripping on an inclined treatment surface, and it can be difficult to apply pressure until the two solutions react and reach a certain hardness. To address these drawbacks, various applicators have been developed to efficiently react the two solutions and maximize efficacy, and treatment methods, such as using them in combination with tissue reinforcement materials, have also been developed. To address the drawbacks of liquid fibrin glue, sheet formulations have been developed (Patent Documents 5-7). Because fibrinogen and thrombin themselves are difficult to process into sheets, fibrin glue sheets have been realized by supporting them on supports such as collagen or synthetic polymers. When the fibrinogen and thrombin are supported on the support, they elute upon contact with body fluids on the treatment surface, reacting to produce fibrin. Similar to the liquid formulation, this produces a gel that covers the treatment site, achieving the intended purpose. Sheet formulations do not require preparation before use, and they can be pressed against the treatment site and applied with pressure, offering improved convenience compared to liquid formulations. However, since it requires support components of non-human origin, it is not necessarily satisfactory from the viewpoint of biocompatibility.

[0003] It has been known that fibrinogen-containing fractions gel or produce insoluble matter during the production process of plasma fraction preparations (Patent Documents 1 and 2, Non-Patent Document 1). The resulting gels and insoluble matter are solubilized and purified, and then used as active pharmaceutical ingredients for various fibrinogen preparations. It has also been known that gels can be obtained by low-temperature treatment of purified fibrinogen or by low-temperature treatment with the addition of divalent metal ions (Non-Patent Documents 2 and 3). These gels (insoluble matter) are formed by the salting-out effect or hydrogen or ionic bonds. It has also been reported that fibrinogen can be covalently crosslinked and polymerized in the presence of FXIII or FXIIIa and calcium ions to produce a gel (Patent Documents 1 to 4, Non-Patent Documents 4 and 5). This reaction is an irreversible reaction in which isopeptide bonds are formed between glutamine and lysine residues in the Aα and γ chains of fibrinogen directly by transglutaminase FXIII or FXIIIa, without the intermediate step of thrombin-mediated fibrin production. Reversible fibrinogen gelation has been utilized in the production of fibrinogen drug substance for fibrinogen preparations and fibrin glue preparations (Patent Documents 1 to 4, Non-Patent Document 1). Conversely, the irreversible fibrinogen gelation reaction is considered a reaction that should be avoided in the production or storage of various fibrinogen preparations, and methods to avoid this reaction have been devised (Patent Documents 1 to 4). It was not known that the fibrinogen gels reported in Non-Patent Documents 4 and 5 could be used for hemostasis, tissue adhesion / closure, or as a cell scaffolding material. Furthermore, no reports have been published to date of this fibrinogen gel as a dry gel, nor have there been any reports of its preparation methods.

[0004] Patent No. 2896235 Publication Special Publication No. 63-40546 Publication Patent No. 3867931 Publication Patent No. 5074750 Publication Special Publication No. 2004-521115 Publication Patent No. 5192254 Publication Special Publication No. 2013-526369 Publication

[0005] Transfusion Medicine Reviews, 21(2), 101-117, 2007J Biorheol, 31(1), 12-15, 2017Gels, 9(3), 175, 2023THROBOSIS RESEARCH, 37, 613-628, 1985Biochemistry, 39, 6698-6705, 2000

[0006] The present invention aims to solve the above problems and to provide a new type of biomaterial that can replace fibrin glue and is useful for hemostasis, tissue adhesion and closure, cell scaffolding, and the like.

[0007] The present inventors have discovered that fibrinogen polymers, which have been suppressed or removed as reaction products to be eliminated in fibrinogen compositions, are actually more potent than monomeric fibrinogen, and have found that fibrinogen gels can be used as biomaterials for hemostasis, tissue adhesion and closure, cell scaffolding, etc. They have also found that fibrinogen dry gels are particularly useful in medical settings, and as a result of extensive research, have completed the present invention. Specific embodiments of the present invention are as follows, but the present invention is not limited to these.

[0008] [1] A dry gel of fibrinogen. [2] The dry gel according to [1] above, which restores to a hydrogel upon addition of water. [3] The dry gel according to [1] above, wherein, when the hydrogel (insoluble matter) obtained after hydration of the dry gel is suspended or dissolved and developed by electrophoresis under reducing conditions, two types of polypeptide chains, Aα and γ, are polymerized and crosslinked, and the ratio to the Bβ chain satisfies either of the following conditions: (Aα polymer) / (Bβ monomer)>0.15 (γ dimer) / (Bβ monomer)>0.2 (where Aα represents the Aα chain constituting fibrinogen, Bβ represents the Bβ chain constituting fibrinogen, and γ represents the γ chain constituting fibrinogen). [4] The dry gel contains transglutaminase and a calcium salt, and the molar ratio of fibrinogen:transglutaminase:calcium salt is 5-300:0.01-10:0.05×10. ３ ~200 x 10 ３The dry gel according to any one of [1] to [3] above, wherein the transglutaminase is FXIII and / or FXIIIa and the calcium salt is calcium chloride. [5] The dry gel according to [4] above, wherein the transglutaminase is FXIII and / or FXIIIa and the calcium salt is calcium chloride. [6] The dry gel according to any one of [1] to [5] above, wherein the dry gel is in the form of a composition containing a fibrinogen dry gel. [7] The dry gel according to [6] above, wherein the composition further contains thrombin. [8] The dry gel according to [6] or [7] above, wherein the composition is a sheet-shaped composition including a layer containing a fibrinogen dry gel as a main component ("fibrinogen dry gel layer"). [9] The dry gel according to [8] above, wherein the composition further includes one or more layers containing a component other than the fibrinogen dry gel layer as a main component, and wherein the composition is a sheet-shaped composition consisting of the fibrinogen dry gel layer and the one or more layers.

[10] The dry gel according to [9] above, wherein the layer other than the fibrinogen dry gel layer is a layer containing thrombin as a main component.

[11] A biomaterial containing a fibrinogen gel.

[12] The biomaterial according to

[11] above, which is used for hemostasis or tissue adhesion / closure.

[13] The biomaterial according to

[11] above, which is used for regenerative medicine.

[14] The biomaterial according to

[11] above, which is a sustained-release material.

[15] The biomaterial according to any of

[11] to

[14] above, wherein the fibrinogen gel is a dry fibrinogen gel.

[16] The biomaterial according to any of

[11] to

[15] above, wherein the dry fibrinogen gel is the dry fibrinogen gel according to any of [2] to

[10] above.

[17] A method for producing a composition containing a fibrinogen dry gel, comprising the step of reacting fibrinogen with transglutaminase in the presence of a calcium salt to obtain a fibrinogen gel.

[18] A method for producing the composition according to

[17] above, wherein the step of obtaining a fibrinogen gel further comprises the step of drying the fibrinogen gel to obtain a dry gel.

[19] A method for producing a composition according to

[17] or

[18] above, wherein in the step of obtaining a fibrinogen gel, a layer containing fibrinogen gel as the main component (a "fibrinogen gel layer") is constructed, and a sheet-like composition constituted by the fibrinogen gel layer is obtained.

[20] A method for producing the composition according to

[19] above, further comprising constructing one or more "layers whose main component is another component" other than the fibrinogen gel layer in addition to the fibrinogen gel layer.

[21] A method for producing the composition according to any one of

[17] to

[20] above, wherein the calcium salt is calcium chloride and the transglutaminase is FXIII and / or FXIIIa.

[0009] The present invention provides a fibrinogen dry gel or the like that is useful as a biomaterial for hemostasis, tissue adhesion / closure, cell scaffolding material, and the like.

[0010] FIG. 1 shows a process for obtaining a fibrinogen dry gel of the present invention, as described below, and the resulting powder composition and sheet composition. Figure A shows a process for obtaining a flowable fibrinogen gel or fibrinogen hydrogel from a fibrinogen gel reaction solution and then drying each to obtain a fibrinogen dry gel. Figure B shows a process for processing the fibrinogen dry gel to obtain a fibrinogen gel powder and a fibrinogen gel sheet. Figure C shows a process for obtaining a two-layered sheet composition containing a fibrinogen gel layer from a flowable fibrinogen gel or fibrinogen hydrogel. Figure D shows a scanning electron microscope image of a fibrinogen gel powder obtained by crushing a fibrinogen dry gel and processing it into a powder. From top to bottom, the images show fibrinogen powder derived from fibrinogen before the formation of a fibrinogen gel, fibrinogen gel powder derived from a flowable fibrinogen gel, and fibrinogen gel powder derived from a non-flowable fibrinogen hydrogel. Figure E shows a micrograph of a two-layered sheet composition consisting of a fibrinogen gel layer and a thrombin layer. The left image shows the sponge-like structure after the drying process before compression, and the right image shows the structure after compression. Both are cross-sectional views, with the fibrinogen gel layer on the left and the thrombin layer on the right. Figure 2 shows the (Aα polymer) / (Bβ monomer) ([2A]) and (γ dimer) / (Bβ monomer) ([2B]) obtained by electrophoresis under reducing conditions on a composition obtained by adding water to the freeze-dried fibrinogen gel preparation obtained in Example 1 described below. The numbers in the figure legend indicate the fibrinogen concentration (mg / mL) during the gelation reaction. Figure 3 shows the results of the characterization of the flowable fibrinogen gel obtained in Example 2 described below. Figure A shows size exclusion chromatograms of a fibrinogen concentrate (top) and a flowable fibrinogen gel (bottom). Figure B shows the fibrinogen concentration and the proportion of fibrinogen polymers measured by the thrombin clotting time method before gel formation (0 hr) and during gel formation over time from 0 to 7 hours. Figure C shows the fibrinogen concentration and the proportion of fibrinogen polymers measured by the thrombin clotting time method.Figure 4 shows size exclusion chromatographs of a fibrinogen concentrate (top) and a flowable fibrinogen gel (bottom) from Example 3, described below, along with the relative fibrinogen titers of the major fractions collected. Fraction numbers are shown below the size exclusion chromatographs, and bar graphs are provided for the fractions for which the relative fibrinogen titers were measured. Figure 5 shows the results of evaluation using a rat liver bleeding model in Example 4, described below. ([5A]: Evaluation results for hemostatic activity (bleeding volume over 10 minutes); [5B]: Evaluation results for adhesiveness (adhesion strength of test substance to wound surface (180° peel adhesive strength)). Shown as (Mean) ± (SE). N = 6-9 per group.) 'Fbg' in the table is an abbreviation for 'fibrinogen powder' in the text. Figure 6 shows the test results (tensile shear adhesive strength) from a rat skin adhesion test in Example 4, described below. Shown as (Mean) ± (SE). N=5 or 6 for each measurement point. 'Fbg' in the table is an abbreviation for 'fibrinogen powder' in the text. Figure 7 shows the evaluation results in a rat liver bleeding model in Example 5 described below ([7A]: evaluation results for hemostatic properties (amount of bleeding in 10 minutes); [7B]: evaluation results for adhesive properties (adhesion strength of test substance to wound surface (180° peel adhesive strength)). Shown as (Mean) ± (SE). N=7 or 8 for each group). 'Fbg', 'Fbg gel', and 'Fbn' in the table are abbreviations for 'fibrinogen', 'fibrinogen gel', and 'fibrin', respectively, in the text. Figure 8 shows histopathological images of various test substances applied to bleeding sites on the surface of a rat liver wound in Example 5 described below. ((A) Thr-fibrinogen powder, (B) Thr-fibrinogen gel powder, (C) Thr-fibrin powder, (D) Thr-fibrinogen sheet, (E) Thr-fibrinogen gel sheet, and (F) Thr-fibrin sheet. In all cases, the liver tissue is underneath and the test substance is placed on top. The liver tissue is gray, the test substance is light gray, and the blood is dark gray.) Figure 9 shows the evaluation results of a rat liver bleeding model in Example 6 described below. ([9A]: Evaluation results for hemostatic properties (amount of bleeding in 10 minutes); [9B]: Evaluation results for adhesive properties (adhesion strength of the test substance to the wound surface (180° peel adhesive strength)). Results are shown as (Mean) ± (SE). N = 7 to 9 for each group. 'Fbg gel' in the table is an abbreviation of 'fibrinogen gel' in the text.Figure 10 shows the growth curve of cultured cells in Example 7, which will be described later. The day the cells were seeded was Day 0, and CCK-8 assays were performed on Days 1, 7, 14, and 28. The horizontal axis represents the number of days, and the vertical axis represents the A450 value, as shown in a line graph. Results are expressed as (Mean) ± (SE). N = 3 for each point. 'Fbg gel' in the table is an abbreviation for 'fibrinogen gel' in the text.

[0011] Various embodiments of the present invention are described in detail below. Unless otherwise defined in context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that might be used in connection with the described invention.

[0012] Fibrinogen is one of the coagulation factors responsible for secondary hemostasis following primary hemostasis via platelet thrombus formation. In the normal hemostasis process, fibrinogen is converted to fibrin (monomer) by the action of thrombin, which then associates non-covalently via hydrogen bonds, ionic bonds, etc. to form a fibrin polymer. Further, covalent bonds (isopeptide bonds between glutamine and lysine residues in the α and γ chains of adjacent fibrin molecules) are formed between the non-covalently associated fibrin molecules by the action of transglutaminase FXIII or FXIIIa, forming a three-dimensional mesh structure microscopically and a gel macroscopically. This three-dimensional mesh structure then covers and solidifies the entire platelet thrombus, completing the hemostasis process. The fibrin gel has the properties of being able to be formed into a paste by compounding fibrinogen with drugs such as thrombin. Therefore, fibrin glue preparations are used in medical settings to generate fibrin gel not only at bleeding sites but also at damaged sites of organs, tissues, bones, etc., to adhere and seal the damaged tissue, ultimately stopping leakage of blood, body fluids, or gas. They are then used as a scaffold for regenerating damaged tissue, for tissue adhesion and closure, cell scaffolding, etc. The present inventors have now unexpectedly discovered that fibrinogen gel, rather than fibrin gel, is useful for hemostasis, tissue adhesion and closure, cell scaffolding, etc. Furthermore, they have also found that, among fibrinogen gels, fibrinogen dry gel is particularly useful from the perspective of its high practicality in medical settings. The present invention is described in detail below.

[0013] [Regarding fibrinogen dry gel] The present invention provides a "fibrinogen dry gel" as one embodiment thereof. The manufacturing method thereof will be described later. The fibrinogen dry gel is a fluid fibrinogen gel formed by covalently polymerizing and crosslinking fibrinogen, for example, by the action of transglutaminase, or a fibrinogen hydrogel formed by further polymerization and crosslinking, from which water, the dispersion medium, is removed by drying (preferably, freeze-drying) (Fig. 1A). The fibrinogen dry gel may contain other components derived from the manufacturing process. Note that the fibrinogen gel-forming reaction does not involve thrombin, and therefore does not produce fibrin.

[0014] As shown in Figure 1A, the viscosity of the fibrinogen solution increases continuously and the fluidity decreases as gel formation progresses due to fibrinogen polymerization and crosslinking (the degree of polymerization becomes more advanced), ultimately resulting in a non-fluid hydrogel. In this specification, a "fluid fibrinogen gel" refers to a fluid fibrinogen gel containing fibrinogen polymers and may also contain unpolymerized fibrinogen monomers. Here, a "fibrinogen polymer" refers to fibrinogen polymerized by covalent bonds, the proportion and degree of polymerization of which increase as the gelation reaction progresses. Such polymers may be included in both fluid fibrinogen gels and fibrinogen hydrogels. In this specification, a "fibrinogen hydrogel" refers to a gel that has progressed to a higher degree of gelation, contains fibrinogen polymers, and has lost its fluidity, and may contain some unpolymerized fibrinogen monomers. As used herein, the term "fibrinogen dry gel" refers to a fluid fibrinogen gel or fibrinogen hydrogel that has been dried (preferably freeze-dried) to remove water, the dispersing medium. The dry gel may contain fibrinogen monomers and fibrinogen polymers.

[0015] The above-mentioned "fibrinogen dry gel" will be described below in terms of its properties. The fibrinogen dry gel is preferably one that can be hydrated to efficiently produce a hydrogel (insoluble matter) (see Example 1 (2) 3) described below). More specifically, it is preferably one that can be hydrated to a fibrinogen concentration of 2.5 to 80 mg / mL and left at room temperature for 1 hour to produce a hydrogel (insoluble matter). A preferred embodiment of a fibrinogen dry gel is one in which, when 1 volume of the resulting hydrogel (insoluble matter) obtained after hydration is suspended or dissolved in 50 mM dithiothreitol in 7.2 M urea (6.7 volumes or more) and developed by electrophoresis under reducing conditions (fibrinogen application amount per well: 0.1 to 10 μg / 3 mm well), two types of polypeptide chains, Aα and γ, are polymerized and crosslinked, and the ratio to Bβ chain satisfies either of the following conditions: (Aα polymer) / (Bβ monomer)>0.15 (γ dimer) / (Bβ monomer)>0.2 (where Aα represents the Aα chain constituting fibrinogen, Bβ represents the Bβ chain constituting fibrinogen, and γ represents the γ chain constituting fibrinogen). The Aα polymer is a polymer in which any number of Aα chains and / or γ chains are covalently bonded. It should be noted that fibrin is composed of α-chains and β-chains, which are generated by cleaving fibrinopeptide A and fibrinopeptide B from the Aα-chain and Bβ-chain of fibrinogen, respectively, with thrombin, and γ-chains.

[0016] The dry gel contains transglutaminase and calcium salt, and the molar ratio of fibrinogen:transglutaminase:calcium salt is 5-300:0.01-10:0.05×10 ３ ~200 x 10 ３ In a preferred embodiment, the dry gel contains transglutaminase FXIII and / or FXIIIa in a ratio of: In this case, the transglutaminase may be FXIII and / or FXIIIa, and the calcium salt may be calcium chloride.

[0017] [Use of Fibrinogen Gel] As described above, the "fibrinogen gel" of the present invention (which refers to a fluid fibrinogen gel immediately after the start of fibrinogen gel formation, a fibrinogen hydrogel, or a fibrinogen dry gel) may be in the form of a composition containing other components derived from the production process. However, it may also be a composition containing "other components" other than the fibrinogen gel, as necessary. Here, the "other components" may be additives such as inorganic salts, amino acids (arginine, glycine, glutamic acid, isoleucine, etc.), surfactants, trehalose, sugar alcohols (glycerol, mannitol, etc.), chelating agents (ethylenediaminetetraacetic acid, sodium citrate, etc.), thickening polysaccharides (soluble starch, carrageenan, locust bean gum, xanthan gum, etc.), gelling polysaccharides (agar, pectin, chitosan, etc.), and animal-derived proteins (albumin, collagen, gelatin, etc.). Examples of "other components" include components that promote blood coagulation (e.g., polyphosphates, phospholipids, protamine), cell cultures, gene therapy materials, physiologically active substances (e.g., growth factors, hormones), steroids, anticancer drugs, antibiotics, anti-inflammatory agents, and analgesics. The fibrinogen dry gel described above can be used alone, for example, as a pulverized powder composition (see Figure 1B and Figure 1D). Because the fibrinogen dry gel is hydrated with blood or body fluids upon use, it essentially remains and functions as a fibrinogen gel in the body. This powder composition may be combined with one or more compositions primarily composed of "other components" other than the fibrinogen gel powder. Examples of powders primarily composed of "other components" include powders containing thrombin. Furthermore, multiple types of powders, including the fibrinogen gel powder, may be used in combination with biomaterials capable of supporting these components. The fibrinogen dry gel can be used alone, for example, as a molded sheet-like composition (see Figure 1B and Figure 1E). The fibrinogen dry gel is hydrated with blood or body fluids when used, so it essentially remains in the body and functions as a fibrinogen gel.The sheet-shaped composition may include one or more layers primarily composed of "other components" other than the fibrinogen gel layer, and may be a sheet-shaped composition consisting of a fibrinogen gel layer and one or more such layers (preferably six or fewer layers). The sheet-shaped composition may have a two-layer structure or more layers. An example of a layer primarily composed of "other components" other than the fibrinogen gel layer is a layer containing thrombin. The order of lamination is not particularly limited, and an appropriate intermediate layer may be provided between each layer, if necessary. In the sheet-shaped composition described above, the fibrinogen gel layer may also serve as a support for the layer primarily composed of "other components." Alternatively, the sheet-shaped composition may be formed in a form including a support commonly used in the art. This convenience is one of the excellent effects of the present invention.

[0018] [Method for producing fibrinogen gel and composition thereof] The above-mentioned "fibrinogen gel" (which refers to a fibrinogen hydrogel or fibrinogen dry gel produced from a fluid fibrinogen gel immediately after the start of fibrinogen gel formation) is not particularly limited, and can be produced, for example, by the following steps. The origin of the fibrinogen used is not particularly limited; for example, fibrinogen fractionated from pooled human plasma may be used, or fibrinogen derived from a commercially available fibrinogen preparation may be used. If necessary, fibrinogen may be subjected to antiviral treatment or the like. Furthermore, fibrinogen derived from genetically modified organisms or from animals other than humans may also be used.

[0019] (1) A step of reacting fibrinogen with transglutaminase in the presence of a calcium salt to obtain a fluid fibrinogen gel and fibrinogen hydrogel. Fibrinogen gel can be produced by reacting fibrinogen with transglutaminase (e.g., FXIII and / or FXIIIa) in the presence of a calcium salt (e.g., calcium chloride) in an appropriate buffer solution (e.g., a buffer solution containing sodium citrate, sodium chloride, and arginine) to polymerize and crosslink the fibrinogen (Fig. 1A). If necessary, an appropriate solvent such as water (distilled water) may be added. The fibrinogen gel reaction solution is preferably allowed to stand during gel formation. The gel formation temperature is not particularly limited, but can be performed, for example, at room temperature or under heating (preferably 10 to 40°C). Although not limited thereto, the blending ratio of fibrinogen:transglutaminase:calcium salt is, in terms of their molar ratio, 5-300:0.01-10:0.05×10. ３ ~200 x 10 ３In one preferred embodiment, the fibrinogen gel is formed under the above conditions to obtain a fibrinogen hydrogel via a fluid fibrinogen gel (Fig. 1A). The rate and extent of fibrinogen gel formation and the physical properties of the fibrinogen gel can be altered by changing the buffer composition, the fibrinogen / transglutaminase / calcium salt ratio, the temperature, the reaction time, etc. The origin of the transglutaminase used in this step is not particularly limited. It may be human-derived, animal-derived, or recombinantly added transglutaminase. Furthermore, if transglutaminase is present in the fibrinogen used as a raw material, it may be the endogenous transglutaminase. Here, the transglutaminase may be FXIII and / or FXIIIa, and the calcium salt may be calcium chloride. The produced hydrogel may be frozen. During the fibrinogen gel formation step, "other components" may be added as needed. For example, additives may include inorganic salts, amino acids (arginine, glycine, glutamic acid, isoleucine, etc.), surfactants, trehalose, sugar alcohols (glycerol, mannitol, etc.), chelating agents (ethylenediaminetetraacetic acid, sodium citrate, etc.), thickening polysaccharides (soluble starch, carrageenan, locust bean gum, xanthan gum, etc.), gelling polysaccharides (agar, pectin, chitosan, etc.), and animal-derived proteins (albumin, collagen, gelatin, etc.). "Other components" may also include components that promote blood coagulation (polyphosphate, phospholipids, protamine, etc.), cell cultures, gene therapy materials, physiologically active substances (growth factors, hormones, etc.), steroids, anticancer drugs, antibiotics, anti-inflammatory agents, analgesics, etc. The produced fluid fibrinogen gel or fibrinogen hydrogel may be frozen, if necessary, and used in the next step.

[0020] (2) Step of drying the fluid fibrinogen gel or fibrinogen hydrogel to obtain a fibrinogen dry gel. The desired fibrinogen dry gel can be produced by distilling off the water, which serves as the dispersant, from the fluid fibrinogen gel or fibrinogen hydrogel obtained in step (1) (FIG. 1A). The drying method is not particularly limited and can be performed by methods known in the art, such as lyophilization, preferably vacuum lyophilization. The conditions for vacuum lyophilization are not particularly limited and can be appropriately determined by those skilled in the art depending on the intended product. For example, vacuum lyophilization can be performed by performing primary drying at −80 to −10°C for 3 to 120 hours and secondary drying at 10 to 50°C for 0 to 120 hours. This method typically produces a dry gel with a water content of less than 5% by weight.

[0021] (3) Step of Obtaining a Powdered Composition The fibrinogen dry gel obtained in step (2) can be pulverized by a method known in the art to produce a powdered composition (see B and D in Figure 1). If necessary, this powder can be mixed with one or more powders primarily composed of "other components" to produce a powdered fibrinogen dry gel composition containing other components. Mixing with multiple types of powders is useful for separating and preventing components that would react with the fibrinogen, transglutaminase, and calcium salt contained in the fibrinogen gel when mixed. The "other components" are selected appropriately depending on the purpose of the composition and may be, for example, thrombin. By mixing with powders composed of other components in this way, it is possible to adjust the physicochemical properties, efficacy, in vivo degradation characteristics, biocompatibility, etc. of the fibrinogen gel powder composition. Here, "other components" may be added to any powder, including the fibrinogen gel powder, as needed. For example, additives may include inorganic salts, amino acids (arginine, glycine, glutamic acid, isoleucine, etc.), surfactants, trehalose, sugar alcohols (glycerol, mannitol, etc.), chelating agents (ethylenediaminetetraacetic acid, sodium citrate, etc.), thickening polysaccharides (soluble starch, carrageenan, locust bean gum, xanthan gum, etc.), gelling polysaccharides (agar, pectin, chitosan, etc.), and animal-derived proteins (albumin, collagen, gelatin, etc.). "Other components" may also include components that promote blood coagulation (polyphosphate, phospholipids, protamine, etc.), cell cultures, gene therapy materials, physiologically active substances (growth factors, hormones, etc.), steroids, anticancer drugs, antibiotics, anti-inflammatory agents, analgesics, etc. Furthermore, multiple types of powders, including these fibrinogen gel powders, may be combined with biomaterials capable of supporting them by methods known in the art.

[0022] (4) Step of Obtaining a Sheet-Like Composition. A sheet-like composition containing a fibrinogen dry gel can be produced by methods known in the art (see Fig. 1B and Fig. 1E). For example, fibrinogen gel formation in step (1) is carried out in an appropriate molding vessel, followed by polymerization and crosslinking reactions to obtain a fluid fibrinogen gel or fibrinogen hydrogel. This gel is then frozen and dried, if necessary, in step (2), and further compressed to form a sheet-like fibrinogen dry gel layer (fibrinogen gel layer), thereby producing a sheet-like composition. The fibrinogen gel layer thus obtained can also function as a support for the sheet-like composition. Alternatively, a sheet-like composition can be constructed by immobilizing the fibrinogen gel layer on a separate support. Examples of substrates that can form the separate support include bioabsorbable materials such as aliphatic polyesters, cellulose derivatives, collagen, gelatin, thickening polysaccharides, and gelling polysaccharides, processed into cotton, sheet, cloth, or sponge shapes. The fibrinogen gel layer and support may contain other components, if necessary. Examples of such components include inorganic salts, amino acids (e.g., arginine, glycine, glutamic acid, isoleucine), surfactants, trehalose, sugar alcohols (e.g., glycerol, mannitol), chelating agents (e.g., ethylenediaminetetraacetic acid, sodium citrate), thickening polysaccharides (e.g., soluble starch, carrageenan, locust bean gum, xanthan gum), gelling polysaccharides (e.g., agar, pectin, chitosan), and animal-derived proteins (e.g., albumin, collagen, gelatin). Examples of other components include components that promote blood coagulation (e.g., polyphosphates, phospholipids, protamine), cell cultures, gene therapy materials, physiologically active substances (e.g., growth factors, hormones), steroids, anticancer drugs, antibiotics, anti-inflammatory agents, and analgesics.

[0023] In the present invention, the sheet-shaped composition may be constructed by adding one or more (preferably six or fewer) "layers whose main component is another component" to the layer whose main component is fibrinogen gel (the "fibrinogen gel layer"). Multi-layering is useful for separating and preventing substances that would react with the fibrinogen, transglutaminase, and calcium salt contained in the fibrinogen gel when mixed. The "other component" is appropriately selected depending on the purpose of the composition, and may be, for example, thrombin. By providing such a "layer whose main component is another component," it is possible to adjust the physicochemical properties, efficacy, in vivo degradation characteristics, biocompatibility, and the like of the fibrinogen gel sheet-shaped composition. Multi-layered sheet-shaped compositions can also be produced by methods known in the art (FIG. 1C). For example, the fluid fibrinogen gel or fibrinogen hydrogel obtained in the molding container in step (1) can be frozen as needed, and then a layer containing other components can be constructed on top of the fibrinogen gel layer. This can then be frozen as needed in step (2), dried, and compressed into a sheet, producing a sheet-like composition in which a "layer mainly composed of other components" is further constructed on the fibrinogen gel layer. Alternatively, a sheet-like composition can be produced by further constructing a "layer mainly composed of other components" on a fibrinogen gel layer constructed by immobilizing the fibrinogen gel layer in a form that includes a separate support. Any of the "fibrinogen gel layer," "layer mainly composed of other components," and layers containing a support may contain "other components" as needed. For example, additives may include inorganic salts, amino acids (arginine, glycine, glutamic acid, isoleucine, etc.), surfactants, trehalose, sugar alcohols (glycerol, mannitol, etc.), chelating agents (ethylenediaminetetraacetic acid, sodium citrate, etc.), thickening polysaccharides (soluble starch, carrageenan, locust bean gum, xanthan gum, etc.), gelling polysaccharides (agar, pectin, chitosan, etc.), and animal-derived proteins (albumin, collagen, gelatin, etc.).Examples of "other components" include components that promote blood coagulation (polyphosphate, phospholipids, protamine, etc.), cell cultures, gene therapy materials, physiologically active substances (growth factors, hormones, etc.), steroids, anticancer drugs, antibiotics, anti-inflammatory agents, analgesics, etc. In a sheet-like composition having a multilayer structure, the order of construction of each layer is not limited, and by appropriately changing the order in production, a sheet-like composition having a layer order suitable for the intended use can be produced. Furthermore, in order to adjust the interaction between each layer, an intermediate layer may be provided by laminating the layers after a freezing step, if necessary.

[0024] [Usefulness of Fibrinogen Gel] The "fibrinogen gel" of the present invention (which refers to a fluid fibrinogen gel immediately after the start of fibrinogen gel formation, a fibrinogen hydrogel, or a fibrinogen dry gel) itself or a composition containing it is useful as a biomaterial. For example, by applying it to the body, it can be widely used as a biomaterial derived from a living organism for hemostasis, tissue adhesion / closure, cell scaffolding, etc. In particular, it has been confirmed that fibrinogen dry gel retains the properties of the fluid fibrinogen gel or fibrinogen hydrogel before drying, and that these properties can be reproduced by adding water. Furthermore, because excess water is removed from the fibrinogen dry gel, it has higher storage stability than fluid fibrinogen gel or fibrinogen hydrogel.

[0025] Fluid fibrinogen gels, fibrinogen hydrogels, and fibrinogen dry gels rehydrated with water contain fluid fibrinogen polymers, and these polymers have been shown to shorten the thrombin clotting time compared to fibrinogen monomers at the same concentration. Thrombin is generated in the blood at the wound site through the normal coagulation mechanism, and this acts on fibrinogen to produce fibrin at the wound site. When fibrinogen gel is applied to the wound, the fibrin produced by the normal coagulation mechanism also serves as an adhesive component between the fibrinogen gel and the wound site. If the fibrinogen gel is a hydrogel, it will immediately return to its pre-drying hydrogel state by itself, and if it is a dry gel, it will immediately return to its pre-drying hydrogel state by water migrated from the blood, and will serve as a wound-clotting component. The fluid fibrinogen gel that may be contained in the fibrinogen gel is subjected to the action of thrombin to become fibrin in a shorter time than normal fibrin formation from fibrinogen, and integrates with the fibrin produced by the normal coagulation mechanism described above. Through these fibrins, the gel-like fibrinogen gel adheres firmly to the wound site in a short time, enabling the wound site to be closed, thereby suppressing bleeding and achieving reliable hemostasis after a certain period of time.

[0026] Fibrinogen gel with the appropriate hardness generally remains at the treatment site even when it absorbs bodily fluids. For example, it has been confirmed that when applied to a wound, it remains there and is less likely to allow blood to pass to the opposite side of the bleeding surface. Therefore, compared to fibrinogen, which first goes through a liquid state at the wound site and then coagulates and gels under the action of thrombin, achieving occlusive properties, fibrinogen gel can encapsulate the wound more uniformly and robustly. Because fibrinogen gel with the appropriate hardness does not soak into gauze or other materials, it can be applied and pressed with gauze or other materials immediately after application to the treatment site. For example, when pressed against the bleeding surface of a wound, excess blood is pushed out, and fibrin produced by the hemostatic mechanism penetrates the unevenness of the wound surface, coagulating and gelling, thereby more firmly anchoring the treated fibrinogen gel to the wound surface. The fibrinogen gel introduced into the body then functions as a scaffold for cells to regenerate damaged tissue, similar to fibrin, and is degraded by the fibrinolytic system, primarily plasmin, and replaced with a collagen scaffold produced by the migrated cells, thereby regenerating the tissue. Therefore, fibrinogen gel is extremely useful as a biomaterial derived from a living organism.

[0027] The application of fibrinogen gel to a living body can be carried out, for example, by applying it to a wound site. The dosage for application can be determined appropriately by a person skilled in the art depending on the intended use. For example, for the purpose of hemostasis, the fibrinogen gel can be applied in a dose of 0.1 to 50 mg / cm in terms of fibrinogen depending on the size of the wound. ２ , preferably 0.5 to 25 mg / cm ２ , more preferably 2 to 15 mg / cm ２ Just apply it.

[0028] While the above-mentioned excellent functions are exhibited by fibrinogen gel alone, their functions can be more effectively exhibited by using it in combination with other components. The other components to be combined can be selected appropriately depending on the intended use. For example, when thrombin is used as the "other component" and applied to a wound site, the thrombin promotes fibrin production in the blood, which, combined with the excellent effects of the fibrinogen gel, enables more effective hemostasis, tissue adhesion, and closure of the wound site. Use in combination with such other components can be achieved, for example, by separately formulating the fibrinogen gel and the other components and applying them simultaneously to the body, or by applying a formulation combining both components in the form of the multilayered sheet composition described above to the body. Furthermore, if the fibrinogen gel contains growth factors or hormones as other components, it can promote the proliferation of specific cell populations and regulate homeostasis. Alternatively, if steroids or anticancer drugs are added, it can also have anti-inflammatory and anticancer effects. Furthermore, the inclusion of an analgesic can also alleviate pain at the treatment site. Based on the above, fibrinogen gel can be used as a scaffold for transplanted cells and cell growth, as well as a sustained-release agent for additives, making it a viable biomaterial for regenerative medicine. Furthermore, the aforementioned fibrinogen dry gel has been confirmed to have the function of imparting physical strength to sheet-shaped preparations. For example, a sheet-shaped composition consisting of multiple layers of human-derived fibrinogen gel and human-derived thrombin can provide a highly biocompatible sheet-shaped bioglue composed solely of human-derived components. It has also been confirmed that fibrinogen gels with the same degree of polymerization have higher plasmin resistance, i.e., fibrinolysis resistance, than the corresponding fibrin gels.

[0029] The various excellent functions of the fibrinogen gel of the present invention described above are specifically demonstrated in the examples below.

[0030] The present invention will be further illustrated by the following examples, which are not intended to limit the scope of the present invention and may be modified without departing from the scope of the present invention.

[0031] Example 1 Fibrinogen Hydrogel and Dry Gel (1) Preparation of Fibrinogen Dry Gel and Hydrogel 1) Fibrinogen (hereinafter also referred to as "fibrinogen concentrate") derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for Intravenous Injection 1g "JB", Japan Blood Products Organization) was used. FXIII activity in the fibrinogen concentrate was measured using a fully automated blood coagulation analyzer CS-2400 (Sysmex) and a Verichrome FXIII (Sysmex). The fibrinogen preparation was dialyzed to exchange the buffer (trisodium citrate dihydrate 2 g / L, sodium chloride 5 g / L, L-arginine 5 g / L) and adjusted to a fibrinogen concentration of 80 mg / mL (the same applies below). The 80 mg / mL fibrinogen concentrate was diluted with water (distilled water) and added to a 48-well plate at 150 μL / well in the arrangement shown in Table 1.

[0032]

[0033] 2) Calcium chloride was added to each well to the concentration shown in Table 1 above, and the plate was shaken and stirred, then allowed to stand at room temperature (approximately 25°C) to initiate fibrinogen gel formation. After 24 hours, KC4 Delta steel balls (Tcoag) were added one by one to the composition in each well (prepared product before lyophilization). The 48-well plate was placed in a vacuum freeze dryer (FZ-6, LABCONCO) pre-cooled to -30°C, and after confirming that it was frozen, the plate was freeze-dried using the following program to obtain a freeze-dried product. Segment 1: -30°C, hold for 25 hours Segment 2: Heat to 32°C at 0.03°C / min, hold for 6 hours Fibrinogen gel formation under the 48 conditions shown in Table 1 was performed using fibrinogen concentrates prepared from three lots of fibrinogen preparations, with each lot containing N=3, for a total of nine plates. Here, the three lots of fibrinogen concentrates contained 0.468-0.613 IU / mL of FXIII per 2 mg / mL of fibrinogen.

[0034] (2) Evaluation of Preparations The gelation and other properties of the preparations were evaluated as follows. 1) Hardness of the preparation before freeze-drying: After fibrinogen gel formation, a KC4 Delta steel ball was placed from above into each well containing the preparation. After freeze-drying, the hardness of the preparation before freeze-drying was visually inspected from the bottom of the 48-well plate. A score of - was given if the steel ball sank to the bottom of the well and was easily visible, ± if it was barely visible, and + if it was not visible. Conditions that yielded a ± or + rating were considered to have resulted in the fibrinogen gel-forming reaction resulting in the formation of a fibrinogen hydrogel. 2) Cracks in the preparation after freeze-drying: The presence or absence of cracks in the preparation obtained by freeze-drying after fibrinogen gel formation was checked. The 48-well plate after freeze-drying was visually inspected, and a score of - was given if there were cracks, and + if there were none. 3) Hardness of freeze-dried preparations when reconstituted in water: 150 μL / well of water was added to the freeze-dried preparations and the preparations were left to reconstitute at room temperature for 1 hour. Evaluation was based on whether a hydrogel (insoluble matter) of a certain hardness was formed. Scores were assigned as - if the hydrogel (insoluble matter) could not be picked up with tweezers after reconstitution in water, ± if it crumbled, and + if it could be picked up. Conditions for which a ± or + rating was obtained were considered to have restored the fibrinogen dry gel to a hydrogel (insoluble matter). For 1) and 3) above, - was assigned 0 points, ± 1 point, and + 2 points. For 2) above, the number of + ratings was tallied. The results for nine plates per condition are shown in Table 2.

[0035] 4) Evaluation of the freeze-dried preparation by electrophoresis: To the composition obtained by adding water to the freeze-dried preparation in 3) above at 150 μL / well, 900 μL of 8M urea and 100 μL of reducing agent (500 mM dithiothreitol-containing sample reducing agent (10x), Invitrogen) were added to each well, and the mixture was loosened with a spatula and allowed to stand at room temperature for 1.5 hours to suspend or dissolve the composition. The progress of fibrinogen gel formation was confirmed from the electrophoretic image as follows: Electrophoresis (Nu-PAGE, Invitrogen) was performed under reducing conditions (fibrinogen applied at a rate of 0.3 μg / 3 mm well per well), followed by Coomassie staining. Digital images were captured using an image analyzer (BIO-RAD, GS-900). Images were analyzed using ImageJ (National Institutes of Health). The fibrinogen bands appearing in each lane were classified into γ-monomer, Bβ-monomer, Aα-monomer, γ-dimer, and bands 1 to 10 higher, from the lowest molecular weight, as bands representing Aα-polymers (polymers in which any number of Aα-chains and / or γ-chains are covalently bonded). Their signal intensities were individually quantified. The signal intensity of the quantified bands was evaluated by calculating the sum of the signal intensities of the Aα polymer bands relative to the Bβ monomer (Aα polymer) and the ratio of γ dimer using the following formulas: (Aα polymer) / (Bβ monomer) and (γ dimer) / (Bβ monomer), respectively.

[0036] (3) Experimental Results 1) Regarding the hardness of the preparations before freeze-drying obtained when fibrinogen gel was formed for 24 hours under the 48 conditions in Table 1, those containing 2.5 to 80 mg / mL of fibrinogen concentrate and 1.56 to 100 mM calcium chloride frequently produced fibrinogen hydrogels hard enough to hold a steel ball (Table 2 below). 2) Regarding cracking in the preparations obtained by freeze-drying after fibrinogen gel formation, those containing 10 to 80 mg / mL of fibrinogen concentrate and 1.56 to 100 mM calcium chloride frequently produced well-formed freeze-dried preparations, while those containing 10 to 40 mg / mL of fibrinogen concentrate and 1.56 to 50 mM calcium chloride frequently produced crack-free freeze-dried preparations (Table 2 below). 3) When freeze-dried preparations were reconstituted in water, those containing 2.5 to 80 mg / mL fibrinogen concentrate and 1.56 to 100 mM calcium chloride frequently yielded hydrogels (insoluble matter) that could be picked up with tweezers (Table 2). 4) When freeze-dried preparations were reconstituted in water, suspended or dissolved compositions were electrophoresed under reducing conditions. As shown in Figure 2, differences in the ratio of Aα polymer and γ dimer relative to Bβ monomer were observed (in Figure 2, [2A] indicates (Aα polymer) / (Bβ monomer), and [2B] indicates (γ dimer) / (Bβ monomer). The ratio of Aα polymer / Bβ monomer for compositions reconstituted in water was less than 0.15 when calcium chloride was 0 mM, but was greater than 0.15 when calcium chloride was 1.56 mM or greater. The (γ-dimer) / (Bβ-monomer) ratio of the composition obtained by reconstituting the freeze-dried preparation with water was less than 0.2 when calcium chloride was 0 mM, but was 0.2 or greater when calcium chloride was 1.56 mM or greater.

[0037]

[0038] *The table shows a 48-well plate, and the fibrinogen and calcium concentrations in each well correspond to the arrangement in Table 1. The numbers in the table are the aggregate scores for three lots of fibrinogen concentrate, N=3 for each, for a total of nine plates. '-' indicates that no shaped freeze-dried preparation was obtained. "Hardness of preparation before freeze-drying" was scored from 0 to 18, with 18 being the hardest, and "cracks in the freeze-dried preparation" was scored from 0 to 9, with 9 being the fewest cracks. "Hardness of freeze-dried preparation when reconstituted in water" was scored from 0 to 18, with 18 being the hardest.

[0039] (4) Arrangement of parameters The various parameters in the preparation of the fibrinogen gel described above are arranged as follows: 1) Conditions under which a fibrinogen hydrogel can be formed If the conditions under which the hardness of the preparation before freeze-drying in Table 2 is scored as 1 or higher with a frequency of 50% or more are met, the concentrations of the following components in the fibrinogen gel reaction solution are fibrinogen (mg / mL): FXIII (IU / mL): calcium chloride (mM) = 2.5-80: 0.595-24.5: 1.56-100 When the above are expressed as molar ratios, fibrinogen: FXIII: calcium chloride = 7.35-235: 0.039-1.61: 1560-100000. 2) Conditions under which a crack-free fibrinogen dry gel can be formed If a fibrinogen hydrogel can be formed and the cracking score of the freeze-dried preparation in Table 2 is 1 or higher at a frequency of 50% or more, the concentrations of the following components in the fibrinogen gel reaction solution are: Fibrinogen (mg / mL): FXIII (IU / mL): calcium chloride (mM) = 10-40: 2.34-12.3: 1.56-50 Expressed as a molar ratio, the following is fibrinogen:FXIII:calcium chloride = 29.4-118: 0.154-0.807: 1560-50000. 3) Conditions for forming a fibrinogen dry gel that restores to a hydrogel when water is added If a fibrinogen hydrogel can be formed and the freeze-dried preparation in Table 2 receives a score of 1 at a frequency of 50% or more when reconstituted in water, the concentrations of the following components in the fibrinogen gel reaction solution are: Fibrinogen (mg / mL): FXIII (IU / mL): calcium chloride (mM) = 2.5-80: 0.595-24.5: 1.56-100 Expressed as a molar ratio, fibrinogen:FXIII:calcium chloride = 7.35-235: 0.039-1.61: 1560-1000004) When the freeze-dried preparation was reconstituted in water under conditions that resulted in a hardness score of 1 or greater, the freeze-dried preparation was hydrated to obtain a composition (1 volume), which was then suspended or dissolved in 50 mM dithiothreitol in 7.2 M urea (6.7 volumes or greater) and subjected to electrophoresis under reducing conditions. The two polypeptide chains that constitute fibrinogen, Aα and γ, were polymerized and crosslinked, and the ratio of Aα to Bβ chains satisfied one of the following conditions: (Aα polymer) / (Bβ monomer)>0.15 (γ dimer) / (Bβ monomer)>0.2 (where Aα polymer is a polymer in which any number of Aα chains and / or γ chains are covalently bonded).

[0040] (5) Implications of Example 1 1) The conditions under which fibrinogen hydrogels could be formed and the conditions under which fibrinogen dry gels could be restored to hydrogels upon addition of water were generally correlated. While these conditions can vary significantly depending on the buffer composition, additives, temperature, and gelation time during gelation, the conditions under which fibrinogen hydrogels could be formed and fibrinogen dry gels obtained in this example were appropriate when the fibrinogen concentrate was in the range of 2.5 to 80 mg / mL, the FXIII was in the range of 0.59 to 24.5 IU / mL, and the calcium chloride was in the range of 1.56 to 100 mM. 2) The conditions under which fibrinogen hydrogels could be formed and the conditions under which fibrinogen dry gels could be restored to hydrogels upon addition of water did not correlate with the conditions under which crack-free fibrinogen dry gels were likely to be formed. 3) The conditions for obtaining a fibrinogen dry gel suitable for forming a sponge or compressing it into a sheet are those that minimize cracking and restore the fibrinogen dry gel to a hydrogel upon addition of water. These conditions can vary significantly depending on the buffer composition, additives, temperature, and gelation time during gelation. However, the fibrinogen gel formation conditions shown in this example were appropriate: a fibrinogen concentrate in the range of 10-40 mg / mL, FXIII in the range of 2.34-12.5 IU / mL, and calcium chloride in the range of 1.56-50 mM. 4) Fibrinogen gel formation occurred under conditions that yielded a score of 1 or higher when the hardness of the preparation before lyophilization or the hardness of the preparation after lyophilization was reconstituted in water. A higher score indicated more advanced fibrinogen gel formation. This was also confirmed by the increase in the proportions of Aα polymer and γ dimer.Although this can vary greatly depending on the buffer composition, additives, temperature, and gel formation time during gel formation, under the fibrinogen gel formation conditions shown in this Example, when fibrinogen is in the range of 2.5 to 80 mg / mL, FXIII is in the range of 0.595 to 24.5 IU / mL, and calcium chloride is in the range of 1.56 to 100 mM, the hydrogel (insoluble matter) (1 volume) obtained after adding water is suspended or dissolved in 50 mM dithiothreitol in 7.2 M urea (6.7 volumes or more) and developed by electrophoresis under reducing conditions, the two polypeptide chains that make up fibrinogen, Aα and γ, are polymerized and crosslinked, and the ratio to the Bβ chain satisfies any of the following conditions: (Aα polymer) / (Bβ monomer)>0.15 (γ dimer) / (Bβ monomer)>0.2 (Here, Aα polymer is a polymer in which any number of Aα chains and / or γ chains are covalently bonded.) 5) Non-Patent Document 2, under conditions different from those used in this example, confirmed that when fibrinogen was in the range of 3 to 24 mg / mL, FXIII was in the range of approximately 0.25 to 2 IU / mL, and the calcium chloride concentration was in the range of 0.05 to 5 mM, the formation of fibrinogen Aα polymer and γ dimer progressed and an insoluble fibrinogen gel was produced. It was also confirmed that a similar reaction proceeded more rapidly when FXIII was replaced with FXIIIa. While FXIII was used as the transglutaminase in this example, the inventors separately confirmed that a similar reaction proceeded when FXIII was replaced with FXIIIa.

[0041] Example 2 Clotting Ability of Fibrinogen Gel (1) (1) Preparation and Evaluation of Fibrinogen Gel Fibrinogen (fibrinogen concentrate) derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for Intravenous Injection 1g "JB," Japan Blood Products Organization) was used. The fibrinogen concentrate contained 5.57 IU / mL (average value) of FXIII per 20 mg / mL of fibrinogen. The fibrinogen preparation was dialyzed to exchange the buffer (trisodium citrate dihydrate 2 g / L, sodium chloride 5 g / L, L-arginine 10 g / L). Calcium chloride was added to 11 mg / mL of the fibrinogen concentrate to a final concentration of 25 mM to initiate fibrinogen gel formation, yielding a fibrinogen polymer. The fibrinogen solution before the addition of calcium chloride and the fibrinogen gel reaction solution from immediately after the addition of calcium chloride until 7 hours later were fluid, but lost fluidity after 8 hours and became a fibrinogen hydrogel. A size-exclusion chromatogram was obtained by using an HPLC system (Shimadzu Seisakusho, CBM-20A) and a column (TOSOH, TSK-gel G4000SWXL) with a mobile phase of 0.3 M sodium chloride, 0.05 M phosphoric acid, pH 7.0, at 0.5 mL / min and 25°C. The samples applied to the column were the fibrinogen solution without calcium chloride (fibrinogen concentrate) and the liquid fibrinogen gel reaction solution (fluid fibrinogen gel) obtained every hour from the addition of calcium chloride until 7 hours later. Furthermore, fibrinogen concentration was measured by the thrombin clotting time method using a clotting time measuring device (KC4 Delta, Tcoag) and Thrombocheck Fib (Sysmex). Measurement of fibrinogen concentration by the thrombin clotting time method was performed on the fibrinogen solution without calcium chloride (fibrinogen concentrate) and the liquid fibrinogen gel reaction solution (fluid fibrinogen gel) 1, 3, 5, and 7 hours after the addition of calcium chloride.

[0042] The fibrinogen gel formation that occurs when calcium chloride is added to a fibrinogen concentrate was confirmed by size exclusion chromatography, indicating the progression of fibrinogen polymerization. The size exclusion chromatogram of fibrinogen concentrate without added calcium chloride showed one peak each for fibrinogen monomer and polymer (Figure 3A, top). At this time, the fibrinogen polymer content was 19%. The chromatogram 7 hours after the addition of calcium chloride showed a fibrinogen monomer peak and multiple fibrinogen polymer peaks, and the fibrinogen polymer content increased to 70% (Figure 3A, bottom). The fibrinogen gel reaction solution, which was fluid up to 7 hours after fibrinogen gel formation, had become a non-fluidic hydrogel by 8 hours. Figure 3B shows the fibrinogen concentrate and the fibrinogen polymer content at hourly intervals from the start of fibrinogen gel formation up to 7 hours. During fibrinogen gel formation in the fibrinogen concentrate, the fibrinogen gel reaction solution remained fluid for up to 7 hours. The fibrinogen concentration, as determined by the thrombin clotting time method, was calculated before the addition of calcium chloride and 1, 3, 5, and 7 hours after the addition. These results are shown in Figure 3B. The percentage of fibrinogen polymer generated and the fibrinogen concentration, as determined by the thrombin clotting time method, increased over time during the 7-hour period when the solution remained fluid (Figure 3B). The fibrinogen concentrate without calcium chloride contained 19% fibrinogen polymer, which increased over time to 70% after 7 hours of calcium chloride addition. The fibrinogen concentration, as determined by the thrombin clotting time method, also increased from 11 mg / mL to 20 mg / mL. All samples were diluted 100-fold for the measurements. The clotting time, as determined by the thrombin clotting time method, was 25 seconds when a fibrinogen concentration of 11 mg / mL was obtained, and 15 seconds when a fibrinogen concentration of 20 mg / mL was obtained. There was a positive correlation between the proportion of fibrinogen polymers in the fibrinogen concentrate and the fibrinogen concentration measured by the thrombin clotting time method (R ２ = 0.9897, Figure 3C).

[0043] (2) Summary of parameters The various parameters used in preparing the fibrinogen gel described above are summarized below. 1) Conditions for forming fibrinogen hydrogel The concentrations of the following components in the fibrinogen gel reaction solution were: fibrinogen (mg / mL): FXIII (IU / mL): calcium chloride (mM) = 20:5.57:25. Expressed as a molar ratio, the fibrinogen:FXIII:calcium chloride = 58.8:0.366:25000. 2) The polymer content in the fibrinogen gel after the addition of calcium chloride was >19% (measured by size exclusion chromatography).

[0044] (3) Implications of Example 2 1) In a fibrinogen gel reaction solution prepared by adding calcium chloride to fibrinogen and FXIII and / or FXIIIa, fibrinogen gel formation begins, and the proportion of fibrinogen polymers increases. Initially, the fibrinogen gel is fluid, but as the polymer content increases, the viscosity increases and it becomes a non-fluid fibrinogen hydrogel. 2) When the thrombin clotting time of the fibrinogen gel reaction solution was measured using a Thrombocheck Fib, it shortened over time, confirming an increase in fibrinogen concentration using the thrombin clotting time method. In other words, as the fibrinogen polymer content of the fibrinogen gel increased, the thrombin clotting time shortened, resulting in an increase in the apparent fibrinogen titer. 3) Because fibrinogen gel contains fluid fibrinogen gel even when it becomes a polymerized hydrogel or dry gel, it can be processed into a glue formulation that coagulates quickly when combined with thrombin.

[0045] Example 3 Clotting Ability of Fibrinogen Gel (2) (1) Preparation of Fibrinogen Gel and Evaluation of Fluidity Fibrinogen (fibrinogen concentrate) derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for Intravenous Injection 1g "JB," Japan Blood Products Organization) was used. The fibrinogen concentrate contained 5.57 IU / mL (average value) of FXIII per 20 mg / mL of fibrinogen. The fibrinogen preparation was dialyzed and exchanged with TBS buffer (TBS Tablets pH 7.6, Takara Bio). Calcium chloride was added to 20 mg / mL of fibrinogen concentrate to concentrations of 0.0625, 0.125, 0.2, 0.25, 0.5, 1, and 32 mM to initiate fibrinogen gel formation, and fluidity was confirmed after 1 to 72 hours. Fluidity was assessed as a fluid fibrinogen gel if it could be pipetted and passed through a 0.45 μm filter, and as a fibrinogen hydrogel if it could not be pipetted or could be pipetted but did not pass through a 0.45 μm filter. If at a certain evaluation point it could not be pipetted or did not pass through a 0.45 μm filter, it was assessed to have become a hydrogel from that point on. (2) Evaluation of the relative potency of fibrinogen with different degrees of polymerization obtained from fluid fibrinogen gel Calcium chloride was added to 20 mg / mL of fibrinogen concentrate to a concentration of 0.4 mM to initiate fibrinogen gel formation, and a fluid fibrinogen gel was obtained after 1 hour. The flowable fibrinogen gel was prepared using a liquid chromatography system (AKTA pure 150, GE Healthcare) with a gel filtration column (Superose 6 Increase 10 / 300GL, GE Healthcare) and a TBS buffer mobile phase at 0.5 mL / min. The sample was applied to obtain a chromatogram, and 0.5 mL aliquots were collected. The fibrinogen concentration of the collected fractions was measured by the thrombin clotting time method using a clotting time analyzer (KC4 Delta, Tcoag) and a Thrombocheck Fib (Sysmex). The fibrinogen concentration of a 1 mg / mL fibrinogen concentrate (calculated by absorbance conversion) using the thrombin clotting time method was defined as 100% relative titer.The relative titer of fibrinogen in the measurement fraction was calculated using the following formula: [Relative titer of fibrinogen in measurement fraction] = [Fibrinogen concentration in measurement fraction at 1 mg / mL in absorbance equivalent, determined by thrombin clotting time method] / [Fibrinogen concentration in fibrinogen concentrate at 1 mg / mL in absorbance equivalent, determined by thrombin clotting time method] × 100%.

[0046] (3) Preparation of fibrinogen gel and evaluation of fluidity The fluidity of the fibrinogen gel reaction solution was not lost even after 72 hours of adding 200 μM or less calcium chloride to the fibrinogen concentrate (Table 3). When 250 μM calcium chloride was added to the fibrinogen concentrate, the fibrinogen gel reaction solution no longer passed through a 0.45 μm filter after 6 hours. Therefore, the solution before this point was considered to be a fluid fibrinogen gel, and the solution after this point was considered to be a fibrinogen hydrogel. When calcium chloride was added in the range of 500 μM to 32 mM, the solution became a fibrinogen hydrogel after 1 hour.

[0047]

[0048] *The fluidity of fibrinogen gel was evaluated as ◯ (fluid fibrinogen gel) if it could be pipetted and passed through a 0.45 μm filter, △ (fibrinogen hydrogel) if it could not be pipetted, and × (fibrinogen hydrogel) if it could not be pipetted, as shown in Table 3. If it could not be pipetted at a certain evaluation point or did not pass through a 0.45 μm filter, it was determined to have become a hydrogel from that point on, and no evaluation was made (NA).

[0049] (4) Evaluation of the relative potency of fibrinogen with different degrees of polymerization obtained from fibrinogen gel. Size exclusion chromatography was performed on a fibrinogen concentrate (top of Figure 4) and a fluid fibrinogen gel (bottom of Figure 4) prepared by adding 0.4 mM calcium chloride to the fibrinogen concentrate and allowing it to stand for 1 hour. From the chromatogram, peaks were designated peak 1 and peak 2, representing fibrinogen polymers, starting with the peak with the shortest column retention time. The subsequent peaks were designated fibrinogen monomer peaks. Fibrinogen polymer (sum of peaks 1 and 2) accounted for 22% of the fibrinogen concentrate and 41% of the fluid fibrinogen gel. The relative potency of fibrinogen before fractionation was calculated by size exclusion chromatography for the fibrinogen concentrate and fluid fibrinogen gel. When the potency of the fibrinogen concentrate and fluid fibrinogen gel was determined as 100%, the potency of the latter increased by 131% (Table 4). The relative fibrinogen titers of the fibrinogen concentrate and flowable fibrinogen gel fractions collected using a size-exclusion chromatography system were calculated. The relative fibrinogen titers of the polymer fraction (fraction A.11) and monomer fraction (fraction B.3) of the fibrinogen concentrate were 289% and 42.5%, respectively (Figure 4, top, Table 4). Similarly, the relative fibrinogen titers of the polymer fraction (fractions A.6-10) and monomer fraction (fraction B.3) of the flowable fibrinogen gel were 202-405% and 40.5%, respectively (Figure 4, bottom, Table 4).

[0050]

[0051] *The relative titer of fibrinogen was determined as 100% when the fibrinogen concentration of a 1 mg / mL fibrinogen concentrate, calculated as absorbance by the thrombin clotting time method, was calculated without adding calcium. The relative titer of fibrinogen in the measurement fraction was calculated using the following formula: [Relative titer of fibrinogen in the measurement fraction] = [Fibrinogen concentration of a 1 mg / mL measurement fraction, calculated as absorbance by the thrombin clotting time method] / [Fibrinogen concentration of a 1 mg / mL fibrinogen concentrate, calculated as absorbance by the thrombin clotting time method] x 100%

[0052] (5) Summary of Parameters The various parameters used in preparing the fibrinogen gel described above are summarized below. 1) Conditions for forming fibrinogen hydrogel The concentrations of the following components in the fibrinogen gel reaction solution were: Fibrinogen (mg / mL): FXIII (IU / mL): Calcium chloride (mM) = 20: 5.57: 0.25-32. Expressed as a molar ratio, the ratio is: Fibrinogen: FXIII: Calcium chloride = 58.8: 0.366: 250-32,000. 2) The fluid fibrinogen gel contained 22%-41% fibrinogen polymer (by size exclusion chromatography). 3) When the fibrinogen polymer content in the fluid fibrinogen gel was 41%, the relative titer of fibrinogen was 131% of that of the fibrinogen concentrate. 4) The relative fibrinogen titers of the monomer fraction in the fibrinogen concentrate and the flowable fibrinogen gel were 42.5% and 40.5%, respectively. 5) The relative fibrinogen titers of the polymer fraction in the fibrinogen concentrate and the flowable fibrinogen gel were 289% and 202-405%, respectively.

[0053] (6) Implications of Example 3 1) The fibrinogen monomer contained in the fibrinogen concentrate and fluid fibrinogen gel has a low fibrinogen relative titer of less than 100%. In contrast, the fibrinogen polymer fraction separated from the fibrinogen concentrate and fluid fibrinogen gel has a fibrinogen relative titer of more than 200%. 2) The fluid fibrinogen gel contains more fibrinogen polymer than the fibrinogen concentrate with a fibrinogen relative titer of 100%, with a fibrinogen relative titer of 131%. Furthermore, by separating the fibrinogen polymer from the fluid fibrinogen gel, a fluid fibrinogen polymer fraction with a fibrinogen relative titer of more than 200% can be efficiently obtained. 3) It was confirmed that the fluid fibrinogen gel obtained by fibrinogen gel formation contains fibrinogen polymer. Furthermore, fibrinogen polymers are also contained in fibrinogen hydrogels in which fibrinogen gel formation has progressed, and in fibrinogen dry gels obtained by drying these. In other words, the relative potency of fibrinogen in both fibrinogen gels is higher than that of fibrinogen before the gelation reaction. Therefore, applying fibrinogen gel to bleeding sites where the coagulation system is activated and thrombin is generated can be expected to promote hemostasis. Furthermore, by combining it with thrombin, it can be processed into a glue preparation that clots in a short time.

[0054] Example 4: Application of fibrinogen gel as a biomaterial for hemostasis / tissue adhesion and closure (1) (1) Preparation of fibrinogen gel powder, etc. 1) Fibrinogen (fibrinogen concentrate) derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for intravenous injection 1g "JB," Japan Blood Products Organization) was used. Here, FXIII was contained at a mean value of 5.57 IU / mL for 20 mg / mL of fibrinogen. The fibrinogen preparation was dialyzed to exchange the buffer (trisodium citrate dihydrate 2 g / L, sodium chloride 5 g / L, L-arginine hydrochloride 10 g / L). Fibrinogen gel was formed by adding calcium chloride to a mixture of 20 mg / mL fibrinogen concentrate, 5 mg / mL albumin (25% intravenous albumin "Venesys", Japan Blood Products Organization), 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, 2.5 mg / mL L-arginine hydrochloride, and 50.7 mg / mL trehalose dihydrate to a concentration of 12.5 mM. The viscosity was measured using a tuning fork viscometer (SV-10A, A&D) and was found to be 2.85, 3.42, 34.1, 134, and 480 mPa*s*g / cm. ３When this time was reached, the resulting solutions were designated as fibrinogen solution (fibrinogen gel not yet formed (gel formation time: 0 min)), pr fibrinogen (fluid fibrinogen gel), pg fibrinogen (fibrinogen hydrogel), g fibrinogen (fibrinogen hydrogel), and og fibrinogen (fibrinogen hydrogel), and each of these was frozen to stop the fibrinogen gel formation. 2) Calcium chloride was added to a mixture containing 21.4 IU / mL of Thr (Human alpha Thrombin, Haematologic Tech. Inc.), 10 mg / mL of albumin, 1 mg / mL of trisodium citrate dihydrate, 2.5 mg / mL of sodium chloride, 5 mg / mL of L-arginine hydrochloride, and 141 mg / mL of trehalose dihydrate to a concentration of 12.5 mM (Thr solution), and the mixture was frozen. 3) The frozen fibrinogen solution, pr fibrinogen, pg fibrinogen, g fibrinogen, og fibrinogen, and Thr solution were freeze-dried using the following program (vacuum freeze dryer, FZ-6, LABCONCO). Segment 1: Hold at -30°C for 100 hours. Segment 2: Heat to 30°C at 0.03°C / min and hold for 6 hours. The freeze-dried fibrinogen product and freeze-dried fibrinogen gel product obtained by freeze-drying, i.e., fibrinogen dry gel, and freeze-dried Thr product, were each powdered using a crusher / classifier (Picoplex, HOSOKAWA MICRON) to obtain fibrinogen powder, pr fibrinogen powder, pg fibrinogen powder, g fibrinogen powder, og fibrinogen powder, and Thr powder. 4) Fibrinogen powder, pr fibrinogen powder, pg fibrinogen powder, g fibrinogen powder, and og fibrinogen powder were mixed with Thr powder in a weight ratio of 1:1, respectively, to obtain Thr-fibrinogen powder, Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder.

[0055] (2) Arrangement of parameters The various parameters used in the preparation of the above fibrinogen gel powder, etc. are arranged as follows: 1) Conditions for forming fibrinogen hydrogel The concentrations of the following components in the fibrinogen gel reaction solution were: Fibrinogen (mg / mL): FXIII (IU / mL): Calcium chloride (mM) = 20: 5.57: 12.5 When expressed as a molar ratio, the fibrinogen: FXIII: calcium chloride = 58.8: 0.366: 12,500.

[0056] (3) Evaluation in a Rat Liver Hemorrhage Model (Test Method) A group of nine rats (strain: SD, sex: male, manufacturer: Charles River Japan, age at time of use: 7 weeks) was used for the test. After sedating the rats with a triple anesthetic (10 mL / kg, i.p.), 250 U / kg heparin was administered intravenously into the tail vein and the rats were fixed in a dorsal position. The abdomen was opened along the midline to expose the liver. Four minutes after heparin administration, a 10 mm diameter template was pressed against the left lateral lobe of the liver, and the surface of the raised area was excised using a razor to create a flat wound (approximately 8 mm in diameter: 0.5 cm ２) were prepared. Once bleeding had subsided, 32 mg of each of Thr-fibrinogen powder, Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder (equivalent to 4.0 mg fibrinogen and 2.1 IU Thr) was applied to the wound and held in close contact with the wound for 5 seconds. The pre-bleeding rate (g / min) was calculated from the amount of bleeding in the 30 seconds immediately before test substance treatment, and the amount of bleeding (g) in the 10 minutes after test substance treatment was calculated (blood was collected with a cotton ball and calculated from the weight difference before and after treatment). Furthermore, the post-bleeding amount was calculated for each animal based on the 30-second bleeding period of 10 to 10.5 minutes (see below), and the hemostasis rate for each group was calculated, with 5 μL or less being considered hemostasis. Only animals with a pre-bleeding rate of 0.01 to 0.1 g / min were included in the evaluation, and data from 6 to 9 animals per group were used. After determining hemostasis, the liver was removed, and the liver and test substance were each secured with clips. Adhesion strength (gf, 180° peel adhesive strength) was measured using a chart (Lab Chart, AD Instruments) obtained via a data acquisition and analysis system (Power Lab 2 / 26, AD Instruments) and a pressure transducer (Force Transducer, AD Instruments). The filter papers used to wipe away bleeding for 30 seconds between 10 and 10.5 minutes after treatment with the test substance as described above were then arranged in a holder. For the calibration curve, two filters each soaked in 8, 4, 2, 1, and 0 μL (blank) of blood were placed in the same holder, for a total of 10 filters, and allowed to dry overnight. After drying, digital images were captured (GT-X970, EPSON). The images were analyzed using ImageJ (National Institutes of Health). Specifically, the amount of blood adhering to each filter paper was quantified as signal intensity. Next, a calibration curve equation was created to quantify the amount of blood from the signal intensity of the calibration filter paper. Finally, the post-bleeding volume of each individual wiped with the filter paper was calculated from the signal intensity of each filter paper using the calibration curve equation.

[0057] (Test Results) The hemostatic properties (assessed by bleeding volume), adhesive properties (assessed by adhesive strength), and occlusive properties (assessed by hemostatic rate) of Thr-fibrinogen powder, Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder were evaluated using a rat liver bleeding model as described above. The results are shown in Figure 5 ([5A]: evaluation results of hemostatic properties, [5B]: evaluation results of adhesive properties, shown as (Mean) ± (SE)) and Table 5 (evaluation results of occlusive properties) (N = 6-9). Thr-fibrinogen powder resulted in a greater amount of bleeding and lower hemostatic properties than the other powders (see [5A] in Figure 5). No significant differences in adhesive properties were observed between the groups (see [5B] in Figure 5). The hemostasis rate of the Thr-fibrinogen powder was also lower than that of the other powders, indicating poor occlusiveness (see Table 5 below).

[0058]

[0059] *'Fbg powder' in the table is an abbreviation for 'fibrinogen powder' in the text.

[0060] (4) Rat Skin Adhesion Test (Test Method) Rat skin was stripped, the subcutaneous fat layer was removed, and a 1 × 2 cm square was cut out. The test substance was applied to a 1 × 1 cm square area at the following doses, and two skin pieces were overlapped. 32 mg each of Thr-fibrinogen powder, Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder (equivalent to 4.0 mg fibrinogen and 2.1 IU / mL Thr) was placed on one skin piece, and 50 μL of human plasma (standard human plasma for blood coagulation reagents, SIEMENS) was placed on the other, and the two pieces were overlapped. After the test substance treatment, the skin was overlapped and compressed with a 100 g weight for 30 seconds. After 10, 30, and 90 minutes, the edge of each skin piece was pinched with a clip, and the adhesive strength (tensile shear adhesive strength) was measured using a chart (Lab Chart, AD Instruments) obtained via a data collection and analysis system (Power Lab 2 / 26, AD Instruments) and a pressure transducer (Force Transducer, AD Instruments).

[0061] (Test Results) The adhesive strengths of Thr-fibrinogen powder, Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder were evaluated using the rat skin adhesion test described above. The test results are shown in Figure 6 (shown as (Mean) ± (SE), N = 5 or 6). The adhesive strength of Thr-fibrinogen powder improved up to 30 minutes, but decreased at 90 minutes. The adhesive strength of the other samples continued to increase up to 90 minutes. The adhesive strength of Thr-g fibrinogen powder was highest after 90 minutes. Note that the adhesive strength of Thr-og fibrinogen powder at 90 minutes was lower than that of the other powders.

[0062] (5) Implications of Example 4 1) Fibrinogen dry gels with different degrees of fibrinogen polymerization were prepared and powdered. The resulting powders, arranged in order of decreasing fibrinogen polymerization degree, were pr fibrinogen powder, pg fibrinogen powder, g fibrinogen powder, and og fibrinogen powder. 2) In a rat liver bleeding model, Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder, which were prepared by combining pr fibrinogen powder, pg fibrinogen powder, g fibrinogen powder, and og fibrinogen powder with Thr powder, had higher hemostatic and occlusive properties than Thr-fibrinogen powder, which was prepared by combining Thr powder with fibrinogen powder with less advanced fibrinogen polymerization. This is thought to be because pr fibrinogen powder, pg fibrinogen powder, g fibrinogen powder, and og fibrinogen powder contain fibrinogen gel with a high degree of polymerization, and this short thrombin clotting time results in a rapid clotting reaction at the treatment surface, resulting in high hemostasis and closure. 3) In rat skin adhesion tests, the adhesive strength of Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder, which were prepared by combining pr fibrinogen powder, pg fibrinogen powder, g fibrinogen powder, and og fibrinogen powder, which have highly polymerized fibrinogen, with Thr powder, continued to increase up to 90 minutes, whereas the adhesive strength of Thr-fibrinogen powder, which was prepared by combining less polymerized fibrinogen powder with Thr powder, decreased from 30 to 90 minutes. It is generally known that the generation of excess thrombin in the blood induces the fibrinolytic system. Specifically, a decrease in adhesive strength in a rat skin adhesion test using plasma indicates the occurrence of fibrinogen. Therefore, the fibrinogen gels contained in pr fibrinogen powder, pg fibrinogen powder, g fibrinogen powder, and og fibrinogen powder were more resistant to fibrinogen than fibrinogen.4) In a rat skin adhesion test, the Thr-og fibrinogen powder, which used the most polymerized fibrinogen gel, showed low adhesive strength after 90 minutes. In other words, excessive polymerization of the fibrinogen gel can lead to a decrease in adhesive strength. 5) Under the conditions shown in this example, the Thr-g fibrinogen powder exhibited the highest overall efficacy when used as a biological glue. In other words, the viscosity of the fibrinogen gel was 2.85 mPa*s*g / cm. ３ When a material with a viscosity at least 1.2 times higher than that of the original material was used as a biological glue, it showed improved hemostatic and occlusive properties and increased resistance to fibrinolysis.

[0063] Example 5 Application of fibrinogen gel as a biomaterial for hemostasis / tissue adhesion and closure (2) (1) Preparation of fibrinogen gel powder, etc. 1) Fibrinogen (fibrinogen concentrate) derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for intravenous injection 1g "JB", Japan Blood Products Organization) was used. Here, FXIII was contained at 5.57 IU / mL (average value) for 20 mg / mL of fibrinogen. 2) After the buffer was exchanged by dialysis, a fibrinogen preparation was prepared by preparing a mixture of 20 mg / mL fibrinogen concentrate, 2.5 mg / mL BSA (Bovine Serum Albumin, SIGMA), 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, 2.5 mg / mL L-arginine hydrochloride, and 50.7 mg / mL trehalose dihydrate. Calcium chloride was added to the mixture to a concentration of 12.5 mM, and the mixture was immediately frozen to obtain a frozen fibrinogen solution. The fibrinogen preparation was prepared by dialysis to exchange the buffer, followed by preparing a mixture of 20 mg / mL fibrinogen concentrate, 2.5 mg / mL BSA, 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, 2.5 mg / mL L-arginine hydrochloride, and 50.7 mg / mL trehalose dihydrate. Calcium chloride was added to this mixture to a concentration of 12.5 mM, and the mixture was left overnight to prepare a fibrinogen hydrogel. The fibrinogen preparation was prepared by dialysis to exchange the buffer, followed by preparing a mixture of 20 mg / mL fibrinogen concentrate, 2.5 mg / mL BSA, 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, 2.5 mg / mL L-arginine hydrochloride, and 50.7 mg / mL trehalose dihydrate. To this mixture, 0.125 IU / mL Thr (Human alpha Thrombin, Haematologic Tech. Inc.) and 12.5 mM calcium chloride were added, and the mixture was left overnight to prepare a fibrin hydrogel.A mixture containing 21.4 IU / mL of Thr, 2.5 mg / mL of BSA, 0.5 mg / mL of trisodium citrate dihydrate, 1.25 mg / mL of sodium chloride, 2.5 mg / mL of L-arginine hydrochloride, and 70.7 mg / mL of trehalose dihydrate was prepared, and calcium chloride was added to the mixture to a concentration of 12.5 mM to prepare a Thr solution. 3) The frozen fibrinogen solution, fibrinogen hydrogel, fibrin hydrogel, and Thr solution were freeze-dried using the following program (vacuum freeze dryer, FZ-6, LABCONCO). Segment 1: Hold at -30°C for 50 hours Segment 2: Heat to 30°C at 0.03°C / min and hold for 6 hours The resulting freeze-dried products were each vigorously shaken in their containers to crush them by the impact, yielding fibrinogen powder, fibrinogen gel powder, fibrin powder, and Thr powder. The fibrinogen powder, fibrinogen gel powder, and fibrin powder were each mixed with Thr powder in a weight ratio of 1:1 to obtain Thr-fibrinogen powder, Thr-fibrinogen gel powder, and Thr-fibrin powder.

[0064] (2) Preparation of fibrinogen gel sheet, etc. 1) After the fibrinogen preparation was dialyzed to exchange the buffer, a mixture of fibrinogen concentrate 20 mg / mL, BSA 2.5 mg / mL, trisodium citrate dihydrate 0.5 mg / mL, sodium chloride 1.25 mg / mL, and L-arginine hydrochloride 2.5 mg / mL was prepared. Calcium chloride was added to this to make a concentration of 12.5 mM, and then the mixture was immediately diluted with 0.2 mL / cm ２ The fibrinogen solution was poured into a tray at 0.2 mL / cm, and immediately frozen at −20° C. A Thr solution prepared by adding calcium chloride to a mixture of 21.4 IU / mL Thr, 2.5 mg / mL BSA, 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, and 2.5 mg / mL L-arginine hydrochloride to a concentration of 12.5 mM was added thereto. ２The fibrinogen preparation was prepared by dialysis to exchange the buffer, followed by a mixture of 20 mg / mL fibrinogen concentrate, 2.5 mg / mL BSA, 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, and 2.5 mg / mL L-arginine hydrochloride. Calcium chloride was added to the mixture to a concentration of 12.5 mM, and the mixture was immediately diluted with 0.2 mL / cm ２ The fibrinogen hydrogel was prepared by pouring the mixture into a tray at 100°C and leaving it overnight. After freezing the mixture at -20°C, the same Thr solution as above was added at 0.2 mL / cm ２ The fibrinogen preparation was prepared by dialysis to exchange the buffer, followed by a mixture of 20 mg / mL fibrinogen concentrate, 2.5 mg / mL BSA, 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, and 2.5 mg / mL L-arginine hydrochloride. 0.125 IU / mL Thr and 12.5 mM calcium chloride were added to the mixture, and the mixture was immediately frozen at 0.2 mL / cm. ２ The fibrin hydrogel was prepared by pouring the mixture into a tray at 0.2 mL / cm and leaving it overnight. After freezing the mixture at -20°C, the same Thr solution as above was added at 0.2 mL / cm. ２ and frozen at -20°C (for Thr-fibrin sheets). 2) The frozen trays for Thr-fibrinogen sheets, Thr-fibrinogen gel sheets, and Thr-fibrin sheets were freeze-dried using the following program (vacuum freeze dryer, FZ-6, LABCONCO). Segment 1: -30°C, hold for 50 hours Segment 2: Heat to 30°C at 0.03°C / min, hold for 6 hours The resulting freeze-dried products were compressed and formed into sheets to produce Thr-fibrinogen sheets, Thr-fibrinogen gel sheets, and Thr-fibrin sheets, respectively.

[0065] (3) Summary of parameters The various parameters used in preparing the fibrinogen gel described above are summarized below. 1) Conditions for forming fibrinogen hydrogel The concentrations of the following components in the fibrinogen gel reaction solution were: fibrinogen (mg / mL): FXIII (IU / mL): calcium chloride (mM) = 20: 5.57: 12.5 When expressed as a molar ratio, the fibrinogen:FXIII:calcium chloride = 58.8: 0.366: 12,500.

[0066] (4) Evaluation of tensile strength of sheet adhesive (Test method) For the Thr-fibrinogen sheet, Thr-fibrinogen gel sheet and Thr-fibrin sheet, a 1 × 2 cm piece was cut out, and both ends were pinched with clips to obtain a 1 cm square sheet. The tensile strength (gf, tensile shear strength) was measured using a chart (Lab Chart, AD Instruments) obtained via a data collection and analysis system (Power Lab 2 / 26, AD Instruments) and a pressure transducer (Force Transducer, AD Instruments).

[0067] (Test Results) 1) The tensile strength of the Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet was 3.3 ± 3.7 gf for the Thr-fibrinogen sheet, 42.8 ± 9.3 gf for the Thr-fibrinogen gel sheet, and 12.7 ± 17.9 gf for the Thr-fibrin sheet. In particular, the Thr-fibrinogen sheet could be molded into a sheet, but it had almost no plasticity and had a texture that would disintegrate on contact with anything.

[0068] (5) Evaluation in a Rat Liver Hemorrhage Model (Test Method) A group of eight rats (strain: SD, sex: male, manufacturer: Charles River Japan, age at time of use: 7 weeks) was used for the test. One minute after intravenous administration of 300 U / kg heparin, the animals were sedated with a triple anesthetic (10 mL / kg, i.p.) and fixed in a supine position. A midline abdominal incision was made to expose the liver, and four minutes after heparin administration, a 10 mm diameter template was pressed against the left lateral lobe, and the surface of the raised area was excised using a razor to create a flat wound (approximately 8 mm in diameter: 0.5 cm ２) were prepared. Once bleeding had subsided, 32 mg of Thr-fibrinogen powder, 32 mg of Thr-fibrinogen gel powder, and 32 mg of Thr-fibrin powder were applied to the wound, and 1 cm squares of Thr-fibrinogen sheets, 32 mg of Thr-fibrinogen gel sheets, and 32 cm squares of Thr-fibrinogen sheets (equivalent to 4 mg fibrinogen and 4.3 IU Thr) were applied to the wound and adhered to the wound for 5 seconds. The pre-bleeding rate (g / min) was calculated from the amount of bleeding in the 30 seconds immediately before treatment with the test substance, and the amount of bleeding (g) in the 10 minutes after treatment with the test substance was calculated (blood was collected with a cotton ball and calculated from the weight difference before and after treatment). Furthermore, the post-bleeding amount was calculated for each animal based on the 30-second bleeding period of 10 to 10.5 minutes (see below), and the hemostasis rate for each group was calculated, with a value of 5 μL or less being considered hemostasis. Only animals with a pre-bleeding rate of 0.01 to 0.1 g / min were used for evaluation, and data from 7 to 8 animals per group were used. After determining hemostasis, the liver was removed, and the liver and test substance were each secured with clips. Adhesion strength (gf, 180° peel adhesive strength) was measured using a chart (Lab Chart, AD Instruments) obtained via a data collection and analysis system (Power Lab 2 / 26, AD Instruments) and a pressure transducer (Force Transducer, AD Instruments). Filter papers used to wipe away bleeding for 30 seconds between 10 and 10.5 minutes after test substance treatment were arranged in a holder. For the calibration curve, 10 filter papers, each containing 8, 4, 2, 1, and 0 μL (blank) of blood, were placed in the same holder, two of each, and allowed to dry overnight. After the filters were dried, digital images were captured (EPSON, GT-X970). The images were analyzed using ImageJ (National Institutes of Health). Specifically, the amount of blood adhering to each filter paper was quantified as signal intensity. Next, a calibration curve equation was created to quantify the amount of blood from the signal intensity of the calibration filter paper. Finally, the amount of post-bleeding bleeding for each individual wiped with the filter paper was calculated from the signal intensity of each filter paper using the calibration curve equation.

[0069] (Test Results) 1) As described above, six test substances, namely, Thr-fibrinogen powder, Thr-fibrinogen gel powder, Thr-fibrin powder, Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet, were applied to bleeding rat liver wounds, and their hemostatic, adhesive, and occlusive properties were evaluated. The evaluation results are shown in Figure 7 ([7A]: Evaluation results for hemostatic properties, [7B]: Evaluation results for adhesive properties, (Mean) ± (SD), N = 7, 8) and Table 6 (Evaluation results for occlusive properties). Regarding hemostatic properties, among Thr-fibrinogen powder, Thr-fibrinogen gel powder, and Thr-fibrin powder, Thr-fibrin powder tended to cause a large amount of bleeding and have weaker hemostatic properties (see [7A] in Figure 7). Among the Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet, the Thr-fibrin sheet tended to produce less bleeding. Regarding adhesiveness, among the Thr-fibrinogen powder, Thr-fibrinogen gel powder, and Thr-fibrin powder, the adhesive strength of the Thr-fibrin powder was lower than the other two types of powder (see [7B] in Figure 7). Among the Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet, the adhesive strength of the Thr-fibrinogen sheet was higher than the other two types of sheets. Regarding occlusiveness, among the Thr-fibrinogen powder, Thr-fibrinogen gel powder, and Thr-fibrin powder, the Thr-fibrinogen gel powder had the highest hemostatic rate, followed by the Thr-fibrinogen powder and Thr-fibrin powder. Among the Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet, the hemostatic rate of the Thr-fibrinogen gel sheet was the highest, followed by the Thr-fibrinogen sheet and the Thr-fibrin sheet (see Table 6 below).

[0070]

[0071] *'Fbg', 'Fbg gel' and 'Fbn' in the table are abbreviations of 'fibrinogen', 'fibrinogen gel' and 'fibrin' in the text, respectively.

[0072] (6) Histological Evaluation (Test Method) In the evaluation of the rat liver hemorrhage model (5) above, liver tissue containing the test substance was collected and immersed in 10% neutral buffered formalin for at least 24 hours. The tissue was then excised to prepare a specimen containing the test substance application site. Paraffin blocks were then prepared according to standard methods, and paraffin sections approximately 5 μm thick were prepared from the paraffin blocks. These sections were stained with Diff Quick to prepare histopathological specimens for optical microscopic observation. Microscopic observation focused on the state of the test substance and blood components.

[0073] (Test Results) Figure 8 shows histopathological images of the test substance on the liver wound surface. In Figure 8, (A) shows the Thr-fibrinogen powder, (B) shows the Thr-fibrinogen gel powder, (C) shows the Thr-fibrin powder, (D) shows the Thr-fibrinogen sheet, (E) shows the Thr-fibrinogen gel sheet, and (F) shows the Thr-fibrin sheet. In all cases, the liver tissue is on the bottom, with the test substance placed on top. Compared to the three types of powder, the three types of sheets had fewer void-like structures in the test substance layer and were thinner, with the Thr-fibrinogen gel sheet in particular having fewer voids and being thinner. Furthermore, in the case of the Thr-fibrinogen powder, Thr-fibrinogen gel powder, Thr-fibrin powder, Thr-fibrinogen sheet, and Thr-fibrin sheet, blood penetrated into the porous structure within the test substance, whereas in the case of the Thr-fibrinogen gel sheet, blood did not penetrate into the test substance and was observed to have accumulated slightly between the test substance and the wound surface.

[0074] (7) Implications of Example 5 1) It was found that Thr-fibrinogen gel powder and Thr-fibrinogen sheet were superior in terms of overall efficacy among powders and sheets, respectively. Of the three sheets, the Thr-fibrinogen gel sheet had the highest tensile strength and the best operability, while the other two, the Thr-fibrinogen sheet and the Thr-fibrin sheet, were not suitable for handling during hemostatic treatment. When considering operability as well as efficacy, the Thr-fibrinogen gel sheet was the most superior sheet. 2) When comparing the Thr-fibrinogen gel powder and the Thr-fibrinogen gel sheet, the hemostatic and occlusive properties were roughly equivalent, but the Thr-fibrinogen gel powder was superior in terms of adhesiveness. If adhesiveness is desired, it is recommended to select Thr-fibrinogen gel powder, while if convenience is prioritized, it is recommended to select the Thr-fibrinogen gel sheet. 3) Figure 8 shows the blood permeating the test substance. The three powder types had thicker sealing layers due to the presence of voids, while the sheet type had a thinner layer. Furthermore, the test substance dissolved in the Thr-fibrinogen powder, Thr-fibrinogen sheet, Thr-fibrinogen gel powder, and Thr-fibrinogen gel sheet, whereas the test substance did not dissolve in the Thr-fibrin powder and Thr-fibrin sheet, and fine voids were observed in the fibrous structure. When pathological images were examined to determine the extent of blood penetration, blood was seen to have penetrated to the opposite side of the wound surface in all but the Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet. In other words, the fibrinogen gel powder and fibrinogen gel sheet reverted to a hydrogel upon absorbing the moisture from the blood, confirming that this function functioned as a barrier to prevent further blood penetration. The Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet were superior in terms of robustly encapsulating and sealing the wound surface. This result was consistent with the results of the hemostatic rate, which indicates closure (see Table 6 above). From the above, it became clear that by using fibrinogen gel instead of fibrinogen in the powder or sheet formulation of bioglue, the wound can be tightly closed and reliable hemostasis can be achieved.4) Powdered bioadhesives are also extremely useful, but applying the entire powder evenly to the wound surface takes time. To solve this problem and turn the powder into a product that can be used in medical settings, a separate applicator or similar device would be required to quickly complete the treatment on the wound surface. In contrast, the sheet could be applied immediately to the bleeding surface, making it easier to use and more convenient.

[0075] Example 6 Application of fibrinogen gel as a biomaterial for hemostasis / tissue adhesion and closure (3) (1) Preparation of fibrinogen gel powder and sheet, etc. (Powder preparation) 1) Fibrinogen (fibrinogen concentrate) derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for intravenous injection 1g "JB", Japan Blood Products Organization) was used. Here, FXIII was contained at 5.57 IU / mL (average value) for 20 mg / mL of fibrinogen. 2) After the fibrinogen preparation was dialyzed to exchange the buffer, a mixture of 20 mg / mL fibrinogen concentrate, 2.5 mg / mL BSA (Bovine Serum Albumin, SIGMA), 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, 2.5 mg / mL L-arginine hydrochloride, and 50.7 mg / mL trehalose dihydrate was prepared. Calcium chloride was added to this mixture to a concentration of 12.5 mM, and the mixture was allowed to stand at 25°C for 16 hours to prepare a fibrinogen hydrogel. A mixture containing 21.4 IU / mL of Thr (Human alpha Thrombin, Haematologic Tech. Inc.), 2.5 mg / mL of BSA, 0.5 mg / mL of trisodium citrate dihydrate, 1.25 mg / mL of sodium chloride, 2.5 mg / mL of L-arginine hydrochloride, and 70.7 mg / mL of trehalose dihydrate was prepared, and calcium chloride was added to the mixture to a concentration of 12.5 mM to prepare a Thr solution. 3) The frozen fibrinogen hydrogel and Thr solution were freeze-dried using the following program (vacuum freeze dryer, FZ-6, LABCONCO). Segment 1: Hold at -30°C for 50 hours. Segment 2: Heat to 32°C at 0.03°C / min and hold for 6 hours. The resulting freeze-dried products were each vigorously shaken together with the container, and crushed by the impact to obtain fibrinogen gel powder and Thr powder. The fibrinogen gel powder was mixed with Thr powder in a weight ratio of 1:1 to obtain Thr-fibrinogen gel powder.(Preparation of Sheet) 1) After the fibrinogen preparation was dialyzed to exchange the buffer, a mixture of fibrinogen concentrate 20 mg / mL, BSA 2.5 mg / mL, trisodium citrate dihydrate 0.5 mg / mL, sodium chloride 1.25 mg / mL, and L-arginine hydrochloride 2.5 mg / mL was prepared, to which calcium chloride was added to make a concentration of 12.5 mM, and then immediately diluted at 0.2 mL / cm. ２ The mixture was poured into a tray at 25°C for 16 hours to prepare a fibrinogen hydrogel. After freezing at -20°C, a mixture of 21.4 IU / mL Thr, 2.5 mg / mL BSA, 0.5 mg / mL trisodium citrate dihydrate, 1.25 mg / mL sodium chloride, and 2.5 mg / mL L-arginine hydrochloride was prepared. Calcium chloride was added to the mixture to a concentration of 12.5 mM, and the mixture was diluted with 0.2 mL / cm of Thr solution. ２ and frozen at -20°C (for Thr-fibrinogen gel sheets). 2) The trays for the frozen Thr-fibrinogen gel sheets were freeze-dried using the following program (vacuum freeze dryer, FZ-6, LABCONCO). Segment 1: -30°C, held for 50 hours Segment 2: Heat to 32°C at 0.03°C / min, held for 6 hours The resulting freeze-dried product was compressed and formed into a sheet to form a Thr-fibrinogen gel sheet.

[0076] (2) Summary of parameters The various parameters used in preparing the fibrinogen gel described above are summarized below. 1) Conditions for forming fibrinogen hydrogel The concentrations of the following components in the fibrinogen gel reaction solution were: fibrinogen (mg / mL): FXIII (IU / mL): calcium chloride (mM) = 20:5.57:12.5 When expressed as a molar ratio, the fibrinogen:FXIII:calcium chloride = 58.8:0.366:12500.

[0077] (3) Evaluation in a Rat Liver Hemorrhage Model (Test Method) A group of nine rats (strain: SD, sex: male, manufacturer: Charles River Japan, age at time of use: 7 weeks) was used for the test. After sedating the animals with a triple anesthetic (10 mL / kg, i.p.), 300 U / kg heparin was administered intravenously into the tail vein and the animals were fixed in a dorsal position. The abdomen was opened along the midline to expose the liver. Four minutes after heparin administration, a 12 mm diameter template was pressed against the left lateral lobe, and the surface of the raised area was excised using a razor to create a flat wound (approximately 10 mm in diameter: 0.8 cm ２After the bleeding subsided, a 1 cm square of Surgicel (Johnson & Johnson), Beriplast (CSL Behring) (a mixture of 62.5 μL of fibrinogen solution and 62.5 μL of Thr solution, equivalent to 5 mg of fibrinogen and 18.8 U of Thr), a 1.1 cm square of TachoSil (CSL Behring) (equivalent to 6.9 mg of fibrinogen and 2.5 IU of Thr), 40 mg of Thr-fibrinogen gel powder (equivalent to 5 mg of fibrinogen and 5.4 U of Thr), and a 1.1 cm square of Thr-fibrinogen gel sheet (equivalent to 5 mg of fibrinogen and 5.1 IU of Thr) were applied to the wound and held in close contact with the wound for 5 seconds. The pre-bleeding rate (g / min) was calculated from the amount of bleeding in the 30 seconds immediately prior to test substance administration, and the amount of bleeding in the 10 minutes following test substance administration (g) was calculated from the weight difference between before and after blood collection with a cotton ball. Furthermore, the post-bleeding rate was calculated for each animal based on the 30-second period of bleeding from 10 to 10.5 minutes. A value of 5 μL or less was considered hemostasis, and the hemostasis rate for each group was calculated. Only animals with a pre-bleeding rate of 0.01 to 0.1 g / min were used for evaluation, with data from 7 to 9 animals per group. After determining hemostasis, the liver was removed, and the liver and test substance were secured with clips. The adhesive strength (mN, 180° peel strength) was measured using a tension-compression testing machine (MCT-2150, A&D). The filter papers used to wipe away the bleeding for 30 seconds from 10 to 10.5 minutes following test substance administration were arranged in a holder. For the calibration curve, two filters each containing 8, 4, 2, 1, and 0 μL (blank) of blood were soaked in each filter paper, for a total of 10 filters, and the filters were arranged in the same holder and allowed to dry overnight. After the filters dried, digital images were captured (GT-X970, EPSON). The images were analyzed using ImageJ (National Institutes of Health). Specifically, the amount of blood adhering to each filter paper was quantified as signal intensity. Next, a calibration curve equation was created to quantify the amount of blood from the signal intensity of the calibration filter paper. Finally, the amount of post-bleeding bleeding for each individual wiped with the filter paper was calculated from the signal intensity of each filter paper using the calibration curve equation.

[0078] (Test Results) The test results are shown in Figure 9. [9A] shows the evaluation results for hemostatic properties. The degree of bleeding 10 minutes after treatment with the test substance was plotted on a graph as the bleeding volume (g / 10 min). [9B] shows the test results for adhesive strength. The test substance was applied to the bleeding surface of the liver, and 10 minutes later, the test substance was peeled off from the liver. The tensile strength at this time was plotted on a graph as adhesive strength (mN, 180° peel adhesive strength). N = 7 and 8 for the Control and TachoSil groups, respectively, and N = 9 for the other groups. 1) In the rat liver bleeding model, the average pre-bleeding rate immediately before treatment with the test substance was 0.04 to 0.05 g / min for all groups. 2) None of the 1.1 cm square Thr-fibrinogen gel sheets used for evaluation had any cracks or other breakages. Furthermore, all test substances were applied by applying pressure to the bleeding surface with a cotton ball, but the test substance did not stick to the cotton ball. Regarding the amount of bleeding, which is an evaluation index of hemostatic properties, the control group had a bleeding rate of 0.61 g / 10 min. In contrast, all groups treated with the test substances had bleeding rates of 0.14 g / 10 min or less, with the Surgicel group showing particularly low values ​​of 0.08 mg / 10 min and the Thr-fibrinogen gel sheet group showing low values ​​of 0.05 g / 10 min. The bleeding rates of all groups were lower than those of the control group (see [9A] in Figure 9). The adhesive strength, an index of adhesiveness, was 30 and 52 mN in the Beriplast and TachoSil groups, respectively, while the Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet groups were 52 and 61 mN, respectively, indicating that the Beriplast group tended to have lower adhesive strength than the other groups (see [9B] in Figure 9). Because Surgicel is not an adhesive, it was not used for comparison of adhesiveness. The hemostasis rate, an index of occlusion, was 66.7% and 75.0% in the Beriplast and TachoSil groups, respectively, while the Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet groups were 44.4% and 88.9%, respectively.Compared to existing drugs, the hemostatic rate of the Thr-fibrinogen gel powder was lower, but the Thr-fibrinogen gel sheet was equal to or better (see Table 7 below).

[0079]

[0080] *'Fbg gel' in the table is an abbreviation of 'fibrinogen gel' in the text.

[0081] (4) Implications of Example 6 1) Fibrinogen dry gel can be crushed into powder, and by combining this with similarly crushed Thr powder, a biological glue can be made. This Thr-fibrinogen gel powder glue exhibited hemostatic and adhesive properties comparable to existing liquid and sheet fibrin glues. 2) A biological glue can be made by compressing a composition consisting of two dry gel layers, a fibrinogen gel layer and a thrombin layer, into a sheet. This Thr-fibrinogen gel sheet glue exhibited hemostatic, adhesive, and occlusive properties comparable to existing liquid and sheet fibrin glues. 3) In Examples 5 and 6, the adhesive and occlusive properties of the Thr-fibrinogen gel powder and the Thr-fibrinogen gel sheet were reversed. In Examples 5 and 6, the liver wound area was 0.5 cm ２ and 0.8 cm ２The hemostasis assessment criteria (post-bleeding volume 5 μL or less) remained unchanged, making Example 6 a more severe model. Furthermore, the treatment amount of the test substance was adjusted according to the wound area. However, with powders in particular, unevenness was more likely to occur as the wound surface became larger, leading to adhesive breakthrough in thinner areas and continued bleeding. Despite these differences, the sheet easily covered the wound surface uniformly, demonstrating consistent, high efficacy across tests. 4) In clinical practice, Thr-fibrinogen gel powder, like Beriplast (liquid), requires an applicator for treatment. However, Thr-fibrinogen gel sheets, unlike Surgicel (sheet formulation) and TachoSil (sheet formulation), do not require an applicator and can be used immediately after opening, making them more convenient. 5) Existing fibrin glue sheet products, such as TachoSil and EVARREST (Johnson & Johnson), use horse collagen or bioabsorbable synthetic polymers as supports in addition to human-derived components. Both of these are considered foreign bodies when used in humans. In contrast, the support of the Thr-fibrinogen gel sheet is a gel of human-derived fibrinogen and fibrinogen obtained with FXIII and / or FXIIIa. Therefore, it can be constructed solely from human-derived components and does not require any components that are foreign to humans. 6) While the Thr-fibrinogen gel sheet adhesive shown here has a two-layer structure, it can be made into a three- or more-layer structure by adding natural or synthetic polymers to each layer or by adding a support layer containing natural or synthetic polymers, thereby changing the physical properties. Furthermore, it is possible to add layers containing components that promote blood coagulation or physiologically active substances other than Thr, FXIII, FXIIIa, and fibrinogen gel to create a three- or more-layer structure to impart different efficacy effects.

[0082] Example 7 Application of Fibrinogen Gel as a Substrate for Regenerative Medicine (1) Preparation of Fibrinogen Gel, etc. 1) Fibrinogen (fibrinogen concentrate) derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for Intravenous Injection 1g "JB", Japan Blood Products Organization) was used. Here, FXIII was contained at 5.57 IU / mL (average value) per 20 mg / mL of fibrinogen. The 20 mg / mL fibrinogen preparation was dialyzed and replaced with 10 mM HEPES, and then concentrated by ultrafiltration to a final concentration of 20 mg / mL. 2) Preparation of fibrinogen hydrogel: 20 mg / mL fibrinogen concentrate, to which calcium chloride had been added to make a 12.5 mM solution, was placed in a 96-well plate at 77 μL / well and allowed to stand at room temperature to form a fibrinogen hydrogel. After 24 hours, the plate was frozen at -20°C. 3) Preparation of Thr-fibrinogen hydrogel: 50 μL of 188 U / mL Thr (Human alpha Throbmin, PROLITIX) was layered on top of the frozen fibrinogen hydrogel prepared as described above, and the plate was then frozen at -20°C. 4) Freeze-drying: The frozen fibrinogen hydrogel and Thr-fibrinogen hydrogel were freeze-dried using the following program (vacuum freeze dryer, FZ-6, LABCONCO). Segment 1: -30°C, hold for 25 hours. Segment 2: Heat to 32°C at 0.03°C / min and hold for 6 hours. The resulting lyophilized products were used as fibrinogen dry gels and Thr-fibrinogen dry gels, respectively, and stored at 4°C. Sterilization was performed by irradiating with 28.7-30.5 kGy of gamma rays prior to use. 5) Preparation of Beriplast (hydrogel). Fibrinogen and thrombin solutions from Beriplast (CSL Behring) were prepared according to the package insert. These were diluted 1:1 with 10 mM HEPES to give fibrinogen concentrations of 40 mg / mL and thrombin concentrations of 150 IU / mL, respectively. 37.5 μL of the diluted fibrinogen solution was placed in a 96-well plate and frozen at -20°C.37.5 μL of diluted thrombin solution was layered on the frozen fibrinogen solution and left to stand at room temperature for 24 hours to allow the fibrinogen and thrombin to react and form fibrin, which became Beriplast (hydrogel). 6) Preparation of TachoSil TachoSil (CSL Behring) was punched out with a 7 mm diameter belt punch, placed into a 96-well plate, and pressed firmly against the bottom of the well.

[0083] (2) Summary of parameters The various parameters used in preparing the fibrinogen gel described above are summarized below. 1) Conditions for forming fibrinogen hydrogel The concentrations of the following components in the fibrinogen gel reaction solution were: fibrinogen (mg / mL): FXIII (IU / mL): calcium chloride (mM) = 20:5.57:12.5 When expressed as a molar ratio, the fibrinogen:FXIII:calcium chloride = 58.5:0.366:12500.

[0084] (3) Cell culture test (Test method) MSCs (human bone marrow-derived mesenchymal stem cells, Takara Bio) were used for the cell culture test, GM2 medium (mesenchymal stem cell growth medium 2, Takara Bio) was used for culture, and Accutase Soln (Accutase in DPBS without Ca, Mg, Nacalai Tesque) was used for cell detachment. Culture was performed in a standard 37°C, 5% CO2 incubator. Cells were detached and suspended in Accutase Soln, and 2 × 10 ４ The cells were seeded in a cell / well format onto fibrinogen dry gel, Thr-fibrinogen dry gel, Beriplast (hydrogel), and Tachosil. The seeding date was designated Day 0, and on Days 1, 7, 14, and 28, A450 was measured for three cases per condition according to the CCK-8 (Cell Counting Kit-8, DOJINDO) protocol to evaluate cell proliferation.

[0085] (Test Results) The test results are shown in Figure 10. MSC proliferation was confirmed in the fibrinogen dry gel, Thr-fibrinogen dry gel, and Beriplast (hydrogel), but not in TachoSil. When a cell suspension was seeded into the dried specimens of fibrinogen dry gel, Thr-fibrinogen dry gel, and TachoSil, the manner in which the cell suspension permeated differed. While the cell suspension permeated the fibrinogen dry gel and Thr-fibrinogen dry gel in a short time, it took a long time for the cell suspension to permeate the TachoSil.

[0086] (4) Implications of Example 7 1) Regarding the state of the cell suspension when seeded on the substrate, TachoSil did not penetrate well, and because Beriplast is a hydrogel, the cell suspension simply sat on top of it. The fibrinogen dry gel and Thr-fibrinogen dry gel were quickly penetrated by the cell suspension. If a larger number of cells are to be seeded, fibrinogen dry gel and Thr-fibrinogen dry gel, which have high permeability to the cell suspension, are advantageous. For example, by placing filter paper under the fibrinogen dry gel and Thr-fibrinogen dry gel and repeatedly adding the cell suspension on top of the dry gel, it becomes possible to filter out the cells through the dry gel that has been restored to a hydrogel by the cell suspension. 2) Of the dried products TachoSil, fibrinogen dry gel, and Thr-fibrinogen dry gel, only fibrinogen dry gel and Thr-fibrinogen dry gel were shown to promote MSC proliferation. Fibrinogen dry gel, alone or in combination with thrombin, functioned as a scaffold for MSCs, an adhesive cultured cell line, and demonstrated better cell proliferation than TachoSil. 3) Lyophilized fibrinogen dry gel and Thr-fibrinogen dry gel were shown to promote MSC proliferation, similar to Beriplast (hydrogel). Beriplast is a two-liquid mixture hydrogel that requires adjustment of the fibrinogen and thrombin solutions prior to use, making it difficult to prepare a uniform fibrin hydrogel with consistent concentration. In other words, fibrinogen solution and thrombin solution begin to solidify locally before being uniformly mixed, so in this example, thrombin solution was layered on top of frozen fibrinogen solution and allowed to solidify at room temperature to obtain a planarly uniform fibrin hydrogel (although not uniform in the vertical direction). On the other hand, because fibrinogen hydrogel reacts slowly, a three-dimensionally uniform hydrogel can be relatively easily prepared by leaving a mixture of fibrinogen, FXIII and / or FXIIIa, and a calcium salt at room temperature. A fibrinogen dry gel, which is obtained by drying this mixture, has better storage stability than fibrin hydrogel or fibrinogen hydrogel.In addition, fibrinogen dry gels are also superior in terms of permeability to culture media and cell suspensions. For these reasons, fibrinogen dry gels are preferred when providing scaffold materials for cells that do not require preparation immediately before use.

[0087] The present invention relates to embodiments of fibrinogen gels and the like that are useful as biomaterials for hemostasis, tissue adhesion / closure, cell scaffolding, etc., and are useful, for example, in the field of medicine. This application is based on Japanese Patent Application No. 2024-021702 (filing date: February 16, 2024), the contents of which are incorporated in full herein.

Claims

1. Dry fibrinogen gel.

2. The dry gel according to claim 1, which is restored to a hydrogel upon addition of water.

3. A dry gel according to claim 1 or 2, in which, when the hydrogel (insoluble matter) obtained after adding water to the dry gel is suspended or dissolved and developed by electrophoresis under reducing conditions, two types of polypeptide chains, Aα and γ, are polymerized and cross-linked, and the ratio to the Bβ chain satisfies either of the following conditions: (Aα polymer) / (Bβ monomer)>0.15 (γ dimer) / (Bβ monomer)>0.2 (where Aα represents the Aα chain that constitutes fibrinogen, Bβ represents the Bβ chain that constitutes fibrinogen, and γ represents the γ chain that constitutes fibrinogen).

4. The dry gel contains transglutaminase and a calcium salt, and the molar ratio of fibrinogen:transglutaminase:calcium salt is 5-300:0.01-10:0.05 x 10. ３ ~200 x 10 ３ The dry gel according to claim 1 or 2, wherein the dry gel is contained in a ratio of:

5. The dry gel according to claim 4, wherein the transglutaminase is FXIII and / or FXIIIa and the calcium salt is calcium chloride.

6. The dry gel of claim 1, wherein the dry gel is in the form of a composition containing a dry gel of fibrinogen.

7. The dry gel of claim 6, wherein the composition further contains thrombin.

8. The dry gel according to claim 6 or 7, wherein the composition is a sheet-shaped composition comprising a layer mainly composed of a dry gel of fibrinogen ("fibrinogen dry gel layer").

9. The dry gel according to claim 8, wherein the composition further comprises one or more layers whose main component is a component other than the fibrinogen dry gel layer, and the composition is in the form of a sheet comprising the fibrinogen dry gel layer and the one or more layers.

10. The dry gel according to claim 9, wherein the layer other than the fibrinogen dry gel layer is a layer containing thrombin as a main component.

11. Biomaterials containing fibrinogen gel.

12. The biomaterial according to claim 11, which is used for hemostasis or tissue adhesion / closure.

13. The biomaterial according to claim 11, which is for use in regenerative medicine.

14. The biomaterial according to claim 11, which is a sustained-release material.

15. The biomaterial according to claim 11, wherein the fibrinogen gel is a dry fibrinogen gel.

16. The biomaterial according to any one of claims 11 to 15, wherein the dry gel of fibrinogen is the dry gel according to claim 2.

17. A method for producing a composition containing a dry fibrinogen gel, comprising the step of reacting fibrinogen with transglutaminase in the presence of a calcium salt to obtain a fibrinogen gel.

18. A method for producing the composition of claim 17, wherein the step of obtaining a fibrinogen gel further comprises a step of drying the fibrinogen gel to obtain a dry gel.

19. A method for producing the composition described in claim 17, wherein in the step of obtaining a fibrinogen gel, a layer containing fibrinogen gel as the main component (a "fibrinogen gel layer") is constructed, and a sheet-like composition constituted by the fibrinogen gel layer is obtained.

20. A method for producing the composition according to claim 19, further comprising constructing, in addition to the fibrinogen gel layer, one or more "layers whose main component is another component" other than the fibrinogen gel layer.

21. A method for producing a composition according to any one of claims 17 to 20, wherein the calcium salt is calcium chloride and the transglutaminase is FXIII and / or FXIIIa.

Images