WO2024053602A1 - Feuille poreuse, film adhésif tissulaire, leur utilisation en tant qu'agent hémostatique ou matériau anti-adhérence, et leurs procédés de production - Google Patents

Feuille poreuse, film adhésif tissulaire, leur utilisation en tant qu'agent hémostatique ou matériau anti-adhérence, et leurs procédés de production Download PDF

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WO2024053602A1
WO2024053602A1 PCT/JP2023/032230 JP2023032230W WO2024053602A1 WO 2024053602 A1 WO2024053602 A1 WO 2024053602A1 JP 2023032230 W JP2023032230 W JP 2023032230W WO 2024053602 A1 WO2024053602 A1 WO 2024053602A1
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sheet
gelatin
porous sheet
raw material
porous
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PCT/JP2023/032230
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English (en)
Japanese (ja)
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哲志 田口
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国立研究開発法人物質・材料研究機構
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins

Definitions

  • the present invention relates to a porous sheet, a tissue adhesive film, the use thereof as a hemostatic agent or an anti-adhesion material, and a method for producing the same.
  • Tissue adhesive membranes are polymeric membranes that can adhere to biological tissues such as blood vessels and skin, and thereby prevent blood leakage during surgeries such as cardiovascular surgery, thereby improving surgical safety. can be increased.
  • the present inventor has reported a non-porous tissue adhesive film formed by integrating gelatin and/or a hydrophobized gelatin derivative and crosslinked molecules (Patent Document 1).
  • the present invention provides a porous sheet that has high biocompatibility and adhesive strength and can be applied to a tissue adhesive membrane, based on the above-mentioned technical level.
  • a hydrophobized gelatin derivative in which a hydrocarbon group is introduced into gelatin which has the formula (1): [In formula (1), Gltn represents a gelatin residue, L represents a single bond or a divalent linking group, R1 is a hydrocarbon group having 1 to 20 carbon atoms, and R2 is a hydrogen atom or a carbon It is a hydrocarbon group of number 1 to 20]
  • the porous sheet according to [1] wherein the porous sheet has a density of 0.01 g/cm 3 to 0.3 g/cm 3 .
  • porous sheet according to any one of [1] to [13] for preparing a hemostatic agent, a sealant (occlusion material) or a dressing.
  • the porous sheet according to any one of [1] to [13] can be used to cover tissues that need to be covered (for example, anastomoses such as nerves and tendons, tissues (pulmonary pleura, gastrointestinal anastomoses, oral cavity/periodontal tissues, etc.) A method of covering tissue to cover wounds caused by ).
  • a method for hemostasis of tissue which comprises adhering the porous sheet according to any one of [1] to [13] to a tissue that requires or is expected to require hemostasis.
  • the porous sheet according to any one of [1] to [13] compensates for the gap between the dura mater and the dura mater, the dura mater suture, or the gap between the dura mater forming material and the dura mater. how to.
  • a tissue adhesive membrane comprising the porous sheet according to any one of [1] to [13].
  • a hemostatic agent, sealant (closure material), or covering material comprising the porous sheet according to any one of [1] to [13].
  • the basic compound is a compound whose pH in an aqueous solution of 0.05 mol/L to 0.5 mol/L is 7.5 to 10.
  • the pH buffer is a mixture of phosphoric acid and sodium phosphate.
  • the at least one sheet (I) comprises two sheets (I); Of the two sheets (I), one is laminated on one side of the sheet (II), and the other is laminated on the other side of the sheet (II), [23] to [33] ]
  • [35] [23] A hemostatic agent, a sealant (closure material), or a covering material comprising the tissue adhesive film or laminate according to any one of [23] to [34].
  • An anti-adhesion material comprising the tissue adhesive film or laminate according to any one of [23] to [34].
  • tissue adhesive film or laminate according to any one of [23] to [34] for coating or covering tissue.
  • 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.
  • tissue adhesive film or laminate according to any one of [23] to [34] for preparing a hemostatic agent, a sealant (occlusion material), or a tissue dressing.
  • tissue adhesive film or laminate according to any one of [23] to [34] for preparing a tissue adhesion prevention material.
  • [41] [23] A method for covering tissue, which comprises covering a tissue that requires covering with the tissue adhesive film or laminate according to any one of [34].
  • the tissue adhesive film or laminate according to any one of [23] to [34] fills the gap between the dura mater and the dura mater, the dura mater suture, or the gap between the dura mater forming material and the dura mater. , how to fill in the gaps.
  • a method for hemostasis of tissue which comprises adhering the tissue adhesive film or laminate according to any one of [23] to [34] to a tissue that requires or is expected to require hemostasis.
  • a method for preventing tissue adhesion which comprises adhering the tissue adhesive film or laminate according to any one of [23] to [34] to a tissue whose adhesion to other tissues needs to be prevented.
  • [45] The method for producing a porous sheet according to any one of [1] to [13], Mixing the hydrophobized gelatin derivative, the crosslinking agent, an acid, and a solvent to prepare a raw material solution (I) with a pH of 5 or less; freeze-drying the raw material liquid (I) to obtain the porous sheet;
  • a method for producing a porous sheet comprising: [46] The method according to [45], wherein the concentration of the hydrophobized gelatin derivative in the raw material solution (I) is 1 w/v% to 20 w/v%.
  • [47] [45] or [46] a method for producing a tissue adhesive membrane, comprising the step of producing a porous sheet.
  • [48] Producing sheet (I), which is the porous sheet, by the method described in [45] or [46]; Producing a sheet (II) containing a crosslinked product of unhydrophobized gelatin; Laminating the sheet (I) and the sheet (II); A method for producing a tissue adhesive film, comprising: [49] The sheet (II), preparing a raw material solution (II) containing the unhydrophobized gelatin, a basic compound, and a solvent; Lyophilizing the raw material liquid (II) into a sheet form to form a precursor sheet; heating the precursor sheet to thermally crosslink the unhydrophobicized gelatin; The method according to [48], which is produced by. [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.
  • FIG. 1 is a schematic cross-sectional view of a porous sheet according to a first embodiment.
  • the photo on the left is the porous sheet before being attached to living tissue
  • the photo on the right is the porous sheet being attached to living tissue.
  • the schematic diagram on the left is a schematic diagram of the hydrophobized gelatin derivative and crosslinking agent existing in an unreacted state in the porous sheet before it is pasted on biological tissue
  • the schematic diagram on the right is a diagram of the porous sheet pasted on biological tissue.
  • FIG. 2 is a schematic diagram showing an example of a crosslinking reaction between a hydrophobized gelatin derivative and a crosslinking agent in a sheet. It is a flow chart showing a manufacturing method of a porous sheet of a 1st embodiment.
  • FIG. 7 is a schematic cross-sectional view of the laminate of Modification 1 of the second embodiment, and is a diagram illustrating how the laminate is attached to living tissue. It is a cross-sectional schematic diagram of the laminated body of the modification 2 of 2nd Embodiment.
  • “Gelatin” generally refers to a polymer whose triple helical structure is unraveled and denatured by treating natural or synthetic collagen with heat, acid, or alkali. "time” means “gelatin” that has not been subjected to “hydrophobization” treatment and has no alkyl group introduced. Therefore, “gelatin” is sometimes expressed as "ApGltn” or “GltnNH 2 " herein.
  • hydrophobicization refers to introducing a hydrocarbon group into “gelatin”, more specifically, by bonding a hydrocarbon group to an amino group via a linking group or directly, A process that increases the hydrophobicity of gelatin.
  • hydrophobicized gelatin derivative refers to the introduction of a hydrocarbon group into “gelatin”
  • hydrophobicized gelatin derivative refers to the introduction of a hydrocarbon group into “gelatin”
  • hydrophobicized gelatin derivative refers to the introduction of a hydrocarbon group into “gelatin”
  • bonding of a hydrocarbon group to an amino group via a linking group or directly This refers to a derivative of "gelatin” with increased hydrophobicity, and is sometimes expressed as "GltnNH-L-CHR 1 R 2 ".
  • the porous sheet 10 includes a hydrophobized gelatin derivative (second gelatin) in which a hydrocarbon group is introduced into raw material gelatin (first gelatin), and a crosslinking agent dispersed in the hydrophobized gelatin derivative.
  • the crosslinking agent is dispersed in the hydrophobized gelatin derivative, and the crosslinking agent and the hydrophobized gelatin derivative are in a substantially unreacted state. be.
  • Moisture is required for the crosslinking reaction (hardening reaction) between the hydrophobized gelatin derivative and the crosslinking agent, and the environment must be neutral to alkaline. Since living tissue (for example, the porcine aorta 20 shown in FIG. 1B) normally contains neutral water, the porous sheet 10 absorbs neutral water by being attached to the living tissue 20. to crosslink and adhere to living tissue 20.
  • the crosslinking reaction typically involves a primary amino group possessed by a hydrophobized gelatin derivative and a crosslinkable group possessed by a crosslinking agent (typically an active ester group, etc.). ) is the reaction.
  • the porous sheet 10 maintains the structure of the raw material gelatin, and therefore has high biocompatibility. Hydrophobic interactions occur between the porous sheet 10 and the living tissue 20 due to the hydrophobic groups, and the hydrophobic groups permeate into the living tissue 20 at a molecular level (anchoring). A physically strong bond is formed between the porous sheet 10 and the porous sheet 20, and the porous sheet 10 can be strongly adhered to the surface of the living tissue 20. In this way, the porous sheet 10 strongly adheres to the living tissue 20 just by pasting it. Furthermore, due to its porous structure, the porous sheet 10 has improved flexibility and/or flexibility compared to non-porous membranes, and can be bent to follow the surface shape of the living tissue 20.
  • the porous sheet 10 is thought to be able to efficiently absorb moisture when attached to the biological tissue 20 and promote the curing reaction (crosslinking reaction) between the hydrophobized gelatin derivative and the crosslinking agent. .
  • the details of the porous sheet 10 will be explained below.
  • the hydrophobized gelatin derivative refers to a gelatin derivative in which a hydrocarbon group is introduced into raw material gelatin (first gelatin), and has a structure represented by the following formula (1).
  • Gltn represents a gelatin residue
  • 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 It is a hydrocarbon group having 1 to 20 carbon atoms.
  • GltnNH- is a structure derived from the above-mentioned raw material gelatin. Therefore, raw gelatin is represented by GltnNH2 . Note that the details of the raw material gelatin will be described later.
  • -CHR 1 R 2 is a hydrocarbon group introduced into the raw gelatin via L (single bond or divalent linking group).
  • the divalent linking group for L is not particularly limited, but includes -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -N(R)- (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 (alkenylene group having 2 to 10 carbon atoms), and combinations thereof, among which -C(O)- is preferred.
  • L is preferably a single bond or -C(O)-.
  • -CHR 1 R 2 (hydrocarbon group) is preferably bonded to the ⁇ -amino group of raw material gelatin, and is bonded to the ⁇ -amino group of lysine (Lys) in raw material gelatin. More preferably, they are bonded.
  • a so-called reductive amination reaction Examples include a method using an aldehyde or a ketone), a Schotten-Baumann reaction (a method using an acid chloride), and the like.
  • the -NH- structure (secondary amino group) of formula (1) can be detected, for example, by a band around 3300 cm -1 in an FT-IR (Fourier transform infrared absorption) spectrum.
  • FT-IR Fastier transform infrared absorption
  • the hydrocarbon group having 1 to 20 carbon atoms for R 1 and R 2 is not particularly limited, and includes, for example, a chain hydrocarbon group having 1 to 20 carbon atoms, and a chain hydrocarbon group having 3 to 20 carbon atoms. Examples include alicyclic hydrocarbon groups, aromatic hydrocarbon groups having 6 to 14 carbon atoms, and groups combining these.
  • R 2 When R 2 is 1 to 20 hydrocarbon groups, R 2 may be the same as or different from R 1 . Moreover, the hydrocarbon groups of R 1 and R 2 may be linear or branched.
  • the chain hydrocarbon group having 1 to 20 carbon atoms is not particularly limited, but includes methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, octyl group (or caprylic group), nonyl group (or pelargonyl group), decyl group, dodecyl group (or lauryl group), and tetradecyl group (or myristyl group).
  • Examples of the alicyclic hydrocarbon group having 3 to 20 carbon atoms include cyclopropyl group, cyclopentyl group, cyclohexyl group, adamantyl group, and norbornyl group.
  • the aromatic hydrocarbon group having 6 to 14 carbon atoms includes, but is not particularly limited to, a phenyl group, a tolyl group, a naphthyl group, and the like.
  • groups combining the above include, but are not particularly limited to, aralkyl groups having 6 to 12 carbon atoms such as a benzyl group, a phenethyl group, a naphthylmethyl group, and a naphthylethyl group.
  • R 1 is a linear alkyl group and R 2 is a hydrogen atom.
  • R 1 is preferably a straight chain alkyl group having 6 to 12 carbon atoms, more preferably a straight chain alkyl group having 8 to 10 carbon atoms.
  • the hydrophobized gelatin derivative represented by formula (1) is preferably at least one selected from the group consisting of the following formulas (2) and (3), and more preferably the gelatin derivative represented by formula (2). preferable.
  • Formula (2) is a case where L in formula (1) is a single bond, and the hydrocarbon group (-CHR 1 R 2 ) is attached to raw material gelatin via an imino bond (-NH-) which is a secondary amine. has been introduced into the gelatin residue Gltn.
  • Formula (3) is a case where L in formula (1) is -C(O)-, and the hydrocarbon group (-CHR 1 R 2 ) is bonded to the gelatin of the raw material gelatin through the amide bond (-NHCO-). The residue Gltn has been introduced.
  • formula (2) and formula (3) the meaning of each symbol is the same as in formula (1) already explained, and the preferred form is also the same.
  • the hydrophobized gelatin derivative may be composed of only one type of hydrophobized gelatin derivative, or may be a mixture of two or more types of hydrophobized gelatin derivatives.
  • the content of imino groups (-NH-CHR 1 R 2 ) to which alkyl groups are bonded in the hydrophobized gelatin derivative relative to the content of amino groups (-NH 2 ) in gelatin (raw material gelatin) before hydrophobization is defined as the "degree of submission” (hereinafter sometimes simply referred to as "DS").
  • the hydrocarbon group introduction rate of the hydrophobized gelatin derivative is preferably, for example, 5 mol% to 50 mol%, 5 mol% to 20 mol%, or 5 mol% to 10 mol%, from the viewpoint of improving the adhesiveness of the porous sheet.
  • the imino group/amino group (molar ratio) in the hydrophobized 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 hydrophobized gelatin derivative is within the above range, it is easy to obtain a porous sheet 10 with high adhesiveness.
  • hydrocarbon group introduction rate is determined from the value obtained by quantifying the number of amino groups in the raw material gelatin and the number of amino groups in the hydrophobized gelatin derivative by the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method). It can be calculated using the following formula.
  • Hydrocarbon group introduction rate of hydrophobized gelatin derivative (mol%) [Number of amino groups in raw material gelatin - Number of amino groups in hydrophobized gelatin derivative] / [Number of amino groups in raw gelatin] x 100
  • the molecular weight of the hydrophobized 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 introduced hydrocarbon groups. Therefore, the possible range of the weight average molecular weight (Mw) of the hydrophobized gelatin derivative is almost the same as the possible range of the weight average molecular weight (Mw) of the raw material gelatin, which will be described later.
  • hydrophobized gelatin derivatives described above may be commercially available products or may be self-synthesized products.
  • Hydrophobized gelatin derivatives can also be synthesized by introducing hydrophobic groups (hydrocarbon groups) into the raw material gelatin (first gelatin) described below, for example, using the synthesis method disclosed in International Publication No. 2020/137903. good.
  • the content regarding the method for synthesizing a hydrophobized gelatin derivative disclosed in WO 2020/137903 is incorporated herein by reference.
  • the raw material gelatin may be obtained from naturally-derived collagen or synthetic collagen (including fermentation, genetic recombination, etc.).
  • gelatin obtained from naturally derived or synthesized collagen may be subjected to some kind of treatment (excluding hydrophobic treatment).
  • gelatin is obtained by treating naturally occurring collagen obtained from the skin, bones, tendons, etc. of mammals, birds, fish, etc. with acid or alkali (heat-extracted if necessary).
  • alkali treatment of naturally-derived collagen is particularly preferable, since a porous sheet having better effects of the present invention can be obtained.
  • naturally derived gelatin means gelatin obtained by heat, acid, or alkali treatment of naturally derived collagen; for example, the term “fish-derived gelatin” refers to It refers to gelatin obtained by heat, acid, or alkali treatment of collagen derived from collagen.
  • gelatin that has undergone low endotoxin treatment and has a reduced endotoxin content Gelatin raw materials vary depending on the climate of the country of manufacture, the manufacturing environment, the animal of origin, the extraction method, the cleanliness of the equipment used, etc., but generally they contain 1,000 to 100,000 EU/g of endotoxin, and Gelatin that has undergone some type of endotoxin reduction treatment is called "low-endotoxin-treated gelatin.”
  • “Low endotoxin treated gelatin” usually contains 5,000 EU/g or less endotoxin, preferably 1,000 EU/g or less, and more preferably 100 EU/g or less endotoxin. More preferably, it contains 10 EU/g or less of endotoxin.
  • Such low-endotoxin-treated gelatin is not particularly limited and any known gelatin can be used, but examples thereof include those described in JP-A No. 2007-231225, the content of which is incorporated herein by reference. Incorporated.
  • gelatin derived from mammals examples include gelatin derived from pigs and cows.
  • Gelatin derived from fish is not particularly limited, but gelatin derived from cold water fish (cold water fish) such as salmon, trout, cod, walleye pollack, sea bream, tilapia, and tuna (hereinafter also referred to as "gelatin derived from cold water fish") may be used. ) is preferred.
  • Gelatin derived from cold water fish can maintain fluidity without solidifying even at low temperatures.
  • the crosslinking agent can be easily and uniformly dispersed even at low temperatures, and the porous sheet of this embodiment can be efficiently formed by freeze-drying (freeze-drying) described below. 10 can be manufactured. Furthermore, it also has the characteristic of easily absorbing moisture when applied to a living body.
  • Cold-water fish-derived gelatin is a polymer in which two or more amino acids are linked in a linear chain, with 190 or less imino acids per 1000 constituent amino acids, more specifically 80 or less hydroxyproline ( Hydroxyproline) and has 110 or less prolines.
  • the room temperature fluidity of gelatin derived from cold water fish is thought to be due to the fact that the number of hydroxyprolines is 80 or less or the number of prolines is 110 or less. If either condition is satisfied, the denaturation temperature will be approximately room temperature or lower, and room temperature fluidity will occur.
  • the number of hydroxyprolines in Thai gelatin is 73, the number of prolines is 108, and the denaturation temperature is 302.5K.
  • Tilapia gelatin has a hydroxyproline number of 82, a proline number of 110, and a denaturation temperature of 309K.
  • porcine gelatin has a hydroxyproline number of 95, a proline number of 121, and a denaturation temperature of 316K.
  • gelatin derived from cold water fish has a similar amino acid sequence to gelatin derived from animals, is easily decomposed by enzymes in living organisms, and has high biocompatibility.
  • the molecular weight of the raw material gelatin is not particularly limited, but the weight average molecular weight (Mw) is preferably 5,000 to 100,000, more preferably 10,000 to 50,000, and even more preferably 20,000 to 40,000. .
  • the weight average molecular weight means the weight average molecular weight calculated
  • the raw material gelatin may be composed of only one type of gelatin, or may be a mixture of two or more types of gelatin.
  • the crosslinking agent is typically a compound having at least two substituents (crosslinking groups) in one molecule that can react with the primary amino group of the hydrophobized gelatin derivative.
  • the crosslinking group possessed by the crosslinking agent is not particularly limited, but active ester groups (activated ester group) is preferred. That is, as a crosslinking agent, a compound having at least two active ester groups in one molecule is preferable.
  • Such crosslinking agents include polybasic acids activated with N-hydroxysuccinimide or N-hydroxysulfosuccinimide.
  • genipin aldehyde compounds, acid anhydrides, maleimide compounds, dithiocarbonates, diisothiocyanates, and the like can be used as crosslinking agents.
  • polybasic acids examples 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, and polyaspartic acid.
  • carboxymethylated dextrin carboxymethylated dextran, carboxymethylated starch, carboxymethylated cellulose, carboxymethylated chitosan, and carboxymethylated pullulan.
  • disuccinimidyl glutarate DSG
  • disuccinimidyl suberate DSS
  • disuccinimidyl tartrate DST
  • 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 converted into an active ester 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:
  • n is a number such that Mw is about 3,000 to 30,000, preferably 5,000 to 27,000, more preferably 15,000 to 25,000);
  • aldehyde compounds include formyl group-introduced polysaccharides in which two or more formyl groups are introduced into one molecule, such as formyl group-introduced starch, formyl group-introduced dextran, formyl group-introduced dextrin, and formyl group-introduced hyaluronic acid. Can be mentioned.
  • Examples of acid anhydrides include glutaric anhydride, maleic anhydride, and succinic anhydride.
  • examples of the diisothiocyanate include hexamethylene diisothiocyanate and the like.
  • activated polyethylene glycol polybasic acid esters and formyl group-introduced polysaccharides are preferred, and activated polyethylene glycol polybasic acid esters are more preferred.
  • crosslinking agent explained above may be a commercially available product or may be synthesized by a known method. Further, 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.
  • the ratio of the crosslinkable groups of the crosslinking agent to the amino groups of the hydrophobized gelatin derivative is not particularly limited. From the viewpoint of efficiently promoting the crosslinking reaction, for example, it is preferable that the crosslinking group possessed by the crosslinking agent is 0.25 to 2 equivalents, with respect to 1 equivalent of the amino group possessed by the hydrophobized gelatin derivative. More preferably 3 equivalents to 1.5 equivalents, even more preferably 0.3 equivalents to 0.8 equivalents.
  • the amino group and the crosslinkable group eg, active ester group
  • the content of the hydrophobized gelatin derivative and the content of the crosslinking agent in the porous sheet 10 are not particularly limited, and may be adjusted as appropriate within a range that satisfies the above-mentioned preferred ratio of crosslinkable groups to amino groups, for example.
  • the content of the hydrophobized 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. It may be 5 to 7.5% by mass.
  • the porous sheet 10 may be composed only of the hydrophobized gelatin derivative and the crosslinking agent, or may contain other components other than the hydrophobized gelatin derivative and the crosslinking agent within the range that achieves the effects of the present invention. .
  • the total content of the hydrophobized 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.
  • Components other than the hydrophobized gelatin derivative and the crosslinking agent are not particularly limited, but include, for example, solvents, buffering agents, colorants, preservatives, functional nanoparticles, and drugs (anticancer drugs, antithrombotic drugs, etc.). , antibacterial agents, growth factors, etc.).
  • the density of the porous sheet 10 is not particularly limited, but may be, for example, 0.01 g/ cm 3 to 0.3 g/cm 3 or 0.02 g/cm 3 to 0.08 g/cm 3 .
  • the density of the porous sheet 10 is preferably 0.02 g/cm 3 to 0.1 g/cm 3 , more preferably 0.05 g/cm 3 to 0.1 g/cm 3 , particularly preferably , 0.05g/cm 3 to 0.08g/cm 3 .
  • the porous sheet can have sufficient voids, which can provide appropriate flexibility and/or flexibility, and further improve efficiency. It can absorb moisture and accelerate the curing reaction. Moreover, if the density is at least the lower limit of the above range, a sufficient amount of cured product (crosslinked product) can be obtained from the porous sheet, and strong adhesiveness can be exhibited to living tissue. Furthermore, the film strength is increased and handling properties are also improved. Note that the density of the porous sheet 10 can be adjusted by, for example, the gelatin concentration of the raw material liquid used in the porous sheet manufacturing method described below.
  • 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.
  • a sufficient amount of cured product (crosslinked product) can be obtained from the porous sheet, and strong adhesiveness can be exhibited to living tissue. Furthermore, the film strength is increased and handling properties are also improved.
  • the upper limit of the thickness of the porous sheet is not particularly limited, the practical range as a tissue adhesive film is, for example, 8 mm or less, or 5 mm or less.
  • the thinner the thickness, the better the flexibility, and the better the processability when used with a tissue adhesive film for example. 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.
  • the porous sheet 10 of this embodiment is a sheet containing an unreacted crosslinking agent dispersed in a hydrophobized gelatin derivative (see FIG. 1B); This means that most of the crosslinking agent contained in the sheet 10 is unreacted. Therefore, for example, a sheet made of a crosslinked hydrophobized gelatin derivative and containing a small amount of unreacted crosslinking agent is different from the porous sheet 10 of this embodiment.
  • Most of the hydrophobized gelatin derivatives contained in the porous sheet of this embodiment are in an uncrosslinked state before use (for example, before adhesion to the living tissue 20), and after use (for example, before adhesion to the living tissue 20), , crosslinked with a crosslinking agent. 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 the present embodiment exist in an unreacted state.
  • the manufacturing method of the porous sheet 10 is not particularly limited, it can be manufactured, for example, by a manufacturing method including steps S1 and S2 described below.
  • the manufacturing method of this embodiment uses freeze-drying to efficiently obtain a sheet with a porous structure (porous sheet).
  • Step S1 Preparation of raw material liquid (acidic)
  • Step S2 Freeze-drying of raw material liquid.
  • Process S1 First, an acidic raw material liquid (raw material liquid (I)) containing a hydrophobized gelatin derivative (second gelatin), a crosslinking agent, an acid, and a solvent is prepared (step S1 in FIG. 2).
  • raw material liquid (I) raw material liquid (raw material liquid (I)) containing a hydrophobized gelatin derivative (second gelatin), a crosslinking agent, an acid, and a solvent is prepared (step S1 in FIG. 2).
  • hydrophobized gelatin derivative examples include the embodiments described above regarding the porous sheet 10, and the preferred embodiments are also the same.
  • a cold-water fish-derived gelatin derivative in which a hydrocarbon group is introduced into cold-water fish-derived gelatin (raw material gelatin) is more preferable.
  • the crosslinking agent can be easily and uniformly dispersed even at low temperatures, and the porous sheet 10 can be obtained by freeze-drying (step S2) while maintaining this state.
  • the concentration of the hydrophobized gelatin derivative in the raw material liquid is not particularly limited, but is, for example, 1 w/v% to 20 w/v%, 1.2 w/v% to 12 w/v%, or 2.5 w/v% to It may be set to 7.5 w/v%. If the concentration of the hydrophobized gelatin derivative in the raw material liquid is within the above range, a porous sheet 10 having a porosity within an appropriate range can be easily obtained.
  • crosslinking agent examples include the forms described as a constituent of the porous sheet 10, and preferred forms are also the same.
  • concentration of the crosslinking agent in the raw material liquid is not particularly limited, and may be adjusted as appropriate, for example, so as to satisfy the above-mentioned preferred ratio of crosslinkable groups to amino groups.
  • concentration of the crosslinking agent in the raw material liquid may be, for example, 1 w/v% to 10 w/v%, or 2.5 w/v% to 7.5 w/v%.
  • the preferable pH of the raw material liquid at room temperature (20° C.) is less than pH 7, less than pH 6, or less than pH 5, and the lower limit is not particularly limited, but is, for example, more than pH 1.
  • the nucleophilicity of the amine is suppressed, and the hydrophobized gelatin derivative and the crosslinking agent can be mixed without reacting.
  • freeze-drying in this state step S2
  • a porous sheet 10 containing the crosslinking agent and the hydrophobized gelatin derivative in a substantially unreacted state is obtained.
  • the type of acid is not particularly limited as long as the pH of the raw material liquid can be adjusted within the above range, but it may evaporate or sublimate during freeze-drying (step S2) and remain in the final product (porous sheet 10). Hydrochloric acid, acetic acid, etc. are preferable, and hydrochloric acid is more preferable from the viewpoint of being difficult to remove.
  • One type of acid may be used alone, or two or more types may be used in combination. Further, the concentration of acid in the raw material liquid may be adjusted as appropriate so that the pH of the raw material liquid is within the above range.
  • the solvent for the raw material liquid is not particularly limited as long as it can dissolve the hydrophobized gelatin derivative, crosslinking agent, and acid; for example, water (ion-exchanged water, distilled water, pure water), water-alcohol mixed solvent, water-acetone. Examples include mixed solvents, among which water is preferred.
  • the raw material liquid may be composed only of a hydrophobized gelatin derivative, a crosslinking agent, an acid, and a solvent, or may contain other components other than these. Other components are the same as the other components (for example, drugs, functional nanoparticles, etc.) that the porous sheet 10 can contain, as described above. Further, the raw material liquid may contain, as another component, a porogen that can serve as a template for the pores of the porous structure.
  • the porogen is preferably a sublimable solid such as ice microcrystals or t-butanol.
  • the raw material liquid may be prepared by uniformly mixing a hydrophobized gelatin derivative, a crosslinking agent, an acid, a solvent, and other components as necessary using a conventionally known method.
  • the prepared raw material liquid is freeze-dried to obtain the porous sheet 10 (step S2 in FIG. 2).
  • the porous sheet 10 of the desired size can be obtained by pouring the raw material liquid into a container having the same volume as the porous sheet 10 of the desired size and evaporating the solvent (and acid) by freeze-drying.
  • the freeze-drying method a conventionally known method may be used.
  • 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
  • the freeze-drying time is, for example, 96 hours. It may be less than 48 hours or less than 48 hours. Note that prior to freeze-drying, so-called preliminary freezing may be performed.
  • tissue adhesive film The porous sheet 10 of this embodiment has both high biocompatibility and adhesive strength, and thus can be used as a tissue adhesive film (tissue adhesive material) for use with living tissues. Since the porous sheet 10 has high flexibility and/or flexibility, it can also be used by being bent to follow the surface shape of living tissue.
  • Tissue adhesive membranes include, for example, hemostatic materials to stop bleeding from biological tissues (blood vessels, etc.), anti-adhesion materials to prevent postoperative adhesions, and biological tissues (pulmonary pleura, gastrointestinal anastomoses, oral and periodontal tissues). Wound dressings for covering wounds caused by (etc.), etc.
  • the porous sheet 10 also has excellent absorbency and biocompatibility, so that it can be used, for example, in the gap between the dura mater and the dura mater, at the dura mater suture area. Alternatively, it can also be used as a sealant (closing material) for filling the gap between the dura mater forming material and the dura mater.
  • the tissue adhesive film may be planar or, for example, cylindrical. Since the porous sheet 10 has high flexibility and/or flexibility, it can be easily processed into a cylindrical shape.
  • the cylindrical tissue adhesive membrane can be used, for example, to cover anastomoses such as nerves and tendons.
  • the tissue adhesive film of this embodiment adheres to living tissue by pasting it on living tissue and leaving it.
  • the standing time is the time required for the tissue adhesive film to absorb moisture in the tissue and crosslink, and may be set as appropriate depending on the proportion of the constituent materials in the tissue adhesive film. The time may be between 10 minutes and 10 minutes. Further, at this time, heating may be performed as long as it is 37° C. or less, or a weak alkaline aqueous solution such as a sodium bicarbonate aqueous solution may be applied.
  • the laminate 50 includes the porous sheet 10 of the first embodiment (hereinafter sometimes referred to as "lower layer 10" or “sheet (I)”), and raw material gelatin (first gelatin) laminated thereon. ) (hereinafter sometimes referred to as "upper layer 30" or “sheet (II)”). That is, the lower layer 10 (sheet (I)) is laminated on one surface (on the lower surface) of the upper layer 30 (sheet (II)).
  • the laminate 50 of this embodiment can be used, for example, as a tissue adhesive film.
  • the laminate 50 is attached with the lower layer 10 side in contact with the living tissue.
  • the lower layer 10 absorbs moisture by being attached to living tissue, crosslinks, and adheres to the tissue. Since the laminate 50 includes the porous sheet 10 (lower layer 10), it has the same effects as the first embodiment. That is, it has high biocompatibility and adhesive strength, and also has flexibility and/or flexibility.
  • the layered structure makes it difficult for moisture to circulate to the upper layer 30, and the stickiness (adhesiveness) of the surface of the layered product 50 (upper layer 30) is suppressed. Therefore, for example, handling properties during surgery are improved. Further, since no hydrophobic group is introduced into the raw material gelatin of the upper layer 30, its adhesiveness to living tissue is low. Therefore, the surface (upper layer 30) of the laminate 50 attached to living tissue is difficult to adhere to other living tissues, and also functions as an anti-adhesion material. The details of the laminate 50 of this embodiment will be explained below.
  • the lower layer 10 of the laminate 50 may have the same form as the porous sheet 10 of the first embodiment, and the preferred form is also the same.
  • 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. Further, the density of the lower layer 10 is preferably 0.05g/cm 3 to 0.2g/cm 3 , more preferably 0.07g/cm 3 to 0.2g/cm 3 . When the density is below the above upper limit value, the laminate 50 can have sufficient voids, which can provide more appropriate flexibility and further reduce the hardening reaction between the hydrophobized gelatin derivative and the crosslinking agent. Can be promoted. Further, if the density is equal to or higher than the lower limit value, the water absorption rate when the laminate 50 is attached to living tissue becomes slow, making it more difficult for water to flow into the upper layer 30.
  • the curing reaction progresses in the lower layer 10 before much water reaches the upper layer 30, allowing strong adhesion to living tissue.
  • the laminate 50 exhibits strong adhesiveness even in an environment with a lot of moisture, such as in a living body where a large amount of blood, body fluids, etc. are present due to bleeding, etc., and the surface (upper layer 30) Stickiness (stickiness) is suppressed.
  • handling properties are improved, and adhesion to other tissues and adhesion to rubber gloves during surgery is suppressed.
  • the thickness of the lower layer 10 of the laminate 50 may be 0.1 mm or more, 0.5 mm or more, 1.5 mm or more, or 2 mm or more. If the thickness is greater than or equal to the above lower limit, the laminate will exhibit strong adhesion even in an environment with a lot of moisture, such as in a living body where there is a large amount of blood or body fluids due to bleeding, etc. The stickiness (adhesiveness) of the surface of 50 (upper layer 30) is suppressed, the handling properties are improved, and the adhesion prevention performance to other tissues is further enhanced. Further, the upper limit of the thickness of the lower layer 10 is not particularly limited, but the practical range as a tissue adhesive film is, for example, 8 mm or less, or 5 mm or less.
  • 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.
  • the raw material gelatin for the upper layer 30 may be the same as the raw material gelatin of the first embodiment described above, and the preferred embodiments are also the same.
  • the crosslinked product of raw material gelatin is not particularly limited, and may be a crosslinked product obtained by crosslinking raw material gelatin by any method, but it is preferably a thermally crosslinked product. According to thermal crosslinking, a crosslinked product of raw material gelatin can be obtained more easily, and it is safe because there is no generation of impurities derived from the crosslinking agent.
  • the thermally crosslinked product for example, amino groups in the gelatin mixture react with other reactive groups (eg, carboxy groups, mercapto groups, etc.) to form a crosslinked structure.
  • the upper layer 30 may be composed only of a crosslinked product of raw gelatin, or may contain other components other than the crosslinked product of raw gelatin as long as the effects of the present invention are achieved.
  • the content of the crosslinked product of raw material gelatin in the upper layer 30 may be 100% by mass, 95% by mass or more, or 90% by mass or more.
  • other components other than the crosslinked raw material gelatin include basic compounds described in Modification 1 below.
  • Other components other than the basic compound are not particularly limited, but include, for example, pigments, photocatalysts, colorants, preservatives, inorganic nanoparticles (gold nanoparticles, iron oxide nanoparticles, calcium phosphate nanoparticles, etc.), and drugs. (anticancer drugs, antithrombotic drugs, antibacterial agents, growth factors, etc.), etc.
  • Top layer 30 is preferably porous and/or thin.
  • the flexibility and/or flexibility of the entire laminate 50 can be further enhanced.
  • the density of the upper layer 30 is not particularly limited, and may be, for example, 0.01 g/cm 3 to 0.3 g/cm 3 . If the density is below the upper limit of the above range, more sufficient flexibility can be easily obtained. Moreover, if the density is at least the lower limit of the above range, the film strength will increase and the handling properties will also improve.
  • 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 at least the above lower limit, handling properties will be further improved, and adhesion to other tissues and adhesion to rubber gloves during surgery will be suppressed. Further, the upper limit of the thickness of the upper layer 30 is not particularly limited, but from the viewpoint of improving flexibility, it is preferably, for example, 2 mm or less, 1 mm or less, or 0.5 mm or less.
  • 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, handling properties are further improved, and adhesion prevention performance to other tissues is further enhanced.
  • the manufacturing method of the laminate 50 of this embodiment is not particularly limited, it can be manufactured, for example, by a manufacturing method including steps S10, S20, and S30 described below (see FIG. 7).
  • Step S10 Preparation of lower layer 10 (sheet (I))
  • Step S20 Preparation of upper layer 30 (sheet (II))
  • Step S30 Lamination of lower layer 10 and upper layer 30.
  • Step S10 The method for manufacturing the lower layer 10 (porous sheet (I)) is not particularly limited, but may be performed by a method similar to that for the porous sheet 10 described in the first embodiment, for example, a method including steps S1 and S2 shown in FIG. Can be manufactured.
  • Step S20 The method for manufacturing the upper layer 30 (sheet (II) containing a crosslinked product of raw material gelatin) is not particularly limited, but may be manufactured by, for example, the method described below. First, a raw gelatin solution (raw material solution (II)) containing raw gelatin 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 upper layer 30 is obtained by heating the sheet to thermally crosslink it.
  • a raw gelatin solution raw material solution (II)
  • solvent is evaporated by freeze-drying to obtain a sheet (precursor sheet) of a desired size.
  • the upper layer 30 is obtained by heating the sheet to thermally crosslink it.
  • Examples of the solvent for dissolving raw material gelatin include those similar to the solvent form of the raw material liquid in the first embodiment, and preferred forms are also the same.
  • the concentration of raw gelatin in the raw 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 manufactured.
  • the raw gelatin concentration in the raw gelatin solution may be, for example, 1 w/v% to 20 w/v%.
  • a conventionally known method may be used for the freeze-drying, and the preferable 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.
  • the method for thermally crosslinking the 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 preferably 80 to 200°C, more preferably 100 to 200°C.
  • the heating time during thermal crosslinking is not particularly limited, but is preferably 0.1 to 20 hours, more preferably 1 to 10 hours.
  • Step S30 The laminate 50 of this embodiment is obtained by stacking the manufactured lower layer 10 and upper layer 30 one on top of the other. When the laminate 50 is attached to living tissue, the lower layer 10 is crosslinked, thereby bonding the lower layer 10 and the upper layer 30 together.
  • the upper layer (sheet (II)) 30 contains a basic compound 31.
  • the basic compound 31 is a compound that exhibits basicity when it comes into contact with moisture. Except for containing the basic compound 31, the laminate 51 has the same configuration as the laminate 50 of the second embodiment described above. Regarding the configuration similar to that of the laminate 50, explanation in this modification will be omitted.
  • the laminate 51 of this modification can be used, for example, as a tissue adhesive film, and has the same effects as the laminate 50. Furthermore, the laminate 51 has the following effects by containing the basic compound 31. As shown in FIG. 8, when the laminate 51 is applied to the biological tissue 20, the laminate 51 absorbs moisture from the biological tissue 20, and the basic compound 31 is eluted by the absorbed moisture. Thereby, the pH within the lower layer 10 increases more quickly, and the crosslinking reaction (hardening reaction) between the hydrophobized gelatin derivative and the crosslinking agent is promoted. That is, it is possible to shorten the time (adhesion time) from when the laminate 51 is attached to the living tissue 20 to when sufficient adhesive strength is obtained.
  • the basic compound 31 is not particularly limited as long as it shows basicity when in contact with water, but for example, if the pH of an aqueous solution of 0.05 mol/L to 0.5 mol/L is 7.5 to 10, The compounds shown below are preferred, and the compounds whose pH in aqueous solution of 0.1 mol/L to 0.3 mol/L is 8.0 to 9.5 are more preferred. A compound having such characteristics can further shorten the adhesion time.
  • the basic compound 31 is a pH buffering agent whose aqueous solution functions as a pH buffering solution. Examples of the pH buffer 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.
  • the pH within the lower layer 10 can be easily maintained within a desired range when the laminate 51 is attached to the biological tissue 20. Moreover, it is possible to suppress the pH of the lower layer 10 from rising too much, and damage to the living tissue 20 can be prevented.
  • pH buffer examples include the components and active ingredients of the pH buffer listed below.
  • 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 phosphoric acid and a phosphate salt (e.g. sodium acetate)
  • citrate buffer a mixture of phosphoric acid and a phosphate salt (e.g. sodium acetate)
  • citrate buffer a mixture of phosphoric acid and a phosphate salt (e.g.
  • citrate phosphate buffer a mixture of citric acid and a phosphate (e.g., sodium phosphate)
  • a borate buffer a mixture of citric acid and a phosphate (e.g., sodium phosphate)) acid and a borate (e.g., sodium borate)
  • tartrate buffer a mixture of tartaric acid and a tartrate (e.g., sodium tartrate)
  • succinate buffer a mixture of succinic acid and a succinate) (e.g., a mixture with sodium succinate), maleic acid (a mixture of maleic acid and a maleate salt (e.g., sodium maleate)), Tris-hydrochloric acid buffer (a mixture of tris-aminomethane and hydrochloric acid), carbonate buffer liquid (a mixture of carbonic acid and a carbonate (e.g.
  • the ratio of each component in the pH buffer may be adjusted as appropriate depending on the desired pH. .
  • Examples of the basic compound 31 other than the pH buffer include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, zinc hydroxide, and the like. These basic compounds 31 may be one type of compound, or may be a mixture of two or more types of compounds.
  • the content of the basic compound 31 in the upper layer 30 is not particularly limited, and may be adjusted as appropriate depending on the basicity strength of the basic compound 31.
  • the content of the basic compound 31 in the upper layer 30 may be, for example, 0.1% to 20% by weight, or 0.3% to 10% by weight.
  • the density and thickness of the upper layer 30 can be within the ranges described in the second embodiment. Further, when the upper layer 30 contains a basic compound, the higher the density and/or the thicker the upper layer 30, the greater the amount of the basic compound contained, so that the bonding time can be further shortened. Guessed. From the viewpoint of further shortening the bonding time, the density of the upper layer 30 may be, for example, 0.08 g/cm 3 to 0.3 g/cm 3 , and the thickness of the upper layer 30 may be, for example, 0.2 mm to 2 mm. .
  • the method of manufacturing the laminate 51 of this modification is not particularly limited, and is similar to the laminate 50 of the second embodiment described above except for using the basic compound 31 in producing the upper layer 30, for example, as shown in FIG. It can be manufactured by a method including steps S10 to S30 shown below.
  • the manufacturing process of the upper layer 30 (step S20 in FIG. 7) will be described below, and the description of the manufacturing process similar to that of the laminate 50 in this modification will be omitted.
  • Step S20 First, a raw gelatin solution (raw material solution (II)) containing raw 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 upper layer 30 is obtained by heating the sheet to thermally crosslink it.
  • raw gelatin solution raw material solution (II)
  • 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 upper layer 30 is obtained by heating the sheet to thermally crosslink it.
  • the basic compound examples include those similar to the basic compound 31 contained in the upper layer 30 described above, and preferred embodiments are also the same.
  • the pH of the raw material liquid (II) becomes alkaline.
  • the raw material liquid (II) preferably has a pH of 8 to 10, and preferably 8 to 9, for example.
  • the mixture of the solvent (e.g., water) and the basic compound is the pH buffer
  • the raw material solution (II) is a solution of raw gelatin dissolved in the pH buffer.
  • the concentration of the pH buffer may be, for example, 0.05 mol/L to 0.5 mol/L.
  • the concentration of the pH buffer is, for example, the concentration of a mixture of a weak acid and a salt of a weak acid, or the concentration of a mixture of a weak base and a salt of a weak base in the pH buffer.
  • the upper layer 30 can be produced by performing step S20 in the same manner as in the second embodiment described above, except for using a basic compound.
  • 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 bottom surface) of the sheet (II) 30, and the other is laminated on the other surface (on the top surface) of the sheet (II). Laminated.
  • the sheet (I) and sheet (II) included in the laminate 52 may be the same as the sheet (I) and sheet (II) described in the second embodiment and modification example 1, respectively.
  • the method for manufacturing the laminate 52 is not particularly limited, and the laminate 50 of the second embodiment and the laminate 51 of the first modification described above are the same as the laminate 50 of the second embodiment and the laminate 51 of the first modification except that three layers are laminated in the lamination step (step S30). Similarly, it can be manufactured by a method including steps S10 to S30 shown in FIG.
  • the two sheets (I) 10 have the same effect as the porous sheet 10 of the first embodiment.
  • the laminate 52 can be used, for example, as a double-sided adhesive tissue adhesive film, and furthermore, the sheet (II) 30 contains a basic compound as in Modification 1. In this case, the adhesion time for both sides of the sheet (I) can be reduced at the same time.
  • nonanal (nonylaldehyde) is reacted with the amino group of raw material gelatin to form a Schiff base, and then the obtained Schiff base is reacted with a reducing agent.
  • a hydrophobized gelatin derivative was obtained by reduction to a stable secondary amine.
  • the obtained precipitate was washed with 250 mL of ethanol (1 hour x 3 times) to remove unreacted nonanal and 2-picoline borane. Thereafter, it was vacuum dried for 3 days to obtain C9-ApGltn in a yield of 90% by mass.
  • the hydrocarbon group introduction rate (nonyl group introduction rate) DS degree of submission (hereinafter sometimes simply referred to as "DS") of C9-ApGltn is determined by the number of amino groups in the raw material gelatin and the hydrophobized gelatin derivative.
  • the number of amino groups in the hydrophobized gelatin derivative was determined by the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method), and from the obtained value, the content of amino groups (-NH 2 ) in the raw material gelatin was determined.
  • hydrophobized gelatin derivative synthesized in this example may be referred to as 7.2C9-ApGltn or 7.2C9.
  • a hydrophobized gelatin derivative liquid (liquid A) was prepared according to the following procedure.
  • the synthesized 7.2C9-ApGltn was dissolved in a 0.1 mM HCl solution, and then the pH was adjusted to 4 using 1M HCl, and then purified water was added so that the concentration of 7.2C9-ApGltn was 15 w/v%.
  • the mixture was diluted with water to obtain Solution A.
  • a crosslinking agent liquid (liquid B) was prepared according to the following procedure.
  • Liquids A and B were thoroughly mixed using a magnetic stirrer at 280 rpm for 30 seconds, and the pH was further adjusted to 4 with a 0.1N HCl solution to obtain a raw material liquid.
  • the gelatin concentration of the obtained raw material liquid was 7.5 w/v%.
  • a gelatin (7.2C9-ApGltn) concentration of 7.5% w/v was prepared in the same manner as in Example 1-1, and diluted with a 0.1mM HCl solution to give a gelatin concentration of 2.
  • the obtained raw material liquid was freeze-dried in the same manner as in Experiment 1-1 to produce a porous sheet.
  • Example 2-1 ⁇ Synthesis of hydrophobized gelatin derivative>
  • a gelatin derivative (hereinafter appropriately referred to as "C8-ApGltn") in which an octyl group was introduced into raw material gelatin (Org-ApGltn) was used.
  • the hydrophobized gelatin derivative is obtained by reacting an alkyl aldehyde (octanal, octyl aldehyde (manufactured by Tokyo Kasei Kogyo Co., Ltd.)) with the amino group of raw material gelatin to form a Schiff base in the same manner as in Example 1-1.
  • the resulting Schiff base was reduced to a stable secondary amine using a reducing agent.
  • Example 3-1 ⁇ Synthesis of hydrophobized gelatin derivative>
  • a gelatin derivative (hereinafter appropriately referred to as "C10-ApGltn") in which a decyl group was introduced into raw material gelatin (Org-ApGltn) was used.
  • the hydrophobized gelatin derivative is obtained by reacting an alkyl aldehyde (decanal, decyl aldehyde (manufactured by Tokyo Kasei Kogyo Co., Ltd.)) with the amino group of raw material gelatin to form a Schiff base in the same manner as in Example 1-1.
  • the resulting Schiff base was reduced to a stable secondary amine using a reducing agent.
  • the hydrophobized gelatin derivative synthesized in this example may be referred to as 9.3C10-ApGltn or 9.3C10.
  • a raw material solution with a gelatin concentration of 7.5 w/v% was prepared by the same method as in Example 1-1, except that 9.3C10-ApGltn was used instead of 7.2C9-ApGltn, and the obtained raw material The liquid was freeze-dried to produce a porous sheet.
  • Example 3-2 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 diluted with 0.1 mM HCl solution to prepare a gelatin derivative. A raw material solution having a concentration of 5 w/v% and a pH of 4 was obtained. The obtained raw material liquid was freeze-dried in the same manner as in Example 3-1 to produce a porous sheet.
  • a hydrophobized gelatin derivative (9.3C10-ApGltn) having a concentration of 7.5 w/v% was prepared in the same manner as in Example 3-1, and it was diluted with a 0.1 mM HCl solution.
  • the obtained raw material liquid was freeze-dried in the same manner as in Example 3-1 to produce
  • 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. Note that 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 prepared in Examples 1-1, 2-1, and 3-1 using a raw material solution with a gelatin concentration of 7.5 w/v% was approximately 0.075 g/ cm3 , and the gelatin concentration
  • the density of the porous sheets prepared in Examples 1-2, 2-2, and 3-2 using 5 w/v% raw material liquid was approximately 0.05 g/ cm3 , and the gelatin concentration was 2.5 w/v%.
  • the density of the porous sheets prepared in Examples 1-3, 2-3, and 3-3 using v% raw material liquids was about 0.025 g/cm 3 .
  • 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 FIG. 4, the porosity (density) of the porous sheet depends on the gelatin concentration (7.2C9-ApGltn concentration or Org-ApGltn concentration) of the raw material solution. The lower the gelatin concentration, the larger the percentage of voids in the porous sheet (lower density), and the higher the gelatin concentration, the smaller the percentage of voids in the porous sheet (higher density).
  • sealing strength pressure strength
  • porcine aorta as a base material.
  • the porous sheet 10 prepared in each example or comparative example was placed so as to cover the central defect 20a (diameter 1 mm) of the porcine aorta 20 (diameter 35 mm), and was fixed by pressing lightly with tweezers. , and left to stand for 10 minutes.
  • Comparative Example 1-3 in which the gelatin concentration of the raw material liquid was 2.5 w/v%, and Examples 2-3, 1-3, and 3-3 are compared.
  • the porous sheet using the raw material gelatin Comparative Example 1-3
  • the porous sheet using the hydrophobized gelatin derivative Examples 2-3, 1-3, and 3-3
  • Peeling pressure pressure resistance
  • the longer the introduced alkyl chain the higher the peeling pressure tended to be.
  • hydrophobized gelatin derivatives are used compared to porous sheets (Org) using raw gelatin.
  • the porous sheets that were used showed high peel pressure.
  • Example 4-1 Comparative Example 2-1, Comparative Examples 2-2 to 4-3, Example 5-1, Comparative Example 3-1, Comparative Example 3-2, and Example 6- described below
  • a laminate was produced in which a porous sheet of a hydrophobized gelatin derivative (lower layer, sheet (I)) and a porous sheet of raw material gelatin (upper layer, sheet (II)) were laminated.
  • sheet (II) was colored with a dye (acid blue). Table 2 shows details of the sheets produced in each experiment.
  • Example 4-1 First, as the sheet (I), a porous sheet of the hydrophobized gelatin derivative 9.3C10-ApGltn prepared in Experiment 3-1 (thickness 2 mm, gelatin concentration in the raw material solution (I) was 7.5 w/v% ) was prepared.
  • a 5 w/v % aqueous solution (raw material solution (II)) of raw material gelatin (Org-ApGltn) was prepared and colored with acid blue (25 ⁇ g/mL).
  • the prepared raw gelatin aqueous solution was cast into a silicone sheet mold (70 mm x 70 mm x depth: 0.5 mm).
  • the sample was first freeze-dried at -30°C overnight. After freeze-drying, it was heated at 150° C. for 5 hours to thermally crosslink Org-ApGltn to obtain sheet (II).
  • Sheet (II) was laminated on sheet (I) to obtain a laminate.
  • the sheet (I) was a porous sheet of the hydrophobized gelatin derivative 9.3C10-ApGltn prepared in Example 3-3 (thickness: 2 mm, the concentration of the hydrophobized gelatin derivative in the raw material liquid was 2.5 w/v%). ) was prepared. Otherwise, a laminate consisting of sheets (I) and (II) was produced in the same manner as in Example 4-1.
  • FIG. 6 shows photographs of the laminates produced in Example 4-1, Comparative Example 2-1, and Comparative Example 2-2.
  • the upper row is a photograph taken from the sheet (II) side
  • the lower row is a photograph taken from the sheet (I) side.
  • 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 sheet (I) was changed from 2 mm to 1 mm.
  • 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.
  • Example 6-2 Sheet (I) and ( A laminate consisting of II) was produced.
  • Example 6-3 Sheet (I) and ( A laminate consisting of II) was produced.
  • the laminate with a two-layer structure had lower adhesiveness than the porous sheet with a single-layer structure (Example 3-1). From this result, it was found that by laminating a porous sheet of raw material gelatin on a porous sheet of hydrophobized gelatin derivative to form a laminate, when the laminate is used as a biological tissue adhesive film, it is possible to adhere to other biological tissues. It can be expected to suppress adhesion to rubber gloves during surgery, etc.
  • Example 5-1 and 6-1 to 6-3 were laminates that obtained good results in pressure resistance test 2, which will be described later.
  • the adhesion 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).
  • the laminate produced in each example was punched out using a dumbbell cutter to produce a circular evaluation sample with a diameter of 15 mm.
  • a dumbbell cutter to produce a circular evaluation sample with a diameter of 15 mm.
  • the central defect (diameter 1 mm) of the porcine aorta (diameter 35 mm) was covered.
  • evaluation samples (laminates) of each Example and Comparative Example were placed thereon.
  • the sheet (I) of the laminate was placed on the lower side (the side in contact with the porcine aorta).
  • 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.
  • the Unipack was peeled off from the evaluation sample, and the sealing strength (pressure strength) was measured in accordance with ASTM (F2392-04).
  • the porcine aorta 20 carrying the evaluation sample 10 is fixed to a jig, and 37°C physiological saline is poured into the defect 20a covered with the evaluation sample 10 (laminate) at a flow rate of 2 mL/min.
  • the pressure at which the evaluation sample 10 was peeled was measured.
  • three sheets were prepared and the pressure resistance test was conducted three times. The results are shown in Table 2.
  • the laminates containing the porous sheets (I) with relatively low raw material liquid gelatin concentrations produced in Comparative Examples 3-2, 2-2, 3-1, and 2-1 did not use Unipack.
  • the laminate also peeled off from the porcine aorta, making it impossible to measure pressure resistance (evaluation result: N.D.). That is, these porous sheets could not provide sufficient adhesiveness. Therefore, the second and subsequent pressure tests were not conducted on the laminates of Comparative Examples 3-2, 2-2, 3-1, and 2-1.
  • the laminate containing the porous sheet (I) having a relatively high raw material liquid gelatin concentration prepared in Examples 5-1, 4-1, and 6-1 to 6-3 was We were able to peel it off from the aorta and conduct a pressure test.
  • Table 2 shows the highest compressive strength obtained for each laminate.
  • the evaluation results (pressure resistance strength) of the same test conducted using a commercially available hemostatic agent are also shown in Table 2.
  • the evaluation results of the laminates of Examples 5-1, 4-1, and 6-1 to 6-3 were better than the evaluation results of commercially available products, and it was confirmed that the laminates had adhesion and pressure resistance without any practical problems. It could be confirmed.
  • Example 7-1 to 7-7, 8-1, and 9-1 described below a pH buffer was used during the production of sheet (II) to produce a laminate.
  • Example 7-8 a laminate was produced under the same conditions as in Example 7-1, except that no pH buffer was used during production of sheet (II). Table 3 shows details of the laminates produced in each experiment.
  • Example 7-1 ⁇ Synthesis of hydrophobized gelatin derivative> Experiment 1-1 except that decanoic anhydride was used as the hydrophobizing reagent and the amount of alkyl acid anhydride was adjusted so that the hydrocarbon group introduction rate (decanoyl group introduction rate) DS was 12 mol%.
  • a hydrophobized gelatin derivative was synthesized using the same method as described above.
  • the hydrophobized gelatin derivative synthesized in this experiment may be referred to as 12C10-ApGltn or 12C10.
  • Sheet (II) was laminated on sheet (I) to obtain a laminate for this experiment.
  • Example 7-4 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 gelatin concentration was 15 w/v% in producing the sheet (I).
  • 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 the production of sheet (I) was 10,000.
  • 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 the production of sheet (I) was 10,000.
  • 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 the production of sheet (I) was 10,000.
  • Example 7-8 A laminate was produced under the same conditions as in Example 7-1, except that a pH buffer was not used during the production of sheet (II) and pure water was used instead.
  • Adhesion comparison test This test was conducted on the laminates prepared in Examples 7-1 to 7-8, 8-1, and 9-1, and on the laminates of Example 4-1, etc. An adhesion comparison test was conducted. The adhesion of the laminates of all the examples shown in Table 3 was lower than that of the single-layer porous sheet (Example 3-1). From this result, when these laminates are used as a living tissue adhesive film, it can be expected to suppress adhesion to other living tissues, suppress adhesion to rubber gloves during surgery, etc.
  • the evaluation results of the laminates in all experiments except Examples 7-8 were better than the evaluation results of commercially available products, indicating that they had adhesion and pressure resistance without any practical problems. It could be confirmed.
  • the sheet (II) was a basic compound (a mixture of phosphoric acid and sodium phosphate). Contains. This causes the pH within the sheet (I) to rise more quickly, promoting the crosslinking reaction (curing reaction) between the hydrophobized gelatin derivative and the crosslinking agent, and resulting in good evaluation results even if the adhesion time was shortened to 3 minutes. It is assumed that it was obtained.
  • the laminate of Examples 7-8 in which no pH buffer was used in the production of sheet (II) i.e., the laminate containing no basic compound
  • the laminate of Example 4-1, etc. in which no pH buffer was used to prepare the sheet (II) shown in Table 2, with the adhesion time shortened to 3 minutes.
  • Sufficient adhesive strength could not be obtained with a short bonding time of 3 minutes, and similarly to Examples 7-8, the pressure resistance could not be measured (evaluation result: N.D.).
  • the porous sheet of the present invention can be used as a tissue adhesive film used in vivo (for example, a hemostatic material, an anti-adhesion material), and has high adhesive strength and high biocompatibility. Therefore, the porous sheet of the present invention can be used in the surgical and internal medicine fields.

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  • Health & Medical Sciences (AREA)
  • Surgery (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Materials For Medical Uses (AREA)

Abstract

L'invention concerne une feuille poreuse ayant une biocompatibilité et une force d'adhérence élevées, et ayant en outre une souplesse et/ou une flexibilité.　La feuille poreuse comprend : une seconde gélatine ayant un groupe hydrocarboné introduit dans une première gélatine ; et un agent de réticulation n'ayant pas réagi dispersé dans la seconde gélatine. La seconde gélatine comprend une structure représentée par la formule (1).
PCT/JP2023/032230 2022-09-06 2023-09-04 Feuille poreuse, film adhésif tissulaire, leur utilisation en tant qu'agent hémostatique ou matériau anti-adhérence, et leurs procédés de production WO2024053602A1 (fr)

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JP2022-141561 2022-09-06
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JP2023071912 2023-04-26

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05176983A (ja) * 1991-12-26 1993-07-20 Kanebo Ltd 二層性蛋白質シート及びその製造方法
JPH10113384A (ja) * 1996-10-14 1998-05-06 Yoshihiko Shimizu 医用代替膜及びその製造方法
JP2009515619A (ja) * 2005-11-17 2009-04-16 ゲリタ アクチェンゲゼルシャフト 特に医療用途のための複合材料、及び材料の製造方法
WO2012046759A1 (fr) * 2010-10-05 2012-04-12 独立行政法人物質・材料研究機構 Film d'adhésif tissulaire et procédé de production de celui-ci
JP2014005211A (ja) * 2012-06-21 2014-01-16 Kagoshima Univ ゼラチンゲルの作製法
WO2015076252A1 (fr) * 2013-11-22 2015-05-28 独立行政法人物質・材料研究機構 Film poreux adhésif tissulaire, son procédé de fabrication, et bande de film poreux adhésif tissulaire
US20200237957A1 (en) * 2017-10-04 2020-07-30 Bio-Change Ltd. Cross-linked protein foams and methods of using thereof a polyvalent cellular scaffold

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05176983A (ja) * 1991-12-26 1993-07-20 Kanebo Ltd 二層性蛋白質シート及びその製造方法
JPH10113384A (ja) * 1996-10-14 1998-05-06 Yoshihiko Shimizu 医用代替膜及びその製造方法
JP2009515619A (ja) * 2005-11-17 2009-04-16 ゲリタ アクチェンゲゼルシャフト 特に医療用途のための複合材料、及び材料の製造方法
WO2012046759A1 (fr) * 2010-10-05 2012-04-12 独立行政法人物質・材料研究機構 Film d'adhésif tissulaire et procédé de production de celui-ci
JP2014005211A (ja) * 2012-06-21 2014-01-16 Kagoshima Univ ゼラチンゲルの作製法
WO2015076252A1 (fr) * 2013-11-22 2015-05-28 独立行政法人物質・材料研究機構 Film poreux adhésif tissulaire, son procédé de fabrication, et bande de film poreux adhésif tissulaire
US20200237957A1 (en) * 2017-10-04 2020-07-30 Bio-Change Ltd. Cross-linked protein foams and methods of using thereof a polyvalent cellular scaffold

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