JP5292025B2 - Carboxylic acid-containing biological tissue adhesive and preparation method thereof - Google Patents

Description

  The present invention relates to a biological tissue adhesive containing fibrinogen and thrombin as active ingredients and a method for preparing the same. In detail, it is related with the biological tissue adhesive agent characterized by containing a fixed ratio of carboxylic acid with respect to the amount of fibrinogen, and its preparation method.

  Fibrinogen and thrombin are factors involved in blood coagulation and play an important role in hemostasis and wound healing. For example, in hemostasis, prothrombin, which is normally in an inactive state in blood, is activated to become thrombin triggered by tissue cell damage, which converts soluble forms of fibrinogen into insoluble forms of fibrin at the final stage of the coagulation cascade. Is achieved by converting to Thrombin and fibrinogen are used as active ingredients of hemostatic agents based on this principle.

  As such an example, there is a report on a biological tissue adhesive containing fibrinogen and factor XIII (see, for example, Patent Document 1). A freeze-dried product of freeze-dried fibrinogen or a plasma protein mixture containing a high proportion of fibrinogen, which is an active ingredient of a biological tissue adhesive obtained by the production method disclosed herein, is enhanced during a lysis operation with a lysis solution. It is described that it dissolves gradually only at a certain temperature.

  In general, it is necessary to use a high-concentration fibrinogen solution in order to sufficiently exert an adhesive action as a biological tissue adhesive. However, it is not easy to obtain a fibrinogen solution with a high concentration when fibrinogen freeze-dried product is dissolved with a small amount of dissolution solution because it takes time to completely dissolve the fibrinogen. If it takes a long time to dissolve, not only can it not be used for emergency use, but there is also a concern that it may adversely affect the patient.

  In order to solve such a problem, a method for producing a biological tissue adhesive containing an inhibitor for suppressing fibrinolytic activity, which contains fibrinogen and factor XIII in a specific ratio, and the biological tissue adhesive A technique for improving the stability by freezing at low temperature has been reported (see, for example, Patent Document 2). In addition, focusing on the solvent for increasing the solubility of fibrinogen (low salt concentration buffer solution), 6 to 10% (W / V) of a solvent obtained by adding various fibrinogen dissolution promoters (monosaccharides) to this. A method for obtaining a fibrinogen solution and a method for cryopreserving the fibrinogen solution have been reported (for example, see Patent Document 3). Although the convenience of using these frozen preparations is improved compared to freeze-dried preparations, there are still problems such as the time required for thawing during use and the need for special equipment for storage in the frozen state. Was included.

  In response to such a problem, a fibrinogen solution that can be stored in a solution state at a low temperature has been developed. This fibrinogen solution contains a substance having a guanidino group such as arginine that can suppress gelation at low temperatures (see, for example, Patent Document 4). However, the addition of this substance causes new problems such as a delay in coagulation time.

Japanese Examined Patent Publication No. 63-40546 Japanese Examined Patent Publication No. 63-40547 JP-A-57-149229 JP-A-7-173073

As described above, conventional hemostats and wound healing agents have been devised, but there is still room for improvement in coagulation time, storage method, and the like.
Accordingly, an object of the present invention is to provide a highly convenient biological tissue adhesive containing fibrinogen and thrombin as active ingredients and a method for producing the same.

As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that when a biological tissue adhesive is prepared by mixing a fibrinogen solution and a thrombin solution, a certain percentage of the amount of carbon is present relative to the amount of fibrinogen. It was discovered that by containing an acid, the coagulation time can be shortened regardless of the coexistence of a coagulation time delay substance such as arginine, and the method for preparing the biological tissue adhesive can also be provided for a sheet formulation. The headline and the present invention have been completed. Therefore, the present invention is as follows.
[1] A biological tissue adhesive containing fibrinogen and thrombin as active ingredients, containing 0.05 mmol or more of carboxylic acid or a salt thereof with respect to 40 mg of fibrinogen.
[2] The biological tissue adhesive according to [1], comprising carboxylic acid or a salt thereof in a proportion of 0.05 to 0.5 mmol with respect to 40 mg of fibrinogen.
[3] The biological tissue adhesive according to [1] or [2], wherein the carboxylic acid is a carboxylic acid having 2 to 6 carbon atoms.
[4] The carboxylic acid is one or a combination of two or more selected from the group consisting of acetic acid, glutamic acid, citric acid, tartaric acid, aspartic acid, malic acid, and succinic acid, [3] The biological tissue adhesive described.
[5] The biological tissue adhesive according to any one of [1] to [4], wherein the biological tissue adhesive is applied to a bioabsorbable sheet.
[6] A biological tissue adhesive comprising a fibrinogen composition and a thrombin composition, containing carboxylic acid or a salt thereof in a proportion of 0.05 mmol or more per 40 mg of fibrinogen.
[7] The biological tissue adhesive according to [6], comprising carboxylic acid or a salt thereof in a proportion of 0.05 to 0.5 mmol with respect to 40 mg of fibrinogen.
[8] The biological tissue adhesive according to [6] or [7], wherein the thrombin composition contains a carboxylic acid or a salt thereof.
[9] The fibrinogen composition contains 0.1-2 mmol of arginine with respect to 60-100 mg of fibrinogen, and the thrombin composition contains 0.075-1.25 mmol of carboxylic acid or a salt thereof with respect to 30-10000 units of thrombin. The biological tissue adhesive according to [6] or [7], which is contained.
[10] The fibrinogen composition contains 0.1 to 0.5 mmol of arginine with respect to 80 mg of fibrinogen, and the thrombin composition contains carboxylic acid or a salt thereof with a ratio of 0.1 to 1 mmol with respect to 30 to 1500 units of thrombin. The biological tissue adhesive according to [6] or [7], wherein
[11] The fibrinogen composition contains arginine in a proportion of 0.5 mmol with respect to 80 mg of fibrinogen, and the thrombin composition contains carboxylic acid or a salt thereof in a proportion of 0.1 to 1 mmol with respect to 30 to 1500 units of thrombin. The biological tissue adhesive according to [6] or [7], characterized in that it is characterized.
[12] The biological tissue adhesive according to any one of [6] to [11], wherein the carboxylic acid is a carboxylic acid having 2 to 6 carbon atoms.
[13] The carboxylic acid is one or a combination of two or more selected from the group consisting of acetic acid, glutamic acid, citric acid, tartaric acid, aspartic acid, malic acid, and succinic acid, [12] The biological tissue adhesive described.
[14] The biological tissue adhesive according to any one of [6] to [13], wherein at least one of a fibrinogen composition or a thrombin composition is applied to a bioabsorbable sheet.
[15] A method for producing a biological tissue adhesive, comprising mixing the fibrinogen composition according to any one of [6] to [14] and a thrombin composition.

According to the present invention, a biological tissue adhesive containing fibrinogen and thrombin as active ingredients and a method for preparing the same are provided. The biological tissue adhesive of the present invention includes, for example, carboxylic acid or a salt thereof in a proportion of 0.05 to 0.5 mmol with respect to 40 mg of fibrinogen. With this configuration, when fibrinogen and thrombin are mixed, the coagulation time is shortened and the adhesive force is increased, so that the hemostatic effect and the tissue closing effect can be enhanced. Furthermore, since the liquid mixture of fibrinogen and thrombin coagulates instantaneously, dripping is prevented and a highly convenient biological tissue adhesive can be provided.

The present invention relates to a method for preparing a biological tissue adhesive containing fibrinogen and thrombin as active ingredients in the presence of a certain proportion of carboxylic acid or a salt thereof relative to the amount of fibrinogen, and biological tissue adhesion obtained by the method Characterized by the agent.

  The thrombin component used in the present invention is generally thrombin commercially available as a reagent (for example, Thrombin from human Plasma (product number T6884 manufactured by Sigma-Aldrich), etc.), low-temperature ethanol fractionation, chromatography operation, etc. Biologically-derived thrombin (for example, JP Thrombin (Chemistry and Serum Therapy Research Institute)) or recombinant thrombin obtained by genetic recombination technology may be used. When a gene recombination technique is used, a thrombin gene obtained from an animal subject to hemostasis is preferably used. In the present invention, JP Thrombin was used.

  On the other hand, the fibrinogen component is, for example, a method combining a cold ethanol precipitation method and a fibrinogen solubility lowering effect by glycine (Blomback, B. and Blomback, M., Arkiv Kemi, 10, p.415-443, 1956), glycine Can be prepared by the glycine precipitation method (Kazal, LA, et al., Proc. Soc. Exp. Biol. Med., 113, p.989-994, 1963) and the gene recombination technique used alone, What was produced by any method may be used. In the present invention, a fibrinogen lyophilized powder contained in a kit of a commercially available biological tissue adhesive (product name: Bolhir, Institute for Chemical and Serum Therapy) was used.

  A biological tissue adhesive containing fibrinogen and thrombin as active ingredients is actually prepared by mixing a fibrinogen composition containing fibrinogen as main components and a thrombin composition containing thrombin as main components. As a usage form, it may be directly applied or sprayed so that the two compositions are mixed in a place requiring hemostasis, or may be used in the form of a bioabsorbable sheet. When used as a bioabsorbable sheet, a method in which one or both of thrombin and fibrinogen is applied to, for example, a bioabsorbable sheet made of polyglycolic acid, and both are combined immediately before use or at the time of use is used. It is done.

  Fibrinogen solution is preferably used at a high concentration in order to obtain a strong adhesive force. However, since fibrinogen has a large molecular weight and takes time to completely dissolve, generally it is 6-10% (W / V), Preferably, it is prepared and used so as to be an 8% (W / V) fibrinogen solution. In order to suppress gelation of liquid fibrinogen during low-temperature storage, 0.1 to 2 M arginine may be added to the fibrinogen composition (Patent Document 4). In addition, blood coagulation factor XIII (Curtis, CG., Lorand, L., Methods Enzymol., 45, p.177-191, 1976) is added for the purpose of cross-linking and fibrinolysis in fibrin gel matrix. And aprotinin may be added. For dissolving fibrinogen lyophilized powder, water for injection, aprotinin solution and the like are used.

  The thrombin solution is generally used in a concentration range that exerts a hemostatic action, for example, a thrombin concentration of 30 to 10,000 units / mL, preferably 30 to 1500 units / mL. The thrombin composition includes proteins (eg, albumin), amino acids (eg, arginine, histidine, lysine, glutamic acid, glycine and aspartic acid), oligosaccharides (eg, sucrose, trehalose, sorbitol, Stabilizers such as erythritol and mannitol), surfactants (eg polysorbate 80) and polyhydric alcohols (eg glycerol, propylene glycol, butylene glycol and sorbitol) may be added. As a diluting solution of the thrombin composition, a citrate buffer solution, a phosphate buffer solution or a Tris buffer solution having a concentration of 0.01 to 0.05 M and pH 6.0 to 8.0 is generally used. Moreover, when using with said bioabsorbable sheet | seat form, the permeability to the sheet | seat of a thrombin or a fibrinogen component can be improved by containing a penetrating agent (for example, Tween 80).

  In the present invention, a certain proportion of carboxylic acid or a salt thereof is further added to the amount of fibrinogen. Preferably, it is prepared so that 0.05 to 0.5 mmol of carboxylic acid or a salt thereof may coexist with 40 mg of fibrinogen. The carboxylic acid is preferably a carboxylic acid having 2 to 6 carbon atoms. More preferably, the carboxylic acid used in the present invention is selected from the group consisting of acetic acid, glutamic acid, citric acid, tartaric acid, aspartic acid, malic acid and succinic acid. These can be used alone or in combination of two or more. These carboxylic acids may be contained in either the thrombin composition or the fibrinogen composition, but are preferably contained in the thrombin composition.

  Therefore, the tissue bioadhesive of the present invention comprises a fibrinogen composition containing 0.1 to 2.0 mmol of arginine in a ratio of 60 to 100 mg of fibrinogen and a carvone of 0.075 to 1.250 mmol in a ratio of 30 to 10,000 units of thrombin. It is prepared by mixing a thrombin composition containing an acid or a salt thereof. Preferably, a fibrinogen composition containing arginine at a ratio of 0.5 mmol to 80 mg of fibrinogen and a thrombin composition containing a carboxylic acid or a salt thereof at a ratio of 0.1 to 1.0 mmol to thrombin 30 to 1500 units are used. Is done. The present invention includes the tissue bioadhesive thus prepared.

The effect of including the carboxylic acid or its salt in the biological tissue adhesive of the present invention is evaluated by the coagulation time and adhesive force from mixing the thrombin composition and fibrinogen composition to coagulation. More specifically, the clotting time is defined as the time from immediately after dropping the fibrinogen composition to the 96-well U-bottom plate containing the thrombin composition until the mixture of both compositions solidifies. The adhesive force is expressed as the maximum tensile strength measured by pulling one skin vertically after a mixture of thrombin composition and fibrinogen composition is sandwiched between two pig skins and pressed for a certain period of time. Alternatively, the specimen is placed between two pig skins, and after pressing for a certain time, the maximum tensile strength measured by pulling one skin horizontally is displayed.
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

《Experimental example》
<Influence of arginine on coagulation time of biological tissue adhesive>
The coagulation time when the fibrinogen composition added with arginine and the thrombin composition were mixed in order to suppress gelation of the liquid fibrinogen during low-temperature storage was measured by the same method as in Example 1 described later. As the fibrinogen composition, the 8% (w / v) fibrinogen solution prepared in Example 1 and the same fibrinogen solution added with 0.1 M, 0.3 M and 0.5 M arginine were used, and the thrombin composition was the same as in Example 1. A thrombin solution having an activity of 50 units / mL prepared by the above method was used. As a result, the delay of coagulation time was recognized by the addition of 0.1 M or more arginine (FIG. 1).

<Influence of carboxylic acid on coagulation time of biological tissue adhesive>
The fibrinogen composition is prepared by dissolving a fibrinogen lyophilized powder contained in a kit of a commercially available biological tissue adhesive (product name: Bolhir, Institute of Chemical and Serum Therapy) with water for injection so that the fibrinogen concentration becomes 8%. Prepared. The fibrinogen composition contains 0.02M sodium citrate.
The thrombin composition consists of a commercially available thrombin solution (product name: JP Thrombin Foundation for Chemistry and Serum Therapy), 1% (W / V) human serum albumin, 0.049 M sodium citrate, 0.001 M citric acid, 0.1 Thrombin concentration in solutions of various concentrations of carboxylate in thrombin solution containing% (W / V) polysorbate 80 (Nippon Oil), 1% glycerol (Nacalai Tesque) and 0.028M mannitol (Wako Pure Chemical Industries) It was prepared by diluting to 30 units / mL. The thrombin composition includes 0.00012M sodium citrate from thrombin lyophilized powder, 0.049M sodium citrate and 0.001M citric acid from thrombin lysate, and a newly added concentration of carboxylic acid. As a control, the same thrombin composition to which no carboxylic acid was added was used.
A 96-well U-bottom plate was filled with 30 μl of the thrombin composition, and an equal amount of the fibrinogen composition was dropped into the 96-well U-bottom plate. This stirring was repeated, and the time until the clot generated from the start of dropping dropped on the microchip and lifted was measured as the coagulation time. As a result, the addition of carboxylic acid shortened the coagulation time, and the shortening time was proportional to the concentration of carboxylic acid (Table 1). The symbol AF in the left column of Table 1 is the type of carboxylic acid (A: sodium acetate, B: sodium glutamate, C: potassium sodium tartrate, D: sodium aspartate, E: disodium malate, F: monosodium succinate) The top row of the table shows the final concentration after mixing the two compositions of each carboxylic acid newly added, the values in the table are the solidification time (seconds) measured 3 times (control is measured 6 times),-is not Show the measurement. All the groups to which carboxylic acid was added were significantly different from those without carboxylic acid by the Mann-Whitney U test (p <0.05).

<Effect of carboxylic acid on adhesive strength of biological tissue adhesive>
200 μl of the thrombin composition obtained in Example 1 and 200 μl of the fibrinogen composition were quickly mixed and placed between the porcine skins (adhesion area 6.25 cm ² ) attached to two inspection jigs, and after 5 minutes of pressure bonding Immediately, one inspection jig was pulled vertically to measure the maximum tensile strength (Newton: N), and the value was displayed as the fibrin gel adhesive strength. As a result, no decrease in adhesive strength due to the addition of carboxylic acid was observed (Table 2). The symbol AC in the left column of Table 2 is the type of carboxylic acid (A: sodium glutamate, B: potassium sodium tartrate, C: disodium malate), and the top row in the table shows the two compositions of each newly added carboxylic acid. The final concentration after mixing, the numerical values in the table are the maximum tensile strengths (N) measured twice (control is measured five times),-indicates unmeasured.

<Influence of carboxylic acid on coagulation time of biological tissue adhesive in the presence of arginine>
(1) The arginine-containing fibrinogen composition was prepared by adding 0.5% fibrinogen lyophilized powder contained in a kit of a commercially available biological tissue adhesive (product name: Bolhir, Foundation for Chemical and Serum Therapy) to a fibrinogen concentration of 8%. Prepared by dissolving in M arginine solution. The fibrinogen composition contains 0.02M sodium citrate.
The thrombin composition was prepared in the same manner as in Example 1 except that the thrombin concentration was 1500 units / mL. The thrombin composition thus includes 0.006M sodium citrate from thrombin lyophilized powder, 0.049M sodium citrate and 0.001M citric acid from thrombin lysate, and a newly added concentration of carboxylic acid.
The coagulation time was measured by the same method as in Example 1. As a result, the effect of shortening the coagulation time by addition of carboxylic acid was recognized even in the presence of arginine (Table 3). The pH of the biological tissue adhesive was 6.49-6.78 (control was 6.75). The symbol AG in the left column of Table 3 indicates the type of carboxylic acid (A: sodium acetate, B: sodium glutamate, C: sodium citrate, D: potassium sodium tartrate, E: sodium aspartate, F: disodium malate, G : Monosodium succinate), the top row in the table shows the final concentration of each carboxylic acid added after mixing the two compositions, and the values in the table are measured 3 times (control is measured 6 times). (Unit),-indicates unmeasured. All the groups to which carboxylic acid was added were significantly different from those without carboxylic acid by the Mann-Whitney U test (p <0.05).

  (2) Tables 4 and 5 show the results of measuring the coagulation time when two types of carboxylic acids, sodium acetate and sodium glutamate, and sodium citrate and potassium sodium tartrate, were added, respectively. Coagulation time was measured by the same method as in Example 3- (1) except that two carboxylic acids were added when preparing the thrombin composition. As a result, even when two carboxylic acids were added in combination, shortening of the coagulation time was recognized as in the case of adding one carboxylic acid. The pH of the biological tissue adhesive was 6.56-6.75 (control was 6.75). The upper two rows and the left two columns in Tables 4 and 5 are the final concentrations after mixing the two compositions of each newly added carboxylic acid, and the values in the table are solidification times measured three times (control is measured six times) (In seconds), Na is sodium,-indicates unmeasured. All the groups to which carboxylic acid was added were significantly different from those without carboxylic acid by the Mann-Whitney U test (p <0.05).

<Influence of carboxylic acid on adhesive strength of biological tissue adhesive in the presence of arginine>
The thrombin composition obtained in Example 3- (1) was filled in polyglycolic acid felt (hereinafter referred to as “PGA sheet”) having a size of 2.0 × 2.5 cm at 50 μL / cm ² and freeze-dried to fix thrombin. A PGA sheet was prepared. A sheet of 250 μl of the fibrinogen composition obtained in Example 3- (1) was dropped onto this sheet and placed between the ^two pig skins (adhesion area 2.5 cm ² ) attached to the two inspection jigs and pressed for 30 seconds. Thereafter, one of the inspection jigs was immediately pulled in the horizontal direction to measure the maximum tensile strength (N), and the value was displayed as the fibrin gel adhesion. As a result, it was confirmed that the adhesive strength by addition of carboxylic acid was significantly higher than that without addition of carboxylic acid (Table 6). The symbol AF in the left column of Table 6 indicates the type of carboxylic acid (A: sodium glutamate, B: sodium citrate, C: potassium sodium tartrate, D: sodium aspartate, E: disodium malate, F: monosodium succinate. ), The top row of the table shows the final concentration of each newly added carboxylic acid when the fibrinogen composition was infiltrated into the thrombin-immobilized PGA sheet, and the values in the table were measured 3 times (control was measured 8 times). Maximum tensile strength (N),-indicates unmeasured. All the groups to which carboxylic acid was added were significantly different from those without carboxylic acid by the Mann-Whitney U test (p <0.05).

<Effect of existing carboxylic acid on coagulation time of biological tissue adhesive in the presence of arginine>
The fibrinogen composition obtained in Example 3- (1) was dialyzed against a 0.5 M arginine solution with a dialysis membrane having a molecular weight cut off of 12000-14000 to prepare a fibrinogen composition containing no carboxylic acid.
The thrombin composition is a thrombin concentration of 1500 units / mL with a commercially available thrombin solution (product name: JP Thrombin Foundation for Chemistry and Serum Therapy) with various concentrations of carboxylate added to water for injection. It was prepared by diluting. The thrombin composition includes 0.006M sodium citrate derived from thrombin lyophilized powder and a newly added concentration of carboxylic acid. As a control, the same thrombin composition to which no carboxylic acid was added was used.
Using the above thrombin composition and fibrinogen composition, the coagulation time of the biological tissue adhesive was measured by the same method as in Example 1. As a result, shortening of the coagulation time was recognized by containing 0.053 M or more of carboxylic acid in the biological tissue adhesive of the present invention (Table 7). That is, regardless of the presence of the existing carboxylic acid calculated from Examples 1 and 3 (final concentration 0.035-0.038M), a new carboxylic acid is added at a final concentration of 0.05M or more after mixing the two compositions. It was shown that the coagulation time was shortened.
The symbol AF in the left column of Table 7 indicates the type of carboxylic acid (A: sodium glutamate, B: sodium citrate, C: potassium sodium tartrate, D: sodium aspartate, E: disodium malate, F: monosodium succinate. ), The top row of the table is the final concentration after mixing the two compositions of each carboxylic acid newly added, the values in the table are the solidification time (unit: seconds) measured three times (control measured six times),- Indicates unmeasured. All the groups to which carboxylic acid was added were significantly different from those without carboxylic acid by the Mann-Whitney U test (p <0.05).

The preparation method of the biological tissue adhesive of the present invention provides a highly convenient hemostatic agent because it forms rapid and strong fibrin.

FIG. 1 is a drawing showing the results of measuring the coagulation time when an arginine-containing fibrinogen composition and a thrombin composition are mixed.

Claims (12)

A biological tissue adhesive characterized by mixing a fibrinogen composition and a thrombin composition at the time of use , characterized by the following (1) and (2) .
(1) The thrombin composition contains carboxylic acid or a salt thereof consisting of one or a combination of two or more selected from the group consisting of acetic acid, glutamic acid, citric acid, tartaric acid, aspartic acid, malic acid and succinic acid. ,as well as
(2) With respect to 40 mg of fibrinogen, the content of the carboxylic acid or the salt thereof of (1) is a ratio of 0.05 to 0.5 mmol. The biological tissue adhesive according to claim 1, wherein the fibrinogen composition contains arginine. The fibrinogen composition contains arginine in the percentage of 0.1~2mmol against fibrinogen 60 to 100 mg, the carboxylic proportion of 0.075~1.25mmol the thrombin composition against thrombin 30-10000 units The biological tissue adhesive according to claim 1 or 2 , comprising an acid or a salt thereof. The fibrinogen composition contains arginine in the percentage of 0.1~0.5mmol against fibrinogen 80 mg, the carboxylic acid fraction of 0.1~1mmol the thrombin composition against thrombin 30-1500 units or The biological tissue adhesive according to claim 1 or 2 , comprising the salt thereof. The fibrinogen composition contains arginine in the percentage of 0.5mmol against fibrinogen 80 mg, containing said carboxylic acid or salt thereof in the proportion of 0.1~1mmol the thrombin composition against thrombin 30-1500 Units The biological tissue adhesive according to claim 1 or 2, wherein At least one is characterized in that applied to the bioabsorbable sheet, a biological tissue adhesive according to any one of claims 1 to 5 of the fibrinogen composition or the thrombin composition. The manufacturing method of the biological tissue adhesive which consists of a fibrinogen composition and a thrombin composition including the process of following (1) to (3) .
(1) a step of preparing a fibrinogen composition;
(2) A thrombin composition containing a carboxylic acid or a salt thereof consisting of one or a combination of two or more selected from the group consisting of acetic acid, glutamic acid, citric acid, tartaric acid, aspartic acid, malic acid and succinic acid is prepared. The process of
(3) The fibrinogen composition of (1) and the thrombin composition of (2) so that the content of carboxylic acid or a salt thereof is 0.05 to 0.5 mmol with respect to 40 mg of fibrinogen when used. Mixing or blending products, The production method according to claim 7, wherein the fibrinogen composition (1) contains arginine. The fibrinogen composition contains 0.1 to 2 mmol of arginine with respect to 60-100 mg of fibrinogen, and the thrombin composition has a ratio of 0.075 to 1.25 mmol with respect to 30 to 10,000 units of thrombin. The method according to claim 7 or 8, comprising an acid or a salt thereof. The fibrinogen composition contains 0.1 to 0.5 mmol of arginine with respect to 80 mg of fibrinogen, and the thrombin composition has a ratio of 0.1 to 1 mmol with respect to 30 to 1500 units of thrombin or The production method according to claim 7 or 8, wherein the salt is contained. The fibrinogen composition contains arginine at a ratio of 0.5 mmol with respect to 80 mg of fibrinogen, and the thrombin composition contains the carboxylic acid or a salt thereof at a ratio of 0.1 to 1 mmol with respect to 30 to 1500 units of thrombin. The manufacturing method according to claim 7 or 8, characterized in that:   The method according to any one of claims 7 to 11, wherein at least one of the fibrinogen composition or the thrombin composition is applied to a bioabsorbable sheet.

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