WO2025173759A1 - Fibrinogen gel as biomaterial for hemostasis, tissue adhesion/closure, cell scaffold material, and so forth - Google Patents

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

The present invention relates to fibrinogen gels and the like that are useful as biomaterials for hemostasis, tissue adhesion and closure, cell scaffolding materials, etc., and are useful, for example, in the pharmaceutical field.

Fibrin glue has been widely used in medical settings for hemostasis and tissue adhesion/closure (see, for example, Patent Documents 1 to 5). Fibrin glue preparations are broadly divided into liquid formulations and sheet formulations.
In liquid fibrin glue, fibrinogen solution and thrombin solution react on the surface of the wound to produce fibrin, which is then polymerized and cross-linked to form a three-dimensional fibrin mesh structure microscopically and a gel macroscopically, which covers the wound and achieves the above-mentioned purpose of use.
Even fibrin glues that are considered liquid preparations are provided as compositions of dried fibrinogen and dried thrombin in order to improve storage stability, and each composition must be dissolved over time before use to obtain two liquids: fibrinogen solution and thrombin solution (Patent Documents 1 and 2).To solve this problem, fibrin glues composed of fibrinogen solution and thrombin solution that can be stored in liquid form have 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, which can lead to dripping if the treatment surface is inclined, and it can also be difficult to apply pressure until the two solutions react and achieve a certain hardness. To compensate for these drawbacks, several applicators have been developed to efficiently react the two solutions and maximize their efficacy, and treatment methods have also been developed that use them in combination with tissue reinforcement materials.
To overcome the drawbacks of liquid fibrin glue, sheet preparations have been developed (Patent Documents 5 to 7). Because it is difficult to process fibrinogen and thrombin themselves into sheets, fibrin glue sheets have been realized by supporting them on a support such as collagen or a synthetic polymer. When the fibrinogen and thrombin supported on the support come into contact with body fluids on the treatment surface, they dissolve and react to produce fibrin, which, like the liquid preparations, forms a gel that covers the treatment site, achieving the intended use.
Sheet formulations do not require preparation immediately before use and can be pressed against the treatment site, making them more convenient than liquid formulations. However, because they require a non-human-derived support component, they are not necessarily satisfactory in terms of biocompatibility.

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 gels and insoluble matter obtained here 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) were formed by the salting-out effect or hydrogen or ionic bonding.
It has also been reported that fibrinogen can be covalently crosslinked and polymerized with FXIII or FXIIIa in the presence of calcium ions to form 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 directly between glutamine and lysine residues in the Aα and γ chains of fibrinogen by transglutaminase FXIII or FXIIIa, without the intermediate step of fibrin production by thrombin.
Reversible fibrinogen gelation has been utilized in the production of fibrinogen drug substances for fibrinogen preparations and fibrin glue preparations (Patent Documents 1 to 4, Non-Patent Document 1). Conversely, the irreversible fibrinogen gelation reaction is considered to be 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 scaffolding material for cells. Furthermore, there have been no reports to date of a dry gel made from this fibrinogen gel or of a method for preparing such a gel.

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

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

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

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. They have also found that fibrinogen gels can be used as biomaterials for hemostasis, tissue adhesion and closure, cell scaffolding, etc., and have found that fibrinogen dry gels in particular are highly practical in medical settings. As a result of extensive research, they have completed the present invention.
Specific embodiments of the present invention are as follows: However, the present invention is not limited to these.

[1] Fibrinogen dry gel.
[2] The dry gel described in [1] above, which restores to a hydrogel when water is added.
[3] 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 crosslinked, and the ratio of Aα to Bβ chains is one of the following:
(Aα polymer)/(Bβ monomer)>0.15
(γ dimer)/(Bβ monomer)>0.2
(Here, Aα represents the Aα chain constituting fibrinogen, Bβ represents the Bβ chain constituting fibrinogen, and γ represents the γ chain constituting fibrinogen.)
The dry gel according to the above [1] or [2], which satisfies the above.
[4] The dry gel according to any one of the above [1] to [3], which contains transglutaminase and a calcium salt, and the molar ratio of fibrinogen:transglutaminase:calcium salt is 5-300:0.01-10:0.05x10 ³ -200x10 ³ .
[5] The dry gel described in [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 the above [1] to [5], wherein the dry gel is in the form of a composition containing a dry gel of fibrinogen.
[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 ("fibrinogen dry gel layer") containing a fibrinogen dry gel as a main component.
[9] The dry gel described in [8] above, wherein the composition further comprises one or more layers whose main component is a component other than the fibrinogen dry gel layer, and is a sheet-like composition constituted by the fibrinogen dry gel layer and the one or more layers.
[10] The dry gel according to the above [9], 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 for use in regenerative medicine.
[14] The biomaterial according to [11] above, which is a sustained-release material.
[15] The biomaterial according to any one of [11] to [14] above, wherein the fibrinogen gel is a dry fibrinogen gel.
[16] The biomaterial according to any one of [11] to [15] above, wherein the fibrinogen dry gel is the dry gel according to any one of [2] to [10] above.
[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 described in [17] above, 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 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 described in [19] above, 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 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.

The present invention provides fibrinogen dry gels and other materials that are useful as biomaterials for hemostasis, tissue adhesion and closure, cell scaffolding materials, etc.

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 sheet before compression after the drying process, and the right image shows the sheet formed into a thin film by compression. Both are cross-sectional views, with the fibrinogen gel layer on the left and the thrombin layer on the right. 2 shows the (Aα polymer)/(Bβ monomer) ([2A]) and (γ dimer)/(Bβ monomer) ([2B]) obtained by electrophoresis under reducing conditions of a composition obtained by adding water to the freeze-dried preparation after gel formation of fibrinogen obtained in Example 1 described below. The numbers in the figure indicate the fibrinogen concentration (mg/mL) during the gelation reaction. Figure 3 shows the results of the analysis of the properties of the fluid fibrinogen gel performed in Example 2, which will be described later. Figure A shows size exclusion chromatography of the fibrinogen concentrate (top) and the fluid fibrinogen gel (bottom). Figure B shows the fibrinogen concentration and fibrinogen polymer ratio measured by the thrombin clotting time method before fibrinogen gel formation (0 hr) and during gel formation over time from 0 to 7 hours. Figure C shows the fibrinogen concentration and fibrinogen polymer ratio measured by the thrombin clotting time method. 4 shows size exclusion chromatographs of the fibrinogen concentrate (top) and the flowable fibrinogen gel (bottom) in Example 3 described below, and the relative fibrinogen titers of the major fractions collected. The fraction numbers are shown below the size exclusion chromatographs, and a bar graph is shown for the fractions for which the relative fibrinogen titers were measured. 5 shows the evaluation results in a rat liver bleeding model in Example 4 described below ([5A]: evaluation results for hemostatic properties (amount of bleeding in 10 minutes); [5B]: evaluation results for adhesive properties (adhesion strength of the test substance to the wound surface (180° peel adhesive strength)). Shown as (Mean) ± (SE). N = 6 to 9 for each group). 'Fbg' in the table is an abbreviation for 'fibrinogen powder' in the text. Figure 6 shows the test results (tensile shear adhesive strength) of the rat skin adhesion test in Example 4 described later. The results are 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. FIG. 8 shows histopathological images of the hemorrhagic sites on the wound surface of a rat liver 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 on the bottom with the test substance placed on top. Liver tissue is gray, test substance is light gray, and blood is dark gray). Figure 9 shows the evaluation results in 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 test substance to wound surface (180° peel adhesive strength))). Results are shown as (Mean) ± (SE). N = 7-9 for each group. 'Fbg gel' in the table is an abbreviation for 'fibrinogen gel' in the text. Figure 10 shows the growth curve of cultured cells in Example 7 described below. 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, shown as 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.

Various embodiments of the present invention are described in detail below. Unless otherwise defined, 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 methodology described in the publications that might be used in connection with the described invention.

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 properties of this fibrin gel mean that it can be made into a paste by combining fibrinogen with drugs such as thrombin, and therefore it is used as a fibrin glue preparation to produce fibrin gel at the site of damage to organs, tissues, bones, etc., not just at the site of bleeding, but also to adhere and seal the damaged site, ultimately stopping the leakage of blood, body fluids, or gas.It is then used in medical settings as a scaffold for the regeneration of damaged tissue, for tissue adhesion and closure, and as a scaffold material for cells, etc.
The present inventors have now unexpectedly discovered that, instead of fibrin gel, fibrinogen gel is useful for hemostasis, tissue adhesion and closure, cell scaffolding, etc. Furthermore, they have also discovered that, among fibrinogen gels, fibrinogen dry gel is particularly useful from the viewpoint of high practicality in medical settings, etc. The present invention will be described in detail below.

[About fibrinogen dry gel]
According to one embodiment of the present invention,
"Fibrinogen dry gel"
is provided.
The production method will be described later, but 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 production process. Note that the fibrinogen gel-forming reaction does not involve thrombin, and therefore does not produce fibrin.

As shown in Figure 1A, the viscosity of the fibrinogen solution increases continuously and the fluidity decreases as gel formation progresses due to the polymerization and cross-linking of fibrinogen (the degree of polymerization becomes more advanced), and the solution eventually becomes a hydrogel with no fluidity.
As used herein, "flowable fibrinogen gel" refers to a flowable fibrinogen gel that contains fibrinogen polymers and may also contain unpolymerized fibrinogen monomers.
Here, the "fibrinogen polymer" refers to fibrinogen polymerized by covalent bonds, and its proportion and degree of polymerization increase as the gelation reaction proceeds. The polymer can be contained in both a fluid fibrinogen gel and a fibrinogen hydrogel.
As used herein, "fibrinogen hydrogel" refers to a substance that has undergone a high degree of gelation, contains fibrinogen polymers, and has lost 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.

The above-mentioned "fibrinogen dry gel" will now be described 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).
In the case of fibrinogen dry gel, when 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 amount of fibrinogen applied per well is 0.1 to 10 μg/3 mm well), two types of polypeptide chains, Aα and γ, are polymerized and cross-linked, and the ratio to Bβ chain is one of the following conditions:
(Aα polymer)/(Bβ monomer)>0.15
(γ dimer)/(Bβ monomer)>0.2
(Here, Aα represents the Aα chain constituting fibrinogen, Bβ represents the Bβ chain constituting fibrinogen, and γ represents the γ chain constituting fibrinogen.)
A preferred embodiment is one having a polymerized/crosslinked form that satisfies the following: Here, 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.

The dry gel described above contains transglutaminase and a calcium salt, and a preferred embodiment of the dry gel contains fibrinogen:transglutaminase:calcium salt in a molar ratio of 5-300:0.01-10:0.05×10 ³ -200×10 ³ .
Here, the transglutaminase may be FXIII and/or FXIIIa, and the calcium salt may be calcium chloride.

[Usage of fibrinogen gel]
The "fibrinogen gel" of the present invention (which refers to the fluid fibrinogen gel immediately after the start of fibrinogen gel formation, to the fibrinogen hydrogel and fibrinogen dry gel) may be in the form of a composition containing other components derived from the production process, as described above, but 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.). "Other components" further 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 fibrinogen dry gel can be used alone, for example, as a pulverized powder composition (see B and D in Fig. 1). The fibrinogen dry gel is hydrated with blood or body fluids when used, and therefore remains and functions substantially as a fibrinogen gel in the body.
The powder composition may be combined with one or more compositions whose main component is "other components" other than the fibrinogen gel powder. Examples of powders whose main component is "other components" include powders containing thrombin. Furthermore, these multiple types of powders, including the fibrinogen gel powder, may be used in combination with biomaterials capable of supporting them.
The fibrinogen dry gel can be used alone, for example, as a molded sheet-like composition (see B and E in Fig. 1). The fibrinogen dry gel is hydrated with blood or body fluids when used, and therefore remains and functions substantially as a fibrinogen gel in the body.
The sheet-shaped composition may contain one or more layers whose main component is "other components" other than the fibrinogen gel layer, and may be a sheet-shaped composition constituted by a fibrinogen gel layer and one or more such layers (preferably six or fewer layers). Here, the sheet-shaped composition may have a two-layer structure or more layers. Further, an example of a layer whose main component is "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, the fibrinogen gel layer can also serve as a support for the layer mainly composed of "other components," but it may also be formed in a form including a support, as is commonly used in the art. This convenience is one of the excellent effects of the present invention.

[Method for producing fibrinogen gel and its composition]
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.

(1) A process for obtaining a fluid fibrinogen gel and a fibrinogen hydrogel by reacting fibrinogen with transglutaminase in the presence of a calcium salt. 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, one preferred embodiment is when the blending ratio of fibrinogen:transglutaminase:calcium salt is within the range of 5-300:0.01-10:0.05×10 ³ -200×10 ³ in terms of molar ratio.
When fibrinogen gel is formed under the above conditions, a fibrinogen hydrogel is obtained via a fluid fibrinogen gel (Figure 1A). However, by changing the buffer composition, the compounding ratio of fibrinogen, transglutaminase, and calcium salt, the temperature, the reaction time, etc., the rate and degree of fibrinogen gel formation and the physical properties of the fibrinogen gel can be changed.
The origin of the transglutaminase used in this step is not particularly limited, and it may be human-derived, animal-derived, or obtained by genetic recombination and added externally. Furthermore, if the transglutaminase is endogenous to the fibrinogen used as the 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 process, "other ingredients" may be added as needed. Examples of such ingredients 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 ingredients" also include blood coagulation-promoting ingredients (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 flowable fibrinogen gel or fibrinogen hydrogel may be frozen, if necessary, and used in the next step.

(2) A step of drying the fluid fibrinogen gel or fibrinogen hydrogel to obtain a dry fibrinogen gel. The desired dry fibrinogen gel can be produced by distilling off the water, which is the dispersion medium, from the fluid fibrinogen gel or fibrinogen hydrogel obtained in step (1) by drying (Fig. 1A).
The drying method is not particularly limited and can be any method known in the art, for example, lyophilization, preferably vacuum lyophilization. The conditions for vacuum lyophilization are not particularly limited and can be appropriately determined by a person skilled in the art depending on the intended product. For example, vacuum lyophilization can be performed by carrying out 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.

(3) Step of obtaining a powdery composition The dry gel of fibrinogen obtained in step (2) can be pulverized by a method known in the art to produce a powdery composition (FIG. 1B and D).
If necessary, this powder can be mixed with one or more powders primarily composed of "other components" to produce a powdery fibrinogen dry gel composition containing the other components. Mixing multiple types of powders is useful for separating and preventing components that would react with the fibrinogen, transglutaminase, and calcium salts 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 also possible to adjust the physicochemical properties, efficacy, in vivo degradation characteristics, biocompatibility, etc. of the fibrinogen gel powder composition.
Any powder, including fibrinogen gel powder, may contain "other ingredients" as needed. Examples of such ingredients 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 ingredients" include blood coagulation promoters (e.g., polyphosphates, phospholipids, protamines), cell cultures, gene therapy materials, physiologically active substances (e.g., growth factors, hormones), steroids, anticancer drugs, antibiotics, anti-inflammatory agents, and analgesics.
Additionally, these types of powders, including fibrinogen gel powder, may be combined with biomaterials capable of supporting them by methods known in the art.

(4) Step of Obtaining a Sheet-Shaped Composition A sheet-shaped composition containing a dry gel of fibrinogen can be produced by a method known in the art (FIG. 1B and E).
For example, a sheet-like composition can be produced by forming a fibrinogen gel in a suitable molding vessel in step (1), obtaining a fluid fibrinogen gel or fibrinogen hydrogel through polymerization and cross-linking reactions, freezing and drying the gel if necessary in step (2), and then compressing it to form a sheet-like fibrinogen dry gel layer (fibrinogen gel layer). The fibrinogen gel layer thus obtained can also function as a support for the sheet-like composition.
Alternatively, the sheet-shaped composition can be constructed by immobilizing the fibrinogen gel layer on a substrate containing another support, such as a bioabsorbable material such as aliphatic polyesters, cellulose derivatives, collagen, gelatin, or thickening polysaccharides or gelling polysaccharides, which has been processed into a cotton-like, sheet-like, cloth-like, or sponge-like form.
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.

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 above-mentioned layer ("fibrinogen gel layer"), which is primarily composed of fibrinogen gel. 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, etc. of the fibrinogen gel sheet-shaped composition.
A sheet-like composition constructed by multilayering can also be produced by methods known in the art (FIG. 1C). For example, the flowable fibrinogen gel or fibrinogen hydrogel obtained in the molding container in step (1) can be frozen as needed, and then a layer containing another component can be constructed on top of the 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 another component" is further constructed on the fibrinogen gel layer. Alternatively, a sheet-like composition can be produced by constructing a "layer mainly composed of another component" on a fibrinogen gel layer constructed by immobilizing the fibrinogen gel layer in a form including another support.
Any of the layers, including the "fibrinogen gel layer,""layers primarily composed of other components," and the support, may contain "other components" as needed. 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.
In the case of a sheet-shaped composition having a multilayer structure, the order of construction of each layer is not limited, and by appropriately changing the order during production, a sheet-shaped 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.

[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 a living body, it can be widely used as a living-derived biomaterial for hemostasis, tissue adhesion/closure, cell scaffolding, etc.
In particular, fibrinogen dry gels retain the properties of the fluid fibrinogen gel or fibrinogen hydrogel before drying, and it has been confirmed that these properties can be reproduced by adding water. Furthermore, because excess water is removed from fibrinogen dry gels, they have higher storage stability than fluid fibrinogen gels or fibrinogen hydrogels.

Fluid fibrinogen gels and fibrinogen hydrogels, or fibrinogen dry gels rehydrated with water, contain fluid fibrinogen polymers, which have been shown to shorten thrombin clotting times compared to fibrinogen monomers at the same concentration.
Thrombin is generated in the blood at the wound site by 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 generated by the normal coagulation mechanism also contributes as an adhesive component between the fibrinogen gel and the wound site. If the fibrinogen gel is a hydrogel, it will return to its original hydrogel state by itself, and if it is a dry gel, it will immediately return to its original state by water migrated from the blood, and contribute as a wound closure 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.

Fibrinogen gel with the appropriate hardness will generally remain at the treatment site without flowing even when it absorbs body fluids. For example, it has been confirmed that when applied to a wound, it will remain there and will not easily 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, thereby exerting its occlusive properties, fibrinogen gel can encapsulate the wound site more uniformly and firmly.
Fibrinogen gel with the appropriate hardness does not soak into gauze, etc., so it can be pressed onto the treatment site immediately after application. For example, when pressed onto the bleeding surface of a wound, excess blood is pushed out, and the fibrin produced by the hemostatic mechanism penetrates into the irregularities on the wound surface, coagulates, and gels, allowing the treated fibrinogen gel to be more firmly attached 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.

Fibrinogen gel can be applied to the body by, for example, spreading it on the 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 hemostasis, fibrinogen gel can be applied in an amount of 0.1 to 50 mg/ ^cm2 , preferably 0.5 to 25 mg/ ^cm2 , more preferably 2 to 15 mg/ ^cm2 , in terms of fibrinogen, depending on the size of the wound.

Although the above-mentioned excellent functions are exhibited by a fibrinogen gel alone, they 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 the wound site, the thrombin promotes fibrin production in the blood, which, combined with the excellent effects of the fibrinogen gel, allows for more effective hemostasis, tissue adhesion, and closure of the wound site.
The use in combination with such other components can be carried out, for example, by applying to the body simultaneously formulations of fibrinogen gel and other components separately, or by applying to the body a formulation combining both components in the form of the above-mentioned multilayered sheet-like composition.
Furthermore, if fibrinogen gel contains other ingredients such as growth factors or hormones, it can promote the proliferation of specific cell groups and regulate homeostasis. Alternatively, if steroids or anticancer drugs are added, it can have anti-inflammatory or anticancer effects. Furthermore, if analgesics are added, it can alleviate pain at the treatment site. For these reasons, fibrinogen gel can be used as a scaffold for transplanted cells and cell growth, as well as a sustained-release material for additives, and can therefore also be used as a biomaterial for regenerative medicine.
Furthermore, it has been confirmed that the fibrinogen dry gel mentioned above also has 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 bioadhesive composed only of human-derived components.
It has also been confirmed that fibrinogen gels with the same degree of polymerization have higher resistance to plasmin, i.e., fibrinolysis, than the corresponding fibrin gels.

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

The present invention will be further explained in detail by the following examples, but these are not intended to limit the scope of the present invention and may be modified without departing from the scope of the present invention.

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 80 mg/mL (fibrinogen concentration; 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.

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, held for 25 hours. Segment 2: Heat to 32°C at 0.03°C/min, held 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. The three lots of fibrinogen concentrates contained 0.468-0.613 IU/mL of FXIII per 2 mg/mL of fibrinogen.

(2) Evaluation of the Prepared Product The gelation and other properties of the prepared product were evaluated as follows.
1) Hardness of the preparation before freeze-drying:
The hardness of the preparation before freeze-drying was determined by placing a KC4 Delta steel ball from above into each well containing the preparation after fibrinogen gel formation, and visually inspecting the 48-well plate from the bottom after freeze-drying. If the steel ball sank to the bottom of the well and was easily visible, it was rated as -, if it was barely visible, it was rated as ±, and if it was not visible, it was rated as +. Conditions that yielded a rating of ± or + were deemed to have yielded a fibrinogen hydrogel through the fibrinogen gel-forming reaction.
2) Cracking of the preparation after freeze-drying:
The presence or absence of cracks in the preparation obtained by freeze-drying after the formation of a fibrinogen gel was confirmed. The 48-well plate after freeze-drying was visually inspected, and the presence or absence of cracks was scored as - or +, respectively.
3) Hardness of freeze-dried preparation when reconstituted with water:
The freeze-dried preparation was added with 150 μL/well of water and left to rehydrate at room temperature for 1 hour, and evaluation was based on whether a hydrogel (insoluble matter) of a certain hardness was formed. The score was determined as - if the hydrogel (insoluble matter) could not be picked up with tweezers after rehydration, ± if it crumbled, and + if it could be picked up. Conditions that yielded a ± or + rating indicated that the fibrinogen dry gel had reconstituted into a hydrogel (insoluble matter).
For 1) and 3) above, - was assigned 0 points, ± was assigned 1 point, and + was assigned 2 points. For 2) above, the number of + points was tallied. The results for 9 plates per condition are shown in Table 2.

4) Evaluation of the preparation after lyophilization by electrophoresis:
To the composition obtained by adding water to the freeze-dried preparation in 3) above at a rate of 150 μL/well, 900 μL of 8M urea and 100 μL of a reducing agent (500 mM dithiothreitol-containing sample reducing agent (10×), 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 electrophoretic images as follows. After electrophoresis (Nu-PAGE, Invitrogen) under reducing conditions (fibrinogen applied at a rate of 0.3 μg/3 mm well), Coomassie staining was performed, and digital images were captured using an image analyzer (BIO-RAD, GS-900). The captured images were analyzed using ImageJ (National Institutes of Health). The fibrinogen bands appearing in each lane were classified, from the lowest molecular weight to the highest, into γ-monomer, Bβ-monomer, Aα-monomer, γ-dimer, and bands 1 to 10 higher, representing Aα-polymers (polymers in which any number of Aα-chains and/or γ-chains are covalently bonded), and 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.

(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, the preparations containing 2.5 to 80 mg/mL of fibrinogen concentrate and 1.56 to 100 mM of calcium chloride frequently produced fibrinogen hydrogels hard enough to prevent a steel ball from sinking into them (Table 2 below).
2) Regarding cracking of the preparations obtained by freeze-drying after fibrinogen gel formation, freeze-dried preparations with a good shape were obtained when fibrinogen concentrate was added at 10 to 80 mg/mL and calcium chloride was added at 1.56 to 100 mM, while freeze-dried preparations without cracking were frequently obtained when fibrinogen concentrate was added at 10 to 40 mg/mL and calcium chloride was added at 1.56 to 50 mM (Table 2 shown below).
3) When the freeze-dried preparations were reconstituted with 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 below).
4) When the compositions obtained by reconstituting the lyophilized preparations with water were suspended or dissolved and subjected to electrophoresis under reducing conditions, differences in the ratio of Aα polymer and γ dimer relative to Bβ monomer were observed, as shown in Figure 2 (in Figure 2, [2A] indicates (Aα polymer)/(Bβ monomer), and [2B] indicates (γ dimer)/(Bβ monomer)). The (Aα polymer)/(Bβ monomer) ratio of the compositions obtained by reconstituting the lyophilized preparations with water was less than 0.15 when calcium chloride was 0 mM, but was 0.15 or greater when calcium chloride was 1.56 mM or greater. The (γ dimer)/(Bβ monomer) ratio of the compositions obtained by reconstituting the lyophilized preparations 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.

*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, "cracking of preparation after freeze-drying" was scored from 0 to 9, with 9 being the fewest cracks. "Hardness of preparation after freeze-drying when reconstituted in water" was scored from 0 to 18, with 18 being the hardest.

(4) Summary of Parameters The various parameters used in the preparation of the fibrinogen gel are summarized below.
1) Conditions under which fibrinogen hydrogel was formed If the conditions were such that the hardness of the preparation before freeze-drying in Table 2 was scored as 1 or higher with a frequency of 50% or more, the concentrations of the following components in the fibrinogen gel reaction solution were: fibrinogen (mg/mL): FXIII (IU/mL): calcium chloride (mM).
= 2.5 to 80: 0.595 to 24.5: 1.56 to 100
The above is expressed as a molar ratio:
Fibrinogen: FXIII: calcium chloride = 7.35-235: 0.039-1.61: 1560-100000.
2) Conditions under which crack-free fibrinogen dry gels can be formed. If fibrinogen hydrogels can be formed and the cracking score of the freeze-dried preparations in Table 2 is 1 or higher at a frequency of 50% or higher, 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
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 29.4-118: 0.154-0.807: 1560-50000
is.
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 is reconstituted in water and scores of 1 or higher are obtained with 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).
= 2.5 to 80: 0.595 to 24.5: 1.56 to 100
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 7.35-235: 0.039-1.61: 1560-100000
is.
4) When the freeze-dried preparation was reconstituted in water under conditions that gave a hardness score of 1 or more, the dry gel was prepared by adding water to the freeze-dried preparation to obtain a composition (1 volume). When this composition was suspended or dissolved in 50 mM dithiothreitol in 7.2 M urea (6.7 volumes or more) and subjected to electrophoresis under reducing conditions, the two polypeptide chains Aα and γ that constitute fibrinogen were polymerized and cross-linked, and the ratio of these to the Bβ chain satisfied one of the following conditions:
(Aα polymer)/(Bβ monomer)>0.15
(γ dimer)/(Bβ monomer)>0.2
(wherein an Aα polymer is a polymer of any number of Aα chains and/or γ chains covalently linked together)

(5) Implications of Example 1: 1) The conditions under which a fibrinogen hydrogel could be formed and the conditions under which a fibrinogen dry gel could be restored to a hydrogel upon addition of water were generally correlated. These conditions can vary greatly depending on the buffer composition, additives, temperature, and gelation time during gelation, but the conditions under which a fibrinogen hydrogel could be formed and a fibrinogen dry gel could be 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 hydrogel could be formed and the conditions under which fibrinogen dry gel restored to a hydrogel when water was added did not correlate with the conditions under which crack-free fibrinogen dry gel was easily 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 when water is added. These conditions can vary greatly depending on the buffer composition, additives, temperature, and gelation time during gelation. However, the fibrinogen gel formation conditions shown in this example were appropriate when the fibrinogen concentrate was in the range of 10 to 40 mg/mL, the FXIII was in the range of 2.34 to 12.5 IU/mL, and the calcium chloride was in the range of 1.56 to 50 mM.
4) When the hardness of the preparation before freeze-drying or the hardness of the preparation after freeze-drying when reconstituted in water was used as an index, conditions that gave a score of 1 or higher indicated that fibrinogen gel formation had occurred, and the higher the score, the more advanced the fibrinogen gel formation. This was also confirmed by an 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
(wherein an Aα polymer is a polymer of any number of Aα chains and/or γ chains covalently linked together)
5) In Non-Patent Document 2, under conditions different from those used in this example, when fibrinogen is in the range of 3 to 24 mg/mL, FXIII is in the range of approximately 0.25 to 2 IU/mL, and the calcium chloride concentration is in the range of 0.05 to 5 mM, it is confirmed that the formation of fibrinogen Aα polymers and γ dimers progresses and an insoluble fibrinogen gel is produced. It has also been confirmed that a similar reaction proceeds more rapidly when FXIII is replaced with FXIIIa. While FXIII was used as the transglutaminase in this example, the inventors have separately confirmed that a similar reaction proceeds when FXIII is replaced with FXIIIa.

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 preparation contained 5.57 IU/mL (average) 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 a fibrinogen concentrate (11 mg/mL) 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 after 8 hours they lost fluidity and became a fibrinogen hydrogel.
A size-exclusion chromatogram was obtained 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 (flowable fibrinogen gel) obtained every hour for 7 hours after the addition of calcium chloride. Furthermore, fibrinogen concentration was measured by the thrombin clotting time method using a clotting time analyzer (KC4 Delta, Tcoag) and a Thrombocheck Fib (Sysmex). The fibrinogen concentration was measured by the thrombin clotting time method for the fibrinogen solution without calcium chloride added (fibrinogen concentrated solution) and the liquid fibrinogen gel reaction solution (fluid fibrinogen gel) 1, 3, 5, and 7 hours after the addition of calcium chloride.

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.
In the fibrinogen gel formation of the fibrinogen concentrate, the fibrinogen gel reaction solution remained fluid up to 7 hours after the addition of calcium chloride. Therefore, the fibrinogen concentration was calculated by the thrombin clotting time method before the addition of calcium chloride and 1, 3, 5, and 7 hours after the addition, and the results are shown in Figure 3B.
The proportion of generated fibrinogen polymers and the fibrinogen concentration measured by the thrombin clotting time method both increased during the fluidity period up to 7 hours (Fig. 3B). The fibrinogen concentrate without calcium chloride contained 19% fibrinogen polymers, which increased over time to 70% after 7 hours of calcium chloride addition. The fibrinogen concentration measured 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 measured 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 determined by the thrombin clotting time method (R ² =0.9897, FIG. 3C).

(2) Summary of Parameters The various parameters used in the preparation of the fibrinogen gel 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
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 58.8:0.366:25000
is.
2) The polymer content of the fibrinogen gel after addition of calcium chloride is >19% (by size exclusion chromatography).

(3) Implications of Example 2: 1) In the 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 polymer 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 Thrombocheck Fib, it was found to shorten over time, confirming an increase in the fibrinogen concentration by the thrombin clotting time method. In other words, as the fibrinogen polymer content in the fibrinogen gel increased, the thrombin clotting time shortened and the apparent fibrinogen titer increased.
3) Fibrinogen gel contains fluid fibrinogen gel even when it has been polymerized into a hydrogel or dry gel, so when combined with thrombin, it can be processed into a glue preparation that solidifies in a short time.

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 preparation contained 5.57 IU/mL (average) 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 a 20 mg/mL 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 it could not be pipetted or did not pass through a 0.45 μm filter at a certain evaluation point, it was assessed as a hydrogel from that point on.
(2) Evaluation of the relative potency of fibrinogen with different degrees of polymerization obtained from a fluid fibrinogen gel. Calcium chloride was added to a 20 mg/mL 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 using a thrombin clotting time assay with a clotting time analyzer (KC4 Delta, Tcoag) and a Thrombocheck Fib (Sysmex). The fibrinogen concentration of a 1 mg/mL fibrinogen concentrate without added calcium, calculated as a relative titer by the thrombin clotting time assay, was defined as 100%. The relative titer of fibrinogen in the measured fractions was calculated using the following formula:
[Relative fibrinogen titer of measurement fraction]
= [fibrinogen concentration in a 1 mg/mL measurement fraction calculated as absorbance by the thrombin clotting time method]/
[Fibrinogen concentration of 1 mg/mL fibrinogen concentrate measured by thrombin clotting time method in absorbance equivalent] x 100%

(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.

*The fluidity of fibrinogen gel was evaluated as follows: if it could be pipetted and passed through a 0.45 μm filter, it was judged as ◯ (fluid fibrinogen gel); if it could not be passed through, it was judged as △ (fibrinogen hydrogel); and if it could not be pipetted, it was judged as × (fibrinogen hydrogel). These results are shown in Table 3. If it could not be pipetted at a certain evaluation point or if it 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).

(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 leaving it for 1 hour. From the chromatogram, the peaks with the shortest column retention times were designated peak 1 and peak 2, which represent fibrinogen polymers, and the subsequent peak was designated the peak of fibrinogen monomer. 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 the fluid fibrinogen gel. When the potency of the former was taken as 100%, the potency of the latter was found to have 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).

*The relative titer of fibrinogen was calculated by converting absorbance to that of a 1 mg/mL fibrinogen concentrate without added calcium, as determined by the thrombin clotting time method, with the relative titer set at 100%. The relative titer of fibrinogen in the measurement fraction was calculated using the following formula.
[Relative fibrinogen titer of measurement fraction]
= [fibrinogen concentration in a 1 mg/mL measurement fraction calculated as absorbance by the thrombin clotting time method]/
[Fibrinogen concentration of 1 mg/mL fibrinogen concentrate as calculated by the thrombin clotting time method in terms of absorbance] x 100%

(5) Summary of Parameters The various parameters used in the preparation of the fibrinogen gel 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
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 58.8:0.366:250-32000
is.
2) The fluid fibrinogen gel contained 22% to 41% fibrinogen polymer (by size exclusion chromatography).
3) When the fibrinogen polymer content of the flowable fibrinogen gel was 41%, the relative fibrinogen titer was 131% of that of the fibrinogen concentrate.
4) The relative titers of fibrinogen in the monomer fraction of the fibrinogen concentrate and the flowable fibrinogen gel were 42.5% and 40.5%, respectively.
5) The relative fibrinogen titers of the fibrinogen polymer fraction in the fibrinogen concentrate and the flowable fibrinogen gel were 289% and 202-405%, respectively.

(6) Implications of Example 3 1) The fibrinogen monomer contained in the fibrinogen concentrate and the flowable fibrinogen gel has a low relative fibrinogen titer of less than 100%. In contrast, the fibrinogen polymer fractions extracted from the fibrinogen concentrate and the flowable fibrinogen gel have a relative fibrinogen titer of more than 200%.
2) The fluid fibrinogen gel contained more fibrinogen polymers than a fibrinogen concentrate with a fibrinogen relative titer of 100%, and had a higher fibrinogen relative titer of 131%. Furthermore, by separating the fibrinogen polymers from the fluid fibrinogen gel, a fluid fibrinogen polymer fraction with a fibrinogen relative titer of 200% or more can be efficiently obtained.
3) It was confirmed that fibrinogen polymers are contained in the fluid fibrinogen gel obtained by fibrinogen gel formation. Furthermore, fibrinogen polymers are also contained in fibrinogen hydrogels in which fibrinogen gel formation has progressed, and in the fibrinogen dry gels obtained by drying these. In other words, the relative fibrinogen titer of each fibrinogen gel is higher than that of fibrinogen before the fibrinogen 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.

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 5.57 IU/mL (average value) 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 hydrochloride 10 g/L).
Calcium chloride was added to a mixture of 20 mg/mL fibrinogen concentrate, 5 mg/mL albumin (25% intravenous albumin donated by blood "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, to form a fibrinogen gel. At this time, the viscosity was measured using a tuning fork viscometer (SV-10A, A&D), and when the viscosities reached 2.85, 3.42, 34.1, 134, and 480 mPa*s*g/ ^cm3 , the 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), respectively, and these were 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.

(2) Summary of Parameters Various parameters for preparing the above-mentioned fibrinogen gel powder etc. 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
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 58.8:0.366:12500
is.

(3) Evaluation in a rat liver hemorrhage model (test method)
The study was conducted on a group of nine rats (strain: SD, sex: male, manufacturer: Charles River Japan, age at time of use: 7 weeks). After sedation with a triple anesthetic (10 mL/kg, i.p.), 250 U/kg heparin was administered intravenously through the tail vein and the rats were fixed in a dorsal position. A midline abdominal incision was made 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 ^cm2 ).
Once the 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 for 5 seconds. The pre-bleeding rate (g/min) was calculated from the amount of bleeding in the 30 seconds immediately prior to treatment with the test substance, and the amount of bleeding (g) in the 10 minutes following treatment with the test substance was calculated from the weight difference between before and after blood collection with a cotton ball. Furthermore, the post-bleeding volume was calculated for each animal based on the 30-second period of bleeding from 10 to 10.5 minutes (see below). A volume of 5 μL or less was considered hemostasis, and the hemostasis rate for each group was calculated. The animals used for the evaluation were only those whose pre-bleeding rate was 0.01 to 0.1 (g/min), and data from 6 to 9 animals per group were used.
After determining that bleeding had stopped, the liver was removed, and the liver and test substance were each fixed with clips. The adhesive 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).
The filter papers used to wipe away bleeding for 30 seconds from 10 to 10.5 minutes after treatment with the test substance as described above were arranged in a holder. For the calibration curve, two pieces of filter paper each soaked in 8, 4, 2, 1, and 0 (Blank) μL of blood were also arranged in the same holder, for a total of 10 pieces, and they were left to dry overnight or more.
After drying the filter paper, digital images were taken (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 so that the amount of blood could be quantified 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.

(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 to 9).
The Thr-fibrinogen powder resulted in more bleeding and poorer hemostatic properties than the other powders (see [5A] in Figure 5). No significant differences in adhesiveness were observed between the groups (see [5B] in Figure 5). The Thr-fibrinogen powder also had a lower hemostatic rate and poorer occlusive properties than the other powders (see Table 5 below).

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

(4) Rat skin adhesion test (test method)
The skin of the rats was stripped, the subcutaneous fat layer 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).

(Test results)
The adhesive strength of Thr-fibrinogen powder, Thr-pr fibrinogen powder, Thr-pg fibrinogen powder, Thr-g fibrinogen powder, and Thr-og fibrinogen powder was 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. The adhesive strength of Thr-og fibrinogen powder at 90 minutes was lower than that of the other powders.

(5) Implications of Example 4 1) Dry gels of fibrinogen with different degrees of 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 in which fibrinogen polymerization was not advanced.
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 because this has a short thrombin clotting time, the clotting reaction at the treatment site is rapid, resulting in high hemostatic and occlusive properties.
3) In a rat skin adhesion test, 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, in which fibrinogen had undergone polymerization, with Thr powder, continued to increase up to 90 minutes, whereas the adhesive strength of Thr-fibrinogen powder, which was prepared by combining Thr powder with less polymerized fibrinogen 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, when fibrinogen gel with a viscosity at least 1.2 times that of the original fibrinogen (2.85 mPa*s*g/cm ³ ) was used as a biological glue, hemostasis and occlusion were enhanced, as was fibrinolysis resistance.

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 a mean value of 5.57 IU/mL 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 this 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 respective containers, and crushed by the impact to obtain fibrinogen powder, fibrinogen gel powder, fibrin powder, and Thr powder.
Fibrinogen powder, fibrinogen gel powder, and fibrin powder were mixed with Thr powder at a weight ratio of 1:1 to prepare Thr-fibrinogen powder, Thr-fibrinogen gel powder, and Thr-fibrin powder, respectively.

(2) Preparation of fibrinogen gel sheets, etc. 1) For fibrinogen preparations, after buffer exchange by dialysis, 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 was prepared, to which calcium chloride was added to make a concentration of 12.5 mM. The mixture was then immediately poured into a tray at 0.2 mL/ ^cm2 , and the resulting fibrinogen solution was 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 layered on ^top of the fibrinogen sheet at 0.2 mL/cm² and frozen at -20°C (for the fibrinogen sheet).
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, and 2.5 mg/mL L-arginine hydrochloride. Calcium chloride was added to this mixture to make a 12.5 mM solution, and the mixture was immediately poured into a tray at 0.2 mL/ ^cm² and left overnight to prepare a fibrinogen hydrogel. This was then frozen at -20°C, and the same Thr solution as above was layered on top at 0.2 mL/ ^cm² , followed by freezing at -20°C (for Thr-fibrinogen gel sheets).
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, and 2.5 mg/mL L-arginine hydrochloride. Thr was added to the mixture at 0.125 IU/mL and calcium chloride to 12.5 mM. The mixture was then immediately poured into a tray at 0.2 mL/ ^cm² and left overnight to prepare a fibrin hydrogel. This was then frozen at -20°C, and the same Thr solution as above was layered on top at 0.2 mL/ ^cm² , followed by freezing at -20°C (for Thr-fibrin sheet).
2) The trays for the frozen Thr-fibrinogen sheets, the trays for the Thr-fibrinogen gel sheets, and the trays for the Thr-fibrin sheets were freeze-dried using the following program (vacuum freeze dryer, FZ-6, LABCONCO).
Segment 1: held at -30°C for 50 hours Segment 2: heated to 30°C at 0.03°C/min and held for 6 hours The resulting freeze-dried products were compressed and formed into sheets to obtain Thr-fibrinogen sheets, Thr-fibrinogen gel sheets, and Thr-fibrin sheets, respectively.

(3) Summary of Parameters The various parameters used in the preparation of the fibrinogen gel 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
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 58.8:0.366:12500
is.

(4) Evaluation of tensile strength of sheet adhesive (test method)
The Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet were cut into 1 × 2 cm pieces, and both ends were pinched with clips to obtain 1 cm square sheets. The tensile strength (gf, tensile shear strength) of each sheet 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).

(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, although it could be formed into a sheet, had almost no plasticity and had a texture that would disintegrate upon contact with anything.

(5) Evaluation in a rat liver hemorrhage model (test method)
The study was conducted using a group of eight rats (strain: SD, sex: male, manufacturer: Charles River Japan, age at time of use: 7 weeks). 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. 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 ^cm2 ).
After the bleeding 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 held in close contact for 5 seconds. The pre-bleeding rate (g/min) was calculated from the amount of bleeding in the 30 seconds immediately before and after test substance treatment, and the amount of bleeding (g) in the 10 minutes following treatment was calculated from the weight difference between before and after blood collection with a cotton ball. Furthermore, the post-bleeding amount was calculated for each animal based on the 30-second period of bleeding from 10 to 10.5 minutes (see below). A volume of 5 μL or less was considered hemostasis, and the hemostasis rate for each group was calculated. The animals used for the evaluation were only those whose pre-bleeding rate was 0.01 to 0.1 (g/min), and data from 7 to 8 animals per group were used.
After determining that bleeding had stopped, the liver was removed, and the liver and test substance were each fixed with clips. The adhesive 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).
The filter papers used to wipe away bleeding for 30 seconds between 10 and 10.5 minutes after treatment with the test substance were arranged in a holder. For the calibration curve, two filter papers each soaked in 8, 4, 2, 1, and 0 (blank) μL of blood were also arranged in the same holder, for a total of 10 pieces, and allowed to dry overnight or more.
After drying the filter paper, digital images were taken (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 so that the amount of blood could be quantified from the signal intensity of the calibration filter paper. Finally, the post-bleeding amount of each animal wiped with the filter paper was calculated from the signal intensity of each filter paper using the calibration curve equation.

(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 the surface of 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 greater amount of bleeding and exhibit weaker hemostatic properties (see [7A] in Figure 7). Among Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet, Thr-fibrin sheet tended to cause a lesser amount of bleeding.
Regarding adhesiveness, among Thr-fibrinogen powder, Thr-fibrinogen gel powder, and Thr-fibrin powder, the adhesive strength of Thr-fibrin powder was lower than the other two types of powder (see [7B] in Figure 7). Among Thr-fibrinogen sheet, Thr-fibrinogen gel sheet, and Thr-fibrin sheet, the adhesive strength of Thr-fibrinogen sheet was higher than the other two types of sheets.
Regarding occlusion, among Thr-fibrinogen powder, Thr-fibrinogen gel powder, and Thr-fibrin powder, Thr-fibrinogen gel powder had the highest hemostatic rate, followed by Thr-fibrinogen powder and Thr-fibrin powder. Among Thr-fibrinogen sheets, Thr-fibrinogen gel sheets, and Thr-fibrin sheets, Thr-fibrinogen gel sheets had the highest hemostatic rate, followed by Thr-fibrinogen sheets and Thr-fibrin sheets (see Table 6 below).

*'Fbg', 'Fbg gel' and 'Fbn' in the table are abbreviations for 'fibrinogen', 'fibrinogen gel' and 'fibrin', respectively, in the text.

(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 fixed in 10% neutral buffered formalin for at least 24 hours, after which it was excised to prepare specimens 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 for evaluation.

(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.

(7) Implications of Example 5 1) It was found that the Thr-fibrinogen gel powder in powder form and the Thr-fibrinogen sheet in sheet form were comprehensively superior in terms of medicinal efficacy.
Of the three sheets, the Thr-fibrinogen gel sheet had the highest tensile strength and the best handling, while the other two sheets, the Thr-fibrinogen sheet and the Thr-fibrin sheet, were not suitable for handling during hemostasis treatment. When handling was taken into account in addition to efficacy, the Thr-fibrinogen gel sheet was the most excellent.
2) When comparing Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet, hemostatic and occlusive properties were nearly equivalent, but Thr-fibrinogen gel powder was superior in adhesive properties. If adhesiveness is desired, Thr-fibrinogen gel powder is recommended, while Thr-fibrinogen gel sheet is recommended if convenience is a priority. 3) Figure 8 shows how blood penetrates the test substance. The three types of powder had thick sealing layers due to voids, while the sheet had a thin layer. Furthermore, the test substance dissolved in Thr-fibrinogen powder, Thr-fibrinogen sheet, Thr-fibrinogen gel powder, and Thr-fibrinogen gel sheet, whereas the test substance did not dissolve in Thr-fibrin powder and Thr-fibrin sheet, and fine voids were observed in the fibrous structure.
When the pathological images were examined to determine how far the blood had penetrated, blood was seen to have reached the opposite side of the wound surface in all cases except for the Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet. This indicates that the fibrinogen gel powder and fibrinogen gel sheet revert to a hydrogel form when they absorb the moisture from the blood, and this functioned as a barrier to prevent further blood penetration to the outside. Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet were superior in terms of robustly encapsulating and closing the wound surface. This result was consistent with the results of the hemostasis rate (see Table 6 above), which indicates closure.
From the above, it has become clear that by using fibrinogen gel instead of fibrinogen in a powder or sheet preparation of biological glue, it is possible to tightly close the wound and achieve reliable hemostasis.
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.

Example 6 Application of fibrinogen gel as a biomaterial for hemostasis/tissue adhesion and closure (3)
(1) Preparation of fibrinogen gel powder and sheet (powder preparation)
1) Fibrinogen used was fibrinogen (fibrinogen concentrate) derived from a fibrinogen preparation containing FXIII (Fibrinogen HT for Intravenous Injection 1g "JB", Japan Blood Products Organization). Here, FXIII was contained at a mean value of 5.57 IU/mL 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.
Separately, 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 this 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: -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 products were each vigorously shaken in their respective containers, and the impact caused them to break down, yielding 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 Sheets)
1) For the fibrinogen preparation, after the buffer was exchanged by dialysis, 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 was prepared, to which calcium chloride was added to make a concentration of 12.5 mM. The mixture was then immediately poured into a tray at 0.2 mL/ ^cm2 and left at 25°C for 16 hours to prepare a fibrinogen hydrogel. After freezing this 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, and calcium chloride was added to this to make a 12.5 mM Thr solution, which was layered at 0.2 mL/ ^cm² 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: held at -30°C for 50 hours. Segment 2: heated to 32°C at 0.03°C/min and held for 6 hours. The freeze-dried product obtained was compressed and formed into a sheet to prepare a Thr-fibrinogen gel sheet.

(2) Summary of Parameters The various parameters used in the preparation of the fibrinogen gel 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
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 58.8:0.366:12500
is.

(3) Evaluation in a rat liver hemorrhage model (test method)
The study was conducted on a group of nine rats (strain: SD, sex: male, manufacturer: Charles River Japan, age at time of use: 7 weeks). After sedation with a triple anesthetic (10 mL/kg, i.p.), 300 U/kg heparin was administered intravenously through the tail vein and the rats were fixed in a dorsal position. A midline abdominal incision was made 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 ^cm2 ).
Once the bleeding had subsided, a 1 cm square of Surgicel (Johnson & Johnson), Beriplast (CSL Behring) (a mixture of 62.5 μL each of fibrinogen solution and 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 treatment with the test substance, and the amount of bleeding (g) in the 10 minutes after treatment with the test substance was calculated from the difference in weight between before and after blood collection with a cotton ball. Furthermore, the post-bleeding amount was calculated for each individual based on the 30-second period of bleeding from 10 to 10.5 minutes, and the hemostatic rate for each group was calculated, with hemostasis being considered as 5 μL or less. Only animals with a pre-bleeding rate of 0.01 to 0.1 (g/min) were used for evaluation, and data from 7 to 9 animals per group were used.
After determining that bleeding had stopped, the liver was removed, and the liver and test substance were fixed with clips, and the adhesive strength (mN, 180° peel adhesive strength) was measured using a tension and compression tester (MCT-2150, A&D).
After 10 to 10.5 minutes of treatment with the test substance, the filter papers used to wipe away bleeding for 30 seconds were arranged in a holder. For the calibration curve, two filter papers each soaked in 8, 4, 2, 1, and 0 (blank) μL of blood were also arranged in the same holder, for a total of 10 pieces, and the pieces were allowed to dry overnight or longer.
After drying the filter paper, digital images were taken (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 so that the amount of blood could be quantified 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.

(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 amount of bleeding (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). The control group and TachoSil group had N = 7 and 8, respectively, and the other groups had N = 9.
1) In the rat liver hemorrhage model, the mean pre-bleeding rate immediately before the treatment with the test substance was 0.04 to 0.05 g/min in 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 to the bleeding surface by applying pressure with a cotton ball, but the test substances did not stick to the cotton ball.
The amount of bleeding, an index of hemostatic activity, was 0.61 g/10 min in the control group. In contrast, all groups treated with the test substance had bleeding 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 amount of bleeding in both groups was lower than that in the control group (see [9A] in Figure 9).
Regarding adhesive strength, an index of adhesiveness, the Beriplast group and TachoSil group had values of 30 and 52 mN, respectively, while the Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet groups had values of 52 and 61 mN, respectively, with the Beriplast group tending to be lower 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% for the Beriplast and TachoSil groups, respectively, while it was 44.4% and 88.9% for the Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet groups, respectively. Compared to existing drugs, the hemostasis rate for Thr-fibrinogen gel powder was lower, but that for Thr-fibrinogen gel sheet was equal to or higher (see Table 7 below).

*'Fbg gel' in the table is an abbreviation for 'fibrinogen gel' in the text.

(4) Implications of Example 6 1) Fibrinogen dry gel can be crushed into powder, and by combining this with similarly crushed Thr powder, a bioglue can be made. This Thr-fibrinogen gel powder glue exhibited hemostatic and adhesive properties comparable to those of existing liquid and sheet fibrin glues.
2) A composition consisting of two dry gel layers, a fibrinogen gel layer and a thrombin layer, was compressed into a sheet to produce a biological glue. This Thr-fibrinogen gel sheet glue exhibited hemostatic, adhesive, and occlusive properties comparable to those of existing liquid and sheet fibrin glues.
3) In Examples 5 and 6, the adhesive and occlusive efficacy of the Thr-fibrinogen gel powder and Thr-fibrinogen gel sheet were reversed. In Examples 5 and 6, the liver wound areas were 0.5 ^cm2 and 0.8 ^cm2 , respectively, and the hemostasis assessment criteria (post-bleeding volume of 5 μL or less) were unchanged, making Example 6 a more severe model. Furthermore, the treatment amount of the test substance was increased or decreased depending on the wound area. However, with the powder in particular, uneven coverage 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, resulting in consistently high efficacy across tests.
4) In clinical practice, Thr-fibrinogen gel powder requires an applicator for treatment, just like Beriplast (liquid), but Thr-fibrinogen gel sheets do not require an applicator like Surgicel (sheets) or TachoSil (sheets), 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 and bioabsorbable synthetic polymers as supports in addition to human-derived components. Both of these would be 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, so it can be composed only of human-derived components and does not require any components that would be foreign bodies to humans.
6) The Thr-fibrinogen gel sheet adhesive shown here has a two-layer structure, but by adding natural or synthetic polymers to each layer or by adding a support layer containing natural or synthetic polymers, it can be made into a three-layer or more structure, thereby changing its physical properties. It is also possible to add a layer containing a component that promotes blood coagulation other than the Thr, FXIII, FXIIIa, and fibrinogen gel, or a physiologically active substance, to make it into a three-layer or more structure, thereby imparting different efficacy and effects.

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: 77 μL/well of a 20 mg/mL fibrinogen concentrate containing 12.5 mM calcium chloride was placed in a 96-well plate 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 Thr (Human alpha Throbmin, PROLITIX) 188 U/mL was layered on the frozen fibrinogen hydrogel prepared as described above, and the layer was 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, held for 25 hours Segment 2: Heat to 32°C at 0.03°C/min, held for 6 hours The resulting freeze-dried products were used as fibrinogen dry gel and Thr-fibrinogen dry gel, respectively, and stored at 4°C. They were sterilized by irradiation with 28.7 to 30.5 kGy of gamma rays before 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 the diluted thrombin solution was layered on top of the frozen fibrinogen solution and allowed to stand at room temperature for 24 hours to allow the fibrinogen and thrombin to react and form fibrin, forming Beriplast (hydrogel).
6) Preparation of TachoSil TachoSil (CSL Behring) was punched out using a 7 mm diameter belt punch, placed in a 96-well plate, and firmly pressed against the bottom of the well.

(2) Summary of Parameters The various parameters used in the preparation of the fibrinogen gel 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
The above is expressed as a molar ratio:
Fibrinogen:FXIII:Calcium chloride = 58.5:0.366:12500
is.

(3) Cell culture test (test method)
MSCs (human bone marrow-derived mesenchymal stem cells, Takara Bio) were used for cell culture tests, and GM2 medium (mesenchymal stem cell growth medium 2, Takara Bio) was used for culture. Accutase Soln (Accutase in DPBS without Ca, Mg, Nacalai Tesque) was used for cell detachment. Culture was performed in a standard incubator at 37°C with 5% _CO2 .
The cells were detached with Accutase Soln, suspended, and seeded at 2 x 10 ⁴ cells/well onto fibrinogen dry gel, Thr-fibrinogen dry gel, Beriplast (hydrogel), and Tachosil, and cultured.
The day of seeding 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.

(Test results)
The test results are shown in FIG.
Proliferation of MSCs 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 way in which the cells permeated differed: the cell suspension permeated into the fibrinogen dry gel and Thr-fibrinogen dry gel in a short time, whereas it took longer for the cell suspension to permeate into TachoSil.

(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) Among the dried products TachoSil, fibrinogen dry gel, and Thr-fibrinogen dry gel, MSC proliferation was confirmed only in fibrinogen dry gel and Thr-fibrinogen dry gel.
Fibrinogen dry gel, either 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 demonstrated MSC proliferation similar to Beriplast (hydrogel). Beriplast is a two-liquid hydrogel that requires adjustment of the fibrinogen and thrombin solutions before use, making it difficult to prepare a uniform fibrin hydrogel with consistent concentration. In other words, fibrinogen and thrombin solutions begin to solidify locally before being uniformly mixed. In this example, thrombin solution was layered on top of frozen fibrinogen solution and allowed to solidify at room temperature, resulting in a planarly uniform fibrin hydrogel (although not uniform in the vertical direction). On the other hand, since fibrinogen hydrogel reacts slowly, a three-dimensionally uniform hydrogel can be prepared relatively easily 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 fibrin hydrogels, has better storage stability than fibrin hydrogels or fibrinogen hydrogels. Furthermore, a fibrinogen dry gel is also superior in terms of permeability to culture media and cell suspensions. For these reasons, a fibrinogen dry gel is preferred when providing a scaffold material for cells that does not require preparation immediately before use.

The present invention relates to embodiments of fibrinogen gels and the like that are useful as biomaterials for hemostasis, tissue adhesion and closure, cell scaffolding, and the like, and are useful, for example, in the field of medicine.
This application is based on patent application No. 2024-021702 filed in Japan (filing date: February 16, 2024), the contents of which are incorporated in their entirety herein.

Claims (21)

Fibrinogen dry gel. The dry gel described in claim 1, which restores to a hydrogel when water is added. 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 of Aα to Bβ chains is one of the following:
(Aα polymer)/(Bβ monomer)>0.15
(γ dimer)/(Bβ monomer)>0.2
(Here, Aα represents the Aα chain constituting fibrinogen, Bβ represents the Bβ chain constituting fibrinogen, and γ represents the γ chain constituting fibrinogen.)
The dry gel according to claim 1 or 2, which satisfies the above. 3. The dry gel according to claim 1, wherein the dry gel contains transglutaminase and a calcium salt, and the molar ratio of fibrinogen:transglutaminase:calcium salt is 5-300:0.01-10:0.05x10 ³ -200x10 ³ . The dry gel of claim 4, wherein the transglutaminase is FXIII and/or FXIIIa, and the calcium salt is calcium chloride. The dry gel according to claim 1, wherein the dry gel is in the form of a composition containing a dry gel of fibrinogen. The dry gel of claim 6, wherein the composition further contains thrombin. The dry gel described in claim 6 or 7, wherein the composition is a sheet-like composition including a layer ("fibrinogen dry gel layer") whose main component is a fibrinogen dry gel. 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 constituted by the fibrinogen dry gel layer and said one or more layers. 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. A biomaterial containing fibrinogen gel. The biomaterial according to claim 11, which is used for hemostasis or tissue adhesion/closure. The biomaterial described in claim 11, which is for use in regenerative medicine. The biomaterial described in claim 11, which is a sustained-release material. The biomaterial according to claim 11, wherein the fibrinogen gel is a dry fibrinogen gel. The biomaterial according to any one of claims 11 to 15, wherein the fibrinogen dry gel is the dry gel described in claim 2. 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. The method for producing the composition described in claim 17, wherein the step of obtaining a fibrinogen gel further comprises a step of drying the fibrinogen gel to obtain a dry gel. The method for producing the composition described in claim 17, wherein in the step of obtaining a fibrinogen gel, a layer containing fibrinogen gel as a main component (a "fibrinogen gel layer") is constructed, and a sheet-like composition composed of the fibrinogen gel layer is obtained. The method for producing the composition described in claim 19 further comprises constructing, in addition to the fibrinogen gel layer, one or more "layers whose main component is another component" other than the fibrinogen gel layer. The method for producing the composition described in any one of claims 17 to 20, wherein the calcium salt is calcium chloride and the transglutaminase is FXIII and/or FXIIIa.