Porous sheets, tissue adhesive films, their use as hemostatic or anti-adhesion materials, and methods for manufacturing these.

Abstract

Provided is a porous sheet having high biocompatibility and adhesive strength, and further having pliability and / or flexibility.　The porous sheet includes: a second gelatin having a hydrocarbon group introduced into a first gelatin; and an unreacted crosslinking agent dispersed in the second gelatin. The second gelatin includes a structure represented by formula (1).

Description

Technical Field

[0001] The present invention relates to a porous sheet, a tissue adhesion film, use thereof as a hemostatic agent or an anti-adhesion material, and a method for producing the same.

Background Art

[0002] A tissue adhesion film is a polymer film capable of adhering to living tissues such as blood vessels and skin. Thereby, for example, during surgery such as cardiovascular surgery, blood leakage and the like can be prevented, and the safety of the surgery can be enhanced. In this regard, the present inventors have reported a non-porous tissue adhesion film formed by accumulating gelatin and / or a hydrophobized gelatin derivative and cross-linking molecules (Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the above technical level, the present invention provides a porous sheet applicable to a tissue adhesion film having high biocompatibility and adhesion strength.

Means for Solving the Problems

[0005] As a result of intensive studies to achieve the above problems, the present inventors have found that the above problems can be achieved by the following configuration.

[0006] [1] A hydrophobized gelatin derivative in which a hydrocarbon group is introduced into gelatin, represented by the formula (1):

Chemical Formula

[10] The porous sheet according to any one of [1] to [9], wherein the gelatin is gelatin derived from cold-water fish.

[11] The porous sheet according to any one of [1] to

[10] , wherein the crosslinking agent is a compound having at least two crosslinkable groups.

[12] The porous sheet according to

[11] , wherein the amount of crosslinking groups in the crosslinking agent is 0.25 to 2 equivalents per equivalent of amino groups in the hydrophobic gelatin derivative.

[13] A porous sheet as described in any of [1] to

[12] , with a thickness of 1 mm or more.

[14] A porous sheet as described in any of [1] to

[13] , for use as a tissue bonding material.

[15] A porous sheet as described in any of [1] to

[13] , for use as a hemostatic agent, sealant (occlusive agent), or covering material.

[16] Use of any of the porous sheets described in [1] to

[13] for preparing tissue bonding materials.

[17] Use of any of the porous sheets described in [1] to

[13] for preparing hemostatic agents, sealants (occlusive materials), or coverings.

[18] A method of covering tissue, which involves covering tissue that needs to be covered (for example, anastomoses of nerves and tendons, wounds in tissues (pulmonary pleura, gastrointestinal anastomoses, oral and periodontal tissues, etc.)) with a porous sheet as described in any of [1] to

[13] .

[19] A method for tissue hemostasis, comprising adhering a porous sheet described in any of [1] to

[13] to tissue where hemostasis is necessary or expected.

[20] A method for filling gaps, such as filling gaps between dura maters, dural sutures, or gaps between dura mater-forming material and dura mater, using a porous sheet as described in any of [1] to

[13] . [twenty one] A tissue adhesive film comprising a porous sheet as described in any of [1] to

[13] . [twenty two] A hemostatic agent, sealant, or covering material comprising a porous sheet as described in any of [1] to

[13] .

[23] At least one sheet (I) which is a porous sheet according to any one of [1] to

[13] , and a sheet (II) containing a crosslinked product of unhydrolyzed gelatin, and a tissue adhesion film, wherein the at least one sheet (I) is laminated on one surface of the sheet (II).

[24] At least one sheet (I) which is a porous sheet according to any one of [1] to

[13] , and a sheet (II) containing a crosslinked product of unhydrolyzed gelatin, and a laminate, wherein the at least one sheet (I) is laminated on one surface of the sheet (II).

[25] The laminate according to

[24] , for use as a tissue adhesion material.

[26] The density of the sheet (I) is 0.07 g / cm 3 or more, the tissue adhesion film according to

[22] , or the laminate according to

[24] or

[25] .

[27] The crosslinked product of unhydrolyzed gelatin is a thermally crosslinked product of unhydrolyzed gelatin, the tissue adhesion film or laminate according to any one of

[23] to

[26] .

[28] The sheet (II) is porous, the tissue adhesion film or laminate according to any one of

[23] to

[27] .

[29] The density of the sheet (I) is 0.07 g / cm 3 to 0.2 g / cm 3 and the tissue adhesion film or laminate according to any one of

[23] to

[28] .

[30] The sheet (II) further contains a basic compound that exhibits basicity when in contact with moisture, the tissue adhesion film or laminate according to any one of

[23] to

[29] .

[31] The basic compound is a compound whose aqueous solution at a concentration of 0.05 mol / L to 0.5 mol / L has a pH of 7.5 to 10, as described in

[30] , for a tissue adhesion film or laminate.

[32] The tissue adhesion film or laminate according to

[30] or

[31] , wherein the basic compound is a pH buffering agent.

[33] The tissue adhesion film or laminate according to

[32] , wherein the pH buffer is a mixture of phosphoric acid and sodium phosphate.

[34] The aforementioned at least one sheet (I) includes two sheets (I), A tissue adhesive film or laminate according to any one of

[23] to

[33] , wherein one of the two sheets (I) is laminated on one surface of sheet (II) and the other is laminated on the other surface of sheet (II).

[35] A hemostatic agent, sealant, or covering material comprising a tissue adhesive film or laminate as described in any of

[23] to

[34] .

[36] An adhesion prevention material comprising a tissue adhesive film or laminate as described in any of

[23] to

[34] .

[37] A tissue adhesive film or laminate according to any one of

[23] to

[34] for use as a hemostatic agent, sealant (occluding agent), or tissue covering material, or for hemostasis, for filling gaps between dura maters, dural sutures, or gaps between dura mater-forming material and dura mater, or for covering tissue.

[38] A tissue adhesive film or laminate according to any of

[22] to

[34] , for use as a material to prevent adhesion to tissue, or for preventing adhesion to tissue.

[39] Use of any of the tissue adhesive films or laminates described in

[23] to

[34] for preparing hemostatic agents, sealants, or tissue coverings.

[40] Use of any of the tissue adhesion films or laminates described in

[23] to

[34] for preparing a tissue adhesion prevention material.

[41] A method for covering tissue, comprising covering tissue that needs to be covered with a tissue adhesive film or laminate described in any of

[23] to

[34] .

[42] A method for filling gaps, such as gaps between dura maters, dura mater sutures, or gaps between dura mater-forming material and dura mater, using a tissue adhesive film or laminate described in any of

[23] to

[34] .

[43] A method for tissue hemostasis, comprising adhering a tissue adhesive film or laminate described in any of

[23] to

[34] to tissue where hemostasis is necessary or expected.

[44] A method for preventing tissue adhesion, comprising adhering a tissue adhesive film or laminate described in any of

[23] to

[34] to tissue that needs to be prevented from adhering to other tissues.

[45] A method for manufacturing a porous sheet as described in any of [1] to

[13] , The hydrophobic gelatin derivative, the crosslinking agent, the acid, and the solvent are mixed to prepare a raw material solution (I) with a pH of 5 or less. The porous sheet is obtained by freeze-drying the raw material liquid (I). A method for manufacturing a porous sheet containing the following.

[46] The method according to

[45] , wherein the concentration of the hydrophobized gelatin derivative in the raw material liquid (I) is 1 w / v% to 20 w / v%.

[47] A method for producing a tissue adhesive film, comprising the step of producing a porous sheet by the method described in

[45] or

[46] .

[48] A porous sheet (I) is manufactured by the method described in

[45] or

[46] , To produce a sheet (II) containing a crosslinked product of unhydrophobized gelatin, Laminating sheet (I) and sheet (II), A method for producing a tissue adhesion film, including the following.

[49] The aforementioned sheet (II) To prepare a raw material solution (II) containing the aforementioned unhydrophobized gelatin, a basic compound, and a solvent, The aforementioned raw material liquid (II) is freeze-dried into a sheet to form a precursor sheet, The precursor sheet is heated to thermally crosslink the unhydrophobic gelatin. The method described in

[48] , which is manufactured by

[48] .

[50] The method according to

[49] , wherein the basic compound is a pH buffer.

[51] The method according to

[50] , wherein the pH buffer is a mixture of phosphoric acid and sodium phosphate. [Brief explanation of the drawing]

[0007] [Figure 1A] This is a schematic cross-sectional view of the porous sheet according to the first embodiment. [Figure 1B] The photograph on the left shows the porous sheet before it is attached to biological tissue, and the photograph on the right shows the porous sheet after it has been attached to biological tissue. The schematic diagram on the left shows the hydrophobic gelatin derivative and crosslinking agent in an unreacted state in the porous sheet before it is attached to biological tissue, and the schematic diagram on the right shows an example of the crosslinking reaction between the hydrophobic gelatin derivative and the crosslinking agent in the porous sheet after it has been attached to biological tissue. [Figure 2] This is a flowchart showing the method for manufacturing a porous sheet according to the first embodiment. [Figure 3] This is a schematic cross-sectional view of the laminate according to the second embodiment. [Figure 4] The images show a photograph (left) of the porous sheet (top view) prepared in the example, and a scanning electron microscope (SEM) image (right). [Figure 5A] This diagram illustrates the pressure resistance strength test method in the example. [Figure 5B] This figure shows the results of pressure resistance test 1 in the example. [Figure 6] This is a photograph of the laminate fabricated in the example. [Figure 7]This is a flowchart showing the method for manufacturing the laminate according to the second embodiment. [Figure 8] This is a schematic cross-sectional view of a laminated body of a modified example 1 of the second embodiment, illustrating how the laminated body is attached to biological tissue. [Figure 9] This is a schematic cross-sectional view of a laminated body of modified example 2 of the second embodiment. [Modes for carrying out the invention]

[0008] The present invention will be described in detail below. The following description of the constituent elements may be based on typical embodiments of the present invention, but the present invention is not limited to such embodiments.

[0009] [Definition of Terms] In this specification, a numerical range represented by "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0010] "Gelatin" generally refers to a polymer whose triple helix structure has been unraveled and denatured by treating natural or synthetic collagen with heat, acid, or alkali. In this specification, when simply referred to as "gelatin," it means "gelatin" that has not undergone "hydrophobization" treatment and has not had alkyl groups introduced. For this reason, "gelatin" may be expressed as "ApGltn" or "GltnNH2" in this specification. Depending on the context, the terms "raw gelatin" or "unhydrophobized gelatin" may be used, but these terms are synonymous with "gelatin." "Raw gelatin" may be expressed as "Org-ApGltn" or "Org," and "unhydrophobized gelatin" may be expressed as "ApGltn." In this specification, "hydrophobization" refers to a process that increases the hydrophobicity of gelatin by introducing hydrocarbon groups into gelatin, more specifically, by bonding hydrocarbon groups to amino groups via linking groups or directly. Therefore, in this specification, "hydrophobized gelatin derivative" refers to a derivative of gelatin in which the hydrophobicity of gelatin has been increased by introducing hydrocarbon groups into gelatin, more specifically, by bonding hydrocarbon groups to amino groups via linking groups or directly, and means "GltnNH-L-CHR 1 R 2 It is sometimes expressed as "

[0011] [First Embodiment] <Porous Sheet> In this embodiment, the porous sheet 10 shown in Figures 1A and 1B will be described. The porous sheet 10 contains a hydrophobic gelatin derivative (second gelatin) in which hydrocarbon groups are introduced into raw material gelatin (first gelatin), and a crosslinking agent dispersed in the hydrophobic gelatin derivative.

[0012] As shown in the schematic diagram on the left side of Figure 1B, within the porous sheet 10, the crosslinking agent is dispersed in the hydrophobic gelatin derivative, and the crosslinking agent and the hydrophobic gelatin derivative are substantially unreacted. Moisture is required for the crosslinking reaction (curing reaction) between the hydrophobic gelatin derivative and the crosslinking agent, and the environment must be neutral to alkaline. Since biological tissue (for example, the pig aorta 20 shown in Figure 1B) usually contains neutral moisture, the porous sheet 10 absorbs neutral moisture when attached to the biological tissue 20, crosslinks, and adheres to the biological tissue 20. As shown in the schematic diagram on the right side of Figure 1B, the crosslinking reaction is typically a reaction between the primary amino group of the hydrophobic gelatin derivative and the crosslinkable group (typically an active ester group, etc.) of the crosslinking agent.

[0013] The porous sheet 10 maintains the structure of the raw material gelatin, thus exhibiting high biocompatibility. Furthermore, hydrophobic interactions occur between the porous sheet 10 and the biological tissue 20 due to the hydrophobic groups, and these hydrophobic groups penetrate the biological tissue 20 at a molecular level (anchoring), forming a physically strong bond between the two, allowing the porous sheet 10 to adhere strongly to the surface of the biological tissue 20. Thus, the porous sheet 10 adheres strongly to the biological tissue 20 simply by being applied. In addition, due to its porous structure, the porous sheet 10 has improved flexibility and / or pliability compared to non-porous membranes, and is expected to be able to be bent to follow the surface shape of the biological tissue 20, or be formed into a tube to cover anastomoses of nerves, tendons, etc. Furthermore, due to its porous structure, the porous sheet 10 is thought to efficiently absorb moisture when applied to the biological tissue 20, thereby promoting the curing reaction (crosslinking reaction) between the hydrophobic gelatin derivative and the crosslinking agent. The details of the porous sheet 10 are described below.

[0014] As described above, hydrophobic gelatin derivatives (second gelatin) refer to gelatin derivatives in which hydrocarbon groups have been introduced into raw material gelatin (first gelatin), and have a structure represented by the following formula (1).

[0015] [ka] In formula (1), Glatn represents a gelatin residue, L represents a single bond or a divalent linking group, and R 1 R is a hydrocarbon group having 1 to 20 carbon atoms. 2 This is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms.

[0016] In formula (1), GltnNH- is the structure derived from the raw material gelatin described above. Therefore, the raw material gelatin is represented as GltnNH2. Details of the raw material gelatin will be described later. -CHR 1 R 2This refers to a hydrocarbon group introduced into the raw material gelatin, which is introduced into the raw material gelatin via L (a single bond or a divalent linking group).

[0017] The divalent linking group of L is not particularly limited, but examples include -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -N(R)- (where R represents a hydrogen atom or a monovalent organic group (preferably a hydrocarbon group having 1 to 20 carbon atoms)), alkylene groups (preferably alkylene groups having 2 to 10 carbon atoms), alkenylene groups (preferably alkenylene groups having 2 to 10 carbon atoms), and combinations thereof, with -C(O)- being the most preferred. L is preferably a single bond or -C(O)-.

[0018] In equation (1), -CHR 1 R 2 The (hydrocarbon group) is preferably bonded to the ε-amino group of the raw material gelatin, and more preferably bonded to the ε-amino group of lysine (Lys) in the raw material gelatin. The amino group, preferably the amino group of lysine, is bonded to *-CHR with or without a linking group (in other words, directly). 1 R 2 Methods for bonding include, for example, the so-called reductive amination reaction (using aldehydes or ketones) and the Schotten-Baumann reaction (using acid chlorides).

[0019] The -NH- structure (secondary amino group) in formula (1) is, for example, 3300 cm⁻¹ in the FT-IR (Fourier transform infrared absorption) spectrum. -1 It can be detected by nearby bands.

[0020] In equation (1), R 1 and R 2 The hydrocarbon group having 1 to 20 carbon atoms is not particularly limited and includes, for example, chain hydrocarbon groups having 1 to 20 carbon atoms, alicyclic hydrocarbon groups having 3 to 20 carbon atoms, aromatic hydrocarbon groups having 6 to 14 carbon atoms, and groups combining these.

[0021] R 2 If R has 1 to 20 hydrocarbon groups, 2 R 1 It may be the same as or different from R. 1 , and R 2 The hydrocarbon group may be a straight chain or a branched chain.

[0022] The chain-like hydrocarbon group having 1 to 20 carbon atoms is not particularly limited, but examples include methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, octyl group (or capryl group), nonyl group (or pelargoryl group), decyl group, dodecyl group (or lauryl group), and tetradecyl group (or myristyl group).

[0023] Examples of alicyclic hydrocarbon groups having 3 to 20 carbon atoms include cyclopropyl, cyclopentyl, cyclohexyl, adamantyl, and norbornyl groups.

[0024] Aromatic hydrocarbon groups having 6 to 14 carbon atoms are not particularly limited, but examples include phenyl groups, tolyl groups, and naphthyl groups.

[0025] The groups that can be combined as described above are not particularly limited, but examples include aralkyl groups with 6 to 12 carbon atoms, such as benzyl groups, phenethyl groups, naphthylmethyl groups, and naphthylethyl groups.

[0026] From the viewpoint of improving the adhesion of the porous sheet 10, R 1 is a linear alkyl group, and R 2 It is preferable that R is a hydrogen atom. 1 It is preferably a linear alkyl group having 6 to 12 carbon atoms, and more preferably a linear alkyl group having 8 to 10 carbon atoms.

[0027] The hydrophobic gelatin derivative represented by formula (1) is preferably at least one selected from the group consisting of formulas (2) and (3), and the gelatin derivative represented by formula (2) is more preferred.

[0028] [ka] [ka]

[0029] Equation (2) is the case where L is a single bond in equation (1), and the hydrocarbon group (-CHR 1 R 2 ) is introduced to the gelatin residue Gltn of the starting gelatin via an imino bond (-NH-) which is a secondary amine. Equation (3) is the case in equation (1) where L is -C(O)-, and the hydrocarbon group (-CHR 1 R 2 ) is introduced to the gelatin residue Gltn of the raw material gelatin via an amide bond (-NHCO-). In formulas (2) and (3), the meaning of each symbol is the same as in formula (1) already explained, and the preferred form is also the same.

[0030] The hydrophobic gelatin derivative may consist of only one type of hydrophobic gelatin derivative, or it may be a mixture of two or more types of hydrophobic gelatin derivatives.

[0031] Here, the amount of amino groups (-NH2) in the gelatin before hydrophobization (raw gelatin) is compared with the amount of imino groups (-NH-CHR) to which alkyl groups are attached in the hydrophobized gelatin derivative. 1 R 2 The molar ratio of the content of ) is defined as the "degree of substitution" (hereinafter sometimes simply referred to as "DS").

[0032] From the viewpoint of improving the adhesion of the porous sheet, the hydrocarbon group introduction rate of the hydrophobic gelatin derivative is preferably, for example, 5 mol% to 50 mol%, 5 mol% to 20 mol%, or 5 mol% to 10 mol%. In other words, the imino group / amino group (molar ratio) of the hydrophobic gelatin derivative may be 5 / 95 to 50 / 50, 5 / 95 to 20 / 80, or 5 / 95 to 10 / 90. If the hydrocarbon group introduction rate of the hydrophobic gelatin derivative is within the above range, it is easy to obtain a porous sheet 10 with high adhesion.

[0033] The hydrocarbon group introduction rate can be calculated by quantifying the number of amino groups in the raw material gelatin and the number of amino groups in the hydrophobic gelatin derivative using the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method), and then using the following formula. Hydrocarbon group introduction rate (mol%) of hydrophobized gelatin derivatives =[Number of amino groups in raw gelatin - Number of amino groups in hydrophobic gelatin derivative] [Number of amino groups in raw gelatin] × 100

[0034] The molecular weight of hydrophobic gelatin derivatives is not particularly limited and is determined by the molecular weight of the raw material gelatin and the type and amount (number) of hydrocarbon groups introduced. Therefore, the range of possible weight-average molecular weight (Mw) of hydrophobic gelatin derivatives is almost the same as the range of possible weight-average molecular weight (Mw) of the raw material gelatin, which will be discussed later.

[0035] The hydrophobic gelatin derivatives described above may be commercially available or in-house synthesized. Hydrophobic gelatin derivatives may also be synthesized, for example, by introducing a hydrophobic group (hydrocarbon group) into the raw material gelatin (first gelatin) described below, using the synthesis method disclosed in International Publication No. 2020 / 137903. The details of the synthesis method for hydrophobic gelatin derivatives disclosed in International Publication No. 2020 / 137903 are incorporated herein by reference.

[0036] The raw material gelatin (first gelatin) may be obtained from naturally derived collagen or from synthetic collagen (including fermented and genetically modified collagen). Alternatively, gelatin obtained from naturally derived or synthetic collagen may be subjected to some treatment (except hydrophobic treatment). Preferably, gelatin obtained by treating naturally derived collagen obtained from the skin, bones, and tendons of mammals, birds, and fish with acid or alkali (heat extraction as necessary) is used, and among these, gelatin obtained by alkali treatment of naturally derived collagen is particularly preferred in that it yields a porous sheet with superior effects of the present invention. In this specification, the term "naturally derived gelatin" means gelatin obtained by heat, acid, or alkali treatment of naturally derived collagen, and for example, the term "fish-derived gelatin" means gelatin obtained by heat, acid, or alkali treatment of fish-derived collagen.

[0037] Furthermore, it is preferable to use gelatin that has been treated to reduce the endotoxin content. Gelatin raw materials vary depending on the climate of the country of manufacture, the manufacturing environment, the source animal, the extraction method, and the cleanliness of the equipment used, but generally, gelatin containing 1,000 to 100,000 EU / g of endotoxin, and which has undergone some treatment to reduce the endotoxin content, is called "low-endotoxin treated gelatin." "Low-endotoxin treated gelatin" usually contains 5,000 EU / g or less of endotoxin, preferably 1,000 EU / g or less, more preferably 100 EU / g or less, and even more preferably 10 EU / g or less. Such low-endotoxin treated gelatin is not particularly limited, and known types can be used, for example, those described in Japanese Patent Application Publication No. 2007-231225, the contents of which are incorporated herein by reference.

[0038] Examples of gelatin derived from mammals include gelatin derived from pigs and cattle. Gelatin derived from fish is not particularly limited, but gelatin derived from cold-water fish (cold-water species) such as salmon, trout, cod, Alaska pollock, sea bream, tilapia, and tuna (hereinafter also referred to as "cold-water fish-derived gelatin") is preferred. Cold-water fish-derived gelatin does not solidify even at low temperatures and can maintain its fluidity. Therefore, by using a hydrophobic derivative of cold-water fish-derived gelatin as the hydrophobic gelatin derivative, the crosslinking agent can be easily and uniformly dispersed even at low temperatures, and the porous sheet 10 of this embodiment can be efficiently manufactured by freeze-drying, which will be described later. Furthermore, it also has the characteristic of easily absorbing moisture when attached to a living body.

[0039] Gelatin derived from cold-water fish is a polymer in which two or more amino acids are linked in a linear chain. For every 1000 constituent amino acids, it contains 190 or fewer imino acids, more specifically, 80 or fewer hydroxyproline molecules and 110 or fewer proline molecules. The room-temperature fluidity of cold-water fish-derived gelatin is thought to be due to the fact that the number of hydroxyproline molecules is 80 or fewer, or the number of proline molecules is 110 or fewer. If either of these conditions is met, the denaturation temperature is approximately below room temperature, resulting in room-temperature fluidity.

[0040] Thai gelatin has 73 hydroxyproline molecules and 108 proline molecules, with a denaturation temperature of 302.5 K. Tilapia gelatin has 82 hydroxyproline molecules and 110 proline molecules, with a denaturation temperature of 309 K. In contrast, porcine gelatin has 95 hydroxyproline molecules and 121 proline molecules, with a denaturation temperature of 316 K.

[0041] Furthermore, gelatin derived from cold-water fish has an amino acid sequence similar to that of animal-derived gelatin, is easily broken down by enzymes in the body, and also has high biocompatibility.

[0042] The molecular weight of the raw material gelatin is not particularly limited, but the weight-average molecular weight (Mw) is preferably 5,000 to 100,000, more preferably 10,000 to 50,000, and even more preferably 20,000 to 40,000. In this specification, the weight-average molecular weight refers to the weight-average molecular weight determined by gel permeation chromatography (GPC).

[0043] The raw material gelatin may consist of only one type of gelatin, or it may be a mixture of two or more types of gelatin.

[0044] Crosslinking agents are typically compounds that have at least two substituents (crosslinking groups) in one molecule that can react with the primary amino groups of hydrophobized gelatin derivatives.

[0045] The crosslinking group of the crosslinking agent is not particularly limited, but an activated ester group is preferred from the viewpoint of selectively reacting with the primary amino group of the hydrophobic gelatin derivative under mild conditions. That is, a compound having at least two activated ester groups in one molecule is preferred as the crosslinking agent. Examples of such crosslinking agents include polybasic acids activated with N-hydroxysuccinimide or N-hydroxysulfosuccinimide.

[0046] In addition to the above, genipin, aldehyde compounds, acid anhydrides, maleimide compounds, dithiocarbonates, and diisothiocyanates can be used as crosslinking agents.

[0047] Examples of polybasic acids include tartaric acid, citric acid, malic acid, glutaric acid, glutamic acid, aspartic acid, oxaloacetate, cis-aconitic acid, 2-ketoglutaric acid, polytartaric acid, polycitric acid, polymalic acid, polyglutamic acid, polyaspartic acid, carboxymethylated dextrin, carboxymethylated dextran, carboxymethylated starch, carboxymethylated cellulose, carboxymethylated chitosan, and carboxymethylated pullulan.

[0048] Other crosslinking agents that can be used include disuccinimidyl glutarate (DSG), disuccinimidyl severate (DSS), and disuccinimidyl tartrate (DST).

[0049] Furthermore, polybasic acid esters of polyethylene glycol or polyethylene glycol ether, in which at least one of the carboxyl groups of the polybasic acid that have not reacted with polyethylene glycol is activated esterified, for example, 4,7,10,13,16-pentaoxanadecanedioic acid di(N-succinimidyl), and polyethylene glycol di(succinimidyl succinate) (SS-PEG-SS) represented by the following formula:

[0050] [ka]

[0051] (n is the number at which the number-average molecular weight is approximately 20,000); Furthermore, pentaerythritol-polyethylene glycol ether tetrasuccinimidyl glutarate (4S-PEG), represented by the following formula:

[0052] [ka]

[0053] (where n is a number such that Mw is approximately 3,000 to 30,000, preferably 5,000 to 27,000, and more preferably 15,000 to 25,000).

[0054] Examples of aldehyde compounds include formyl-introduced polysaccharides, in which two or more formyl groups are introduced into a single molecule, such as formyl-introduced starch, formyl-introduced dextran, formyl-introduced dextrin, and formyl-introduced hyaluronic acid.

[0055] Examples of acid anhydrides include glutaric anhydride, maleic anhydride, and succinic anhydride. Examples of diisothiocyanates include hexamethylene diisothiocyanate.

[0056] Preferred crosslinking agents include activated polyethylene glycol polybasic acid esters and formyl group-introduced polysaccharides, with activated polyethylene glycol polybasic acid esters being more preferred.

[0057] The crosslinking agents described above may be commercially available products or may be synthesized by known methods. Furthermore, the crosslinking agent may consist of only one type of crosslinking agent or a mixture of two or more types of crosslinking agents.

[0058] In the porous sheet 10, the ratio of the crosslinking groups of the crosslinking agent to the amino groups of the hydrophobic gelatin derivative is not particularly limited. From the viewpoint of efficiently promoting the crosslinking reaction, for example, it is preferable that the amount of crosslinking groups of the crosslinking agent is 0.25 to 2 equivalents, more preferably 0.3 to 1.5 equivalents, and even more preferably 0.3 to 0.8 equivalents, for every 1 equivalent of amino groups of the hydrophobic gelatin derivative. If the ratio of amino groups to crosslinking groups (e.g., active ester groups) is as described above, the hydrophobic gelatin derivative can be sufficiently crosslinked, and the adhesive strength of the porous sheet can be further increased.

[0059] The content of the hydrophobic gelatin derivative and the crosslinking agent in the porous sheet 10 is not particularly limited and may be appropriately adjusted within a range that satisfies the preferred ratio of crosslinkable groups to amino groups as described above. The content of the hydrophobic gelatin derivative in the porous sheet may be, for example, 1 to 15% by mass or 5 to 10% by mass, and the content of the crosslinking agent may be, for example, 1 to 10% by mass or 2.5 to 7.5% by mass.

[0060] The porous sheet 10 may consist only of a hydrophobic gelatin derivative and a crosslinking agent, or it may contain other components other than the hydrophobic gelatin derivative and the crosslinking agent, within the range that achieves the effects of the present invention. The total content of the hydrophobic gelatin derivative and the crosslinking agent in the porous sheet 10 may be 1% by mass or more, 10% by mass or more, 90% by mass or more, or 100% by mass.

[0061] Other components besides hydrophobic gelatin derivatives and crosslinking agents are not particularly limited, but include, for example, solvents, buffering agents, colorants, preservatives, functional nanoparticles, and pharmaceuticals (such as anticancer agents, antithrombotic agents, antibacterial agents, and growth factors).

[0062] Because the porous sheet 10 has voids, its density (apparent density) is lower compared to a non-porous gelatin sheet. The density of the porous sheet 10 is not particularly limited, but for example, 0.01 g / cm³ 3 ~0.3g / cm 3 , or 0.02 g / cm³ 3 ~0.08 g / cm³ 3 It may be so. The density of the porous sheet 10 is preferably 0.02 g / cm³. 3 ~0.1g / cm 3 More preferably, 0.05 g / cm³ 3 ~0.1g / cm 3 And, particularly preferably, 0.05 g / cm³ 3 ~0.08 g / cm³ 3 If the density is below the upper limit of the above range, the porous sheet can have sufficient voids, thereby obtaining appropriate flexibility and / or pliability, and furthermore, it can absorb moisture more efficiently and promote the curing reaction. If the density is above the lower limit of the above range, the porous sheet can obtain a sufficient amount of cured material (crosslinked material) and exhibit strong adhesion to biological tissue. Furthermore, the film strength is also increased and handling is improved. The density of the porous sheet 10 can be adjusted, for example, by the gelatin concentration of the raw material liquid used in the porous sheet manufacturing method described later.

[0063] The thickness of the porous sheet 10 is not particularly limited, but may be, for example, 0.1 mm or more, 0.5 mm or more, 1 mm or more, or 1.5 mm or more. If the thickness is above the lower limit, the porous sheet can produce a sufficient amount of cured material (crosslinked material) and exhibit strong adhesion to biological tissue. In addition, the film strength is increased and handling is improved. The upper limit of the thickness of the porous sheet is not particularly limited, but the practical range as a tissue adhesion film is, for example, 8 mm or less, or 5 mm or less. Also, the thinner the thickness, the more flexible it becomes, and for example, the processability when used as a tissue adhesion film is improved. From the viewpoint of improving flexibility, for example, the thickness of the porous sheet may be 2 mm or less, 1 mm or less, or 0.5 mm or less.

[0064] As described above, the porous sheet 10 of this embodiment contains an unreacted crosslinking agent dispersed in a hydrophobic gelatin derivative (see Figure 1B), which means that the majority of the crosslinking agent contained in the porous sheet 10 is unreacted. Therefore, for example, a sheet made of a crosslinked hydrophobic gelatin derivative that contains a small amount of unreacted crosslinking agent is different from the porous sheet 10 of this embodiment. The majority of the hydrophobic gelatin derivative contained in the porous sheet of this embodiment is in an uncrosslinked state before use (for example, before adhesion to biological tissue 20), and is crosslinked by the crosslinking agent upon use (for example, adhesion to biological tissue 20). For example, it is preferable that 60 mol% to 100 mol%, or 80 mol% to 100 mol%, of the total crosslinking agent contained in the porous sheet 10 of this embodiment is present in an unreacted state.

[0065] [Method for manufacturing porous sheets] The method for manufacturing the porous sheet 10 is not particularly limited, but for example, it can be manufactured by a manufacturing method including steps S1 and S2 described below. In this embodiment, the manufacturing method uses freeze-drying to efficiently obtain a sheet with a porous structure (porous sheet). Step S1: Preparation of raw material solution (acidic), and Step S2: Freeze-drying the raw material liquid.

[0066] Process S1: First, an acidic raw material solution (raw material solution (I)) is prepared, containing a hydrophobic gelatin derivative (second gelatin), a crosslinking agent, an acid, and a solvent (step S1 in Figure 2).

[0067] Examples of hydrophobic gelatin derivatives include the embodiments described above for the porous sheet 10, and the preferred embodiments are similar. Among these, a cold-water fish-derived gelatin derivative, obtained by introducing hydrocarbon groups into cold-water fish-derived gelatin (raw material gelatin), is more preferred as the hydrophobic gelatin derivative. By using a cold-water fish-derived gelatin derivative as the hydrophobic gelatin derivative, the crosslinking agent can be easily and uniformly dispersed even at low temperatures, and the porous sheet 10 can be obtained by freeze-drying (step S2) while maintaining this state.

[0068] The concentration of the hydrophobic gelatin derivative in the raw material solution is not particularly limited, but may be, for example, 1 w / v% to 20 w / v%, 1.2 w / v% to 12 w / v%, or 2.5 w / v% to 7.5 w / v%. If the concentration of the hydrophobic gelatin derivative in the raw material solution is within the above range, a porous sheet 10 having an appropriate porosity can be easily obtained.

[0069] Examples of crosslinking agents include those described as components of the porous sheet 10, and the preferred form is also the same. The concentration of the crosslinking agent in the raw material liquid is not particularly limited and may be adjusted as appropriate, for example, to satisfy the preferred ratio of crosslinkable groups to amino groups as described above. The concentration of the crosslinking agent in the raw material liquid may be, for example, 1 w / v% to 10 w / v%, or 2.5 w / v% to 7.5 w / v%.

[0070] The acid is added to adjust the starting material solution to an acidic state. The preferred pH of the starting material solution at room temperature (20°C) is less than pH 7, pH 6 or less, or pH 5 or less, and the lower limit is not particularly limited, but for example, pH 1 or higher. By making the starting material solution acidic, the nucleophilicity of the amine is suppressed, and the hydrophobic gelatin derivative and the crosslinking agent can be mixed without reacting. By freeze-drying in this state (step S2), a porous sheet 10 containing the crosslinking agent and the hydrophobic gelatin derivative in a substantially unreacted state is obtained.

[0071] The type of acid is not particularly limited as long as the pH of the raw material solution can be adjusted within the above range. However, from the viewpoint of evaporating or sublimating during freeze-drying (step S2) and not remaining in the final product (porous sheet 10), hydrochloric acid, acetic acid, etc. are preferred, and hydrochloric acid is more preferred. One type of acid may be used alone, or two or more types may be used in mixture. Furthermore, the concentration of the acid in the raw material solution may be adjusted as appropriate so that the pH of the raw material solution is within the above range.

[0072] The solvent for the raw material solution is not particularly limited as long as it can dissolve the hydrophobic gelatin derivative, crosslinking agent, and acid. Examples include water (ion-exchanged water, distilled water, pure water), water-alcohol mixed solvent, water-acetone mixed solvent, etc., with water being preferred.

[0073] The raw material solution may consist only of a hydrophobic gelatin derivative, a crosslinking agent, an acid, and a solvent, or it may contain other components. These other components are the same as the other components that the porous sheet 10 can contain as described earlier (e.g., drugs, functional nanoparticles, etc.). The raw material solution may also contain a pologen as another component, which can serve as a template for the pores of the porous structure. As the pologen, a sublimable solid such as ice microcrystals or t-butanol is preferred.

[0074] The raw material solution may be prepared by uniformly mixing a hydrophobic gelatin derivative, a crosslinking agent, an acid, a solvent, and other components as needed, using conventionally known methods.

[0075] Process S2: Next, the prepared raw material liquid is freeze-dried to obtain a porous sheet 10 (step S2 in Figure 2). For example, a porous sheet 10 of the desired size can be obtained by pouring the raw material liquid into a container having the same volume as the porous sheet 10 of the desired size and evaporating the solvent (and acid) by freeze-drying. A conventionally known method may be used for freeze-drying. The vacuum level during freeze-drying may be, for example, 100 mmTorr or less, or 50 mmTorr or less, the temperature during freeze-drying may be, for example, -80°C or less, or -10°C or less, and the freeze-drying time may be, for example, 96 hours or less, or 48 hours or less. Prior to freeze-drying, so-called pre-freezing may be performed.

[0076] [Tissue adhesive membrane] The porous sheet 10 of this embodiment has high biocompatibility and adhesive strength, making it suitable for use as a tissue adhesive film (tissue adhesive) for biological tissues. Because the porous sheet 10 has high flexibility and / or pliability, it can also be used by bending it to conform to the surface shape of biological tissues. Examples of tissue adhesive films include hemostatic materials to stop bleeding from biological tissues (blood vessels, etc.), adhesion prevention materials to prevent postoperative adhesions, and wound dressings to cover wounds in biological tissues (pulmonary pleura, gastrointestinal anastomoses, oral and periodontal tissues, etc.).

[0077] Furthermore, the porous sheet 10 possesses excellent adhesion, as well as excellent absorbency and biocompatibility. Therefore, it can be used, for example, as a sealant (closure material) to fill gaps between dura maters, dura mater suture sites, or gaps between dura mater-forming materials and dura mater during dura mater suturing.

[0078] Furthermore, the tissue adhesion film may be planar or, for example, tubular. Since the porous sheet 10 has high flexibility and / or pliability, it is easy to process into a tubular shape. A tubular tissue adhesion film can be used, for example, to cover anastomoses of nerves or tendons.

[0079] The tissue adhesive film of this embodiment adheres to biological tissue by being attached to the tissue and left to stand. The standing time (adhesion time) is the time required for the tissue adhesive film to absorb moisture in the tissue and crosslink, and can be set appropriately depending on the proportion of constituent materials in the tissue adhesive film, but for example it may be 1 to 10 minutes. In addition, heating may be performed at 37°C or below, or a weak alkaline aqueous solution such as sodium bicarbonate aqueous solution may be applied.

[0080] [Second Embodiment] <Laminate> In this embodiment, the laminate 50 shown in Figure 3 will be described. The laminate 50 includes the porous sheet 10 of the first embodiment (hereinafter sometimes referred to as "lower layer 10" or "sheet (I)") and a sheet 30 (hereinafter sometimes referred to as "upper layer 30" or "sheet (II)") containing a crosslinked material of raw gelatin (first gelatin) laminated on top of it. That is, the lower layer 10 (sheet (I)) is laminated on one surface (the bottom surface) of the upper layer 30 (sheet (II)).

[0081] The laminate 50 of this embodiment can be used, for example, as a tissue adhesive film, similar to the porous sheet 10 of the first embodiment. In this case, the laminate 50 is attached to the biological tissue with the lower layer 10 side in contact. The lower layer 10 absorbs moisture upon contact with the biological tissue, crosslinks, and adheres to the tissue. Since the laminate 50 includes the porous sheet 10 (lower layer 10), it exhibits the same effects as the first embodiment. That is, it has high biocompatibility and adhesive strength, and further possesses flexibility and / or pliability.

[0082] Furthermore, the laminated structure makes it difficult for moisture to penetrate the upper layer 30, suppressing stickiness (adhesion) of the surface of the laminate 50 (upper layer 30). This improves handling, for example, during surgery. In addition, since no hydrophobic groups are introduced into the gelatin raw material of the upper layer 30, it has low adhesion to biological tissue. Therefore, when the laminate 50 is attached to biological tissue, its surface (upper layer 30) does not easily adhere to other biological tissues, and it also functions as an adhesion prevention material. The details of the laminate 50 of this embodiment will be described below.

[0083] The lower layer 10 of the laminate 50 can be the same as the porous sheet 10 of the first embodiment, and the preferred form is also the same.

[0084] The density of the lower layer 10 of the laminate 50 may be the same as that of the porous sheet 10 in the first embodiment. Alternatively, the density of the lower layer 10 may be 0.05 g / cm³. 3 ~0.2g / cm 3 Preferably, it is 0.07 g / cm³. 3 ~0.2g / cm 3 It is more preferable that the density is below the above upper limit. If the density is below the above upper limit, the laminate 50 can have sufficient voids, thereby providing more appropriate flexibility and further promoting the curing reaction between the hydrophobic gelatin derivative and the crosslinking agent. If the density is above the above lower limit, the water absorption rate when the laminate 50 is attached to biological tissue will be slower, and moisture will not easily reach the upper layer 30. As a result, the curing reaction will proceed in the lower layer 10 before a lot of moisture reaches the upper layer 30, allowing for strong adhesion to the biological tissue. This means that, for example, even in environments with a lot of moisture around it, such as inside a living body where a large amount of blood and bodily fluids are present due to bleeding, the laminate 50 will exhibit strong adhesion, and the stickiness (adhesion) of the surface (upper layer 30) will be suppressed. As a result, handling is improved, and adhesion to other tissues and adhesion to rubber gloves during surgery are suppressed.

[0085] The thickness of the lower layer 10 of the laminate 50 may be 0.1 mm or more, 0.5 mm or more, 1.5 mm or more, or 2 mm or more. If the thickness is above the lower limit, for example, even in environments with a lot of moisture around it, such as inside a living organism where a large amount of blood or bodily fluids are present due to bleeding, it will exhibit strong adhesion, and the stickiness (adhesion) of the surface of the laminate 50 (upper layer 30) will be suppressed, improving handling and further enhancing the ability to prevent adhesion to other tissues. Furthermore, there is no particular upper limit to the thickness of the lower layer 10, but the practical range as a tissue adhesive film is, for example, 8 mm or less, or 5 mm or less. Also, the thinner the thickness, the more flexible it becomes, for example, improving processability when used as a tissue adhesive film. From the viewpoint of improving flexibility, for example, the thickness of the lower layer 10 may be 2 mm or less, 1 mm or less, or 0.5 mm or less.

[0086] The raw material gelatin for the upper layer 30 can be the same as that used in the first embodiment described above, and the preferred embodiment is also the same. The crosslinked raw material gelatin is not particularly limited and may be a crosslinked product obtained by any method of crosslinking the raw material gelatin, but it is preferably a thermally crosslinked product. Thermal crosslinking makes it easier to obtain a crosslinked raw material gelatin and is safe as it does not generate impurities derived from the crosslinking agent. In a thermally crosslinked product, for example, amino groups in the gelatin mixture react with other reactive groups (e.g., carboxyl groups and mercapto groups, etc.) to form a crosslinked structure.

[0087] The upper layer 30 may consist only of crosslinked raw gelatin, or it may contain other components besides crosslinked raw gelatin to the extent that it achieves the effects of the present invention. The content of crosslinked raw gelatin in the upper layer 30 may be 100% by mass, 95% by mass or more, or 90% by mass or more. Other components besides crosslinked raw gelatin include, for example, basic compounds as described in Modification 1 below. Other components besides basic compounds are not particularly limited, but include, for example, dyes, photocatalysts, colorants, preservatives, inorganic nanoparticles (gold nanoparticles, iron oxide nanoparticles, calcium phosphate nanoparticles, etc.), and drugs (anticancer agents, antithrombotic agents, antibacterial agents, and growth factors, etc.).

[0088] The upper layer 30 is preferably porous and / or thin film. If the upper layer 30 is porous and / or thin film, the overall flexibility and / or pliability of the laminate 50 can be further enhanced. If the upper layer 30 is porous, its density is not particularly limited, but for example, 0.01 g / cm³. 3 ~0.3g / cm 3 This is also acceptable. If the density is below the upper limit of the above range, it is easier to obtain sufficient flexibility. Also, if the density is above the lower limit of the above range, the membrane strength will increase and handling will improve.

[0089] The lower limit of the thickness of the upper layer 30 is not particularly limited, but for example, 0.01 mm or more, or 0.1 mm or more, is preferred. If the thickness is above the above lower limit, handling is further improved, adhesion to other tissues and adhesion to rubber gloves during surgery are suppressed. The upper limit of the thickness of the upper layer 30 is also not particularly limited, but from the viewpoint of improving flexibility, for example, 2 mm or less, 1 mm or less, or 0.5 mm or less, is preferred.

[0090] The ratio of the film thickness of the lower layer 10 to the film thickness of the upper layer 30 is not particularly limited, but may be, for example, 1 to 6 or 2 to 4. If the ratio is within the above range, handling performance will be further improved and the performance of preventing adhesion to other tissues will be further enhanced.

[0091] <Method for manufacturing laminates> The manufacturing method for the laminate 50 of this embodiment is not particularly limited, but for example, it can be manufactured by a manufacturing method including steps S10, S20, and S30 described below (see Figure 7). Process S10: Preparation of lower layer 10 (sheet (I)), Process S20: Preparation of the upper layer 30 (sheet (II)), and Process S30: Lamination of lower layer 10 and upper layer 30.

[0092] Process S10: The method for manufacturing the lower layer 10 (porous sheet (I)) is not particularly limited, but it can be manufactured by a method similar to that of the porous sheet 10 described in the first embodiment, for example, by a method including steps S1 and S2 shown in Figure 2.

[0093] Process S20: The method for producing the upper layer 30 (sheet (II) containing a crosslinked raw material gelatin) is not particularly limited, but may be produced by, for example, the method described below. First, a raw material gelatin solution (raw material solution (II)) containing raw material gelatin and a solvent is prepared. Next, the raw material gelatin solution is poured into a mold of the desired size, and the solvent is evaporated by freeze-drying to obtain a sheet of the desired size (precursor sheet). The upper layer 30 is obtained by heating the sheet and thermal crosslinking it.

[0094] The solvent used to dissolve the raw material gelatin is the same as the solvent used in the raw material solution in the first embodiment, and the preferred form is also the same. The concentration of raw material gelatin in the raw material gelatin solution is not particularly limited and may be adjusted as appropriate depending on the desired porosity, density, etc. of the upper layer 30 to be produced. The concentration of raw material gelatin in the raw material gelatin solution may be, for example, 1 w / v% to 20 w / v%. A conventionally known method may be used for freeze-drying, and the preferred ranges for vacuum, temperature, and time during freeze-drying are the same as those for freeze-drying in the first embodiment.

[0095] The method for thermal crosslinking the raw gelatin is not particularly limited, and conventionally known methods can be used. The heating temperature during thermal crosslinking is not particularly limited, but for example, 80 to 200°C is preferred, and 100 to 200°C is more preferred. The heating time during thermal crosslinking is not particularly limited, but for example, 0.1 to 20 hours is preferred, and 1 to 10 hours is more preferred.

[0096] Process S30: The laminate 50 of this embodiment is obtained by stacking the manufactured lower layer 10 and the upper layer 30. When the laminate 50 is attached to biological tissue, the lower layer 10 crosslinks, causing the lower layer 10 and the upper layer 30 to adhere together.

[0097] <Modification 1, laminated body 51> As shown in Figure 8, the laminate 51 of this modified example has an upper layer (sheet (II)) 30 containing a basic compound 31. The basic compound 31 is a compound that exhibits basicity when in contact with moisture. Except for containing the basic compound 31, the laminate 51 has the same configuration as the laminate 50 of the second embodiment described above. The description of the configuration similar to that of the laminate 50 will be omitted in this modified example.

[0098] The laminate 51 of this modified example can be used, for example, as a tissue adhesive film, similar to the laminate 50 of the second embodiment, and provides the same effects as the laminate 50. Furthermore, the laminate 51 provides the following effects by containing the basic compound 31. As shown in Figure 8, when the laminate 51 is attached to biological tissue 20, the laminate 51 absorbs the moisture from the biological tissue 20, and the basic compound 31 is eluted by the absorbed moisture. As a result, the pH in the lower layer 10 rises more quickly, and the crosslinking reaction (curing reaction) between the hydrophobic gelatin derivative and the crosslinking agent is promoted. That is, the time (adhesion time) from attaching the laminate 51 to the biological tissue 20 until sufficient adhesive strength is obtained can be shortened.

[0099] The basic compound 31 is not particularly limited as long as it is a compound that exhibits basicity upon contact with water, but for example, a compound whose aqueous solution of 0.05 mol / L to 0.5 mol / L has a pH of 7.5 to 10 is preferred, and a compound whose aqueous solution of 0.1 mol / L to 0.3 mol / L has a pH of 8.0 to 9.5 is more preferred. If a compound has such properties, the bonding time can be further shortened. Furthermore, it is preferable that the basic compound 31 is a pH buffering agent, such that its aqueous solution functions as a pH buffer. Examples of pH buffering agents include a mixture of a weak acid and a salt of a weak acid, or a mixture of a weak base and a salt of a weak base. Using a pH buffering agent makes it easier to maintain the pH in the lower layer 10 within a desired range when the laminate 51 is attached to the biological tissue 20. It also prevents the pH of the lower layer 10 from rising too high, thus preventing damage to the biological tissue 20.

[0100] Examples of pH buffers include the following components and active ingredients of pH buffers: phosphate buffer (a mixture of phosphoric acid and phosphate (e.g., sodium phosphate)), acetate buffer (a mixture of acetic acid and acetate (e.g., sodium acetate)), citrate buffer (a mixture of citric acid and citrate (e.g., sodium citrate)), citrate phosphate buffer (a mixture of citric acid and phosphate (e.g., sodium phosphate)), borate buffer (a mixture of boric acid and borate (e.g., sodium borate)), tartaric acid buffer (a mixture of tartaric acid and tartrate (e.g., sodium tartrate)), succinic acid buffer (a mixture of succinic acid and succinate (e.g., sodium succinate)), maleic acid (a mixture of maleic acid and maleate (e.g., sodium maleate)), Tris hydrochloric acid buffer (a mixture of tris-aminomethane and hydrochloric acid), and carbonate buffer (a mixture of carbonic acid and carbonate (e.g., sodium carbonate)). Furthermore, the ratio of each component in the pH buffer (for example, the ratio of a weak acid to a salt of a weak acid, or the ratio of a weak base to a salt of a weak base) may be adjusted as appropriate according to the desired pH.

[0101] Examples of basic compounds 31 other than pH buffers include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and zinc hydroxide. These basic compounds 31 may be a single compound or a mixture of two or more compounds.

[0102] The content of the basic compound 31 in the upper layer 30 is not particularly limited and may be adjusted as appropriate depending on the strength of the basicity of the basic compound 31. When a pH buffer is used as the basic compound 31, the content of the basic compound 31 in the upper layer 30 may be, for example, 0.1% to 20% by weight or 0.3% to 10% by weight.

[0103] The density and thickness of the upper layer 30 can be within the range described in the second embodiment. Furthermore, if the upper layer 30 contains a basic compound, it is presumed that the greater the density and / or thickness of the upper layer 30, the greater the amount of basic compound it contains, thus shortening the bonding time. From the viewpoint of further shortening the bonding time, the density of the upper layer 30 can be, for example, 0.08 g / cm³. 3 ~0.3g / cm 3 The thickness of the upper layer 30 may be, for example, 0.2 mm to 2 mm.

[0104] <Method for manufacturing the laminate 51> The manufacturing method for the laminate 51 of this modified example is not particularly limited, and except for the use of the basic compound 31 in the preparation of the upper layer 30, it can be manufactured in the same manner as the laminate 50 of the second embodiment described above, for example, by a method including steps S10 to S30 shown in Figure 7. The manufacturing process for the upper layer 30 (step S20 in Figure 7) will be described below, and the manufacturing process that is the same as that for the laminate 50 will not be described in this modified example.

[0105] Process S20: First, a raw material gelatin solution (raw material solution (II)) is prepared, containing raw material gelatin, a basic compound, and a solvent. Next, the raw material gelatin solution is poured into a mold of the desired size, and the solvent is evaporated by freeze-drying to obtain a sheet of the desired size (precursor sheet). The sheet is heated and thermally crosslinked to obtain the upper layer 30.

[0106] Examples of basic compounds include those similar to the basic compound 31 contained in the upper layer 30 described above, and the preferred embodiment is also the same. The presence of a basic compound causes the pH of the raw material solution (II) to be alkaline. From the viewpoint of shortening the bonding time of the resulting laminate 51, the raw material solution (II) is preferably pH 8 to pH 10, and preferably pH 8 to pH 9.

[0107] When a pH buffer is used as the basic compound, the mixture of the solvent (e.g., water) and the basic compound is a pH buffer solution, and the starting material solution (II) is a solution in which the starting material gelatin is dissolved in the pH buffer solution. The concentration of the pH buffer solution may be, for example, 0.05 mol / L to 0.5 mol / L. The concentration of the pH buffer solution is, for example, the concentration of the mixture of a weak acid and a salt of the weak acid, or the concentration of the mixture of a weak base and a salt of the weak base in the pH buffer solution.

[0108] The upper layer 30 can be prepared by carrying out step S20 in the same manner as in the second embodiment described above, except that a basic compound is used.

[0109] <Modified example 2, laminated body 52> As shown in Figure 9, the laminate 52 of this modified example has a three-layer structure including two sheets (I) 10 and a sheet (II) 30. Of the two sheets (I) 10, one is laminated on one surface (bottom surface) of sheet (II) 30, and the other is laminated on the other surface (top surface) of sheet (II).

[0110] The sheets (I) and (II) included in the laminate 52 may be the same as those described in the second embodiment and modified example 1, and the preferred form is also the same. Furthermore, the manufacturing method of the laminate 52 is not particularly limited, and it can be manufactured by a method including steps S10 to S30 shown in Figure 7, similar to the laminate 50 of the second embodiment and the laminate 51 of modified example 1, except that it is laminated into three layers in the lamination process (step S30).

[0111] The two sheets (I) 10 provide the same effects as the porous sheet 10 of the first embodiment. By having sheets (I) 10 on both sides, the laminate 52 can be used, for example, as a double-sided adhesive tissue bonding film. Furthermore, if sheet (II) 30 contains a basic compound, similar to the first modification, the bonding time of sheets (I) on both sides can be shortened simultaneously.

[0112] The embodiments and modifications described above may be combined with each other, as long as they do not exclude one another. [Examples]

[0113] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0114] <<Porous Sheet (Single Layer)>> In Examples 1-1 to 1-3, Comparative Examples 1-1 to 1-3, Examples 2-1 to 2-3, and Examples 3-1 to 3-3 described below, porous sheets (sheet (I), single layer) of hydrophobic gelatin derivatives were prepared. Table 1 shows the details of the sheets prepared in each experiment.

[0115] [Example 1-1] <Synthesis of hydrophobic gelatin derivatives> As the raw material gelatin, pollock-derived gelatin (manufactured by Nitta Gelatin Co., Ltd., weight-average molecular weight (Mw): 84,000, amino group content: 305 μmol / g) (hereinafter referred to as "Org-ApGltn" or "Org" as appropriate) was prepared, and a nonyl group was introduced into Org-ApGltn to obtain a hydrophobized gelatin derivative (hereinafter referred to as "C9-ApGltn" as appropriate). Specifically, as disclosed in International Publication No. 2020 / 137903, nonanal (nonyl aldehyde) was reacted with the amino group of the raw material gelatin to form a Schiff base, and the obtained Schiff base was then reduced to a stable secondary amine with a reducing agent to obtain a hydrophobized gelatin derivative.

[0116] 10 g of Org-ApGltn was dissolved in 35 mL of ultrapure water. While stirring at 55°C, a solution of nonanal (manufactured by Tokyo Chemical Industry Co., Ltd., 0.305 mmol) dissolved in ethanol (manufactured by Junsei Chemical Co., Ltd.) in an amount equal to 0.1 times the amount of amino groups of Org-ApGltn (305 μmol / g) was added to form an imine bond between the nonanal and the amino groups of Org-ApGltn. After stirring at the same temperature for 1 hour, a solution of 2-picolinborane (manufactured by Junsei Chemical Co., Ltd., 0.4575 mmol) dissolved in ethanol was added to reduce the imine. The resulting mixed solution (Org-ApGltn concentration: 20 mass / vol%, water:ethanol = 35:15 mL) was stirred at 55°C for 17 hours to allow the reaction to proceed. Subsequently, the reaction solution (50 mL) was added dropwise to 500 mL of cold ethanol (-7 to 4°C) to purify C9-ApGltn. The resulting precipitate was washed with 250 mL of ethanol (1 hour x 3 times) to remove unreacted nonanal and 2-picolinborane. The precipitate was then vacuum-dried for 3 days to obtain C9-ApGltn in 90% by mass yield.

[0117] The introduction of a nonyl group into the obtained C9-ApGltn was confirmed by Fourier transform infrared spectroscopy, and 1 This was confirmed by 1H-NMR. Furthermore, the degree of substitution (DS) of C9-ApGltn (nonyl group substitution rate) was determined by quantifying the number of amino groups in the raw material gelatin and the number of amino groups in the hydrophobic gelatin derivative using the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method). From the obtained values, the molar ratio of the content of imino groups to which nonyl groups are attached in the hydrophobic gelatin derivative to the content of amino groups (-NH2) in the raw material gelatin was calculated and determined. The DS was 7.2 mol%. Hereinafter, the hydrophobic gelatin derivative synthesized in this example may be referred to as 7.2C9-ApGltn or 7.2C9.

[0118] <Preparation of porous sheets> (1) Preparation of raw material liquid First, a hydrophobic gelatin derivative solution (Solution A) was prepared using the following procedure. The synthesized 7,2C9-ApGltn was dissolved in a 0.1 mM HCl solution, then the pH was adjusted to 4 using 1 M HCl, and finally, the solution was made up with pure water to a concentration of 15 w / v% of 7,2C9-ApGltn to obtain Solution A.

[0119] Next, the crosslinking agent solution (Solution B) was prepared using the following procedure. Pentaerythritol-polyethylene glycol ether tetrasuccinimidyl glutarate (4S-PEG, weight-average molecular weight 20,000, manufactured by NOF Corporation) was used as the crosslinking agent. For every 1 equivalent of the amino group (-NH2 group) of the hydrophobized gelatin derivative in Solution A, 0.5 equivalents of the active ester group (N-hydroxysuccinimide group, NHS group) in Solution B were weighed out (i.e., NHS / NH2 = 50 mol%) of 4S-PEG, and this was dissolved in the same volume of 0.1 mM HCl solution as Solution A to obtain Solution B.

[0120] Solution A and Solution B were thoroughly mixed using a magnetic stirrer at 280 rpm for 30 seconds, and then adjusted with a 0.1 N HCl solution to a pH of 4 to obtain the raw material solution. The gelatin concentration of the obtained raw material solution was 7.5 w / v%.

[0121] (2) Freeze-drying of raw material liquid First, 5 mL of the prepared raw material solution was poured into a silicone sheet mold (50 mm x 50 mm x depth: 2 mm), and a silicone sheet plate was placed on top to fix the thickness of the raw material solution. Using a tabletop shelf-type freeze-dryer (SP Industries, AdVantage Pro, ADP+S), the sample was first freeze-dried overnight at -30°C to produce a porous sheet.

[0122] [Examples 1-2] First, a raw material solution with a gelatin (7,2C9-ApGltn) concentration of 7.5 w / v% was prepared using the same method as in Example 1-1, and this was diluted with a 0.1 mM HCl solution to obtain a raw material solution with a gelatin concentration of 5 w / v% and pH=4. The obtained raw material solution was freeze-dried using the same method as in Example 1-1 to produce a porous sheet.

[0123] [Examples 1-3] First, a raw material solution with a gelatin (7,2C9-ApGltn) concentration of 7.5 w / v% was prepared using the same method as in Example 1-1. This solution was then diluted with a 0.1 mM HCl solution to obtain a raw material solution with a gelatin concentration of 2.5 w / v% and a pH of 4. The obtained raw material solution was freeze-dried using the same method as in Experiment 1-1 to produce a porous sheet.

[0124] [Comparative Examples 1-1 to 1-3] Porous sheets were prepared in the same manner as in Examples 1-1 to 1-3, except that raw material gelatin (Org-ApGltn) was used instead of the hydrophobic gelatin derivative (7.2C9-ApGltn). Specifically, in Comparative Examples 1-1 to 1-3, the concentrations of Org-ApGltn in the raw material solution were 7.5 w / v%, 5 w / v%, and 2.5 w / v%, respectively.

[0125] [Example 2-1] <Synthesis of hydrophobic gelatin derivatives> As the hydrophobic gelatin derivative, a gelatin derivative in which an octyl group was introduced into the raw material gelatin (Org-ApGltn) (hereinafter referred to as "C8-ApGltn" as appropriate) was used. The hydrophobic gelatin derivative was obtained in the same manner as in Example 1-1, by reacting an alkyl aldehyde (octanal, octyl aldehyde (manufactured by Tokyo Chemical Industry Co., Ltd.)) with the amino group of the raw material gelatin to form a Schiff base, and then reducing the obtained Schiff base to a stable secondary amine with a reducing agent.

[0126] The introduction of an octyl group into the obtained C8-ApGltn was confirmed by Fourier transform infrared spectroscopy, and 1 This was confirmed by 1H-NMR. Furthermore, the hydrocarbon group introduction rate (octyl group introduction rate) DS of C8-ApGltn was calculated using the same method as in Example 1-1. The DS was 7.6 mol%. Hereafter, the hydrophobized gelatin derivative synthesized in this example may be referred to as 7.6C8-ApGltn or 7.6C8.

[0127] <Preparation of porous sheets> A raw material solution with a gelatin concentration of 7.5 w / v% was prepared by the same method as in Example 1-1, except that 7.6C8-ApGltn was used instead of 7.2C9-ApGltn. The resulting raw material solution was freeze-dried to produce a porous sheet.

[0128] [Example 2-2] First, a raw material solution with a gelatin concentration of 7.5 w / v% was prepared using the same method as in Example 2-1. This solution was then diluted with a 0.1 mM HCl solution to obtain a raw material solution with a hydrophobized gelatin derivative concentration of 5 w / v% and a pH of 4. The obtained raw material solution was freeze-dried using the same method as in Example 2-1 to produce a porous sheet.

[0129] [Examples 2-3] First, a raw material solution with a gelatin concentration of 7.5 w / v% was prepared using the same method as in Example 2-1. This solution was then diluted with a 0.1 mM HCl solution to obtain a raw material solution with a hydrophobized gelatin derivative concentration of 2.5 w / v% and a pH of 4. The obtained raw material solution was freeze-dried using the same method as in Example 2-1 to produce a porous sheet.

[0130] [Example 3-1] <Synthesis of hydrophobic gelatin derivatives> As the hydrophobic gelatin derivative, a gelatin derivative in which a decyl group was introduced into the raw material gelatin (Org-ApGltn) (hereinafter referred to as "C10-ApGltn" as appropriate) was used. The hydrophobic gelatin derivative was obtained in the same manner as in Example 1-1, by reacting an alkyl aldehyde (decanal, decyl aldehyde (manufactured by Tokyo Chemical Industry Co., Ltd.)) with the amino group of the raw material gelatin to form a Schiff base, and then reducing the obtained Schiff base to a stable secondary amine with a reducing agent.

[0131] The introduction of a decyl group into the obtained C10-ApGltn was confirmed by Fourier transform infrared spectroscopy, and 1This was confirmed by 1H-NMR. Furthermore, the hydrocarbon group introduction rate (decyl group introduction rate) DS of C10-ApGltn was calculated using the same method as in Example 1-1. The DS was 9.3 mol%. Hereinafter, the hydrophobized gelatin derivative synthesized in this example may be referred to as 9.3C10-ApGltn or 9.3C10.

[0132] <Preparation of porous sheets> A raw material solution with a gelatin concentration of 7.5 w / v% was prepared by the same method as in Example 1-1, except that 9.3C10-ApGltn was used instead of 7.2C9-ApGltn. The resulting raw material solution was freeze-dried to produce a porous sheet.

[0133] [Example 3-2] First, a raw material solution with a gelatin (9,3C10-ApGltn) concentration of 7.5 w / v% was prepared using the same method as in Example 3-1. This solution was then diluted with a 0.1 mM HCl solution to obtain a raw material solution with a gelatin derivative concentration of 5 w / v% and a pH of 4. The obtained raw material solution was freeze-dried using the same method as in Example 3-1 to produce a porous sheet.

[0134] [Example 3-3] First, a raw material solution containing a hydrophobic gelatin derivative (9,3C10-ApGltn) at a concentration of 7.5 w / v% was prepared using the same method as in Example 3-1. This solution was then diluted with a 0.1 mM HCl solution to obtain a raw material solution containing a hydrophobic gelatin derivative at a concentration of 2.5 w / v% and a pH of 4. The obtained raw material solution was freeze-dried using the same method as in Example 3-1 to produce a porous sheet.

[0135] The density (apparent density) of the porous sheets prepared in each example or comparative example was determined by dividing the weight of the porous sheet by its volume. The volume of the porous sheet was determined from the silicone sheet mold (50 mm × 50 mm × depth: 2 mm). The density of the porous sheets prepared in Examples 1-1, 2-1, and 3-1 using a raw material solution with a gelatin concentration of 7.5 w / v% was approximately 0.075 g / cm³. 3Therefore, the density of the porous sheets prepared in Examples 1-2, 2-2, and 3-2 using a raw material solution with a gelatin concentration of 5 w / v% was approximately 0.05 g / cm³. 3 Therefore, the density of the porous sheets prepared in Examples 1-3, 2-3, and 3-3 using a raw material solution with a gelatin concentration of 2.5 w / v% was approximately 0.025 g / cm³. 3 That was the case.

[0136] [Table 1]

[0137] [evaluation] (1) Shape observation The shape of the sheets prepared in Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-3 was observed visually and by scanning microscope (SEM) observation. Figure 4 shows photographs (left) and SEM images (right) of the porous sheets obtained in each example and comparative example.

[0138] The sheets prepared in Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-3 were confirmed to be porous (porous sheets). Furthermore, as shown in Figure 4, the porosity (density) of the porous sheets depended on the gelatin concentration of the raw material liquid (7.2C9-ApGltn concentration or Org-ApGltn concentration). The lower the gelatin concentration of the raw material liquid, the larger the proportion of voids in the porous sheet (lower density), and the higher the gelatin concentration, the smaller the proportion of voids in the porous sheet (higher density).

[0139] (2) Pressure resistance test 1 The porous sheets prepared in Examples 1-1 to 1-3, Comparative Examples 1-1 to 1-3, Examples 2-1 to 2-3, and Examples 3-1 to 3-3 were subjected to the pressure resistance tests described below.

[0140] Following ASTM (F2392-04), the sealing strength (pressure resistance) was measured using porcine aorta as the substrate. As shown in Figure 5A, the porous sheet 10 prepared in each example or comparative example was placed over the central defect 20a (1 mm in diameter) of the porcine aorta 20 (35 mm in diameter), lightly pressed in place with tweezers, and left to stand for 10 minutes. After standing, the porcine aorta 20 with the porous sheet 10 was fixed to a jig, and 37°C physiological saline was flowed at a flow rate of 2 mL / min towards the defect 20a covered by the porous sheet 10 (in the direction indicated by the arrow in Figure 5A), and the pressure at which the porous sheet 10 peeled off was measured. The results are shown in Figure 5B. In this experiment, a higher burst pressure indicates higher pressure resistance and higher adhesion of the porous sheet to biological tissue (porcine aorta).

[0141] In Figure 5B, we first compare Comparative Example 1-3, in which the gelatin concentration of the raw material solution was 2.5 w / v%, with Examples 2-3, 1-3, and 3-3. Compared to the porous sheet using raw material gelatin (Comparative Example 1-3), the porous sheets using hydrophobic gelatin derivatives (Examples 2-3, 1-3, and 3-3) had higher peel pressure (pressure resistance). Furthermore, there was a tendency for the peel pressure to increase as the length of the introduced alkyl chain increased. Furthermore, when the raw material gelatin concentration was 5 w / v%, and also when the raw material gelatin concentration was 7.5 w / v%, the porous sheet using the hydrophobic gelatin derivative showed a higher peeling pressure compared to the porous sheet using raw material gelatin (Org).

[0142] In this test (pressure resistance test 1), the porcine aorta was in a neutral (pH=7~7.4) moist state. It is presumed that the porous sheet placed on it absorbed the neutral moisture through capillary action and crosslinked with the active ester group (NHS group) of the crosslinking agent and the amino group (NH2) of the gelatin. It is also presumed that the porous sheet using the hydrophobic gelatin derivative formed hydrophobic interactions between the porous sheet and the tissue (porcine aorta) due to the hydrophobic groups introduced into Org, resulting in strong adhesion to the tissue surface (see Figure 1B).

[0143] <<Laminate>> In Examples 4-1, Comparative Examples 2-1, 2-2, 4-3, 5-1, 3-1, 3-2, and 6-1 to 6-3 described below, laminates were prepared by laminating a porous sheet of hydrophobic gelatin derivative (lower layer, sheet (I)) with a porous sheet of raw material gelatin (upper layer, sheet (II)). To clearly distinguish between sheet (I) and (II), sheet (II) was colored with a dye (acid blue). Table 2 shows the details of the sheets prepared in each experiment.

[0144] [Example 4-1] First, as sheet (I), a porous sheet of the hydrophobic gelatin derivative 9.3C10-ApGltn, which was prepared in Experiment 3-1 (thickness 2 mm, gelatin concentration in raw material solution (I) 7.5 w / v%), was prepared.

[0145] Next, a 5 w / v% aqueous solution of raw material gelatin (Org-ApGltn) (raw material solution (II)) was prepared and colored with acid blue (25 μg / mL). The prepared raw material gelatin aqueous solution was cast into a silicone sheet mold (70 mm × 70 mm × depth: 0.5 mm). Using the same freeze-drying oven as used in Example 1-1, the sample was first freeze-dried overnight at -30°C. After freeze-drying, it was heated at 150°C for 5 hours to thermally crosslink the Org-ApGltn and obtain sheet (II).

[0146] A laminate was obtained by stacking sheet (II) on top of sheet (I).

[0147] [Comparative Example 2-1] As sheet (I), a porous sheet of the hydrophobic gelatin derivative 9.3C10-ApGltn, prepared in Example 3-2 (thickness 2 mm, hydrophobic gelatin derivative concentration in the raw material solution 5 w / v%), was prepared. Otherwise, a laminate consisting of sheets (I) and (II) was prepared by the same method as in Example 4-1.

[0148] [Comparative Example 2-2] As sheet (I), a porous sheet of the hydrophobic gelatin derivative 9.3C10-ApGltn, prepared in Example 3-3 (thickness 2 mm, concentration of hydrophobic gelatin derivative in the raw material solution 2.5 w / v%), was prepared. Otherwise, a laminate consisting of sheets (I) and (II) was prepared by the same method as in Example 4-1.

[0149] Figure 6 shows photographs of the laminates prepared in Example 4-1, Comparative Example 2-1, and Comparative Example 2-2. The upper row shows photographs from the sheet (II) side, and the lower row shows photographs from the sheet (I) side.

[0150] [Example 5-1] A laminate consisting of sheets (I) and (II) was fabricated using the same method as in Example 4-1, except that sheet (I) was made with a thickness changed from 2 mm to 1 mm.

[0151] [Comparative Example 3-1] A laminate consisting of sheets (I) and (II) was prepared using the same method as in Comparative Example 2-1, except that sheet (I) was prepared with a thickness changed from 2 mm to 1 mm.

[0152] [Comparative Example 3-2] A laminate consisting of sheets (I) and (II) was prepared using the same method as in Comparative Example 2-2, except that sheet (I) was prepared with a thickness changed from 2 mm to 1 mm.

[0153] [Example 6-1] A laminate consisting of sheets (I) and (II) was fabricated using the same method as in Example 4-1, except that the thickness of sheet (I) was changed from 2 mm to 1.5 mm.

[0154] [Example 6-2] A laminate consisting of sheets (I) and (II) was prepared by the same method as in Example 6-1, except that sheet (II) was prepared by changing the gelatin concentration in raw material liquid (II) from 5 w / v% to 10 w / v%.

[0155] [Example 6-3] A laminate consisting of sheets (I) and (II) was prepared by the same method as in Example 6-1, except that sheet (II) was prepared by changing the gelatin concentration in raw material liquid (II) from 5 w / v% to 20 w / v%.

[0156] [Table 2]

[0157] [evaluation] (1) Adhesion comparison test This test was conducted using the porous sheet (single layer) prepared in Example 3-1 and the laminate prepared in Example 4-1. Sheet (I) of the laminate in Example 4-1 and the porous sheet in Example 3-1 have the same composition and structure. First, the porous sheet in Example 3-1 and the laminate in Example 4-1 were placed on the porcine aorta, lightly pressed down with tweezers to secure them, and left to stand for 10 minutes. At this time, sheet (I) of the laminate was placed on the bottom side (the side in contact with the porcine aorta). After standing, the tackiness of the porous sheet and the surface of the laminate (sheet (II)) attached to the porcine aorta was compared by touching them with a gloved hand.

[0158] The results of this test showed that the two-layer laminate (Example 4-1) had lower adhesiveness than the single-layer porous sheet (Example 3-1). From these results, it can be expected that by laminating a porous sheet of raw gelatin onto a porous sheet of hydrophobic gelatin derivative to form a laminate, when this laminate is used as a biotissue adhesion film, adhesion to other biotissues and adhesion to rubber gloves during surgery can be suppressed.

[0159] Furthermore, this test was conducted using the laminates prepared in Examples 5-1 and 6-1 to 6-3. These laminates are the ones that yielded good results in the pressure resistance test 2 described later. The tackiness of the laminates in Examples 5-1 and 6-1 to 6-3 was also lower compared to the single-layer porous sheet (Example 3-1).

[0160] (2) Pressure resistance test 2 In this test, simulating a living environment where a large amount of blood and bodily fluids are present due to bleeding, etc., the laminates prepared in Examples 4-1, Comparative Example 2-1, Comparative Example 2-2, Example 5-1, Comparative Example 3-1, Comparative Example 3-2, and Examples 6-1 to 6-3 were adhered to biological tissue (porcine aorta) in physiological saline solution, and the adhesion was then evaluated by a pressure resistance test.

[0161] First, the laminates prepared in each example were punched out with a dumbbell cutter to create circular evaluation samples with a diameter of 15 mm. Next, on a hot plate heated to 37°C, the evaluation samples (laminateds) from each example and comparative example were placed in a petri dish (35 mm in diameter) containing 3 mL of physiological saline, so as to cover the central defect (1 mm in diameter) of the porcine aorta (35 mm in diameter). At this time, sheet (I) of the laminate was placed on the bottom side (the side in contact with the porcine aorta). Next, the evaluation sample on the porcine aorta was covered from above with a Unipack (made of polyethylene), a 50 g weight was placed on top of the Unipack, and it was left to stand for 10 minutes.

[0162] After standing, the Unipack was peeled off the evaluation sample, and the sealing strength (pressure resistance) was measured according to ASTM (F2392-04). As shown in Figure 5A, the pig aorta 20 on which the evaluation sample 10 was placed was fixed to a jig, and 37°C physiological saline solution was flowed at a flow rate of 2 mL / min towards the defect 20a covered by the evaluation sample 10 (laminated structure) (in the direction indicated by the arrow in Figure 5A), and the pressure at which the evaluation sample 10 peeled off was measured. In each experiment, three sheets were prepared and the pressure resistance test was performed three times. The results are shown in Table 2.

[0163] As shown in Table 2, the laminates containing porous sheets (I) with relatively low raw material liquid gelatin concentrations, prepared in Comparative Examples 3-2, 2-2, 3-1, and 2-1, peeled off from the porcine aorta when the Unipack was removed, making it impossible to measure the pressure resistance strength (evaluation result: ND). In other words, sufficient adhesion could not be obtained with these porous sheets. For this reason, pressure resistance tests were not performed on the laminates of Comparative Examples 3-2, 2-2, 3-1, and 2-1 from the second test onward.

[0164] On the other hand, the laminates prepared in Examples 5-1, 4-1, and 6-1 to 6-3, which included porous sheets (I) with relatively high gelatin concentrations in the raw material liquid, were able to undergo pressure resistance testing by peeling the Unipack from the aorta at least twice. Table 2 shows the highest pressure resistance strength obtained for each laminate. For reference, Table 2 also shows the evaluation results (pressure resistance strength) of the same test performed using commercially available hemostatic agents. The evaluation results of the laminates in Examples 5-1, 4-1, and 6-1 to 6-3 were better than the evaluation results of the commercially available products, confirming that they possess adhesiveness and pressure resistance that are not problematic for practical use.

[0165] The cause of the above test results is presumed to be as follows: In this study, it is presumed that the porous sheet (I) absorbed physiological saline and moisture from the tissue, becoming neutral to slightly alkaline, which allowed the hydrophobic taragelatin to penetrate the tissue, and simultaneously promoted the cross-linking reaction of the hydrophobic taragelatin by the cross-linking agent. In this case, the porous sheets (I) produced in Examples 3-2, 2-2, 3-1, and 2-1, which have a low gelatin concentration in the raw material solution, have low density and many voids. Therefore, when a large amount of moisture (physiological saline) is present in the surrounding area, it rapidly absorbs a large amount of moisture. As a result, before the lower sheet (I) can sufficiently adhere to the biological tissue (porcine aorta), moisture spreads to the upper sheet (II), causing the upper sheet (II) to become sticky. When the Unipack was peeled off, the entire laminate was likely removed from the porcine aorta. On the other hand, the porous sheets (I) prepared in Examples 5-1, 4-1, and 6-1 to 6-3, which have a high gelatin concentration in the raw material solution, have a high density and few voids. Therefore, even when there is a large amount of moisture in the surroundings (physiological saline and moisture in the tissue), the rate of moisture absorption is relatively slow. For this reason, moisture does not reach the upper sheet (II), and the lower sheet (I) is thought to have adhered to the biological tissue (porcine aorta) with sufficient strength.

[0166] <<Laminate>> In Examples 7-1 to 7-7, 8-1, and 9-1 described below, a pH buffer was used to prepare the laminate when preparing sheet (II). For comparison, in Example 7-8, a laminate was prepared under the same conditions as in Example 7-1, except that a pH buffer was not used when preparing sheet (II). Table 3 shows the details of the laminates prepared in each experiment.

[0167] [Example 7-1] <Synthesis of hydrophobic gelatin derivatives> The hydrophobized gelatin derivative was synthesized using the same method as in Experiment 1-1, except that decanoic acid anhydride was used as the hydrophobizing agent and the amount of alkyl acid anhydride used was adjusted so that the hydrocarbon group introduction rate (decanoyl group introduction rate) DS was 12 mol%. Hereinafter, the hydrophobized gelatin derivative synthesized in this experiment may be referred to as 12C10-ApGltn or 12C10.

[0168] <Creating Sheet (I)> The raw material solution (I) was prepared in the same manner as in Experiment 1-1, except that 12C10-ApGltn was used as the hydrophobic gelatin derivative (gelatin concentration: 7.5 w / v%, NHS / NH2 = 50 mol%). A porous sheet (sheet (I)) was then prepared by freeze-drying the prepared raw material solution in the same manner as in Experiment 1-1, except that the thickness was set to 0.5 mm.

[0169] <Preparation of Sheet (II)> First, 3.39 g of disodium hydrogen phosphate dodecahydrate and 0.08 g of sodium dihydrogen phosphate were dissolved in 100 mL of pure water to prepare a 0.1 mol / L phosphate buffer solution (pH 8). Next, raw material gelatin (Org-ApGltn) was dissolved in the phosphate buffer solution to prepare a 10 w / v% raw material solution (II). The prepared raw material solution (II) was cast into a silicone sheet mold (70 mm × 70 mm × depth: 0.5 mm). Using the same freeze-drying oven as used in Experiment 1-1, the sample was first frozen overnight at -30°C and then freeze-dried. After freeze-drying, it was heated at 150°C for 5 hours to thermally crosslink the Org-ApGltn and obtain sheet (II).

[0170] <Fabrication of laminates> The laminate for this experiment was obtained by stacking sheet (II) on top of sheet (I).

[0171] [Example 7-2] The laminates used in this experiment were prepared in the same manner as in Example 7-1, except that the raw material solution (I) was prepared so that NHS / NH2 = 75 mol% in the preparation of sheet (I).

[0172] [Example 7-3] The laminates used in this experiment were prepared in the same manner as in Example 7-1, except that the raw material solution (I) was prepared so that NHS / NH2 = 100 mol% in the preparation of sheet (I).

[0173] [Example 7-4] In preparing sheet (I), the laminate for this experiment was prepared in the same manner as in Example 7-1, except that the raw material solution (I) was prepared so that the gelatin concentration was 15 w / v%.

[0174] [Examples 7-5] In the preparation of sheet (I), the laminate for this experiment was prepared using the same method as in Example 7-1, except that the weight-average molecular weight of the crosslinking agent (4S-PEG) used was set to 10,000.

[0175] [Examples 7-6] In the preparation of sheet (I), the laminate for this experiment was prepared in the same manner as in Example 7-2, except that the weight-average molecular weight of the crosslinking agent (4S-PEG) used was set to 10,000.

[0176] [Example 7-7] In the preparation of sheet (I), the laminate for this experiment was prepared using the same method as in Example 7-3, except that the weight-average molecular weight of the crosslinking agent (4S-PEG) used was set to 10,000.

[0177] [Examples 7-8] A laminate was produced under the same conditions as in Example 7-1, except that pure water was used instead of a pH buffer solution during the production of Sheet (II).

[0178] [Experiment 8-1] <Synthesis of Hydrophobized Gelatin Derivative> A hydrophobized gelatin derivative was synthesized in the same manner as in Experiment 1-1, except that lauric anhydride was used as the hydrophobizing reagent and the amount of the alkyl acid anhydride was adjusted so that the hydrocarbon group introduction rate (lauroyl group introduction rate) DS was 9 mol%. Hereinafter, the hydrophobized gelatin derivative synthesized in this experiment may be referred to as 9C12-ApGltn or 9C12.

[0179] <Production of Laminate> In the production of Sheet (I), the laminate of this experiment was produced in the same manner as in Example 7-1, except that the hydrophobized gelatin derivative 9C12-ApGltn was used.

[0180] [Experiment 9-1] <Synthesis of Hydrophobized Gelatin Derivative> A hydrophobized gelatin derivative was synthesized in the same manner as in Experiment 1-1, except that octanoic anhydride was used as the hydrophobizing reagent and the amount of the alkyl acid anhydride was adjusted so that the hydrocarbon group introduction rate (octanoyl group introduction rate) DS was 13 mol%. Hereinafter, the hydrophobized gelatin derivative synthesized in this experiment may be referred to as 13C8-ApGltn or 13C8.

[0181] <Production of Laminate> In the production of Sheet (I), the laminate of this experiment was produced in the same manner as in Example 7-1, except that the hydrophobized gelatin derivative 13C8-ApGltn was used.

[0182] [Table 3]

[0183] [Evaluation] (1) Adhesiveness Comparative Test The adhesiveness comparison test performed on the laminates prepared in Examples 7-1 to 7-8, 8-1, and 9-1 was carried out for the laminates such as Example 4-1 described above. The adhesiveness of the laminates of all the examples shown in Table 3 was lower compared to the porous sheet of the single-layer body (Example 3-1). From this result, when these laminates are used as a bio-tissue adhesion film, suppression of adhesion to other bio-tissues, suppression of adhesion to rubber gloves during surgery, etc. can be expected.

[0184] (2) Pressure resistance test 2 The pressure resistance test 2 performed on the laminates prepared in Examples 7-1 to 7-8, 8-1, and 9-1 was carried out for the laminates such as Example 4-1 described above. This test is a test conducted in physiological saline assuming a living body in which bleeding etc. occurs and a large amount of blood, body fluid, etc. are present. In the test for the laminates such as Example 4-1, the adhesion time to a bio-tissue (porcine aorta) was set to 10 minutes (see Table 2), whereas in the test for the laminates such as Example 7-1, the same adhesion time was shortened to 3 minutes. The results are shown in Table 3. For reference, the evaluation results (pressure resistance) of the same test conducted using a commercially available hemostatic agent with an adhesion time of 10 minutes are also shown in Table 3.

[0185] As shown in Table 3, the evaluation results of the laminates of all the experiments except Example 7-8 were better than the evaluation results of the commercially available product, and it was confirmed that they had adhesiveness and pressure resistance without practical problems. In all the examples except Example 7-8, since a pH buffer solution (pH 8) was used for the preparation of Sheet (II), Sheet (II) contains a basic compound (a mixture of phosphoric acid and sodium phosphate). As a result, it is presumed that the pH inside Sheet (I) rises earlier, the crosslinking reaction (hardening reaction) between the hydrophobized gelatin derivative and the crosslinking agent is promoted, and good evaluation results were obtained even when the adhesion time was shortened to 3 minutes.

[0186] On the other hand, in the laminates of Example 7-8, where pH buffer was not used in the preparation of sheet (II) (i.e., laminates that do not contain basic compounds), sufficient adhesive strength could not be obtained with a short bonding time of 3 minutes, and the pressure resistance could not be measured (evaluation result: ND). Furthermore, when the same test was performed on laminates such as Example 4-1, which were not prepared using pH buffer as shown in Table 2, with the bonding time shortened to 3 minutes, sufficient adhesive strength could not be obtained with such a short bonding time, and, similar to Example 7-8, the pressure resistance could not be measured (evaluation result: ND). [Industrial applicability]

[0187] The porous sheet of the present invention can be used as a tissue adhesion membrane (e.g., hemostatic agent, adhesion prevention agent) for use in living organisms, possessing high adhesive strength and high biocompatibility. For this reason, the porous sheet of the present invention can be used in surgical and internal medicine fields. [Explanation of Symbols]

[0188] 10 Porous Sheet (Sheet (I)) 20. Living tissue (porcine aorta) 20a Defect 30. Sheet containing cross-linked raw gelatin (Sheet (II)) 31 Basic Compounds 50, 51, 52 laminated bodies

Claims

1. A tissue adhesive film, At least one sheet (I) and A sheet (II) containing a crosslinked product of unhydrophobized gelatin, The aforementioned at least one sheet (I) is A hydrocarbon group is introduced into gelatin, as shown in formula (1) below. A hydrophobic gelatin derivative containing the structure represented by, Unreacted crosslinking agent dispersed in the hydrophobic gelatin derivative, It is a porous sheet containing, The density of at least one sheet (I) is 0.07 g / cm³. ３ It is ~0.2 g / cm³, A tissue adhesive film in which at least one sheet (I) is laminated on one surface of sheet (II). 【Chemistry 1】 In formula (1), Gltn represents a gelatin residue, L represents a single bond or a divalent linking group, R1 is a hydrocarbon group having 1 to 20 carbon atoms, and R2 is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms.

2. The tissue adhesion film according to claim 1, wherein the crosslinked product of the unhydrophobic gelatin is a thermally crosslinked product of the unhydrophobic gelatin.

3. The tissue adhesion film according to claim 1, wherein the sheet (II) is porous.

4. The tissue adhesion film according to claim 1, wherein the sheet (II) further contains a basic compound that exhibits basicity upon contact with moisture.

5. The tissue adhesion film according to claim 4, wherein the basic compound is a compound whose aqueous solution at a concentration of 0.05 mol / L to 0.5 mol / L has a pH of 7.5 to 10.

6. The tissue adhesion film according to claim 4, wherein the basic compound is a pH buffering agent.

7. The tissue adhesion film according to claim 6, wherein the pH buffer is a mixture of phosphoric acid and sodium phosphate.

8. The aforementioned at least one sheet (I) includes two sheets (I), Of the two sheets (I), one is laminated on one surface of sheet (II). The tissue adhesive film according to claim 1, wherein the other is laminated on the other surface of the sheet (II).

9. A tissue adhesion film according to claim 1, for use as a hemostatic agent.

10. A tissue adhesive film according to claim 1, for preventing adhesion to other tissues when adhered to tissue.

11. The tissue adhesion film according to claim 1, wherein in formula (1), L is a single bond or -C(O)-.

12. The tissue adhesion film according to claim 11, wherein L is a single bond.

13. The tissue adhesion film according to claim 1, wherein in formula (1), R1 is a linear alkyl group and R2 is a hydrogen atom.

14. The tissue adhesion film according to claim 13, wherein R1 is a linear alkyl group having 6 to 12 carbon atoms.

15. The tissue adhesion film according to claim 1, wherein the hydrocarbon group introduction rate of the hydrophobic gelatin derivative is 5 mol% to 50 mol%.

16. The tissue adhesion film according to claim 1, wherein the gelatin is gelatin derived from cold water fish.

17. The tissue adhesion film according to claim 1, wherein the crosslinking agent is a compound having at least two crosslinkable groups.

18. The tissue adhesion film according to claim 17, wherein the amount of crosslinking groups in the crosslinking agent is 0.25 to 2 equivalents relative to 1 equivalent of amino groups in the hydrophobic gelatin derivative.

19. The tissue adhesive film according to claim 1, wherein the thickness of the porous sheet is 1 mm or more.

20. A method for manufacturing a porous sheet, The porous sheet is A hydrophobic gelatin derivative having a structure represented by the following formula (1), in which a hydrocarbon group is introduced into gelatin, Unreacted crosslinking agent dispersed in the hydrophobic gelatin derivative, Includes, The aforementioned manufacturing method is To prepare a raw material solution (I) with a pH of 5 or less, comprising the hydrophobic gelatin derivative, the crosslinking agent, an acid, and a solvent. The aforementioned raw material liquid (I) is freeze-dried in sheet form to obtain the porous sheet. A method for manufacturing a porous sheet, including the following. 【Chemistry 2】 In formula (1), Gltn represents a gelatin residue, L represents a single bond or a divalent linking group, R1 is a hydrocarbon group having 1 to 20 carbon atoms, and R2 is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms.

21. The method for producing a porous sheet according to claim 20, wherein the concentration of the hydrophobic gelatin derivative in the raw material liquid (I) is 1 w / v% to 20 w / v%.

22. A method for producing a tissue adhesive film, comprising the step of producing a porous sheet by the method described in claim 20 or 21.

23. A method for manufacturing tissue adhesion films, A porous sheet (I) is manufactured by the method described in claim 20 or 21, To manufacture a sheet (II) containing a crosslinked product of unhydrophobized gelatin, Laminating the aforementioned sheet (I) and the aforementioned sheet (II), A method for producing a tissue adhesion film, including the following.

24. Manufacturing the aforementioned sheet (II) To prepare a raw material solution (II) containing the aforementioned unhydrophobized gelatin, a basic compound, and a solvent, The aforementioned raw material liquid (II) is freeze-dried into a sheet to form a precursor sheet, The precursor sheet is heated to thermally crosslink the unhydrophobic gelatin to obtain the sheet (II), A method for producing a tissue adhesion film according to claim 23, comprising the above.

25. The method for producing a tissue adhesion film according to claim 24, wherein the basic compound is a pH buffering agent.

26. The method for producing a tissue adhesion film according to claim 25, wherein the pH buffer is a mixture of phosphoric acid and sodium phosphate.

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