JP2004049921A - Wound dressing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a wound dressing capable of retaining the form and condition of the dressing even in possible contact with, an exudate, and the like, and minimizing contamination with microorganisms and substances originated therefrom which adversely affect a human body. <P>SOLUTION: This wound dressing comprises a cell-adhering polypeptide (P) containing not greater than 0.15 EU/mg of endotoxin based on the weight of the cell-adhering polypeptide (P) and a coating material (N). The coating material (N) is preferably a material which is difficult to decompose (N2), and the polypeptide (P) is preferably a cell-adhering synthetic polypeptide (P2). <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a wound dressing. More particularly, it relates to a wound dressing having a cell adhesive polypeptide.

Conventionally, as a wound dressing having a cell-adhesive polypeptide, a wound dressing obtained by processing naturally-derived collagen into a sheet or sponge (Patent Document 1), and a skin-derived fibroblast on a collagen sheet or in a collagen sheet And a fibroblast-containing collagen sheet (Patent Document 2) obtained by culturing the same.

JP-A-7-59812 JP-A-9-47502

Conventional wound dressings having cell-adhesive polypeptides have a problem such as infection due to contamination of the wound dressing with microorganisms or microorganism-derived substances that adversely affect the human body because the main constituent is a protein, In addition, there has been a problem that the wound dressing is lost by exudation from the wound and loses the form of the wound dressing, and cannot be replaced when the wound dressing is applied to a wound surface. That is, an object of the present invention is to provide a wound dressing material in which contamination of microorganisms and microorganism-derived substances that adversely affect the human body is extremely low, and the shape of the wound dressing material is maintained even when there is contact with exudate or the like. To provide.

As a result of intensive studies, the present inventors have found that the above objects can be achieved by using specific peptides and coating materials, and have reached the present invention. That is, the feature of the wound dressing of the present invention is that the content of the endotoxin is less than 0.15 EU / mg based on the weight of the cell adhesive polypeptide (P) and the coating material ( N).

The wound dressing of the present invention has a very low endotoxin content and can adhere cells to the wound dressing very efficiently. Furthermore, when the wound dressing of the present invention is applied to a wound surface, the wound surface can be regenerated in an extremely favorable state without infection or fusion. Therefore, the wound dressing of the present invention has a low risk of contamination by microorganisms and substances derived from microorganisms, can easily replace the wound dressing, and can promote wound healing.

The cell adhesive polypeptide (P) contains a minimal amino acid sequence (X) representing a cell adhesion signal. Examples of the minimum amino acid sequence (X) that represents a cell adhesion signal include, for example, “Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990” and “Osaka Prefectural Mother and Child Medical Center, Vol. 8, No. 1”. No. 58-66, 1992 ".

Among these minimum amino acid sequences (X), Arg Gly Asp sequence, Leu Asp Val sequence, Arg Glu Asp Val sequence (1), Tyr Ile Gly Ser Arg sequence (2), Pro Asp Ser Gly Arg sequence (3) Arg Tyr Val Val Leu Pro Arg sequence (4), Leu Gly Thr Ile Pro Gly sequence (5), Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile sequence (6), Ile Lys Val Ala Val sequence (7), Leu Arg Glu sequence, Asp Gly Glu Ala sequence (8), Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9), Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys sequence (10) , His Ala Val sequence and Tyr Lys Leu Asn Val Asn Asp Ser sequence (11) are preferable, and from the viewpoint of cell adhesion, more preferably Arg Gly Asp sequence, Tyr Ile Gly Ser Arg sequence (2) and Ile Lys Val Ala Val sequence (7), particularly preferably Arg Gly Asp sequence. The amino acid sequence is represented by three letters, and the sequence number corresponding to the sequence listing is described in () (the same applies hereinafter).

The cell adhesive polypeptide (P) may have at least one minimum amino acid sequence (X) in one molecule, but preferably has two or more in one molecule from the viewpoint of cell adhesion, Those having 3 or more are more preferable, those having 5 or more are particularly preferable, those having 50 or less in one molecule are preferable, those having 30 or less are more preferable, and those having 20 or less are particularly preferable. Further, two or more types of sequences may be included in one molecule.

The number average molecular weight (Mn) of the cell adhesive polypeptide (P) is preferably 3,000,000 or less, more preferably 1,000,000 or less, particularly preferably 300,000 or less, from the viewpoint of cell adhesion. It is preferably at least 300, more preferably at least 1,000, particularly preferably at least 3,000. The Mn of the cell adhesive polypeptide (P) was determined by separating the cell adhesive polypeptide (P) by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and comparing the migration distance with a standard substance. (The same applies hereinafter).

As the cell-adhesive polypeptide (P), a cell-adhesive natural polypeptide (P1) and a cell-adhesive artificial polypeptide (P2) can be used. Cell-adhesive artificial polypeptide (P2) is characterized in that it can freely design the number and arrangement of the amino acid sequence (Y), enhance cell adhesiveness, and facilitate sterilization and endotoxin degradation by heat treatment. preferable.

As the cell-adhesive natural polypeptide (P1), glycoproteins present in the basement membrane (eg, laminin, entactin (nidogen), tenascin, agrin, osteonectin, osteocalcin, osteopontin, fibrin, fibrinogen, vitronectin, ancarin, bamin and Thrombospondin, etc.), proteoglycans (eg, aggrecan, perlecan, biglycan, decorin, fibromodulin, versican, durin, neurocan, brevican, lumican, serglycine, syndecan, CD44, beta-glycan, thrombomodulin, glypican, Celebroglycan and NG2 proteoglycan, etc., glycoproteins present in cell membranes (eg, integrins, integrin superfamily, cadherin Fine cadherin superfamily etc.), and tight junction related substances (for example, occludin, etc.) and the like.

Examples of the artificial cell-adhesive polypeptide (P2) include a polypeptide consisting of the Tyr Ile Gly Ser Arg sequence (2), a polypeptide consisting of the Ile Lys Val Ala Val sequence (7), and an Arg Gly Asp Ser sequence (14). A polypeptide consisting of the GlyArg Gly Asp Ser sequence (15), a polypeptide consisting of the Gly Arg Gly Asp Ser Pro sequence (16), and a polypeptide consisting of the Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro sequence (17) Polypeptide, polypeptide consisting of Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala sequence (18), Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro Ser polypeptide (19), Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Polypeptide consisting of Val Ala Ser Ala Asp Arg sequence (20), Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp polypeptide (21), and polypeptides thereof At least one type of polypeptide For example, a polymer made of a metal can be used. In addition to these, as a polymer, for example, a polymer consisting of (Arg Gly Asp Ser) ₄ sequence (22), a polymer consisting of (Arg Gly Asp Ser) ₈ sequence (23), (Arg Gly Asp Ser) ₁₆ A polymer consisting of the sequence (24), a polymer consisting of the (Gly Arg Gly Asp Ser) ₈ sequence (25), a polymer consisting of the (Gly Arg Gly Asp Ser Pro) ₈ sequence (26), a (Arg Gly Asp Ser Pro) (Ala Ser Ser Lys Pro) A polymer consisting of _four sequences (27), (Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ala) A polymer consisting of _four sequences (28), (Pro Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gly Pro _{Ser) 4} consisting of the sequence (29) polymer, polymer comprising (Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val Ser Ala Asp Arg) 4 sequence (30), or, ( Val Cys Glu Pro Gly Tyr Ile Gly Ser Arg Cys Asp) Polymers consisting of ₄ sequences (31). From the viewpoint of cell adhesion, the polymerization degree (number of repeating units) of this polymer is preferably 2 or more, more preferably 3 or more, particularly preferably 4 or more, and preferably 50 or less, and more preferably 30 or less. Particularly preferred is 20 or less, most preferably 16 or less.

The artificial cell-adhesive polypeptide (P2) preferably further contains a heat-resistant amino acid sequence (Y) other than the minimum amino acid sequence (X) that represents a cell adhesion signal. Examples of the thermostable amino acid sequence (Y) include a Gly Ala Gly Ala Gly Ser sequence (12), a Gly Val Gly Val Pro sequence (13), a Gly Pro Pro sequence, a Gly Ala Gln Gly Pro Ala Gly Pro Gly sequence (32), Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln sequence (33), Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro sequence (34), Gly Ala sequence, Gly Ala Gly Ala Gly Tyr (35) Sequence, Gly Ala Gly Val Gly Tyr sequence (36), Gly Ala Gly Tyr Gly Val sequence (37), Asp Gly Gly (Ala) ₁₂ Gly Gly Ala sequence (38), Gly Val Pro Gly Val sequence (39 ), Gly, Ala, and Gly Gly Ala sequences. When the heat-resistant amino acid sequence (Y) is contained, the stability to heat is further increased, and the cell-adhesive polypeptide and the substrate containing the cell-adhesive polypeptide are easily heat-sterilized by an autoclave or the like. Among these heat-resistant amino acid sequences (Y), Gly Ala Gly Ala Gly Ser sequence (12), Gly Val Gly Val Pro sequence (13) and Gly Pro Pro sequence are preferable because excellent heat resistance is obtained. Preferably, it is a Gly Ala Gly Ala Gly Ser sequence (12).

The heat-resistant amino acid sequence (Y) is preferably the same or different (Y) in order to further improve the stability to heat. The polymerization degree (number of repeating units) of the heat-resistant amino acid sequence (Y) is preferably 2 to 100, more preferably 3 to 50, particularly preferably 4 to 30, and most preferably 4 to 20. is there.
When the thermostable amino acid sequence (Y) is contained, (Y) may be contained at any position of the artificial cell-adhesive polypeptide (P2). It is preferable that the polymer (Y) and the polymer (X) are alternately located, from the viewpoint of the ease of taking the β-turn structure of the artificial cell-adhesive polypeptide (P2).

When the cell-adhesive artificial polypeptide (P2) has a heat-resistant amino acid sequence (Y), the content of the heat-resistant amino acid sequence (Y) is determined from the viewpoint of heat stability. The compound having 3 or more in one molecule of (P2) is preferable, more preferably 10 or more, particularly preferably 30 or more, and preferably 10,000 or less, more preferably It has 3,000 or less, particularly preferably 1,000 or less.

Examples of the cell-adhesive artificial polypeptide (P2) having a heat-resistant amino acid sequence (Y) are described in, for example, JP-A-3-502935 and Handbook of Biodegradable Polymers, Harwood Academic Publishers, Amsterdam. And the following polypeptides: That is, a polypeptide (SLPF) of about 100,000 Mn having about 13 each of (Gly Ala Gly Ala Gly Ser) ₉ sequence (40) and Arg Gly Asp sequence, (Gly Ala Gly Ala Gly Ser) ₉ sequence ( (Gly Ala Gly Ala Gly Ser) ₉ sequence (40) and Ile Lys Val Ala Val sequence (7), each having about 100,000 polypeptides each having about 13 Tyr Ile Gly Ser Arg sequences (2). And (Gly Val Gly Val Pro) ₈ sequence (41), (Gly Ala Gly Ala Gly Ser) ₁₂ sequence (42) and Arg Gly Asp sequence A polypeptide of about 100,000 Mn having about 12 each, and about 6 each of (Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro) ₂ (43) sequence and Arg Gly Asp sequence And polypeptides having an Mn of about 50,000.

In addition to the above, as a cell-adhesive artificial polypeptide (P2) having a heat-resistant amino acid sequence (Y), the following minimum amino acid sequence (X) and heat-resistant amino acid sequence (1) to (3): A polypeptide having a structure in which Y and Y are alternately chemically bonded can also be used.
(1) When the minimum amino acid sequence (X) is the Arg Gly Asp sequence (x1), five (x1) and Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly sequence (44) (y1 A) a polypeptide (45) having 4 of (x1), a polypeptide (46) having 10 of (x1) and 9 of (y1), 15 of (x1) and 14 of (y1). 4 polypeptides (47) and (x1) and 3 Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly sequence (48) (y2) (49), a polypeptide (50) having eight (x1) and seven (y2), a polypeptide (12) having (x1) and eleven (y2) ( 51), 5 of (x1) and 4 of Ser Pro Ala Ala Ser Asp Gly Gly (Ala) ₁₂ Gly Gly Ala Ala Ser Thr Gly sequence (52) (y3) (53), a polypeptide (54) having 10 of (x1) and 9 of (y3), a polypeptide (55) having 15 of (x1) and 14 of (y3) ), A polypeptide (57) having 5 of (x1) and 4 of (Gly Ala) ₁₃ sequences (y4) (56), and a polypeptide having 10 of (x1) and 9 of (y4). Peptide (58), polypeptide (59) having 15 of (x1) and 14 of (y4), and the like.

(2) When the minimum amino acid sequence (X) is the Tyr Ile Gly Ser Arg sequence (2) (x2) (x2) and 5 Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly sequence (44) Polypeptide (60) having 4 (y1), polypeptide (61) having 10 (x2) and 9 (y1), 15 (x2) and (y1) Polypeptide (62) having 14 of (x2) and 4 of Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly sequence (48) ( a polypeptide (63) having three of y2), a polypeptide (64) having eight of (x2) and seven of (y2), twelve of (x2) and eleven of (y2). polypeptide (65) having a five and Ser Pro Ala Ala Ser Asp Gly Gly (Ala) 12 Gly Gly Ala Ala Ser Thr Gly sequence (52) (y in (x2) A) a polypeptide (66) having four of (x2), a polypeptide (67) having ten of (x2) and nine of (y3), fifteen of (x2) and fourteen of (y3). Polypeptide (69) having 5 of polypeptides (68) and (x2) and 4 of (Gly Ala) ₁₃ sequence (y4) (56), 10 of (x2) and 9 of (y4) And a polypeptide (71) having 15 (x2) and 14 (y4).

(3) When the minimum amino acid sequence (X) is the Ile Lys Val Ala Val sequence (7) (x3) (x3) and 5 Ser Pro Ala Gly Gly (Ala Gly Ala Gly Ser Gly) ₃ Ala Ser Thr Gly sequence (44) Polypeptide (72) having four (y1), polypeptide (73) having ten (x3) and nine (y1), fifteen (x3) and (y1) (74), 4 polypeptides of (x3) and Ser Pro Ala (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Ala Ser Thr Gly sequence (48) ( a polypeptide (75) having three of y2), a polypeptide (76) having eight of (x3) and seven of (y2), twelve of (x3) and eleven of (y2) polypeptide (77) having a five and Ser Pro Ala Ala Ser Asp Gly Gly (Ala) 12 Gly Gly Ala Ala Ser Thr Gly sequence (52) (y a (x3) A) a polypeptide (78) having four (x3) and a polypeptide (79) having ten (x3) and nine (y3); fifteen (x3) and fourteen (y3) Polypeptide (81) having 5 of polypeptides (80) and (x3) and 4 of (Gly Ala) ₁₃ sequence (y4) (21), 10 of (x3) and 9 of (y4) (82), and a polypeptide (83) having 15 (x3) and 14 (y4).

Commercially available artificial cell-adhesive polypeptides (P2) that can be obtained from the market include, for example, RGDS [polypeptide consisting of Arg Gly Asp Ser sequence (14), Mn about 400, manufactured by Peptide Institute], GRGDS [Polypeptide consisting of Gly Arg Gly Asp Ser sequence (15), Mn about 500, manufactured by Peptide Institute], Pronectin F [Arg Gly Asp sequence and ₉ (Gly Ala Gly Ala Gly Ser) sequence (40), respectively] A polypeptide produced by genetically modified Escherichia coli, Mn about 100,000, manufactured by Sanyo Kasei Kogyo Co., Ltd., and Pronectin F plus [pronectin F was made water soluble by reacting it with dimeraminoethyl chloride. things, manufactured by Sanyo Chemical Industries, Ltd.] and ProNectin L [Ile Lys Val Ala Val sequence (7) (Gly Ala Gly Ala Gly Ser) 9 sequences (40) and a each have about 13 genetically Polypeptides produced by recombinant Escherichia coli, Mn about 100,000, include Sanyo Chemical Industries, Ltd.] and the like.

The cell-adhesive artificial polypeptide (P2) is produced artificially and can be easily prepared by, for example, an organic synthesis method (enzymatic method, solid-phase synthesis method, liquid-phase synthesis method, etc.), a gene recombination method, or the like. Can be manufactured. Regarding the organic synthesis method, for example, Biochemistry Experiment Course 1, Protein Chemistry IV (July 1, 1981, edited by The Japanese Biochemical Society, published by Tokyo Chemical Co., Ltd.) or Seizoku Chemistry Experiment Course 2, Protein Chemistry ( Below) (May 20, 1987, edited by The Japanese Biochemical Society, published by Tokyo Chemical Doujin Co., Ltd.). Regarding the gene recombination method, for example, a method described in Japanese Patent Publication No. 3-502935 can be applied. In the case of using the genetic recombination method, since it may contain impurities derived from recombinant microorganisms, it is preferable to purify the polypeptide by affinity purification using an anti-polypeptide antibody or the like to make the purity of the polypeptide 80% by weight or more, It is more preferably at least 90% by weight, particularly preferably at least 95% by weight. Among them, the genetic recombination method is preferred from the viewpoint that the amino acid sequence of the cell adhesive polypeptide can be easily designed and manufactured.

Examples of the cell-adhesive artificial polypeptide obtained by the organic synthesis method include a polypeptide composed of an Arg Gly Asp Ser sequence (14) (Mn approximately 400) and a polypeptide composed of a Gly Arg Gly Asp Ser sequence (15) (Mn approximately 500). ), A polypeptide (Mn about 600) consisting of the Gly Arg Gly Asp Ser Pro sequence (16) or a polypeptide (Mn about 1000) consisting of the Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro sequence (17). Peptides and other polymers are used. Examples of the polymer include a polymer (Mn about 1700) consisting of (Arg Gly Asp Ser) ₄ sequence (22), a polymer (Mn about 3000) consisting of (Arg Gly Asp Ser) ₈ sequence (23), (Arg Gly Asp Ser) A polymer consisting of ₁₆ sequences (24) (Mn about 7000), a polymer consisting of (Gly Arg Gly Asp Ser) ₈ (25) (Mn about 4000), (Gly Arg Gly Asp Ser Pro) ₈ (Mn about 5000) or (Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro) ₄ (27) polymer (Mn about 4000). The polymerization degree (number of repeating units) of these polymers is preferably 2 to 50, more preferably 3 to 30, particularly preferably 4 to 20, and most preferably 4 to 16.

Examples of the cell-adhesive artificial polypeptide obtained by the genetic recombination method include, for example, a polypeptide (SLPF) having an Mn of about 100,000 having (Gly Ala Gly Ala Gly Ser) ₉ sequence (40) and about 13 Arg Gly Asp sequences, respectively. ), A polypeptide of about 100,000 Mn having about 13 each of (Gly Ala Gly Ala Gly Ser) ₉ sequence (40) and Tyr Ile Gly Ser Arg sequence (2), (Gly Ala Gly Ala Gly Ser) ₉ A polypeptide of about 100,000 Mn having about 13 each of the sequence (40) and the Ile Lys Val Ala Val sequence (7), (Gly Val Gly Val Pro) ₈ sequence (41) and (Gly Ala Gly Ala Gly Ser ) A polypeptide of about 100,000 Mn having about 12 each of ₁₂ sequences (42) and ₁₂ Arg Gly Asp sequences, and (Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro) ₂ (43) sequence And arg Gly Asp sequence, each having about 6 Mn De, and the like.

The cell adhesive polypeptide (P) is modified with a compound (AM) containing an amino group and / or an ammonio group from the viewpoint that many cells can be retained on a wound dressing and the healing period can be further reduced. Is preferred.

As the compound (AM) containing an amino group and / or an ammonio group, polyamine, amino alcohol, a halide having an amino group, an amino group-containing monomer, a polymer of an amino group-containing monomer, and a quaternized product thereof are used. it can.
As the polyamine, a polyamine having at least one primary amino group or a secondary amino group (2-56 carbon atoms) or the like is used, such as an aliphatic polyamine, an alicyclic polyamine, a heterocyclic polyamine, and an aromatic polyamine. Is used.

Examples of the aliphatic polyamine include alkylenediamines (ethylenediamine, propylenediamine, trimethylenediamine, tetramethylenediamine, hexamethylenediamine, and the like), and polyalkylenepolyamines having 2 to 6 carbon atoms in an alkylene group (diethylenetriamine, iminobispropylamine, Triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine and the like, and their alkyl (C1-18) substituted products (dimethylaminopropylamine, diethylaminopropylamine, dipropylaminopropylamine, methylethylaminopropylamine, Trimethylhexamethylenediamine, N, N-dioctadecylethylenediamine, trioctadecylethylenediamine, methyliminobispropylamine, etc.) And the like.

Examples of the alicyclic polyamine include 1,3-diaminocyclohexane, 1,3-bis (methylamino) cyclohexane, 1,3-bis (dihydroxyamino) cyclohexane, isophoronediamine, menthanediamine, and 4,4′-methylenedicyclohexane. And diamines.
Examples of the heterocyclic polyamine include piperazine, N-methylpiperazine, N-aminoethylpiperazine, 1,4-diaminoethylpiperazine, and the like.
Examples of the aromatic polyamine include phenylenediamine, N, N′-dimethylphenylenediamine, N, N, N′-trimethylphenylenediamine, diphenylmethanediamine and 2,6-diaminopyridine, tolylenediamine, diethyltolylenediamine, 4'-bis (methylamino) diphenylmethane, 1-methyl-2-methylamino-4-aminobenzene, and the like.

As the amino alcohol, an amino alcohol having 2 to 58 carbon atoms or the like is used, and an alkanolamine having 2 to 10 carbon atoms [monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, monobutanolamine, triethanolamine, triethanolamine Propanolamine, tributanolamine, N, N-bis (hydroxyethyl) ethylenediamine and N, N, N ′, N′-tetrakis (hydroxyethyl) ethylenediamine and the like], and alkyl (C1-18) substituted products thereof [ N, N-dimethylethanolamine, N, N-diethylethanolamine, N-ethyldiethanolamine, N-octadecyldiethanolamine, N, N-diethyl-N ′, N′-bis (hydroxyethyl) ethylenediamine, - dioctadecyl -N ', N'-bis (hydroxyethyl) ethylenediamine and N, N, N'trioctadecyl -N'- hydroxyethyl ethylenediamine] and the like.

Examples of the halide having an amino group include halogenated (eg, chlorine and bromine) alkylamines having 2 to 17 carbon atoms, such as aminoethyl chloride, N-methylaminopropyl chloride, dimethylaminoethyl chloride, and diethylaminoethyl chloride. , Dibenzylaminoethyl bromide, dimethylaminopropyl bromide, diethylaminopropyl chloride and dibenzylaminopropyl chloride.

Examples of the amino group-containing monomer include an amino group-containing vinyl compound having 5 to 21 carbon atoms, ethyleneimine, and an amino acid having 2 to 20 carbon atoms.
As the amino group-containing vinyl compound, amino group-containing (meth) acrylate, amino group-containing (meth) acrylamide, amino group-containing aromatic vinyl hydrocarbon, amino group-containing allyl ether, and the like are used. In addition, (meth) acrylamide means acrylamide and / or methacrylamide.

Examples of the amino group-containing (meth) acrylate include aminoethyl (meth) acrylate, N-methylaminoethyl (meth) acrylate, N, N-dimethylaminoethyl (meth) acrylate, N, N-diethylaminopropyl (meth) acrylate, N, N-dipropylaminoethyl (meth) acrylate, N-benzyl-N-methylaminoethyl (meth) acrylate, N, N-dibenzylaminoethyl (meth) acrylate, N, N-dibenzylaminopropyl (meth) ) Acrylate, morpholinoethyl (meth) acrylate and N-methylpipetidinoethyl (meth) acrylate.

Examples of the amino group-containing (meth) acrylamide include aminoethylacrylamide, N-methylaminopropylacrylamide, N, N-dimethylaminoethyl (meth) acrylamide, N, N-diethylaminopropyl (meth) acrylamide, N, N-dipropyl Examples include aminoethyl (meth) acrylamide, N-benzyl-N-methylaminoethyl (meth) acrylamide, morpholinoethyl (meth) acrylamide, and N-methylpipetidinoethyl (meth) acrylamide.

Examples of the amino group-containing aromatic vinyl hydrocarbon include aminoethylstyrene, N-methylaminoethylstyrene, N, N-dimethylaminostyrene, N, N-dipropylaminostyrene and N-benzyl-N-methylaminostyrene. No.
Examples of the amino group-containing allyl ether include aminoethyl allyl ether, N-methylaminoethyl allyl ether, N, N-dimethylaminoethyl allyl ether, N, N-diethylaminoethyl allyl ether, and the like.

As amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, proline, cysteine, lysine, serine, glycine, 3-aminopropionic acid , 8-aminoactanoic acid and 20-aminoeicosanoic acid.

Examples of the polymer of the amino group-containing monomer include a vinyl polymer comprising an amino group-containing vinyl compound, polyethyleneimine, and a polypeptide (not including the cell adhesive polypeptide (P)). The weight average molecular weight of the polymer of the amino group-containing monomer is preferably 500 or more, more preferably 1,000 or more, particularly preferably 2,000 or more, and preferably 1,000,000 or less, more preferably 800,000. Or less, particularly preferably 500,000 or less. The weight average molecular weight can be measured by gel permeation chromatography (GPC). As the reference substance, for example, a polystyrene standard having a molecular weight of 420 to 20,600,000 (manufactured by Tosoh Corporation) can be used.

Examples of these quaternized compounds include those obtained by quaternizing these amino groups with a quaternizing agent (eg, methyl chloride, ethyl chloride, benzyl chloride, dimethyl carbonate, dimethyl sulfate, and ethylene oxide).

As a method of modifying with a compound (AM) containing an amino group and / or an ammonio group, for example, a compound (AM) containing an amino group and / or an ammonio group is reacted with a cell adhesion polypeptide before modification. A method and a method of physically adsorbing a compound (AM) containing an amino group and / or an ammonio group to a cell-adhesive polypeptide before modification and the like can be applied. The method includes the cell-adhesive polypeptide (P ) And the coating material (N), the same chemical bonding and / or physical adsorption as in (1) to (3) can be used, and the preferable chemical bonding and / or physical adsorption are also the same.

The average number (number) of amino groups of the cell adhesive polypeptide modified with the compound (AM) containing an amino group and / or an ammonio group is 100,000 or less per molecule of the cell adhesive polypeptide (P). It is more preferably 10,000 or less, particularly preferably 1,000 or less, and is preferably 0.001 or more, more preferably 0.01 or more, and particularly preferably 0.1 or more.

The average number of the amino groups can be quantified by a known method. Examples of the known methods include a trinitrobenzenesulfonic acid (TNBS) method [Chemical IV of Protein (Tokyo Kagaku Dojin, 1981) and the like] A method for measuring the total amine value by an indicator (such as bromophenol blue) titration [JIS K7237-1986, ASTM D2074-66, etc.].
Specifically, for example, a compound (AM) containing a known number of amino groups and / or containing an amino group is measured by the TNBS method while changing the concentration per unit volume, and a calibration curve (amino group Graph of the number and the absorbance). The cell adhesion polypeptide (P) before modification is measured by the TNBS method, and the obtained absorbance is converted into the number of amino groups using a calibration curve. At the same time, the modified cell adhesive polypeptide is measured by the TNBS method, and the obtained absorbance is converted into the number of amino groups using a calibration curve. The difference between the number of amino groups before and after the modification is calculated, and the value is divided by the number of molecules of the cell adhesive polypeptide (P) used for the measurement, whereby the cell is modified into the cell adhesive polypeptide (P). Average number of amino groups.

The compound (AM) containing an amino group and / or an ammonio group may be bound to the coating material (N). As a method of bonding the compound (AM) containing an amino group and / or an ammonio group to the coating material (N), for example, a compound (AM) containing an amino group and / or an ammonio group and the coating material (N) And a method in which a compound (AM) containing an amino group and / or an ammonio group is physically adsorbed to the coating material (N), and the like. The method includes the cell adhesion polypeptide (P ) And the coating material (N), the same chemical bonding and / or physical adsorption as in (1) to (3) can be used, and the preferable chemical bonding and / or physical adsorption are also the same, The same applies to the quantification method.

The average number (number) of amino groups of the coating material (N) modified with the compound (AM) containing an amino group and / or an ammonio group is 10 ⁸ / cm 2 per unit area of the wound dressing of the present invention. ² or more, more preferably ^{10 10} / cm ² or more, particularly preferably ^{10 12} / cm ² or more, and is preferably ^{10 22} / cm ² or less, more preferably ^{10 20} / cm ² The number is particularly preferably 10 ¹⁸ / cm ² or less.

Since the cell adhesive polypeptide is composed of amino acids like proteins, it becomes a nutrient for microorganisms and is susceptible to contamination by microorganisms. In addition, when the cell-adhesive polypeptide is produced by a genetically modified microorganism, the cell-adhesive polypeptide accumulated in the microorganism is purified and extracted, so that a microorganism-derived substance is easily mixed into the cell-adhesive polypeptide. Therefore, it is necessary to more strictly control and prevent the incorporation of microorganisms and microorganism-derived substances into the cell adhesive polypeptide than in the case of the wound material (N). As an indicator of the contamination of microorganisms and substances derived from microorganisms, endotoxin is appropriate because it is a harmful substance itself. Endotoxin is a toxin released after death from bacteria that are toxic to the cell components themselves, and has a basic structure in which glycoproteins and lipids are linked (Genetic Engineering Keyword Book Revised 2nd Edition, published by Yodosha Co., Ltd., 2000) .

The endotoxin content (EU / mg) in the cell adhesive polypeptide (P) of the present invention is preferably less than 0.15 from the viewpoint of safety, based on the weight of the cell adhesive polypeptide (P). , More preferably less than 0.015, particularly preferably less than 0.0015.

As a method of measuring the endotoxin content, a Limulus test method utilizing the coagulation of a horseshoe crab blood cell extract in response to endotoxin and the like can be applied. Limulus test reagent kits that can be easily obtained from the market include, for example, Limulus F Single Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) and Limulus ES-2 Single Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). And the like. Preparation of the sample solution used for the reagent kit is performed by dissolving the cell-adhesive polypeptide in water that does not contain endotoxin, such as demineralized distilled water (sterile), endotoxin test water, or water for local injection. In addition, as the standard substance, an endotoxin standard product determined by the Japanese Pharmacopoeia and a standard substance tested with this endotoxin standard product can be used.

As a method of measuring the endotoxin content using a commercially available Limulus test reagent kit, for example, when using a Limulus test reagent kit having an endotoxin detection sensitivity of 0.015 EU / mL, 1 mg of a measurement sample (cell adhesive polypeptide) is used. 0.2 mL of a sample solution (1 mg / mL) dissolved in 1 mL of endotoxin test water was mixed with the LAL reagent, and visually determined whether or not a water-insoluble gel was formed. When the gel was formed, the endotoxin content was determined. Can be determined to be 0.015 EU / mg or more, and if no gel is formed, the endotoxin content can be determined to be less than 0.015 EU / mg. When 0.1 mg of the measurement sample is used instead of 1 mg of the measurement sample (0.1 mg / mL of the sample solution), it can be similarly determined whether or not the content is 0.15 EU / mg or more. Similarly, by using 10 mg of the measurement sample (10 mg / mL sample solution), it can be determined whether the content is 0.0015 EU / mg or more.

Because endotoxin is contained in bacterial cell walls, etc., when cell-adhesive polypeptides are produced by genetic recombination with bacteria or when cell-adhesive polypeptides are handled in environments other than aseptic environments, endotoxin is May be mixed in. In such a case, as a method of removing endotoxin mixed in the cell adhesive polypeptide, for example, a column method of separating endotoxin using an endotoxin adsorption affinity column, a gel filtration column or a column for hydrophobic chromatography, an autoclave Alternatively, a heating method in which endotoxin is inactivated by heat using a dry heat sterilizer or the like, a combination of these methods, and the like can be applied. Among these, the heating method is one in which the sterilization operation is simple and the endotoxin is surely removed. The heating temperature is preferably 40 ° C. or higher, more preferably 60 ° C. or higher, particularly preferably 80 ° C. or higher, and preferably 500 ° C. or lower, more preferably 300 ° C. or lower, particularly preferably 200 ° C. or lower. . The heating time is preferably 1 second or more, more preferably 10 seconds or more, particularly preferably 1 minute or more, and preferably 5000 minutes or less, more preferably 500 minutes or less, and particularly preferably 100 minutes or less. The method for removing these endotoxins can be used for the cell-adhesive natural polypeptide (P1) and the cell-adhesive artificial polypeptide (P2).

The coating material (N) of the present invention is a material that is easily dispersed, dissolved or absorbed in a culture solution or a wound surface when used in cell culture or applied to a wound surface (hereinafter, easily biodegradable material (N1)). In addition, when used in cell culture or applied to a wound surface, a material that is difficult to disperse, dissolve, or absorb in a culture solution or a wound surface (hereinafter, hardly biodegradable material (N2)) can be used. Further, the readily biodegradable material (N1) and the hardly biodegradable material (N2) can be used in combination. Among these materials, the hardly biodegradable material (N2) is preferable because it can be easily replaced when applied to a wound surface and is easy to handle. As the coating material (N), those containing a toxic substance having a serious adverse effect on the human body cannot be used.

As the readily biodegradable material (N1), a natural polymer (N1A), a synthetic polymer (N1B), an inorganic substance (N1C) and the like can be used.
Examples of the natural polymer (N1A) include collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, elastin, chitin, chitosan, fibrin, alginic acid, starch, dextran, albumin, Examples include polyhydroxybutyric acid, pectin, pectic acid, galactan, pullulan, agarose, cellulose, gluten and fibroin.

As the synthetic polymer (N1B), for example, a monomer selected from the group consisting of lactic acid, leucine, glycolic acid, ε-caprolactone, dioxanone, malic acid, lactide and glycolide is used as an essential monomer (co). Examples include a polymer (polyglycolic acid), and a synthetic polypeptide not containing the minimum amino acid sequence (X) representing a cell adhesion signal.

As the inorganic substance (N1C), for example, calcium carbonate and calcium phosphate are used. Examples of the calcium carbonate include light calcium carbonate and heavy calcium carbonate. Examples of the calcium phosphate include hydroxyapatite, tricalcium phosphate, and a mixture thereof with another calcium phosphate (for example, monocalcium hydrogen phosphate and the like).

Of these, natural polymers (N1A) and synthetic polymers (N1B) are preferred, and synthetic polymers (N1B) are more preferred, and lactic acid, leucine, glycolic acid, ε-caprolactone, dioxanone, malic acid, lactide are particularly preferred. And a (co) polymer (polyglycolic acid) comprising a monomer selected from the group consisting of glycolide and glycolide as an essential monomer.

As the hardly biodegradable material (N2), a natural polymer (N2A), a synthetic polymer (N2B), an inorganic substance (N2C) and the like can be used. Examples of the natural polymer (N2A) include natural fibers (such as cotton, wool, hemp, and silk).

Examples of the synthetic polymer (N2B) include polyolefins (polyethylene, polypropylene and modified products thereof), olefin copolymers (ethylene-vinyl acetate copolymer, ethylene-ethyl (meth) acrylate copolymer, ethylene- Methyl (meth) acrylate copolymer and ethylene- (meth) acrylic acid copolymer), polyurethane, polyester, polyacrylic acid, polyamide, polyvinyl chloride, polyvinylidene chloride, polystyrene, fluororesin, silicone resin, cellulose and Chemical fibers (viscose rayon, cupra rayon, polynosic, acetate, triacetate, polyethylene, polypropylene, polyamide, polyester, polyacrylonitrile, vinylon, polyvinyl chloride, vinylidene and polyurethane ) Or the like is used. In addition, (meth) acrylate means acrylate and / or methacrylate.

As the inorganic substance (N2C), for example, metal (gold, silver, platinum, titanium, nickel, and the like) and ceramics (alumina, zirconia, aluminum nitride, and the like) are used.
Among these, from the viewpoint of handleability, etc., the hardly biodegradable material (N2) is preferable, more preferably natural polymer (N2A) and synthetic polymer (N2B), particularly preferably polyolefin, polyurethane, polyester and polyacryl. Acids, most preferably polyurethanes.

The coating material (N) is selected in consideration of flexibility, stretchability, appropriate water vapor permeability, fungus barrier property and sterility. The hardness of the coating material (N) is preferably 40 or more, more preferably 50 or more, particularly preferably 60 or more, and preferably 100 or less, more preferably 90 or less, particularly preferably 90 or more in terms of easy handling. Is 80 or less. In addition, hardness is measured based on JISK6301-1995, 5.2 spring-type hardness test (A type). The moisture permeability (g / m ² , 24 hr) of the coating material (N) is preferably at least 200, more preferably at least 400, particularly preferably at least 2,000, in order to keep the amount of exudate in the wound moderate. Further, it is preferably 15,000 or less, more preferably 10,000 or less, particularly preferably 7000 or less. The moisture permeability is measured according to JIS Z0208-1976 (40 ° C., 90% RH).

The contact angle (degree) of water on the surface of the coating material (N) is preferably 15 or more, more preferably 30 or more, particularly preferably 50 or more, and preferably 120 or less, from the viewpoint of promoting wound healing. More preferably, it is 110 or less, particularly preferably 100 or less. The contact angle of the surface of the coating material (N) with water can be measured using a contact angle meter (for example, Model CA-S150, manufactured by Kyowa Interface Science Co., Ltd.). The measurement conditions are as follows: measurement atmosphere temperature: 25 ± 1 ° C., measurement atmosphere relative humidity: 65 ± 5%, measurement target temperature: 25 ± 1 ° C., liquid droplet volume: 1.8 ± 2 μL, droplet dripping needle : 18 G, reading of contact angle: reading 15 15 seconds after dropping. The calculation method of the contact angle is calculated from the following equation.
(Contact angle) = 2 tan ^-1 {(height of droplet after drop) / (radius of droplet after drop)}

The contact angle of the surface of the coating material (N) with water can be controlled by subjecting the coating material (N) to a surface treatment, and includes, for example, a functional group (perfluoroalkyl group, polyoxyethylene group, carboxyl group, carbonyl group). , A hydroxyl group and an amino group), a physical treatment for forming irregularities on the surface, and an adsorption treatment by blocking proteins and the like. Of these, the chemical treatment and the adsorption Treatment is preferred, more preferably chemical treatment.

Examples of the chemical treatment include SG (Soil Guard) processing and SR (Soil Release) processing (introduction to polymer drugs, supervised by Takehiko Fujimoto, issued by Sanyo Chemical Industries, Ltd.), silane coupling agent treatment, ozone treatment, electron beam Processing, oxidizing agent processing, plasma processing, corona discharge processing, Goro discharge processing and the like can be used. As the physical treatment, for example, a method of polishing the surface with a diamond file (DT-101N) can be used, and a desired surface shape can be obtained at the time of molding the coating material (N). As the adsorption treatment, for example, a method of immersing the coating material (N) in a protein-containing solution to adsorb the protein or the like can be used. Examples of proteins include serum-derived proteins such as albumin and milk-derived proteins such as casein.

The shape of the coating material (N) is not particularly limited as long as it can be used for treating wounds (pressure ulcers, ulcers, burns, etc.), and examples thereof include sheet-like and thread-like shapes. The sheet shape is preferable from the viewpoint of easy handling in the application of. The thickness of the sheet is preferably 1 μm or more, more preferably 5 μm or more, particularly preferably 15 μm or more, most preferably 30 μm or more, and preferably 5 cm or less, more preferably 1 cm or less, particularly preferably 3 mm or less. Most preferably, it is 500 μm or less.

Examples of the sheet form include a film, a foam (sponge), a nonwoven fabric, a woven fabric, a knitted fabric, and a gel shape. Of these, films and foams are preferred, and films are more preferred.
The basis weight (g / m ² ) of the film is not particularly limited, but is preferably 10 or more, more preferably 20 or more, particularly preferably 30 or more, and preferably 150 or less, more preferably 100 or less, particularly preferably 100 or less. Preferably it is 75 or less. The density (Kg / m ³ ) of the foam is preferably 15 or more, more preferably 40 or more, particularly preferably 60 or more, and preferably 500 or less, more preferably 250 or less, and particularly preferably 150 or less. The basis weight (g / m ² ) of the nonwoven fabric, woven fabric and knitted fabric is not particularly limited, but is preferably 20 or more, more preferably 30 or more, particularly preferably 50 or more, and preferably 300 or less, more preferably It is 200 or less, particularly preferably 100 or less.

The film and the foam may have fine holes on the entire surface or a part thereof. The size of the holes is preferably such that air and water vapor can easily pass therethrough. The size of the hole is preferably 0.005 or more, more preferably 0.01 or more, particularly preferably 0.05 or more, and preferably 25 or less, as the hole opening area (mm ² ). It is more preferably 10 or less, particularly preferably 5 or less. The shape of the hole may be circular, elliptical, triangular, square, polygonal, linear (slit) and the like, and any shape can be used as long as the desired amount of exudate stored in the wound can be maintained appropriately. May be used.

In the wound dressing material of the present invention, the cell adhesive polypeptide (P) and the coating material (N) usually contain a chemical bond (such as an ionic bond, a hydrogen bond and / or a covalent bond) and / or a physical adsorption (van der Waals). (Adsorption by force). From the viewpoint that the cell adhesive polypeptide (P) and the coating material (N) are firmly bonded, a chemical bond is preferable, and a covalent bond is more preferable.

As a method for covalently bonding the cell adhesive polypeptide (P) and the coating material (N), for example, those having a primary amino group or a secondary amino group (for example, arginine, Histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, proline, cysteine, lysine, serine, glycine, ornithine, histidine, 3-aminopropionic acid, 8 -Aminooctanoic acid, cell adhesion polypeptide containing 20-aminoeicosanoic acid as a constituent unit) and those having a carboxyl group among (N) (for example, polyglycolic acid, the minimum amino acid sequence representing a cell adhesion signal) (X) -free synthetic poly Peptide, modified polyethylene or polypropylene, ethylene- (meth) acrylic acid copolymer, polyacrylic acid, collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin, elastin, fibrin, alginic acid, (2) a method of reacting (P) with a primary amino group or a secondary amino group in (P) and a hydroxyl group in (N). Having (e.g., polyglycolic acid, ethylene-vinyl acetate copolymer, polyurethane, polyester, cellulose, chemical fiber, collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, (3) (P) And N) having a carboxyl group (for example, a cell-adhesive polypeptide containing aspartic acid, glutamic acid, serine, threonine, tyrosine, tyrosine, hydroxypurine and the like as constituent units) and a carboxyl group of (N). (4) (P) having a halogen atom (for example, a cell-adhesive polypeptide modified with a halide having an amino group) and (N) having a hydroxyl group or a mercapto group. (Eg, the above hydroxy A method of reacting with a coating material having a structure in which part or all of hydroxyl groups are replaced with mercapto groups other than those having a sil group; and (5) (P) having a vinyl group (for example, , A cell-adhesive polypeptide modified with an amino group-containing monomer) and (N) having an amino group, a hydroxyl group or a carboxyl group.

These reactions can be performed by a known method (for example, a method described in “Basic and Experimental Peptide Synthesis, Oct. 5, 1997, published by Maruzen Co., Ltd.”). Specifically, it is as follows (1) to (6).
(1) When reacting a compound having a primary amino group or a secondary amino group in (P) with a compound having a carboxyl group in (N), the carboxyl group of (N) is reacted with a carbodiimide compound in advance. , An acyl isourea {R'-N = C (OCOR) -NH-R '(where -OCOR is derived from (N))}, and then a primary amino group or a secondary amino group of (P) (P) can be introduced into (N) by forming an amide bond. Examples of the carbodiimide compound include N, N'-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

(2) When reacting a compound having a primary amino group or a secondary amino group in (P) with a compound having a hydroxyl group in (N), the hydroxyl group of (N) is previously reacted with a carbonyldiimidazole compound. After the reaction, the imidazole derivative {R-Im, Im is an imidazoline ring and R is derived from (N)} is added by adding a compound having a primary amino group or a secondary amino group of (P) ( (P) can be introduced into N) by forming an NC bond. Examples of the carbonyldiimidazole compound include N, N'-carbonyldiimidazole and the like.

(3) When reacting (P) having a hydroxyl group with (N) having a carboxyl group, the carboxyl group of (N) is reacted with a carbodiimide compound in advance to obtain an acyl isourea, By adding (P) having a hydroxyl group, (P) can be introduced into (N) by forming an ester bond.

(4) When reacting (P) having a halogen atom with (N) having a hydroxyl group or a mercapto group, by mixing both in the presence or absence of an alkali compound, (P) can be introduced into (N) by forming an ether bond or a thioether bond. Examples of the alkali compound include inorganic alkali compounds (such as sodium hydroxide, potassium hydroxide, and lithium hydroxide) and organic alkali compounds (such as dimethylaminopyridine, ammonia, triethylamine, sodium methoxide, and DBU (registered trademark of San Apro Corporation)). Is mentioned.

(5) When reacting (P) having a vinyl group with (N) having an amino group, a hydroxyl group or a carboxyl group, mixing both in the presence or absence of an alkali compound Then, by adding Michael, it is possible to introduce (P) into (N).

As a method for physically adsorbing, ion-bonding and / or hydrogen-bonding the cell-adhesive polypeptide (P) to the coating material (N), for example, (P) and (N) are charged into a solvent or the like and mixed. And the like. Although the solvent is not particularly limited, 0.001 to 50% by weight (preferably 0.01 to 10% by weight) of inorganic salt, organic acid salt, amino acid, vitamin, alcohol, lipid / sugar, acid and / or base is used. The contained aqueous solution, water, body fluid and the like can be used.

As the inorganic salt, metal halides, metal sulfates, metal phosphates, metal nitrates, metal carbonates, metal perhalates and the like can be used. For example, sodium chloride, sodium sulfate, sodium phosphate, calcium chloride , Iron nitrate, potassium chloride, magnesium sulfate, sodium carbonate, sodium hydrogen phosphate, potassium phosphate, potassium hydrogen phosphate, copper sulfate, iron sulfate, lithium chloride, sodium bromide, lithium bromide, sodium perchlorate Lithium chlorate and the like.
Examples of the organic acid salt include sodium formate, sodium acetate, lithium acetate and sodium tartrate.

Examples of the amino acids include arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine, glycine, and the like.
As vitamins, for example, choline, inositol, nicotinamide, glutamine, vitamin A, include vitamins _{B 12} and vitamin C and the like.
As the alcohol, an alcohol having 1 to 4 carbon atoms can be used, and examples thereof include methanol, ethanol, isopropyl alcohol, and butanol.
Examples of lipids / sugars include lipids, monosaccharides, disaccharides, oligosaccharides, amino sugars, and acidic sugars.

As the acid, an inorganic acid and an organic acid having 1 to 6 carbon atoms can be used, and examples thereof include hydrochloric acid, phosphoric acid, acetic acid, formic acid, phenol and sulfuric acid.
As the base, an inorganic base or an organic base having 2 to 6 carbon atoms can be used, and examples thereof include sodium hydroxide, potassium hydroxide, ammonia, and triethylamine.
Examples of the water include distilled water, ion-exchanged water, tap water, and ion-exchanged distilled water.
Body fluids include blood, plasma, serum, urine, and the like.
Among these solvents, an aqueous solution containing an inorganic salt, an acid and / or a base, and water are preferable, and an aqueous solution containing an inorganic salt, an acid and / or a base is more preferable.

The content of the cell adhesive polypeptide (P) in the wound dressing of the present invention is preferably 0.1 ng / cm ² or more, more preferably, per unit area of the wound dressing, from the viewpoint of improving cell adhesion. Is at least 1 ng / cm ² , particularly preferably at least 10 ng / cm ² , most preferably at least 100 ng / cm ^2, preferably at most 100 mg / cm ² , more preferably at most 10 mg / cm ² , particularly preferably at most 1 mg / cm ^2. / Cm ² or less, most preferably 100 μg / cm ² or less.
The method for measuring the content of the cell adhesive polypeptide (P) per unit area is not particularly limited, and for example, an immunological measurement method can be used. Specifically, a part (for example, 1 cm × 1 cm square) of the surface of the wound dressing is cut out, and an antibody that binds to the cell adhesive polypeptide (P) is labeled with an enzyme (hereinafter referred to as an enzyme-labeled antibody). By reacting 1) and measuring the amount of enzyme of the reacted enzyme-labeled antibody 1, the content of the cell adhesive polypeptide (P) per unit area can be measured.
The enzyme-labeled antibody 1 is usually obtained by chemically bonding an enzyme and a specific antibody, and can be chemically bonded by a known method. For example, an enzyme (for example, peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, etc.) and a specific antibody are subjected to a glutaraldehyde method, a periodate method, a maleimide method, a pyridyl disulfide method, or the like. Methods for chemical binding (Ultrasensitive enzyme immunoassay, written by Eiji Ishikawa, Gakkai Shuppan Center, 1993; Enzyme immunoassay, translated by Eiji Ishikawa, Tokyo Kagaku Dojin, 1989; and enzyme antibody method, Keiichi Watanabe Ed., Interdisciplinary Planning Co., Ltd., 1992). The specific antibody is an antibody that specifically binds to the cell adhesive polypeptide (P), and can be prepared by a known method. For example, a polyclonal antibody preparation method and a monoclonal antibody preparation method (enzyme immunoassay, translated by Eiji Ishikawa, Tokyo Kagaku Dojin, 1989; and an enzyme antibody method, edited by Keiichi Watanabe et al., Interdisciplinary Planning Co., Ltd., 1992) can be applied. . The affinity constant of the specific antibody for the cross-reactive antigen is preferably as small as possible. For example, when the affinity constant of the specific antibody for the cell adhesion polypeptide (P) is 1, the affinity constant for the cross-reactive antigen is 1 It is preferably at most 0.1, more preferably at most 0.1, particularly preferably at most 0.01. These affinity constants can be obtained by a method described in an enzyme immunoassay (translated by Eiji Ishikawa, Tokyo Chemical Dojin, 1989).

The wound dressing of the present invention may be subjected to a sterilization treatment as needed. Examples of the sterilization method include radiation sterilization, ethylene oxide gas sterilization, plasma sterilization, γ-ray sterilization, alcohol sterilization, autoclave sterilization, and dry heat sterilization. Of these, autoclave sterilization and dry heat sterilization are preferred because the sterilization operation is simple.
The heating temperature in autoclave sterilization and dry heat sterilization is preferably 40 ° C or higher, more preferably 60 ° C or higher, particularly preferably 80 ° C or higher, and preferably 180 ° C or lower, more preferably 160 ° C or lower. And particularly preferably 140 ° C. or lower.
The heating time in the autoclave sterilization and dry heat sterilization is preferably 1 second or more, more preferably 10 seconds or more, particularly preferably 1 minute or more, and 5000 minutes or less, more preferably 500 minutes or less. And particularly preferably 100 minutes or less.
The pressure in the tank when autoclaving is preferably 0.002 MPa or more, more preferably 0.01 MPa or more, particularly preferably 0.05 MPa or more, and preferably 5 MPa or less, more preferably 1 MPa or less, particularly Preferably it is 0.2 MPa or less.

The wound dressing of the present invention preferably contains a cell growth factor (G1) and / or a cell growth factor binding substance (G2) in order to promote wound healing.
Examples of the cell growth factor (G1) include substances that promote cell proliferation, such as fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, Vascular endothelial cell growth factor, nerve growth factor, stem cell factor, leukemia inhibitory factor, bone morphogenetic factor, heparin-binding epidermal growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, chondromodulin, tenomodulin, interferon, interleukin, Bioactive polypeptides such as tumor necrosis factor, colony stimulating factor, adrenamodulin and natriuretic peptide and the like are used (for example, described in Nagoya University Press, “Med. Ueda Tissue Engineering” (1999)).
Among these cell growth factors (G1), fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte proliferation, from the viewpoint that a wide range of tissue cells can be applied and the healing period can be shortened. Factors, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, osteogenic factor, interleukin and tumor necrosis factor are preferred, more preferably fibroblast growth factor, epidermal growth factor, insulin-like growth factor, Vascular endothelial growth factor, interleukin and tumor necrosis factor.

The cell growth factor binding substance (G2) is a substance that can bind to a cell growth factor by ionic bonding or the like, and includes, for example, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, gelatin, collagen, poly Lactic acid, agarose, alginic acid, and the like are used (for example, Nagoya University Press “Tissue Engineering, Minoru Ueda” (1999), and Yodosha Co., Ltd., “Mechanisms and Diseases of Cell Adhesion” (1998)) Described).
Heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, gelatin, collagen, polylactic acid or alginic acid include alkali metal (such as lithium, potassium and sodium) salts, alkaline earth metals (magnesium) And calcium salts) or ammonium salts.
Among these cell growth factor-binding substances, heparin, heparan sulfate, chondroitin sulfate, hyaluronic acid, and gelatin are preferable, and heparin is more preferable, from the viewpoint that the range of applicable tissue cells is wide and the healing period can be shortened. , Hyaluronic acid and gelatin.

The cell growth factor (G1) and / or the cell growth factor binding substance (G2) is usually present in a state of being bound to the coating material (N). For the binding, the same chemical bonding and / or physical adsorption as the above-mentioned bonding between the cell adhesive polypeptide (P) and the coating material (N) can be used, and the preferable chemical bonding and / or physical adsorption are also the same.

The content of the cell growth factor (G1) and / or the cell growth factor-binding substance (G2) in the wound dressing of the present invention is 0 per unit area of the wound dressing of the present invention from the viewpoint of shortening the healing period. .01pg / cm ² or more, more preferably 0.1 pg / cm ² or more, particularly preferably 1 pg / cm ² or more, and most preferably at 10 pg / cm ² or more, and is preferably 100 [mu] g / cm ² or less, It is more preferably at most 10 μg / cm ² , particularly preferably at most 1 μg / cm ² , most preferably at most 0.1 μg / cm ² . When the cell growth factor (G1) and the cell growth factor-binding substance (G2) are contained, their content ratio (G1 / G2) is preferably at least 0.00001, more preferably at least 0.0001, particularly preferably at least 0.0001. It is preferably 0.001 or more, most preferably 0.01 or more, and is preferably 1000 or less, more preferably 100 or less, particularly preferably 10 or less, and most preferably 1 or less.
The method for measuring the content of the cell growth factor (G1) and / or the cell growth factor-binding substance (G2) per unit area is not particularly limited, and for example, an immunological measurement method can be used. Specifically, a part (for example, a 1 cm × 1 cm square) of the surface of the wound dressing is cut out, and an antibody that binds to (G1) and / or (G2) is labeled with an enzyme (hereinafter referred to as enzyme labeling). By reacting the antibody 2) and measuring the amount of enzyme of the reacted enzyme-labeled antibody 2, the content of (G1) and / or (G2) per unit area can be measured. The enzyme-labeled antibody 2 can be prepared in the same manner as the enzyme-labeled antibody 1 described above.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
<Example 1>
Preparation of Cell Adhesive Polypeptide [P2-1] Arg Gly Asp sequence and (Gly Ala Gly Ala Gly Ser) ₉ sequence according to the method described in Examples in JP-A-3-502935. (40), and a peptide "SLPF" having a number average molecular weight of about 100,000 was produced by genetically modified Escherichia coli and purified by column chromatography to obtain a cell adhesive polypeptide [P2-0]. Obtained. Further, this [P2-0] was subjected to autoclave sterilization (120 ° C., 20 minutes) to obtain a cell adhesive polypeptide [P2-1].

Preparation of non-biodegradable material [N2] 6.67 g of aqueous urethane (trade name: Permarin UA200, manufactured by Sanyo Chemical Industries, Ltd.) and 3.33 g of ion-exchanged water are mixed to prepare a urethane aqueous solution. did. This urethane aqueous solution was put on a polypropylene sheet (manufactured by Medical Agent Co., Ltd.) having a length of 20 cm × a width of 20 cm × a height of 1 mm, and was left at room temperature (about 25 ° C.). After leaving at room temperature for 24 hours, it was dried in a normal air dryer at 120 ° C. for 1 hour. After drying, the urethane film formed on the polypropylene sheet was peeled off from the polypropylene sheet to obtain a hardly biodegradable material [N2].

Preparation of Wound Dressing Material [S1] 1 mg of the cell adhesive polypeptide [P2-1] was dissolved in 1 mL of a 4.5 M aqueous solution of lithium perchlorate, and 99.5% of sodium chloride was added to 0 mL of the same solution. It was diluted 20-fold with a 0.02 M, pH 7.2 phosphate buffer solution (hereinafter, PBS) containing 0.85% by weight to prepare a P2-1 aqueous solution (50 μg / mL). 50 mL of this P2-1 aqueous solution and 10 cm × 10 cm of the hardly biodegradable material [N2] were put into a glass Petri dish, allowed to stand at 25 ° C. for 1 hour, and [P2-1] was adsorbed on [N2]. . Finally, [N2] to which [P2-1] was adsorbed was washed 5 times with 100 mL of ion-exchanged water, and dried in a normal-air dryer at 37 ° C. for 12 hours to prepare a wound dressing [S1]. (Amount (content) of P2-1: 0.5 μg / cm ² )

The adhesion amount (content) of the cell adhesive polypeptide [P2-1] of the wound dressing [S1] was measured by the following procedure.
(1) A 1 cm × 1 cm square wound dressing [H1] with a known amount of adhesion (content) of the cell adhesive polypeptide [P2-1] and a wound dressing [S1] with an unknown amount of adhesion were used. , And one piece was put into 3 mL of PBS containing 1% by weight of bovine serum albumin, and immersed at room temperature (25 ° C.) for 2 hours.
The preparation of the standard wound dressing is performed in the same manner as the preparation of the wound dressing [S1] described above. The weight of the unadhered cell-adhesive polypeptide was determined, and the weight of the unadhered cell-adhesive polypeptide was subtracted from the weight of the cell-adhesive polypeptide before the attachment.

(2) Take out each wound dressing and put each wound dressing 1 in 2 mL of PBS containing peroxidase-labeled anti-P2-1 antibody at 10 μg / mL, bovine serum albumin at 1% by weight and Tween 20 at 0.2% by weight. The sheets were charged and reacted at 37 ° C. for 2 hours. After the reaction, each wound dressing was washed three times with 5 mL of PBS containing Tween 20 at 0.2% by weight.
In addition, peroxidase-labeled anti-P2-1 antibody was prepared by immunizing rabbits with cell-adhesive polypeptide [P2-1] according to a polyclonal antibody preparation method (enzyme immunoassay, translated by Eiji Ishikawa, Tokyo Chemical Dojin, 1989). Then, an anti-P2-1 antibody is obtained, and the anti-P2-1 antibody and peroxidase (manufactured by Toyobo Co., Ltd.) are bound by a maleimide method (enzyme immunoassay, translated by Eiji Ishikawa, Tokyo Chemical Dojin, 1989). Thus, a peroxidase-labeled anti-P2-1 antibody was obtained.

(3) Take out each wound dressing and put one wound dressing in a mixture of 0.2 mL of OLYDAS-specific reagent coloring solution set (manufactured by Sanyo Chemical Industries, Ltd.) and 1.8 mL of ion-exchanged water. It was charged and reacted at 37 ° C. for 1 hour. After the reaction, the absorbance was measured at a wavelength of 380 nm.
(4) A calibration curve was prepared using the absorbance of the standard wound dressing [H1], and the amount of the wound dressing [S1] attached was obtained from the calibration curve. Hereinafter, the amount of attached polypeptide was measured in the same manner.

<Example 2>
Preparation of Wound Dressing Material [S2] 0.479 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Sigma) was dissolved in 50 mL of ion-exchanged water to prepare an aqueous carbodiimide solution. 50 mL of this carbodiimide aqueous solution and 10 cm × 10 cm of the hardly biodegradable material [N2] were charged into a glass petri dish and allowed to stand at 25 ° C. for 1 hour. Then, it was washed 5 times with 100 mL of ion-exchanged water. Next, 50 mL of an aqueous solution of P2-1 was charged and allowed to stand at 25 ° C. for 1 hour, so that [P2-1] was bound to [N2]. Finally, [N2] to which [P2-1] was bound was washed 5 times with 100 mL of ion-exchanged water, and dried in a normal-air dryer at 37 ° C for 12 hours to prepare a wound dressing [S2]. (Amount (content) of P2-1: 1 μg / cm ² )

<Example 3>
Preparation of Wound Dressing Material [S3] 0.479 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Sigma) was dissolved in 50 mL of ion-exchanged water to prepare an aqueous carbodiimide solution. 50 mL of this carbodiimide aqueous solution and 10 cm × 10 cm of the hardly biodegradable material [N2] were charged into a glass petri dish and allowed to stand at 25 ° C. for 1 hour. Then, it was washed 5 times with 100 mL of ion-exchanged water. Next, 50 mL of an aqueous solution of P2-1 was charged and allowed to stand at 25 ° C. for 1 hour, so that [P2-1] was bound to [N2]. Then, it was washed 5 times with 100 mL of ion-exchanged water. Further, 50 mL of a cell growth factor-containing aqueous solution [GS11] containing 50 ng / mL of fibroblast growth factor (manufactured by Becton Dickinson) [G1-1], which is a cell growth factor (G1), was added thereto, and the mixture was heated at 25 ° C. for 1 hour. After allowing to stand, [G1-1] was bound to [N2]. Then, it was washed five times with 100 mL of ion-exchanged water to prepare a wound dressing [S3]. (Adhesion amount of P2-1: 1 μg / cm ² )

<Example 4>
Preparation of Wound Dressing Material [S4] Instead of the cell growth factor-containing aqueous solution [GS11] of Example 3, interleukin 2 (manufactured by Becton Dickinson) which is a cell growth factor (G1) [G1-2] Was used in the same manner as in Example 3 using a cell growth factor-containing aqueous solution [GS12] containing 5 ng / mL to prepare a wound dressing [S4]. (Adhesion amount of P2-1: 1 μg / cm ² )

<Example 5>
Preparation of Wound Dressing Material [S5] Instead of the cell growth factor-containing aqueous solution [GS11] of Example 3, heparin sodium (manufactured by Nacalai Tesque, Inc.) which is a cell growth factor-binding substance (G2) [G2- 1] was used in the same manner as in Example 3 using an aqueous solution [GS21] containing a cell growth factor-binding substance containing 50 ng / mL to prepare a wound dressing [S5]. (Adhesion amount of P2-1: 1 μg / cm ² )

<Example 6>
Preparation of Wound Dressing Material [S6] Instead of the cell growth factor-containing aqueous solution [GS11] of Example 3, a cell growth factor binding substance, hyaluronic acid [G2-2] (manufactured by ICN Biomedical) was used. A wound dressing [S6] was prepared in the same manner as in Example 3, using an aqueous solution [GS22] containing a cell growth factor-binding substance containing 50 ng / mL. (Adhesion amount of P2-1: 1 μg / cm ² )

<Example 7>
Preparation of Cell Adhesive Polypeptide [P2-2] Obtained in Example 1 was obtained by dissolving 0.096 g of N, N'-carbonyldiimidazole (manufactured by Wako Pure Chemical Industries, Ltd.) in 5 mL of dimethyl sulfoxide. 10 mg of the cell adhesion polypeptide [P2-1] thus obtained was added thereto, and reacted at 37 ° C. for 10 minutes.
Next, 0.107 g of ethylenediamine was added and reacted at 37 ° C. for 20 hours. After the reaction, the mixture was put into a dialysis tube, and dialyzed for 2 hours five times with 1 L of ion-exchanged water to obtain a cell adhesive polypeptide [P2-2]. (Number of modified amino groups of P2-2: 60 / molecule)

The number of modified amino groups of P2-2 was measured by the following procedure.
(1) 0 mg, 10 mg, 30 mg, and 100 mg of L-lysine were each dissolved in 1 L of a 0.6 M, pH 9.5 carbonate buffer (hereinafter, CB) to obtain a standard series. Also, 1 mg of P2-1 and P2-2 were dissolved in 1 mL of CB, respectively, to obtain Sample Liquid 1 and Sample Liquid 2, respectively.
(2) 100 μL of the standard series, sample liquid 1 and sample liquid 2 were added to a glass test tube at a rate of 100 μL / test tube, and 7.2% by weight of sodium 2,4,6-trinitrobenzenesulfonate (TNBS) was added. The aqueous solution containing the solution was added at 20 μL / test tube, stirred, and left at room temperature (25 ° C.) for 2 hours.
(3) After 2 hours, ion-exchanged water was added at 1 mL / test tube, and the absorbance was measured at 367 nm.
(4) A standard curve is prepared for the absorbance of the standard series, the number of amino groups, and a calibration curve. From the calibration curves, the number of amino groups in the sample liquid 1 and the sample liquid 2 is calculated. The number was subtracted to obtain the number of modified amino groups. Hereinafter, the number of amino groups was quantified in the same manner.

Preparation of Wound Dressing Material [S7] Instead of the aqueous solution of P2-1 in Example 2, PBS (aqueous solution of P2-2) containing 50 μg / mL of the cell adhesive polypeptide [P2-2] was used. In the same manner as in Example 2, a wound dressing [S7] was prepared. (Adhesion amount of P2-2: 1 μg / cm ² )

Example 8
Preparation of Wound Dressing Material [S11] Instead of the cell growth factor-containing aqueous solution [GS11] of Example 3, a polyethyleneimine aqueous solution [GS13] containing polyethyleneimine (manufactured by Wako Pure Chemical Industries, Ltd.) at 100 μg / mL. And a wound dressing [S11] was prepared in the same manner as in Example 3. (Adhesion amount of P2-1: 1 μg / cm ² )

<Example 9>
Preparation of Cell Adhesive Polypeptide [P2-3] The solution obtained in Example 1 was obtained by dissolving 0.096 g of N, N'-carbonyldiimidazole (manufactured by Wako Pure Chemical Industries, Ltd.) in 5 mL of dimethyl sulfoxide. 10 mg of the cell adhesion polypeptide [P2-1] thus obtained was added thereto, and reacted at 37 ° C. for 10 minutes. Next, 0.357 g of polyethyleneimine was added and reacted at 37 ° C. for 20 hours. After the reaction, the mixture was put into a dialysis tube, and dialyzed for 2 hours five times with 1 L of ion-exchanged water to obtain a cell adhesive polypeptide [P2-3]. (Number of modified amino groups of P2-3: 3 / molecule)

Preparation of Wound Dressing Material [S12] Instead of the aqueous solution of P2-1 in Example 2, PBS (aqueous P2-3 solution) containing 100 μg / mL of the cell adhesive polypeptide [P2-3] was used. In the same manner as in Example 2, a wound dressing [S12] was prepared. (Adhesion amount of P2-3: 1 μg / cm ² )

<Comparative Example 1>
Preparation of wound dressing [S8] The hardly biodegradable material [N2] of Example 1 was used as a wound dressing [S8].

<Comparative Example 2>
Preparation of wound dressing [S9] A collagen film (manufactured by Koken Co., Ltd., about 1 mm in thickness) was used as a wound dressing [S9].

<Comparative Example 3>
Preparation of wound dressing [S10] Example 1 was repeated except that the autoclave sterilization of the cell adhesive polypeptide [P2-0] was omitted in “Preparation of wound dressing [S1]” of Example 1. To prepare a wound dressing [S10].

<Evaluation 1 (Endotoxin concentration)>
Cell adhesion polypeptide [P2-0], cell adhesion polypeptide [P2-1], cell adhesion polypeptide [P2-2] and cell adhesion polypeptide [P2-3] were used for measuring endotoxin content. Was used as a measurement sample. For each of the measurement samples, 1 mg and 0.1 mg were poured into 1 mL of demineralized distilled water (sterile) (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare 1 mg / mL and 0.1 mg / mL sample solutions. . In addition, a sample solution was measured using Limulus ES-2 Single Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) having an endotoxin detection sensitivity of 0.015 EU / mL according to the instruction manual for the measurement kit.

<Evaluation result 1>
As for the cell adhesive polypeptide [P2-0], gels were formed in both the 1 mg / mL and 0.1 mg / mL sample solutions (endotoxin positive), and the endotoxin content of the measurement sample was 0.015 EU / 0. It was 1 mg or more, that is, 0.15 EU / mg or more.
For the cell adhesion polypeptides [P2-1], [P2-2] and [P2-3], no gel was formed in both the 1 mg / mL and 0.1 mg / mL sample solutions (endotoxin negative), and the measurement was performed. The endotoxin content of the sample was less than 0.015 EU / mg.

<Evaluation 2 (Cell culture A)>
Each of the wound dressing materials [S1] to [S10] of Examples 1 to 7 and Comparative Examples 1 to 3 was cut into a size of 1 cm × 1 cm and placed on the bottom of a 24-hole polystyrene plate (manufactured by Becton Dickinson). The wound dressing was put in the form of a hole, and the four corners of the wound dressing were affixed to the bottom surface with a vinyl tape, and then subjected to UV irradiation for 8 hours in a clean bench for sterilization. In addition, the same operation was performed for four holes per one kind of wound dressing material. The same applies hereinafter.
Next, 1 mL of a low serum medium for normal human skin fibroblast proliferation (manufactured by Kurashiki Boseki Co., Ltd.) and 20,000 normal human skin fibroblasts (manufactured by Kurashiki Boseki Co., Ltd.) were put into one well of a 24-well polystyrene plate. The cells were cultured for 3 days in an incubator at 37 ° C. and a CO ₂ concentration of 5% by volume.
Three days after the culture, the wound dressing materials [S1] to [S10] were peeled off from the bottom of the plate, and the wound dressing materials [S1] to [S10] were loaded into the bottom surface of a new 24-hole polystyrene plate at a rate of 1 sheet / hole. Further, a 0.25% by weight / volume of trypsin solution (a solution in which 0.25 g of trypsin is dissolved in 100 mL, trade name: Trypsin-EDTA, manufactured by Invitrogen Corporation) is charged at 200 μL / well, and 25 ° C. For 3 minutes.
Three minutes later, fetal calf serum (manufactured by Gibco BRL) was added at a rate of 20 μL / well, and the mixture was made into a mixed solution by sucking and discharging with a pipette. 20 μL of the mixed solution, 30 μL of a phosphate buffer solution (0.02 M, pH 7.2) containing 0.85% by weight of NaCl and 10 μL of Tetra Color One (manufactured by Seikagaku Corporation) were placed in a 96-well polystyrene plate. (Manufactured by Becton Dickinson) and placed in an incubator at 37 ° C. and a CO ₂ concentration of 5% by volume for 4 hours.
Four hours later, the amount of formazan produced was measured using a spectrophotometer at an absorbance of 492 nm (control wavelength: 630 nm), and this value was defined as the cell activity. The cell activity is proportional to the height of the absorbance. The results are shown in Table 1 (these results are average data for 4 holes). It should be noted that the tetrazolium salt of tetracolor one is reduced by intracellular mitochondrial dehydrogenase to form formazan, thereby developing color. In the wound dressing [S9] of Comparative Example 2, the wound dressing could not be peeled off from the bottom of the plate after 3 days of culture due to the collapse of the shape, and the cell activity could not be measured.

From the results in Table 1, it can be seen that the wound dressings S1 to S7 of the present invention have extremely high cell activity of the cells adhered to the wound dressing as compared with the wound dressings S8 to S10 of the comparative example. In addition, the wound dressing of the present invention did not undergo shape collapse as in Comparative Example 2.

<Evaluation 3 (Cell culture B)>
Each of the wound dressing materials [S2], [S11], [S12], and [S8] of Examples 2, 8, 9 and Comparative Example 1 was cut into a size of 1 cm ² in diameter, and subjected to UV irradiation in a clean bench for 8 days. Performed for hours and sterilized.
On the other hand, cultured dermis was prepared using PreTissue-Dermal (manufactured by Toyobo Co., Ltd.), a three-dimensional cultured tissue construction kit, according to the instruction manual of the kit. After preparation, the serum medium used in this kit was replaced with a serum-free medium, DMEM medium (manufactured by ICN Biomedicals), to obtain a cultured dermis in a serum-free environment.
The wound dressing material [S2], [S11], [S12], [S8] is put on the upper surface of the cultured dermis, and cell culture is performed for 5 days in an incubator at 37 ° C. and a CO ₂ concentration of 5% by volume. Was. After 5 days of culture, the wound dressings [S2], [S11], [S12] and [S8] were peeled off from the upper surface of the cultured dermis, respectively, and the wound dressings [S2] and [S11] were placed on the bottom of a new 24-hole polystyrene plate. , [S12], and [S8] are charged at 1 sheet / hole, and PBS is further charged at 125 μL / hole and Tetracolor One (manufactured by Seikagaku Corporation) at 25 μL / hole at 37 ° C. and 5 volumes of CO ₂ concentration. % In an incubator for 4 hours. Four hours later, the amount of formazan produced was measured using a spectrophotometer at an absorbance of 492 nm (control wavelength: 630 nm). Cell activity 2 is proportional to the height of the absorbance. The results are shown in Table 2 (these results are average data for 4 holes, respectively).

From the results in Table 2, it can be seen that the wound dressings S2, S11, and S12 of the present invention have extremely high cell activity of the cells migrated from the cultured dermis to the wound dressing as compared with the wound dressing S8 of the comparative example. .

<Evaluation 4 (animal experiment A)>
DM mice (C57BLK Jcl db / db, manufactured by CLEA Japan, Inc.) were subjected to inhalation anesthesia with diethyl ether, the entire back was shaved with a feather razor, and a circular (1 cm diameter) whole was formed in the center. A layered skin defect wound was created. In addition, the DM mouse used was a diabetic confirmation strip (Uro Peace, manufactured by Fujisawa Pharmaceutical Co., Ltd.), for which the onset of diabetes was confirmed.
The wound dressing [S2] of Example 2, the wound dressing [S8] of Comparative Example 1, and the wound dressing [S9] of Comparative Example 2 were each cut into a size of 2 cm × 2 cm. Name: MultiFix, manufactured by Alcare Corporation, and the wound dressing material side is affixed to the wound surface, and further covered with absorbent cotton to improve the adhesion to the wound surface, and an adhesive bandage (trade name: Silky Tex) , Manufactured by Alcare Co., Ltd.).
The growth environment was room temperature 24 ° C, and the feed and water were freely taken. The observation point was 14 days. On the 14th day, the wound treatment material was removed from the wound surface, and the whole wound including the target regenerated tissue was collected from the experimental animal, including the muscle layer existing under the regenerated tissue, and fixed, paraffin-wrapped. An embedding process was performed to prepare a tissue section. The prepared tissue sections were subjected to hematoxylin and eosin staining (HE staining) to evaluate regenerated tissues. Pictures of this regenerated tissue are shown in FIG. 1 (Wound dressing [S2]) and FIG. 2 (Wound dressing [S8]).
In the macroscopic observation, no infection occurred on the wound surface using the wound dressing [S2] and the wound dressing [S8], but the infection occurred on the wound surface using the wound dressing [S9]. . In addition, the wound dressing [S9] was unable to collect tissue due to fusion with the tissue.
From FIG. 1, it can be said that the use of the wound dressing [S2] of the present invention in Example 2 shows a good regenerating state in which regenerated tissue and cell infiltration are much observed and a large amount of matrix production in the regenerated tissue is recognized.
From FIG. 2, it can be said that the one using the wound dressing [S8] of Comparative Example 1 has poor regenerated tissue and cell infiltration, and the regenerated state is poor.

<Evaluation 5 (animal experiment B)>
(1) Application of the wound dressing material [S11] of Example 8 Instead of the wound dressing materials [S2], [S8] and [S9], the wound dressing material [S11] prepared in Example 8 was used. Evaluation 4 (animal experiment A) except that the size of the skin defect wound was changed from 1 cm in diameter to 1.4 cm in diameter, and that the adhesive film was changed to a bioclusive (trade name) manufactured by Johnson & Johnson Co. Similarly, DM mice were grown and animal experiments for wound healing were started. On the third day after the start, the wound dressing was removed from the wound surface, and the wound dressing [S11] was applied again together with the adhesive film.
(2) Application of the wound dressing [S8] of Comparative Example 1 and a trafermin solution:
250 μg of Fiblast Spray 250 (manufactured by Kaken Pharmaceutical Co., Ltd.), a therapeutic agent for pressure ulcers and skin ulcers, was dissolved in 2.5 mL of physiological saline to prepare a 100 μg / mL trafermin solution.
In place of the wound dressing [S11], the wound dressing [S8] prepared in Comparative Example 1 was used. Before laminating the wound dressing together with the adhesive film, 0.2 mL of the trafermin solution (equivalent to 20 μg of trafermin) was used. In the same manner as in the above (1), DM mice were raised and an animal experiment on wound healing was started, except that the above (1) was dropped onto the above-mentioned full-thickness skin defect wound with a pipette.
After the wound dressing was removed from the wound surface three days after the start, 0.2 mL of trafermin solution was dropped on the defective wound, and the wound dressing [S8] was applied again together with the adhesive film.
(3) Observation of Wound Healing The observation days were day 7 and day 14, and the wound dressing was removed from the wound on the day of observation, and the wound was visually observed. The photographs are shown in FIG. 3 (day 7, wound dressing [S11]), FIG. 4 (day 7, wound dressing [S8] and trafermin solution), and FIG. 5 (day 14, wound dressing [S11]). ) And FIG. 6 (Day 14, wound dressing [S8] and trafermin solution).
As a result on the 7th day, in the case of using the wound dressing material [S11] of the present invention of Example 8, epidermis formation was observed from all around the wound margin (FIG. 3). On the other hand, in the case of using the wound dressing [S8] of Comparative Example 1 and the trafermin solution, no epidermis was formed (FIG. 4).
As a result on the 14th day, in the case of using the wound dressing [S11] of the present invention of Example 8, epidermis formation was observed over the entire wound surface (FIG. 5). On the other hand, in the case of using the wound dressing [S8] and the trafermin solution of Comparative Example 1, epidermis formation was observed on a part of the wound surface, but no epidermis formation was observed on the entire wound surface (FIG. 6). .

The wound dressing of the present invention exerts a good wound treatment effect by adhering the cells contained in the wound dressing and the cells contained in the exudate exuding from the wound to the wound dressing of the present invention. In particular, it shows a surprising effect in terms of skin formation.
Suitable cells that can be adhered to the wound dressing of the present invention are human-derived cells, such as cells involved in skin (e.g., epithelial cells, fibroblasts, vascular endothelial cells, and smooth muscle cells), and blood vessels. Cells involved (such as vascular endothelial cells, smooth muscle cells and fibroblasts), cells involved in muscle (such as muscle cells), cells involved in fat (such as fat cells), cells involved in nerves (such as nerve cells) , Cells involved in the liver (such as hepatocytes), cells involved in the pancreas (such as pancreatic islet cells), cells involved in the kidney (such as renal epithelial cells, proximal tubular epithelial cells and mesangial cells), and lungs. Cells involved in bronchi (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in eyes (photoreceptors, corneal epithelial cells, corneal endothelial cells, etc.), cells involved in prostate (epithelial cells) , Stromal cells and smooth muscle cells, etc.) Cells involved in bone (osteoblasts, osteocytes, osteoclasts, etc.), cells involved in cartilage (chondroblasts, chondrocytes, etc.), cells involved in teeth (periodontal cells, osteoblasts, etc.), Blood-related cells (such as leukocytes and erythrocytes) and stem cells, such as bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells (oval cells, small hepatocytes, etc.), adipose tissue stem cells , Embryonic stem (ES) cells, epidermal stem cells, intestinal stem cells, sperm stem cells, embryonic germ stem (EG) cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphoid stem cells, corneal stem cells, progenitor cells (Lipid precursor cells, vascular endothelial precursor cells, cartilage precursor cells, lymphoid precursor cells, NK precursor cells, etc.) and the like.

The application of the wound dressing material of the present invention to the wound surface can be applied without limitation as long as it can cover the wound surface.For example, a cover with an adhesive or a pressure-sensitive adhesive, a bandage, an elastic mesh or an eye patch is used for fixing. And may be sutured or glued. In addition, when the wound is a chronic wound such as an ulcer, it is preferably applied to a wound from the yellow stage to the red stage.If the wound is an acute wound such as a burn or traumatic skin defect, the wound from the inflammatory stage to the granulation stage Application to is preferred.
Furthermore, the wound dressing of the present invention can also be applied as a substrate for culturing the above cells outside the body.

5 is a photomicrograph (45 × magnification) of a tissue section in <Evaluation 4 (animal experiment A)> using the wound dressing [S2] of the present invention obtained in Example 2. FIG. 4 is a micrograph (45 × magnification) of a tissue section in <Evaluation 4 (animal experiment A)> using the comparative wound dressing [S8] obtained in Comparative Example 1. FIG. 11 is a photograph (magnification: 3 ×) of the wound surface on the 7th day in <Evaluation 5 (animal experiment B)> using the wound dressing [S11] of the present invention obtained in Example 8. 9 is a photograph (3 times magnification) of a wound surface on day 7 in <Evaluation 5 (animal experiment B)> using the comparative wound dressing [S8] obtained in Comparative Example 1. 11 is a photograph (three times magnification) of a wound surface on day 14 in <Evaluation 5 (animal experiment B)> using the wound dressing [S11] of the present invention obtained in Example 8. 9 is a photograph (magnification: 3 times) of the wound surface on day 14 in <Evaluation 5 (animal experiment B)> using the comparative wound dressing [S8] obtained in Comparative Example 1.

Claims (9)

Wound covering comprising a cell adhesive polypeptide (P) having an endotoxin content of less than 0.15 EU / mg based on the weight of the cell adhesive polypeptide (P) and a coating material (N) Wood. The wound dressing according to claim 1, wherein the dressing material (N) is a hardly biodegradable material (N2). The wound dressing according to claim 1 or 2, wherein the cell adhesive polypeptide (P) is an artificial cell adhesive polypeptide (P2). Cell adhesion polypeptide (P) is composed of Arg Gly Asp sequence, Leu Asp Val sequence, Arg Glu Asp Val sequence (1), Tyr Ile Gly Ser Arg sequence (2), Pro Asp Ser Gly Arg sequence (3), Arg Tyr Val Val Leu Pro Arg sequence (4), Leu Gly Thr Ile Pro Gly sequence (5), Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile sequence (6), Ile Lys Val Ala Val sequence (7), Leu Arg Glu Sequence, Asp Gly Glu Ala sequence (8), Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9), Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys sequence (10), His The wound according to any one of claims 1 to 3, comprising at least one kind of minimum amino acid sequence (X) selected from the group consisting of an Ala Val sequence and a Tyr Lys Leu Asn Val Asn Asp Ser sequence (11). Coating material. The wound dressing according to any one of claims 1 to 4, wherein the cell adhesive polypeptide (P) is chemically bonded to the coating material (N). The wound dressing according to any one of claims 1 to 5, further comprising a cell growth factor (G1) and / or a cell growth factor binding substance (G2). The wound dressing according to any one of claims 1 to 6, wherein the cell adhesive polypeptide (P) is modified with a compound (AM) containing an amino group or an ammonium group. The wound dressing according to any one of claims 1 to 7, wherein the cell adhesive polypeptide (P) further comprises a heat-resistant amino acid sequence (Y). The wound dressing according to claim 8, wherein the heat-resistant amino acid sequence (Y) is a Gly Ala Gly Ala Gly Ser sequence (12) and / or a Gly Val Gly Val Pro sequence (13).