Porous sheet, tissue adhesive film, use thereof as hemostatic agent or Anti-adhesion material, and methods for producing same

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

Porous sheet, tissue adhesive film, use thereof as a hemostatic agent or anti-adhesion material, and method for producing the same

[0001] The present invention relates to a porous sheet, a tissue adhesive film, their use as a hemostatic agent or an adhesion barrier, and methods for producing them.

[0002] The tissue adhesive film is a polymer film that can adhere to biological tissues such as blood vessels and skin, thereby preventing blood leakage during, for example, cardiovascular surgery, thereby enhancing the safety of the surgery. In this regard, the present inventor has reported a non-porous tissue adhesive film comprising an accumulation of gelatin and / or a hydrophobized gelatin derivative and crosslinking molecules (Patent Document 1).

[0003] Patent No. 5747264

[0004] The present invention provides a porous sheet that has high biocompatibility and adhesive strength and is applicable to tissue adhesive films, in accordance with the above-mentioned state of the art.

[0005] As a result of extensive research into achieving the above object, the present inventors have found that the above object can be achieved by the following configuration.

[0006] [1] A hydrophobic gelatin derivative in which a hydrocarbon group is introduced into gelatin, the hydrophobic gelatin derivative having the formula (1): [In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, R1 represents a hydrocarbon group having 1 to 20 carbon atoms, and R2 represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms], and an unreacted crosslinking agent dispersed in the hydrophobic gelatin derivative. [2] A porous sheet comprising: a hydrophobic gelatin derivative having a structure represented by the following formula (1): [In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, R1 represents a hydrocarbon group having 1 to 20 carbon atoms, and R2 represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms], and an unreacted crosslinking agent dispersed in the hydrophobic gelatin derivative. ３ ~0.3 g / cm ３ [3] The porous sheet according to [1], wherein the density of the at least one sheet (I) is 0.05 g / cm ３ ~0.08g / cm ３[4] The tissue adhesive film according to [2], wherein L is a single bond or —C(O)—. [5] The porous sheet according to [4], wherein L is a single bond. [6] R １ is a straight chain alkyl group, and R ２ [7] The porous sheet according to any one of [1] to [5], wherein R is a hydrogen atom. １is a linear alkyl group having 6 to 12 carbon atoms. [8] The porous sheet according to any one of [1] to [7], wherein the hydrocarbon group introduction rate of the hydrophobized gelatin derivative is 5 mol% to 50 mol%. [9] The porous sheet according to [8], wherein the hydrocarbon group introduction rate of the hydrophobized gelatin derivative is 5 mol% to 10 mol%.

[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 crosslinkable groups of the crosslinking agent are 0.25 equivalents to 2 equivalents per equivalent of amino groups in the hydrophobized gelatin derivative.

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

[12] , wherein the thickness is 1 mm or more.

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

[13] for use as a tissue adhesive material.

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

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

[16] Use of the porous sheet according to any one of [1] to

[13] for preparing a tissue adhesive material.

[17] Use of the porous sheet according to any one of [1] to

[13] for preparing a hemostatic agent, sealant (closure material), or dressing material.

[18] A method for covering tissue requiring coverage (e.g., anastomoses of nerves, tendons, etc., or wounds occurring in tissues (pulmonary pleura, gastrointestinal anastomoses, oral and periodontal tissues, etc.)) with the porous sheet according to any one of [1] to

[13] .

[19] A method for hemostasis of tissue, comprising adhering the porous sheet according to any one of [1] to

[13] to tissue requiring or expected to require hemostasis.

[20] A method for filling a gap between dura maters, a dura mater suture, or a gap between a dura mater formation material and the dura mater, using the porous sheet according to any one of [1] to

[13] .

[21] A tissue adhesive film comprising the porous sheet according to any one of [1] to

[13] .

[22] A hemostatic agent, sealant (closure material), or covering material comprising the porous sheet according to any one of [1] to

[13] .

[23] A tissue adhesive film comprising at least one sheet (I) which is the porous sheet according to any one of [1] to

[13] , and a sheet (II) comprising a crosslinked product of non-hydrophobized gelatin, wherein the at least one sheet (I) is laminated on one side of the sheet (II).

[24] A laminate comprising at least one sheet (I) which is the porous sheet according to any one of [1] to

[13] , and a sheet (II) comprising a crosslinked product of non-hydrophobized gelatin, wherein the at least one sheet (I) is laminated on one side of the sheet (II).

[25] The laminate according to

[24] for use as a tissue adhesive material.

[26] The density of the sheet (I) is 0.07 g / cm. ３

[27] The tissue adhesive film or laminate according to any one of

[23] to

[26] , wherein the crosslinked product of non-hydrophobized gelatin is a thermally crosslinked product of non-hydrophobized gelatin.

[28] The tissue adhesive film or laminate according to any one of

[23] to

[27] , wherein the sheet (II) is porous.

[29] The density of the sheet (I) is 0.07 g / cm or more. ３ ~0.2 g / cm ３

[30] The tissue adhesive film or laminate according to any one of

[23] to

[29] , wherein the sheet (II) further contains a basic compound that exhibits basicity upon contact with water.

[31] The tissue adhesive film or laminate according to

[30] , wherein the basic compound is a compound whose aqueous solution of 0.05 mol / L to 0.5 mol / L exhibits a pH of 7.5 to 10.

[32] The tissue adhesive film or laminate according to

[30] or

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

[33] The tissue adhesive film or laminate according to

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

[34] The tissue adhesive film or laminate according to any one of

[23] to

[33] , wherein the at least one sheet (I) comprises two sheets (I), one of which is laminated on one side of the sheet (II) and the other is laminated on the other side of the sheet (II).

[35] A hemostatic agent, sealant (closure material), or dressing material comprising the tissue adhesive film or laminate according to any one of

[23] to

[34] .

[36] An adhesion barrier comprising the tissue adhesive film or laminate according to any one of

[23] to

[34] .

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

[23] to

[34] , for use as a hemostatic agent, sealant (closure material), or tissue dressing material, or for filling a gap between dura maters, a dura suture, or a gap between a dura mater-forming material and the dura mater, or for covering a tissue, for hemostasis.

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

[22] to

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

[39] Use of the tissue adhesive film or laminate according to any one of

[23] to

[34] for preparing a hemostatic agent, a sealant (closure material) or a tissue dressing material.

[40] Use of the tissue adhesive film or laminate according to any one of

[23] to

[34] for preparing a tissue adhesion preventive material.

[41] A method of covering tissue, comprising covering a tissue that requires coverage with the tissue adhesive film or laminate according to any one of

[23] to

[34] .

[42] A method for filling gaps, comprising filling gaps between dura maters, dura mater sutures, or gaps between a dura mater and a dura mater formation material, with the tissue adhesive film or laminate according to any one of

[23] to

[34] .

[43] A method for tissue hemostasis, comprising adhering the tissue adhesive film or laminate according to any one of

[23] to

[34] to tissue that requires or is expected to require hemostasis.

[44] A method for preventing tissue adhesion, comprising adhering the tissue adhesive film or laminate according to any one of

[23] to

[34] to tissue that requires prevention of adhesion to other tissues.

[45] A method for producing a porous sheet according to any one of [1] to

[13] , comprising mixing the hydrophobicized gelatin derivative, the crosslinking agent, an acid, and a solvent to prepare a raw material solution (I) having a pH of 5 or less, and freeze-drying the raw material solution (I) to obtain the porous sheet.

[46] The method according to

[45] , wherein the concentration of the hydrophobized gelatin derivative in the starting material solution (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 according to

[45] or

[46] .

[48] A method for producing a tissue adhesive film, comprising the steps of producing sheet (I), the porous sheet, by the method according to

[45] or

[46] , producing sheet (II), which contains a crosslinked product of non-hydrophobized gelatin, and laminating sheet (I) and sheet (II).

[49] The method according to

[48] , wherein sheet (II) is produced by: preparing starting material solution (II), which contains the non-hydrophobized gelatin, a basic compound, and a solvent; freeze-drying the starting material solution (II) into a sheet form to form a precursor sheet; and heating the precursor sheet to thermally crosslink the non-hydrophobized gelatin.

[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.

[0007] 1 is a cross-sectional schematic diagram of a porous sheet of a first embodiment. The photograph on the left is a porous sheet before being attached to biological tissue, and the photograph on the right is a porous sheet attached to biological tissue. The schematic diagram on the left is a schematic diagram of a hydrophobic gelatin derivative and a cross-linking agent existing in an unreacted state in the porous sheet before being attached to biological tissue, and the schematic diagram on the right is a schematic diagram showing an example of a cross-linking reaction between a hydrophobic gelatin derivative and a cross-linking agent in the porous sheet attached to biological tissue.

[0033] FIG. 1 is a flowchart showing a method for manufacturing a porous sheet of a first embodiment.

[0034] FIG. 1 is a cross-sectional schematic diagram of a laminate of a second embodiment.

[0035] FIG. 1 is a photograph (left) and a scanning electron microscope (SEM) photograph (right) of a porous sheet (top surface) prepared in an example.

[0036] FIG. 2 is a diagram explaining a pressure resistance strength test method in an example.

[0037] FIG. 3 is a diagram showing the results of pressure resistance strength test 1 in an example.

[0038] FIG. 4 is a photograph of a laminate prepared in an example.

[0039] FIG. 5 is a flowchart showing a method for manufacturing a laminate of a second embodiment.

[0039] FIG. 6 is a cross-sectional schematic diagram of a laminate of a first modified example of the second embodiment, explaining how the laminate is attached to biological tissue. FIG. 10 is a cross-sectional schematic view of a laminate according to Modification 2 of the second embodiment.

[0008] The present invention will be described in detail below. The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment.

[0009] [Definition of Terms] In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0010] "Gelatin" generally refers to a polymer that has been denatured by treating natural or synthetic collagen with heat, acid, or alkali to unravel the triple helix structure. In this specification, the term "gelatin" refers to gelatin that has not been subjected to a "hydrophobic" treatment and to which no alkyl groups have been introduced. Therefore, in this specification, "gelatin" refers to "ApGltn" or "GltnNH ２" Depending on the context, the terms "raw gelatin" or "unhydrophobized gelatin" are 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." As used herein, "hydrophobization" refers to a process of increasing the hydrophobicity of "gelatin" by introducing a hydrocarbon group into "gelatin," more specifically, by binding a hydrocarbon group to an amino group via a linking group or directly. Therefore, as used herein, "hydrophobized gelatin derivative" refers to a derivative of "gelatin" in which the hydrophobicity of "gelatin" has been increased by introducing a hydrocarbon group into "gelatin," more specifically, by binding a hydrocarbon group to an amino group via a linking group or directly, and is "GltnNH-L-CHR １ R ２ " is sometimes expressed as ".

[0011] 1A and 1B, a porous sheet 10 will be described. The porous sheet 10 includes a hydrophobic gelatin derivative (second gelatin) in which a hydrocarbon group has been 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 FIG. 1B , within the porous sheet 10, the crosslinking agent is dispersed in the hydrophobicized gelatin derivative, and the crosslinking agent and the hydrophobicized gelatin derivative are substantially unreacted. Moisture is required for the crosslinking reaction (hardening reaction) between the hydrophobicized gelatin derivative and the crosslinking agent, and the porous sheet 10 must be in a neutral to alkaline environment. Since biological tissue (e.g., the porcine aorta 20 shown in FIG. 1B ) typically contains neutral moisture, the porous sheet 10 absorbs neutral moisture when attached to the biological tissue 20, crosslinking occurs, and the porous sheet 10 adheres to the biological tissue 20. As shown in the schematic diagram on the right side of FIG. 1B , the crosslinking reaction typically occurs between a primary amino group in the hydrophobicized gelatin derivative and a crosslinkable group (typically, an active ester group, etc.) in the crosslinking agent.

[0013] The porous sheet 10 maintains the structure of the original gelatin, resulting in high biocompatibility. Furthermore, hydrophobic groups create hydrophobic interactions between the porous sheet 10 and the biological tissue 20. Furthermore, the hydrophobic groups penetrate the biological tissue 20 at the molecular level (anchoring), forming a strong physical bond between the porous sheet 10 and the biological tissue 20, 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. Furthermore, the porous structure of the porous sheet 10 provides improved flexibility and / or pliability compared to non-porous films, making it possible to bend the sheet to conform to the surface shape of the biological tissue 20 or to form a cylindrical shape to cover anastomotic sites such as nerves and tendons. Furthermore, the porous structure of the porous sheet 10 allows it to efficiently absorb moisture when applied to the biological tissue 20, which is thought to promote the hardening reaction (crosslinking reaction) between the hydrophobic gelatin derivative and the crosslinking agent. The porous sheet 10 is described in detail below.

[0014] As mentioned above, the hydrophobic gelatin derivative (second gelatin) means a gelatin derivative in which a hydrocarbon group has been introduced into raw material gelatin (first gelatin), and has a structure represented by the following formula (1).

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

[0016] In formula (1), GltnNH- is a structure derived from the raw gelatin described above. Therefore, the raw gelatin is GltnNH ２ The raw material gelatin will be described in detail later. １ R ２ is a hydrocarbon group introduced into the starting gelatin, and is introduced into the starting gelatin via L (a single bond or a divalent linking group).

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

[0018] In formula (1), -CHR １ R ２ The (hydrocarbon group) is preferably bonded to the ε-amino group of the starting gelatin, and more preferably to the ε-amino group of lysine (Lys) in the starting gelatin. The *-CHR １ R ２ Examples of the method for bonding include a method utilizing the so-called reductive amination reaction (a method using an aldehyde or a ketone) and a method utilizing the Schotten-Baumann reaction (a method using an acid chloride).

[0019] The —NH— structure (secondary amino group) of formula (1) has, for example, a peak at 3300 cm in the FT-IR (Fourier transform infrared) spectrum. －１ It can be detected by nearby bands.

[0020] In formula (1), R １ and R ２ The hydrocarbon group having 1 to 20 carbon atoms is not particularly limited, and examples thereof include a chain hydrocarbon group having 1 to 20 carbon atoms, an alicyclic hydrocarbon group having 3 to 20 carbon atoms, an aromatic hydrocarbon group having 6 to 14 carbon atoms, and groups combining these.

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

[0022] Examples of the chain hydrocarbon group having 1 to 20 carbon atoms include, but are not limited to, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an octyl group (or a capryl group), a nonyl group (or a pelargornyl group), a decyl group, a dodecyl group (or a lauryl group), and a tetradecyl group (or a myristyl group).

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

[0024] The aromatic hydrocarbon group having 6 to 14 carbon atoms is not particularly limited, but examples thereof include a phenyl group, a tolyl group, and a naphthyl group.

[0025] The group obtained by combining the above is not particularly limited, but examples thereof include aralkyl groups having 6 to 12 carbon atoms, such as a benzyl group, a phenethyl group, a naphthylmethyl group, and a naphthylethyl group.

[0026] From the viewpoint of enhancing the adhesiveness of the porous sheet 10, R １ is a straight chain alkyl group, and R ２ is preferably a hydrogen atom. １ 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 the following formulas (2) and (3), and the gelatin derivative represented by formula (2) is more preferred.

[0028]

[0029] Formula (2) is the case where L is a single bond in formula (1), and １ R ２) is introduced into the gelatin residue Gltn of the raw gelatin through an imino bond (-NH-), which is a secondary amine. Formula (3) is the case where L is -C(O)- in formula (1), and a hydrocarbon group (-CHR １ R ２ ) is introduced into the gelatin residue Gltn of the starting gelatin via an amide bond (—NHCO—). In formulas (2) and (3), the meanings of each symbol are the same as those in formula (1) already explained, and the preferred embodiments are also the same.

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

[0031] Here, the amino group (-NH ２ ) content in the hydrophobicized gelatin derivative relative to the content of imino groups (—NH—CHR １ R ２ The molar ratio of the content of each of the hydrocarbon groups is defined as the "degree of substitution" (hereinafter, sometimes simply referred to as "DS").

[0032] From the viewpoint of enhancing the adhesiveness of the porous sheet, the hydrocarbon group introduction rate of the hydrophobicized 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) in the hydrophobicized 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 hydrophobicized gelatin derivative is within the above range, a porous sheet 10 having high adhesiveness is easily obtained.

[0033] The hydrocarbon group introduction rate can be calculated from the values ​​obtained by quantifying the number of amino groups in raw gelatin and the number of amino groups in the hydrophobicized gelatin derivative by the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method) using the following formula: Hydrocarbon group introduction rate (mol %) of hydrophobicized gelatin derivative = [number of amino groups in raw gelatin - number of amino groups in hydrophobicized gelatin derivative] / [number of amino groups in raw gelatin] x 100

[0034] The molecular weight of the hydrophobicized gelatin derivative 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 the weight average molecular weight (Mw) of the hydrophobicized gelatin derivative is almost the same as the range of the weight average molecular weight (Mw) of the raw material gelatin, which will be described later.

[0035] The hydrophobic gelatin derivatives described above may be commercially available products or may be self-synthesized products. The hydrophobic gelatin derivatives may 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 WO 2020 / 137903. The content of the synthesis method for hydrophobic gelatin derivatives disclosed in WO 2020 / 137903 is incorporated herein by reference.

[0036] The raw material gelatin (first gelatin) may be obtained from naturally occurring collagen or from synthetic collagen (including fermented and recombinant collagen). It may also be gelatin obtained from naturally occurring or synthetic collagen that has been subjected to some kind of treatment (excluding hydrophobic treatment). Gelatin obtained by treating naturally occurring collagen obtained from the skin, bones, tendons, etc. of mammals, birds, fish, etc. with acid or alkali (and optionally heat-extracting) is preferred. Among these, gelatin obtained by alkali-treating naturally occurring collagen is particularly preferred, as it provides a porous sheet with superior effects of the present invention. In this specification, the term "naturally occurring gelatin" refers to gelatin obtained by treating naturally occurring collagen with heat, acid, or alkali. For example, the term "fish-derived gelatin" refers to gelatin obtained by treating fish-derived collagen with heat, acid, or alkali.

[0037] It is also preferable to use endotoxin-reduced gelatin, which has a reduced endotoxin content. Gelatin raw materials vary depending on the climate and production environment of the country of production, the animal of origin, the extraction method, and the cleanliness of the equipment used. Generally, "endotoxin-reduced gelatin" refers to gelatin that contains 1,000 to 100,000 EU / g of endotoxin and has been subjected to some kind of endotoxin-reducing treatment. "Endotoxin-reduced gelatin" typically 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 endotoxin-reduced gelatin is not particularly limited, and known gelatins can be used, including, for example, the gelatin described in JP 2007-231225 A, the contents of which are incorporated herein by reference.

[0038] Examples of mammalian-derived gelatin include porcine and bovine gelatin. Fish-derived gelatin is not particularly limited, but gelatin derived from cold-water fish (cold-water fish) such as salmon, trout, Pacific 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 and maintains fluidity even at low temperatures. Therefore, by using a hydrophobic derivative of cold-water fish-derived gelatin as the hydrophobic gelatin derivative, the crosslinker can be easily and uniformly dispersed even at low temperatures, and the porous sheet 10 of this embodiment can be efficiently produced by lyophilization (freeze-drying), as described below. Furthermore, it has the characteristic of easily absorbing moisture when applied to a living body.

[0039] Cold-water fish-derived gelatin is a polymer in which two or more amino acids are linked in a linear chain, and contains 190 or less imino acids, more specifically, 80 or less hydroxyprolines and 110 or less prolines per 1000 constituent amino acids. The room temperature fluidity of cold-water fish-derived gelatin is thought to be due to the number of hydroxyprolines being 80 or less or the number of prolines being 110 or less. If either of these conditions is met, the denaturation temperature is thought to be below room temperature, resulting in room temperature fluidity.

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

[0041] Furthermore, gelatin derived from cold-water fish has an amino acid sequence similar to that of gelatin derived from animals, is easily decomposed by enzymes in the body, and is highly biocompatible.

[0042] The molecular weight of the starting 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 means 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 may be a mixture of two or more types of gelatin.

[0044] The crosslinking agent is typically a compound having at least two substituents (crosslinkable groups) in one molecule that can react with the primary amino groups of the hydrophobicized gelatin derivative.

[0045] The crosslinkable group possessed by the crosslinking agent is not particularly limited, but an active ester group (activated ester group) is preferred from the viewpoint of being easily reactive selectively with the primary amino group of the hydrophobicized gelatin derivative under mild conditions. That is, the crosslinking agent is preferably a compound having at least two active ester groups in one molecule. 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, diisothiocyanates, and the like can be used as crosslinking agents.

[0047] Examples of polybasic acids include tartaric acid, citric acid, malic acid, glutaric acid, glutamic acid, aspartic acid, oxaloacetic acid, 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] As the crosslinking agent, disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST), etc. can also be used.

[0049] Also available are polybasic acid esters of polyethylene glycol or polyethylene glycol ether in which at least one carboxyl group of the polybasic acid that has not reacted with polyethylene glycol has been activated, such as 4,7,10,13,16-pentaoxanonadecanedioic acid di(N-succinimidyl) and polyethylene glycol di(succinimidyl succinate) (SS-PEG-SS) represented by the following formula:

[0050]

[0051] (n is a number that results in a number average molecular weight of about 20,000); and further, pentaerythritol-polyethylene glycol ether tetrasuccinimidyl glutarate (4S-PEG) represented by the following formula:

[0052]

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

[0054] Aldehyde compounds include formyl group-introduced polysaccharides, in which two or more formyl groups have been introduced into one molecule, such as formyl group-introduced starch, formyl group-introduced dextran, formyl group-introduced dextrin, and formyl group-introduced hyaluronic acid.

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

[0056] As the crosslinking agent, activated polyethylene glycol polybasic acid esters and formyl group-introduced polysaccharides are preferred, and activated polyethylene glycol polybasic acid esters are more preferred.

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

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

[0059] The contents of the hydrophobic gelatin derivative and the crosslinking agent in the porous sheet 10 are not particularly limited and may be adjusted appropriately within the range that satisfies the above-mentioned preferred ratio of crosslinkable groups to amino groups. 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 be composed only of a hydrophobic gelatin derivative and a crosslinking agent, or may contain other components in addition to the hydrophobic gelatin derivative and the crosslinking agent as long as the effects of the present invention are achieved. 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] The components other than the hydrophobic gelatin derivative and the crosslinking agent are not particularly limited, but examples thereof include solvents, buffering agents, coloring agents, preservatives, functional nanoparticles, and drugs (anticancer drugs, antithrombotic drugs, antibacterial agents, growth factors, etc.).

[0062] The porous sheet 10 has voids, and therefore has a lower density (apparent density) than a non-porous gelatin sheet. The density of the porous sheet 10 is not particularly limited, but may be, for example, 0.01 g / cm ３ ~0.3g / cm ３ , or 0.02 g / cm ３ ~0.08g / cm ３ The density of the porous sheet 10 may be preferably 0.02 g / cm ３ ~0.1 g / cm ３ and more preferably 0.05 g / cm ３ ~0.1 g / cm ３ and particularly preferably 0.05 g / cm ３ ~0.08g / cm ３When the density is equal to or less than the upper limit of the above range, the porous sheet can have sufficient voids, thereby obtaining appropriate flexibility and / or pliability, and can more efficiently absorb moisture to promote the curing reaction. Furthermore, when the density is equal to or greater than the lower limit of the above range, the porous sheet can obtain a sufficient amount of cured product (crosslinked product) and exhibit strong adhesiveness to biological tissue. Furthermore, the film strength is also increased, and handling properties are improved. The density of the porous sheet 10 can be adjusted, for example, by the gelatin concentration of the raw material solution used in the porous sheet manufacturing method described below.

[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 equal to or greater than the above-mentioned lower limit, the porous sheet can obtain a sufficient amount of cured product (crosslinked product) and exhibit strong adhesiveness to biological tissue. Furthermore, the film strength is increased, and handling properties are improved. The upper limit of the thickness of the porous sheet is not particularly limited, but a practical range for a tissue adhesive film is, for example, 8 mm or less or 5 mm or less. Furthermore, the thinner the thickness, the more improved the flexibility, and, for example, the better the processability when used as a tissue adhesive film. From the viewpoint of improving flexibility, the thickness of the porous sheet may be, for example, 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 unreacted crosslinking agent dispersed in the hydrophobicized gelatin derivative (see FIG. 1B ). This means that most of the crosslinking agent contained in the porous sheet 10 is unreacted. Therefore, for example, a sheet made of a crosslinked hydrophobicized gelatin derivative containing a small amount of unreacted crosslinking agent is different from the porous sheet 10 of this embodiment. Most of the hydrophobicized gelatin derivative contained in the porous sheet of this embodiment is in an uncrosslinked state before use (e.g., before adhesion to the biological tissue 20) and is crosslinked by the crosslinking agent upon use (e.g., adhesion to the 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 sheet] The method for manufacturing the porous sheet 10 is not particularly limited, but it can be manufactured, for example, by a manufacturing method including steps S1 and S2 described below. In the manufacturing method of this embodiment, a sheet with a porous structure (porous sheet) can be efficiently obtained by using freeze-drying. Step S1: Preparation of a raw material solution (acidic), and Step S2: Freeze-drying the raw material solution.

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

[0067] Examples of hydrophobic gelatin derivatives include the embodiments described above for the porous sheet 10, and the same applies to preferred embodiments. Among them, a cold-water fish-derived gelatin derivative in which a hydrocarbon group has been introduced into cold-water fish-derived gelatin (raw material gelatin) is more preferred as the hydrophobic gelatin derivative. If a cold-water fish-derived gelatin derivative is used 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 while maintaining this state (step S2).

[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 a porosity within an appropriate range can be easily obtained.

[0069] The crosslinking agent may be in the form described as a constituent of the porous sheet 10, and the preferred form is also the same. The concentration of the crosslinking agent in the raw material solution is not particularly limited and may be appropriately adjusted to, for example, satisfy the preferred ratio of crosslinkable groups to amino groups described above. The concentration of the crosslinking agent in the raw material solution 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 raw material solution to an acidic state. The preferred pH of the raw material solution at room temperature (20°C) is less than pH 7, pH 6 or less, or pH 5 or less, with no particular lower limit, but for example, pH 1 or more. By making the raw material solution acidic, the nucleophilicity of the amine is suppressed, and the hydrophobicized 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 hydrophobicized gelatin derivative in a substantially unreacted state is obtained.

[0071] The type of acid is not particularly limited as long as it can adjust the pH of the raw material solution to within the above range, but hydrochloric acid, acetic acid, etc. are preferred, and hydrochloric acid is more preferred, from the viewpoint that they evaporate or sublimate during freeze-drying (step S2) and are unlikely to remain in the final product (porous sheet 10). One type of acid may be used alone, or two or more types may be mixed and used. The concentration of the acid in the raw material solution may be appropriately adjusted 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, the crosslinking agent, and the acid. Examples thereof include water (ion-exchanged water, distilled water, pure water), a water-alcohol mixed solvent, a water-acetone mixed solvent, and the like, and among these, water is preferred.

[0073] The raw material liquid may consist of only the hydrophobic gelatin derivative, the crosslinking agent, the acid, and the solvent, or may contain other components. The other components are the same as the other components that can be contained in the porous sheet 10 (e.g., drugs, functional nanoparticles, etc.) that have already been described. The raw material liquid may also contain a porogen as another component that can serve as a template for the pores of the porous structure. As the porogen, a sublimable solid such as ice crystals or t-butanol is preferred.

[0074] The raw material solution may be prepared by uniformly mixing the hydrophobic gelatin derivative, the crosslinking agent, the acid, the solvent, and, if necessary, other components, using a conventionally known method.

[0075] Step S2: Next, the prepared raw material liquid is freeze-dried to obtain a porous sheet 10 (step S2 in FIG. 2). For example, the raw material liquid is poured into a container having the same volume as the desired size of the porous sheet 10, and the solvent (and acid) is evaporated by freeze-drying to obtain a porous sheet 10 of the desired size. The freeze-drying method may be a conventionally known method. The degree of vacuum 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. Note that so-called pre-freezing may be performed prior to freeze-drying.

[0076] [Tissue adhesive film] The porous sheet 10 of this embodiment has high biocompatibility and adhesive strength, and can therefore be used as a tissue adhesive film (tissue adhesive material) for use on biological tissue. Because the porous sheet 10 has high flexibility and / or pliability, it can be bent to conform to the surface shape of biological tissue. Examples of tissue adhesive films include hemostatic materials for stopping bleeding from biological tissue (such as blood vessels), anti-adhesion materials for preventing postoperative adhesions, and wound dressings for covering wounds in biological tissue (such as pulmonary pleura, gastrointestinal anastomoses, oral and periodontal tissues).

[0077] Furthermore, since the porous sheet 10 has excellent adhesive properties as well as excellent absorbency and biocompatibility, it can also be used as a sealant (closure material) to fill gaps between dura maters, dura mater sutures, or gaps between dura mater formation material and the dura mater, for example, when suturing the dura mater.

[0078] The tissue adhesive film may be flat or cylindrical. The porous sheet 10 has high flexibility and can be easily processed into a cylindrical shape. A cylindrical tissue adhesive film can be used to cover an anastomosis site of a nerve, tendon, or the like.

[0079] The tissue adhesive film of this embodiment adheres to biological tissue by being applied 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 form crosslinks, and may be set appropriately depending on the proportions of the constituent materials in the tissue adhesive film, but may be, for example, 1 to 10 minutes. At this time, the tissue adhesive film may be heated to 37°C or below, or a weak alkaline aqueous solution such as a sodium bicarbonate aqueous solution may be poured over it.

[0080] [Second embodiment] <Laminate> In this embodiment, a laminate 50 shown in Fig. 3 will be described. The laminate 50 includes the porous sheet 10 of the first embodiment (hereinafter, may be referred to as "lower layer 10" or "sheet (I)") and a sheet 30 (hereinafter, may be referred to as "upper layer 30" or "sheet (II)") containing a crosslinked product of raw material gelatin (first gelatin) laminated thereon. That is, the lower layer 10 (sheet (I)) is laminated on one surface (the lower 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 with the lower layer 10 side in contact with biological tissue. When attached to the biological tissue, the lower layer 10 absorbs moisture and crosslinks to adhere to the tissue. Because the laminate 50 includes the porous sheet 10 (lower layer 10), it exhibits the same effects as the first embodiment. That is, it has both high biocompatibility and adhesive strength, and further has flexibility and / or pliability.

[0082] Furthermore, the laminated structure makes it difficult for moisture to migrate to the upper layer 30, suppressing stickiness (adhesion) of the surface (upper layer 30) of the laminate 50. This improves handling during surgery, for example. Also, since no hydrophobic groups are introduced into the raw material gelatin of the upper layer 30, it has low adhesiveness to biological tissue. Therefore, when the laminate 50 is attached to biological tissue, its surface (upper layer 30) is less likely to adhere to other biological tissues, and it also functions as an adhesion inhibitor. Details of the laminate 50 of this embodiment are described below.

[0083] The lower layer 10 of the laminate 50 may have the same configuration as the porous sheet 10 of the first embodiment, and the preferred configuration 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 of the first embodiment. ３ ~0.2 g / cm ３ It is preferable that the density is 0.07 g / cm ３ ~0.2 g / cm ３ It is more preferable that the density is less than the upper limit. If the density is less than the upper limit, the laminate 50 can have sufficient voids, thereby providing more appropriate flexibility and further accelerating the curing reaction between the hydrophobic gelatin derivative and the crosslinking agent. Furthermore, if the density is greater than the lower limit, the water absorption rate when the laminate 50 is attached to biological tissue is slowed, making it more difficult for moisture to reach the upper layer 30. Therefore, the curing reaction progresses in the lower layer 10 before much moisture reaches the upper layer 30, allowing for strong adhesion to biological tissue. This allows the laminate 50 to exhibit strong adhesive properties and reduce stickiness (tackiness) of the surface (upper layer 30) even in environments with high moisture content, such as in a living body where large amounts of blood and body fluids are present due to bleeding, etc. As a result, handling is improved and adhesion to other tissues and to rubber gloves during surgery is 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. A thickness equal to or greater than the above-mentioned lower limit value provides strong adhesiveness, even in a humid environment, such as in a living body where a large amount of blood or body fluids is present due to bleeding, while suppressing stickiness (tackiness) of the surface (upper layer 30) of the laminate 50, improving handleability, and further enhancing the ability to prevent adhesion to other tissues. The upper limit of the thickness of the lower layer 10 is not particularly limited, but a practical range for a tissue adhesive film is, for example, 8 mm or less or 5 mm or less. Furthermore, a thinner thickness improves flexibility, improving processability, for example, when used as a tissue adhesive film. From the viewpoint of improving flexibility, the thickness of the lower layer 10 may be, for example, 2 mm or less, 1 mm or less, or 0.5 mm or less.

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

[0087] The upper layer 30 may be composed solely of the cross-linked product of raw gelatin, or may contain other components in addition to the cross-linked product of raw gelatin as long as the effects of the present invention are achieved. The content of the cross-linked product of raw gelatin in the upper layer 30 may be 100% by mass, 95% by mass or more, or 90% by mass or more. Examples of other components in addition to the cross-linked product of raw gelatin include basic compounds, which will be described in Modification 1 below. Examples of other components in addition to basic compounds are not particularly limited, and include, for example, dyes, photocatalysts, colorants, preservatives, inorganic nanoparticles (gold nanoparticles, iron oxide nanoparticles, calcium phosphate nanoparticles, etc.), and pharmaceuticals (anticancer agents, antithrombotic agents, antibacterial agents, growth factors, etc.).

[0088] The upper layer 30 is preferably porous and / or thin. When the upper layer 30 is porous and / or thin, the flexibility of the entire laminate 50 can be further increased. When the upper layer 30 is porous, the density of the upper layer 30 is not particularly limited, but may be, for example, 0.01 g / cm ３ ~0.3 g / cm ３ If the density is equal to or less than the upper limit of the above range, more sufficient flexibility is likely to be obtained. On the other hand, if the density is equal to or more than the lower limit of the above range, the film strength is increased and the handling properties are improved.

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

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

[0091] <Method for Manufacturing Laminate> The method for manufacturing the laminate 50 of this embodiment is not particularly limited, but the laminate 50 can be manufactured, for example, by a manufacturing method including steps S10, S20, and S30 described below (see FIG. 7 ): step S10: manufacturing a lower layer 10 (sheet (I)), step S20: manufacturing an upper layer 30 (sheet (II)), and step S30: laminating the lower layer 10 and the upper layer 30 together.

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

[0093] Step S20: The method for producing the upper layer 30 (sheet (II) containing a cross-linked product of raw material gelatin) is not particularly limited, and it may be produced, for example, by the method described below. First, a raw material gelatin liquid (raw material liquid (II)) containing raw material gelatin and a solvent is prepared. Next, the raw material gelatin liquid is poured into a mold of the desired size, and the solvent is evaporated by freeze-drying to obtain a sheet (precursor sheet) of the desired size. The sheet is heated to thermally cross-link it, thereby obtaining the upper layer 30.

[0094] The solvent for dissolving the raw gelatin may be the same as the solvent for the raw material solution in the first embodiment, and the preferred forms are also the same. The concentration of the 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 the 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 as the freeze-drying method, and the preferred ranges of the degree of vacuum, temperature, and time during freeze-drying are the same as those for the freeze-drying in the first embodiment.

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

[0096] Step 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 is crosslinked, thereby adhering the lower layer 10 and the upper layer 30 together.

[0097] <Modification 1, Laminate 51> As shown in Fig. 8 , in a laminate 51 of this modification, the upper layer (sheet (II)) 30 contains a basic compound 31. The basic compound 31 is a compound that exhibits basicity when in contact with moisture. Other than containing the basic compound 31, the laminate 51 has the same configuration as the laminate 50 of the second embodiment described above. Description of the same configuration as the laminate 50 will be omitted in this modification.

[0098] The laminate 51 of this modification 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 contains the basic compound 31, which provides the following effects. As shown in FIG. 8 , when the laminate 51 is attached to the biological tissue 20, the laminate 51 absorbs moisture from the biological tissue 20, and the absorbed moisture causes the basic compound 31 to elute. This causes the pH in the lower layer 10 to rise more quickly, accelerating the crosslinking reaction (hardening reaction) between the hydrophobic gelatin derivative and the crosslinking agent. In other words, the time (adhesion time) required for obtaining sufficient adhesive strength after the laminate 51 is attached to the biological tissue 20 can be shortened.

[0099] The basic compound 31 is not particularly limited as long as it exhibits basicity upon contact with water. For example, a compound that exhibits a pH of 7.5 to 10 in a 0.05 mol / L to 0.5 mol / L aqueous solution is preferred, and a compound that exhibits a pH of 8.0 to 9.5 in a 0.1 mol / L to 0.3 mol / L aqueous solution is more preferred. A compound with such properties can further shorten the adhesion time. Furthermore, the basic compound 31 is preferably a pH buffer whose aqueous solution functions as a pH buffer. Examples of pH buffers 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 buffer 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. Furthermore, excessive increases in the pH of the lower layer 10 can be prevented, thereby preventing damage to the biological tissue 20.

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

[0101] Examples of the basic compound 31 other than the pH buffer agent include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, zinc hydroxide, etc. These basic compounds 31 may be one type of compound or a mixture of two or more types of compounds.

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

[0103] The density and thickness of the upper layer 30 can be within the ranges described in the second embodiment. Furthermore, when the upper layer 30 contains a basic compound, it is presumed that the higher the density and / or thickness of the upper layer 30, the greater the amount of basic compound contained, and therefore the shorter the bonding time. From the viewpoint of further shortening the bonding time, the density of the upper layer 30 is, for example, 0.08 g / cm 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 laminate 51> The method for manufacturing laminate 51 of this modified example is not particularly limited, and laminate 51 can be manufactured by a method including, for example, steps S10 to S30 shown in Fig. 7, similar to laminate 50 of the second embodiment described above, except that basic compound 31 is used in producing upper layer 30. The manufacturing step for upper layer 30 (step S20 in Fig. 7) will be described below, and a description of the same manufacturing steps as those for laminate 50 will be omitted in this modified example.

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

[0106] Examples of the basic compound include the same compounds as the basic compound 31 contained in the upper layer 30 described above, and preferred embodiments are also the same. By containing a basic compound, the pH of the raw material solution (II) is alkaline. From the viewpoint of shortening the bonding time of the obtained laminate 51, the pH of the raw material solution (II) is preferably, for example, 8 to 10, and more preferably 8 to 9.

[0107] When a pH buffer is used as the basic compound, a mixture of a solvent (e.g., water) and a basic compound is a pH buffer solution, and the raw material solution (II) is a solution in which raw 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 a mixture of a weak acid and a salt of the weak acid, or the concentration of a mixture of a weak base and a salt of the weak base, in the pH buffer solution.

[0108] Except for using a basic compound, step S20 is carried out in the same manner as in the second embodiment described above, and the upper layer 30 can be produced.

[0109] 9, the laminate 52 of this modification has a three-layer structure including two sheets (I) 10 and a sheet (II) 30. One of the two sheets (I) 10 is laminated on one surface (on the lower surface) of the sheet (II) 30, and the other is laminated on the other surface (on the upper surface) of the sheet (II).

[0110] The sheets (I) and (II) included in the laminate 52 may be the same as the sheets (I) and (II) described in the second embodiment and Modification 1, and the preferred embodiments are also the same. The method for producing the laminate 52 is not particularly limited, and the laminate 52 can be produced by a method including steps S10 to S30 shown in Fig. 7, similar to the laminate 50 of the second embodiment and the laminate 51 of Modification 1, except that three layers are laminated in the lamination step (step S30).

[0111] The two sheets (I) 10 have the same effect as the porous sheet 10 of the first embodiment. By having the sheets (I) 10 on both sides, the laminate 52 can be used, for example, as a double-sided adhesive tissue adhesive film. Furthermore, when the sheet (II) 30 contains a basic compound as in the first modification, the adhesion time of the sheets (I) on both sides can be shortened at the same time.

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

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

[0115] [Example 1-1] <Synthesis of hydrophobic gelatin derivative> As raw gelatin, Alaska pollack-derived gelatin (manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight (Mw): 84,000, amino group content: 305 μmol / g) (hereinafter, as appropriate, referred to as "Org-ApGltn" or "Org") was prepared, and a nonyl group was introduced into Org-ApGltn to obtain a hydrophobic gelatin derivative (hereinafter, as appropriate, referred to as "C9-ApGltn"). Specifically, as disclosed in WO 2020 / 137903, nonanal (nonyl aldehyde) was reacted with the amino group of the raw gelatin to form a Schiff base, and the resulting Schiff base was reduced to a stable secondary amine using a reducing agent to obtain a hydrophobic gelatin derivative.

[0116] 10 g of Org-ApGltn was dissolved in 35 mL of ultrapure water and stirred at 55°C. A solution of nonanal (Tokyo Chemical Industry Co., Ltd., 0.305 mmol) dissolved in ethanol (Junsei Kagaku Co., Ltd.) in an amount 0.1 times the amino group amount (305 μmol / g) of Org-ApGltn was added to form an imine bond between the nonanal and the amino group of Org-ApGltn. After stirring at the same temperature for 1 hour, a solution of 2-picoline borane (Junsei Kagaku Co., Ltd., 0.4575 mmol) dissolved in ethanol was added to reduce the imine. The resulting mixed solution (Org-ApGltn concentration: 20% by mass / volume, water:ethanol = 35:15 mL) was stirred at 55°C for 17 hours to allow the reaction to proceed. The reaction solution (50 mL) was then 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-picoline borane. The resulting precipitate was then dried in vacuo for 3 days to obtain C9-ApGltn in a yield of 90% by mass.

[0117] The introduction of a nonyl group into the obtained C9-ApGltn was confirmed by Fourier transform infrared spectroscopy and １ The hydrocarbon group introduction rate (nonyl group introduction rate) DS (degree of substitution) (hereinafter sometimes simply referred to as "DS") of C9-ApGltn was determined by quantifying the number of amino groups in the raw gelatin and the number of amino groups in the hydrophobicized gelatin derivative by the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method). ２ The DS was determined by calculating the molar ratio of the content of imino groups bonded to nonyl groups in the hydrophobicized gelatin derivative to the content of imino groups bonded to nonyl groups in the hydrophobicized gelatin derivative. The DS was 7.2 mol%. Hereinafter, the hydrophobicized gelatin derivative synthesized in this example may be referred to as 7.2C9-ApGltn or 7.2C9.

[0118] <Preparation of porous sheet> (1) Preparation of raw material solution First, a hydrophobic gelatin derivative solution (solution A) was prepared by the following procedure: The synthesized 7.2C9-ApGltn was dissolved in 0.1 mM HCl solution, and then the pH was adjusted to 4 using 1 M HCl. Then, pure water was added to the solution to make the concentration of 7.2C9-ApGltn 15 w / v % to obtain solution A.

[0119] Next, a crosslinking agent solution (solution B) was prepared by the following procedure. Pentaerythritol-polyethylene glycol ether tetrasuccinimidyl glutarate (4S-PEG, weight-average molecular weight 20,000, manufactured by NOF Corp.) was used as the crosslinking agent. The amino groups (-NH ２ The amount of the active ester group (N-hydroxysuccinimide group, NHS group) in solution B was adjusted to 0.5 equivalents per equivalent of the active ester group (N-hydroxysuccinimide group, NHS group) in solution B (i.e., NHS / NH ２ (=50 mol%) 4S-PEG was weighed out and dissolved in the same amount of 0.1 mM HCl solution as in Solution A to obtain Solution B.

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

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

[0122] [Example 1-2] First, a starting solution of gelatin (7.2C9-ApGltn) with a concentration of 7.5 w / v % was prepared in the same manner as in Example 1-1, and this was diluted with 0.1 mM HCl solution to obtain a starting solution with a gelatin concentration of 5 w / v % and a pH of 4. The obtained starting solution was freeze-dried in the same manner as in Example 1-1 to produce a porous sheet.

[0123] [Example 1-3] First, a starting solution of gelatin (7.2C9-ApGltn) with a concentration of 7.5 w / v % was prepared in the same manner as in Example 1-1, and this was diluted with 0.1 mM HCl solution to obtain a starting solution with a gelatin concentration of 2.5 w / v % and a pH of 4. The obtained starting solution was freeze-dried in the same manner 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 gelatin (Org-ApGltn) was used instead of the hydrophobic gelatin derivative (7.2C9-ApGltn). That is, 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 derivative> A gelatin derivative in which an octyl group was introduced into raw gelatin (Org-ApGltn) (hereinafter referred to as "C8-ApGltn" as appropriate) was used as the hydrophobic gelatin derivative. 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 gelatin to form a Schiff base, and then reducing the resulting 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 could be confirmed by H-NMR. The hydrocarbon group introduction rate (octyl group introduction rate) DS of C8-ApGltn was calculated in the same manner as in Example 1-1. The DS was 7.6 mol%. Hereinafter, the hydrophobicized gelatin derivative synthesized in this example may be referred to as 7.6C8-ApGltn or 7.6C8.

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

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

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

[0130] [Example 3-1] <Synthesis of hydrophobic gelatin derivative> A gelatin derivative (hereinafter referred to as "C10-ApGltn" as appropriate) in which a decyl group was introduced into raw gelatin (Org-ApGltn) was used as the hydrophobic gelatin derivative. 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 gelatin to form a Schiff base, and then reducing the resulting 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 １ This could be confirmed by H-NMR. The hydrocarbon group introduction rate (decyl group introduction rate) DS of C10-ApGltn was calculated in the same manner as in Example 1-1. The DS was 9.3 mol%. Hereinafter, the hydrophobicized gelatin derivative synthesized in this example may be referred to as 9.3C10-ApGltn or 9.3C10.

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

[0133] [Example 3-2] First, a raw material solution of gelatin (9.3C10-ApGltn) with a concentration of 7.5 w / v% was prepared in the same manner as in Example 3-1, and then diluted with 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 in the same manner as in Example 3-1 to prepare a porous sheet.

[0134] [Example 3-3] First, a starting solution of hydrophobic gelatin derivative (9.3C10-ApGltn) with a concentration of 7.5 w / v% was prepared in the same manner as in Example 3-1, and then diluted with 0.1 mM HCl solution to obtain a starting solution with a hydrophobic gelatin derivative concentration of 2.5 w / v% and pH = 4. The resulting starting solution was freeze-dried in the same manner as in Example 3-1 to prepare a porous sheet.

[0135] The density (apparent density) of the porous sheet produced in each Example or Comparative Example was determined by dividing the weight of the porous sheet by the volume. The volume of the porous sheet was determined from a silicone sheet mold (50 mm x 50 mm x depth: 2 mm). The density of the porous sheets produced 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 / cm. ３ The density of the porous sheets produced in Examples 1-2, 2-2, and 3-2 using a raw material solution with a gelatin concentration of 5 w / v% was about 0.05 g / cm ３ The density of the porous sheets produced in Examples 1-3, 2-3, and 3-3 using a raw material solution with a gelatin concentration of 2.5 w / v% was about 0.025 g / cm ３ It was.

[0136]

[0137] [Evaluation] (1) Shape observation The shapes of the sheets produced in Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-3 were observed visually and by scanning electron microscope (SEM). Figure 4 shows a photograph (left) and an SEM image (right) of the porous sheet obtained in each Example and Comparative Example.

[0138] It was confirmed that the sheets produced in Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-3 were porous (porous sheets). Furthermore, as shown in Figure 4, the porosity (density) of the porous sheet depended on the gelatin concentration of the raw material solution (7.2C9-ApGltn concentration or Org-ApGltn concentration). The lower the gelatin concentration of the raw material solution, 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 Strength Test 1 The porous sheets produced 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 a pressure resistance test as described below.

[0140] According to ASTM (F2392-04), the sealing strength (compression strength) was measured using a porcine aorta as a substrate. As shown in FIG. 5A, the porous sheet 10 prepared in each Example or Comparative Example was placed on the central defect 20a (diameter 1 mm) of a porcine aorta 20 (diameter 35 mm), gently pressed with tweezers to fix it, and left to stand for 10 minutes. After standing, the porcine aorta 20 with the porous sheet 10 placed on it was fixed to a jig, and 37°C saline was flowed at a flow rate of 2 mL / min toward the defect 20a covered by the porous sheet 10 (in the direction indicated by the arrow in FIG. 5A), and the pressure when the porous sheet 10 peeled off was measured. The results are shown in FIG. 5B. In this experiment, the higher the burst pressure, the higher the compression strength, which means that the porous sheet has a higher adhesiveness to the biological tissue (porcine aorta).

[0141] In Figure 5B, first, Comparative Example 1-3, in which the gelatin concentration of the raw material solution was 2.5 w / v%, is compared 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 the hydrophobicized gelatin derivatives (Examples 2-3, 1-3, and 3-3) had higher peel pressures (pressure resistance). Furthermore, the longer the alkyl chains introduced, the higher the peel pressure tended to be. Furthermore, when the raw material gelatin concentration was 5 w / v% and when the raw material gelatin concentration was 7.5 w / v%, the porous sheets using the hydrophobicized gelatin derivatives showed higher peel pressures compared to the porous sheet using raw material gelatin (Org).

[0142] In this test (compression strength test 1), the porcine aorta was in a neutral (pH = 7 to 7.4) wet state, and the porous sheet placed on top of it absorbed neutral water by capillary action, and the active ester group (NHS group) of the cross-linking agent and the amino group (NH ２ It is presumed that the hydrophobic groups introduced into Org caused a hydrophobic interaction between the porous sheet and the tissue (porcine aorta), resulting in strong adhesion to the tissue surface (see Figure 1B).

[0143] <<Laminates>> In the following Examples 4-1, Comparative Examples 2-1, 2-2, 3-3, 4-3, 5-1, 3-1, 3-2, and 6-1 to 6-3, laminates were prepared by laminating a porous sheet of a hydrophobic gelatin derivative (lower layer, sheet (I)) and a porous sheet of raw gelatin (upper layer, sheet (II)). To clearly distinguish between sheets (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, a porous sheet of hydrophobic gelatin derivative 9.3C10-ApGltn (thickness: 2 mm, gelatin concentration in raw material solution (I) was 7.5 w / v%) produced in Experiment 3-1 was prepared as sheet (I).

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

[0146] Sheet (II) was laminated on sheet (I) to obtain a laminate.

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

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

[0149] Photographs of the laminates produced in Example 4-1, Comparative Example 2-1, and Comparative Example 2-2 are shown in Figure 6. The upper photograph is from the sheet (II) side, and the lower photograph is from the sheet (I) side.

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

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

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

[0153] Example 6-1 A laminate consisting of sheets (I) and (II) was produced in the same manner 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 produced in the same manner as in Example 6-1, except that the gelatin concentration in the raw material solution (II) was changed from 5 w / v% to 10 w / v% to produce sheet (II).

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

[0156]

[0157] [Evaluation] (1) Comparative Adhesion 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 of Example 4-1 and the porous sheet of Example 3-1 have the same composition and structure. First, the porous sheet of Example 3-1 and the laminate of Example 4-1 were each placed on a porcine aorta, gently pressed with tweezers to fix them, and left to stand for 10 minutes. At this time, sheet (I) of the laminate was on the bottom side (the side in contact with the porcine aorta). After leaving to stand, the porous sheet adhered to the porcine aorta and the surface of the laminate (sheet (II)) were touched with a rubber gloved hand to compare their adhesiveness.

[0158] As a result of this test, the two-layer laminate (Example 4-1) had lower adhesiveness than the single-layer porous sheet (Example 3-1). From this result, by laminating a porous sheet of raw gelatin on a porous sheet of hydrophobicized gelatin derivative to form a laminate, when the laminate is used as a biological tissue adhesive film, it can be expected to suppress adhesion to other biological tissues and adhesion to rubber gloves during surgery.

[0159] Furthermore, this test was carried out using the laminates prepared in Examples 5-1 and 6-1 to 6-3. The laminates of these Examples were laminates that obtained good results in the pressure resistance strength test 2 described below. The adhesiveness of the laminates of Examples 5-1 and 6-1 to 6-3 was also lower than that of the single-layer porous sheet (Example 3-1).

[0160] (2) Pressure Resistance Strength Test 2 In this test, assuming the inside of a living body where a large amount of blood, body fluids, etc. are present due to bleeding or the like, the laminates produced in Example 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 living tissue (porcine aorta) in physiological saline, and then the adhesiveness was evaluated by a pressure resistance strength test.

[0161] First, the laminate prepared in each example was punched with a dumbbell cutter to prepare a circular evaluation sample with a diameter of 15 mm. Next, on a hot plate heated to 37°C, in a Petri dish (diameter 35 mm) containing 3 mL of physiological saline, the evaluation sample (laminate) of each example and comparative example was placed so as to cover the central defect (diameter 1 mm) of a porcine aorta (diameter 35 mm). At this time, the sheet (I) of the laminate was placed on the bottom (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 the Unipack, and the sample was left to stand for 10 minutes.

[0162] After standing, the unipack was peeled off from the evaluation sample, and the sealing strength (pressure resistance) was measured according to ASTM (F2392-04). As shown in FIG. 5A, the porcine aorta 20 with the evaluation sample 10 placed on it was fixed to a jig, and 37°C saline was flowed at a flow rate of 2 mL / min toward the defect 20a covered by the evaluation sample 10 (laminate) (in the direction indicated by the arrow in FIG. 5A), and the pressure when 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, for the laminates prepared in Comparative Examples 3-2, 2-2, 3-1, and 2-1, including the porous sheet (I) with a relatively low gelatin concentration in the raw material solution, the laminate also peeled off from the porcine aorta when the Unipack was peeled off, making it impossible to measure the pressure resistance (evaluation result: N.D.). In other words, these porous sheets did not have sufficient adhesiveness. For this reason, the second and subsequent pressure resistance tests were not performed on the laminates of Comparative Examples 3-2, 2-2, 3-1, and 2-1.

[0164] On the other hand, the laminates containing porous sheets (I) prepared in Examples 5-1, 4-1, and 6-1 to 6-3, which had a relatively high gelatin concentration in the raw material solution, were able to undergo pressure resistance tests by peeling the Unipack from the aorta at least twice. Table 2 shows the highest pressure resistance obtained for each laminate. For reference, Table 2 also shows the evaluation results (pressure resistance) of the same test conducted using a commercially available hemostatic agent. The evaluation results of the laminates of Examples 5-1, 4-1, and 6-1 to 6-3 were better than those of the commercially available product, confirming that they had adhesiveness and pressure resistance that were satisfactory for practical use.

[0165] The cause of the above test results is presumed to be as follows. In this test, it is presumed that the porous sheet (I) absorbed the saline solution and the moisture in the tissue, becoming neutral to slightly alkaline, allowing the hydrophobicized cod gelatin to penetrate the tissue and promoting the crosslinking reaction of the hydrophobicized cod gelatin with the crosslinking agent. The porous sheets (I) produced in Examples 3-2, 2-2, 3-1, and 2-1, which had a low gelatin concentration in the raw material solution, had low density and many voids, and therefore rapidly absorbed large amounts of moisture (saline) when surrounded by a large amount of moisture. Therefore, moisture was transferred to the upper sheet (II) before the lower sheet (I) was sufficiently adhered to the biological tissue (porcine aorta), causing the upper sheet (II) to become adhesive, and thus the entire laminate was detached from the porcine aorta when the Unipack was peeled off. On the other hand, the porous sheets (I) produced in Examples 5-1, 4-1, and 6-1 to 6-3, which had a high gelatin concentration in the raw material solution, had high density and few voids, and therefore absorbed water relatively slowly even when a large amount of water (saline solution and water in tissue) was present around them. This is thought to be why water did not reach the upper sheet (II), and the lower sheet (I) was able to adhere to the biological tissue (porcine aorta) with sufficient strength.

[0166] <<Laminates>> In Examples 7-1 to 7-7, 8-1, and 9-1 described below, laminates were prepared using a pH buffer solution when preparing the sheet (II). For comparison, in Example 7-8, a laminate was prepared under the same conditions as in Example 7-1, except that no pH buffer solution was used when preparing the sheet (II). Table 3 shows details of the laminates prepared in each experiment.

[0167] [Example 7-1] <Synthesis of hydrophobic gelatin derivative> A hydrophobic gelatin derivative was synthesized in the same manner as in Experiment 1-1, except that decanoic anhydride was used as the hydrophobic reagent 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 hydrophobic gelatin derivative synthesized in this experiment may be referred to as 12C10-ApGltn or 12C10.

[0168] <Preparation of Sheet (I)> A 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 / NH ２ = 50 mol%). In addition, the prepared raw material solution was freeze-dried to produce a porous sheet (sheet (I)) 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 gelatin (Org-ApGltn) was dissolved in the phosphate buffer 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 x 70 mm x depth: 0.5 mm). Using the same freeze dryer as used in Experiment 1-1, the sample was first frozen overnight at -30 °C and then freeze-dried. After freeze-drying, the sample was heated at 150 °C for 5 hours to thermally crosslink the Org-ApGltn, yielding Sheet (II).

[0170] <Preparation of Laminate> Sheet (II) was laminated on sheet (I) to obtain a laminate for this experiment.

[0171] [Example 7-2] In the production of sheet (I), NHS / NH ２ A laminate for this experiment was produced in the same manner as in Example 7-1, except that the raw material solution (I) was prepared so that the mol ratio of the hydroxyl group was 75 mol %.

[0172] [Example 7-3] In the production of sheet (I), NHS / NH ２ A laminate for this experiment was produced in the same manner as in Example 7-1, except that the raw material solution (I) was prepared so that the mol ratio of the raw material solution (I) was 100 mol %.

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

[0174] Example 7-5 A laminate for this experiment was produced in the same manner as in Example 7-1, except that the weight-average molecular weight of the crosslinking agent (4S-PEG) used in producing the sheet (I) was 10,000.

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

[0176] Example 7-7 A laminate for this experiment was produced in the same manner as in Example 7-3, except that the weight-average molecular weight of the crosslinking agent (4S-PEG) used in producing the sheet (I) was 10,000.

[0177] Example 7-8 A laminate was produced under the same conditions as in Example 7-1, except that no pH buffer solution was used in producing the sheet (II) and pure water was used instead.

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

[0179] <Preparation of Laminate> A laminate for this experiment was prepared in the same manner as in Example 7-1, except that the hydrophobic gelatin derivative 9C12-ApGltn was used in the preparation of the sheet (I).

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

[0181] <Preparation of Laminate> A laminate for this experiment was prepared in the same manner as in Example 7-1, except that the hydrophobic gelatin derivative 13C8-ApGltn was used in the preparation of the sheet (I).

[0182]

[0183] [Evaluation] (1) Comparative Adhesion Test The laminates prepared in Examples 7-1 to 7-8, 8-1, and 9-1 were subjected to a comparative adhesiveness test similar to that performed on the laminates of Example 4-1 and the like. The adhesiveness of the laminates of all Examples shown in Table 3 was lower than that of the single-layer porous sheet (Example 3-1). From these results, it can be expected that when these laminates are used as biological tissue adhesive films, they will be able to suppress adhesion to other biological tissues and adhesion to rubber gloves during surgery.

[0184] (2) Pressure Resistance Strength Test 2 The laminates produced in Examples 7-1 to 7-8, 8-1, and 9-1 were subjected to the same pressure resistance strength test 2 as that performed on the laminates of Example 4-1 and the like. This test was conducted in physiological saline, simulating the inside of a living body where a large amount of blood, body fluids, etc. is present due to bleeding or the like. Note that, while the test on the laminates of Example 4-1 and the like involved a 10-minute adhesion time to biological tissue (porcine aorta) (see Table 2), the test on the laminates of Example 7-1 and the like involved a shorter adhesion time of 3 minutes. The results are shown in Table 3. For reference, Table 3 also shows the evaluation results (pressure resistance) of the same test, which was conducted using a commercially available hemostatic agent for a 10-minute adhesion time.

[0185] As shown in Table 3, the evaluation results of the laminates in all experiments except for Examples 7-8 were better than those of commercially available products, confirming that they had adhesiveness and pressure resistance that were satisfactory for practical use. In all Examples except for Examples 7-8, a pH buffer solution (pH 8) was used to prepare Sheet (II), so Sheet (II) contained a basic compound (a mixture of phosphoric acid and sodium phosphate). This caused the pH in Sheet (I) to rise more quickly, accelerating the crosslinking reaction (hardening reaction) between the hydrophobicized gelatin derivative and the crosslinking agent, presumably resulting in good evaluation results even when the adhesion time was shortened to 3 minutes.

[0186] On the other hand, the laminate of Example 7-8, in which no pH buffer solution was used in the preparation of sheet (II) (i.e., a laminate not containing a basic compound), did not obtain sufficient adhesive strength at a short adhesion time of 3 minutes, and the pressure resistance could not be measured (evaluation result: N.D.). Note that, when the same test was performed on the laminate of Example 4-1, etc., in which no pH buffer solution was used in the preparation of sheet (II) shown in Table 2, with the adhesion time shortened to 3 minutes, sufficient adhesive strength was not obtained at a short adhesion time of 3 minutes, and the pressure resistance could not be measured, as in Examples 7-8 (evaluation result: N.D.).

[0187] The porous sheet of the present invention can be used as a tissue adhesive film for use in vivo (e.g., hemostatic material, anti-adhesion material), and has high adhesive strength and high biocompatibility, making it suitable for use in the fields of surgery and internal medicine.

[0188] REFERENCE SIGNS LIST 10 Porous sheet (Sheet (I)) 20 Biological tissue (porcine aorta) 20a Defective portion 30 Sheet containing crosslinked raw material gelatin (Sheet (II)) 31 Basic compound 50, 51, 52 Laminate

Claims

1. A porous sheet comprising: A hydrocarbyl group is introduced into gelatin, represented by the following formula (1): 【Chemistry 1】 In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, R １ is a hydrocarbon group having 1 to 20 carbon atoms, R ２ is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms. A hydrophobized gelatin derivative comprising a structure represented by the formula: an unreacted crosslinking agent dispersed in the hydrophobized gelatin derivative; A porous sheet comprising:

2. The density of the porous sheet is 0.01 g / cm ３ ~0.3g / cm ３ The porous sheet according to claim 1 ,

3. The porous sheet according to claim 1, wherein L is a single bond or -C(O)-.

4. The porous sheet according to claim 3 , wherein L is a single bond.

5. R １ is a straight chain alkyl group, R ２ The porous sheet according to claim 1 , wherein is a hydrogen atom.

6. R １ The porous sheet according to claim 5, wherein is a linear alkyl group having 6 to 12 carbon atoms.

7. 2. The porous sheet according to claim 1, wherein the hydrophobic gelatin derivative has a hydrocarbon group introduction rate of 5 mol % to 50 mol %.

8. The porous sheet according to claim 1 , wherein the gelatin is gelatin derived from cold-water fish.

9. The porous sheet according to claim 1 , wherein the crosslinking agent is a compound having at least two crosslinkable groups.

10. The porous sheet according to claim 9, wherein the crosslinking group of the crosslinking agent is 0.25 to 2 equivalents per equivalent of the amino group of the hydrophobic gelatin derivative.

11. The porous sheet according to claim 1, having a thickness of 1 mm or more.

12. A tissue adhesive film comprising the porous sheet according to any one of claims 1 to 11.

13. At least one sheet (I) which is a porous sheet according to any one of claims 1 to 11; A sheet (II) containing a crosslinked product of non-hydrophobized gelatin, The density of the at least one sheet (I) is 0.07 g / cm ３ That's all. The tissue adhesive membrane, wherein the at least one sheet (I) is laminated on one side of the sheet (II).

14. The tissue adhesive film according to claim 13, wherein the crosslinked product of the non-hydrophobized gelatin is a thermally crosslinked product of the non-hydrophobized gelatin.

15. The tissue adhesive membrane of claim 13, wherein the sheet (II) is porous.

16. The density of the at least one sheet (I) is 0.07 g / cm ３ ~0.2g / cm ３ The tissue adhesive film of claim 13, wherein

17. The tissue adhesive film according to claim 13, wherein the sheet (II) further contains a basic compound that exhibits basicity when in contact with moisture.

18. The tissue adhesive film according to claim 17, wherein the basic compound is a compound whose aqueous solution of 0.05 mol / L to 0.5 mol / L has a pH of 7.5 to 10.

19. 18. The tissue adhesive film of claim 17, wherein the basic compound is a pH buffering agent.

20. 20. The tissue adhesive film of claim 19, wherein the pH buffer is a mixture of phosphoric acid and sodium phosphate.

21. The at least one sheet (I) includes two sheets (I), The tissue adhesive film according to claim 13, wherein one of the two sheets (I) is laminated on one side of the sheet (II) and the other is laminated on the other side of the sheet (II).

22. The tissue adhesive film of claim 12 for use as a hemostatic agent.

23. The tissue adhesive film according to claim 12, which prevents adhesion to other tissues when adhered to the tissue.

24. A method for producing the porous sheet according to any one of claims 1 to 11, preparing a raw material solution (I) having a pH of 5 or less, the raw material solution (I) containing the non-hydrophobized gelatin, the crosslinking agent, an acid, and a solvent; freeze-drying the raw material liquid (I) in a sheet form to obtain the porous sheet; and

25. The method according to claim 24, wherein the concentration of the non-hydrophobized gelatin in the raw material liquid (I) is 1 w / v % to 20 w / v %.

26. 25. A method for producing a tissue adhesive film comprising the step of producing a porous sheet by the method of claim 24.

27. A method for producing a tissue adhesive film, comprising: Producing a porous sheet (I) according to the method of claim 24, Producing a sheet (II) containing a crosslinked product of non-hydrophobized gelatin; laminating the sheet (I) and the sheet (II); A method for producing a tissue adhesive film comprising:

28. Producing the sheet (II) preparing a raw material liquid (II) containing the non-hydrophobized gelatin, a basic compound, and a solvent; freeze-drying the raw material liquid (II) into a sheet shape to form a precursor sheet; heating the precursor sheet to thermally crosslink the non-hydrophobicized gelatin to obtain the sheet (II); The method of claim 27, comprising:

29. 29. The method of claim 28, wherein the basic compound is a pH buffering agent.

30. 30. The method of claim 29, wherein the pH buffer is a mixture of phosphoric acid and sodium phosphate.

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