Tissue formation agent

Abstract

To provide a tissue formation agent causing no inflammation.SOLUTION: Provided is a tissue scaffold agent containing alginic acid or a salt thereof and a nonwoven fabric, wherein the tissue is any tissue of an epithelial tissue, a tissue lining an epithelial tissue layer, or a tissue located deeper than these tissues (excluding brain, nerve, heart, or hard tissue).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to tissue forming agents, tissue fillers, and tissue scaffolding agents. [Background technology]

[0002] Scaffolding agents and fillers are used clinically to treat abnormalities in morphology and function, including damage and wound healing, of the skin, mucous membranes, pleura, peritoneum, etc. caused by surgery, trauma, disease, etc. Known active ingredients of these agents include gelatin, collagen, hyaluronic acid, etc. (Non-Patent Documents 1 to 5).

[0003] Alginic acid is a polysaccharide found in algae. It is a polymer composed of two uronic acid monomers, mannuronic acid (M) and guluronic acid (G). Polymers with various properties exist depending on the ratio of these two uronic acids (M / G ratio). Alginic acid is used in a variety of fields, including food, pharmaceuticals, cosmetics, and adhesives. In particular, in the pharmaceutical field, it is known to be used as a tablet disintegrant, a stomach wall protectant, and a dental impression material. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hajime Matsumine et al. “Full-thickness skin reconstruction with basic fibroblast growth factor-impregnated collagen-gelatin sponge”Regenerative Therapy 11 81-87 2019 [Non-patent document 2] Kuroda et al. “Clinical application of injectable growth factor for bone regeneration: a systematic review” Inflammation and Regeneration 39(20)2019 [Non-patent document 3] Chi H.Lee a, Anuj Singla, Yugyung Lee”Biomedical applications of collagen”International Journal of Pharmaceutics 221 1-22 2001 [Non-patent document 4] Sayani Chattopadhyay and Ronald T.Raines “Collagen-Based Biomaterials for Wound Healing”Biopolymers.101(8)821-833 2014 [Non-Patent Document 5] Alastair Carruthers, Jean Carruthers “Soft Tissue Augmentation E-Book: Procedures in Cosmetic Dermatology Series Chapter 6 Belotero and Teosyal by Berthold Rzany” page 35-4 Summary of the Invention [Problem to be solved by the invention]

[0005] The scaffolding agents and fillers containing gelatin, collagen, hyaluronic acid, etc. as active ingredients have the problem that they do not exhibit a sufficiently satisfactory regenerative ability because the cells infiltrate and proliferate at the injected site only weakly.Furthermore, they are recognized as foreign bodies at the injected site, causing an inflammatory reaction. [Means for solving the problem]

[0006] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that alginic acid or its sodium salt is useful as a tissue-forming agent. They have also found that calcium-crosslinking the alginic acid or its salt makes it useful as a tissue-forming agent. They have further found that alginic acid or its sodium salt is useful as a tissue filler and tissue scaffold. The present invention was completed based on these findings and broadly encompasses the inventions described in the following sections.

[0007] Item 1. A tissue-forming agent containing alginic acid or a salt thereof.

[0008] Item 2. The forming agent according to Item 1, wherein the tissue is any one of epithelial tissue, tissue lining an epithelial tissue layer, and tissue located deeper than these tissues.

[0009] Item 3. The forming agent according to Item 1 or 2, wherein the amount of endotoxin contained is 1500 EU / mL or / mg or less.

[0010] Item 4. The forming agent according to any one of Items 1 to 3, wherein the viscosity of the alginic acid or salt thereof is 0.1 to 1500 mPa / s.

[0011] Item 5. The agent according to any one of items 1 to 4, wherein the alginic acid or a salt thereof has an M / G ratio of 0.001 to 0.999.

[0012] Item 6. The agent according to any one of items 1 to 5, wherein the alginic acid or a salt thereof is cross-linked with calcium or polylysine.

[0013] Item 7. The agent according to any one of items 1 to 6, wherein the alginic acid or a salt thereof is sulfated.

[0014] Item 8. The forming agent according to any one of items 1 to 7, further comprising a sulfate.

[0015] Item 9. A tissue filler containing alginic acid or a salt thereof.

[0016] Item 10. A tissue scaffold containing alginic acid or a salt thereof.

[0017] Item 11. A method comprising the step of administering a composition containing alginic acid or a salt thereof to a patient in need of tissue formation.

[0018] Item 12. A method comprising the step of administering a composition containing alginic acid or a salt thereof to a patient in need of tissue regeneration.

[0019] Item 13. A method comprising the step of administering a composition containing alginic acid or a salt thereof to a patient in need of wound healing.

[0020] Item 14. Use of alginic acid or a salt thereof in the manufacture of a tissue-forming agent.

[0021] Item 15. Use of alginic acid or a salt thereof in the manufacture of a tissue filler.

[0022] Item 16. Use of alginic acid or a salt thereof in the production of a tissue scaffolding agent.

[0023] Item 17. A composition for tissue formation containing alginic acid or a salt thereof.

[0024] Item 18. A composition for tissue regeneration containing alginic acid or a salt thereof.

[0025] Item 19. A wound healing composition containing alginic acid or a salt thereof. [Effects of the Invention]

[0026] The present invention can provide a tissue-forming agent. Furthermore, the present invention can provide a tissue-forming agent that does not cause inflammation. [Brief explanation of the drawings]

[0027] [Figure 1]Figure 1 shows the results of Example 1. (A) shows HE-stained and SOFG-stained images of saline three weeks after implantation. (B) shows HE-stained and SOFG-stained images of AL2 three weeks after implantation. [Figure 2] Figure 2 shows the results of Example 2. (A) shows HE-stained and SOFG-stained images of ALG20 12 weeks after implantation. (B) shows HE-stained and SOFG-stained images of AL20 12 weeks after implantation. [Figure 3] 3 shows the results of Example 3. (A) shows HE-stained and SOFG-stained images of ALG500 12 weeks after implantation. (B) shows HE-stained and SOFG-stained images of AL500 12 weeks after implantation. [Figure 4] 4 shows the results of Example 4. (A) shows HE-stained and SOFG-stained images of ALG20 24 weeks after implantation. (B) shows HE-stained and SOFG-stained images of AL20 24 weeks after implantation. [Figure 5] 5 shows the results of Example 5. (A) shows HE-stained and SOFG-stained images of ALG500 24 weeks after implantation. (B) shows HE-stained and SOFG-stained images of AL500 24 weeks after implantation. [Figure 6] Figure 6 shows the results of Example 6. (A) shows HE-stained, SOFG-stained, MTC-stained, and Col-I-stained images of ALG20 24 weeks after implantation. (B) shows HE-stained, SOFG-stained, MTC-stained, and Col-I-stained images of ALG20 24 weeks after implantation. [Figure 7] Figure 7 shows the results of Example 7. (A) shows HE-stained images, Col-I-stained images, vWF-stained images, and alfa-SMA-stained images 24 weeks after implantation of ALG20. (B) shows HE-stained images, Col-I-stained images, vWF-stained images, and alfa-SMA-stained images 24 weeks after implantation of AL20. [Figure 8]Figure 8 shows the results of Example 8. (A) shows HE-stained, SOFG-stained, and MTC-stained images of AL10 one week after implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images of AL100 one week after implantation. [Figure 9] 9 shows the results of Example 9. (A) shows HE-stained, SOFG-stained, and MTC-stained images of AL10 three weeks after implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images of AL100 three weeks after implantation. [Figure 10] 10 shows the results of Example 10. (A) shows HE-stained, SOFG-stained, and MTC-stained images of AL10 6 weeks after implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images of AL100 6 weeks after implantation. [Figure 11] Figure 11 shows the results of Example 11. (A) shows HE-stained, SOFG-stained, and MTC-stained images of AL10 12 weeks after implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images of AL100 12 weeks after implantation. [Figure 12] Figure 12 shows the results of Example 12. (A) shows HE-stained, SOFG-stained, and MTC-stained images one week after implantation of high-endotoxin AL20. (B) shows HE-stained, SOFG-stained, and MTC-stained images one week after implantation of low-endotoxin AL20. [Figure 13] Figure 13 shows the results of Example 13. (A) shows HE-stained, SOFG-stained, and MTC-stained images one week after implantation of high-endotoxin AL500. (B) shows HE-stained, SOFG-stained, and MTC-stained images one week after implantation of low-endotoxin AL500. [Figure 14] Figure 14 shows the results of Example 14. (A) shows HE-stained, SOFG-stained, and MTC-stained images 3 weeks after implantation of high-endotoxin AL20. (B) shows HE-stained, SOFG-stained, and MTC-stained images 3 weeks after implantation of low-endotoxin AL20. [Figure 15]Figure 15 shows the results of Example 15. (A) shows HE-stained, SOFG-stained, and MTC-stained images 3 weeks after implantation of high-endotoxin AL500. (B) shows HE-stained, SOFG-stained, and MTC-stained images 3 weeks after implantation of low-endotoxin AL500. [Figure 16] Figure 16 shows the results of Example 16. (A) shows HE-stained, SOFG-stained, and MTC-stained images 6 weeks after implantation of high-endotoxin AL20. (B) shows HE-stained, SOFG-stained, and MTC-stained images 6 weeks after implantation of low-endotoxin AL20. [Figure 17] Figure 17 shows the results of Example 17. (A) shows HE-stained, SOFG-stained, and MTC-stained images 6 weeks after implantation of high-endotoxin AL500. (B) shows HE-stained, SOFG-stained, and MTC-stained images 6 weeks after implantation of low-endotoxin AL500. [Figure 18] Figure 18 shows the results of Example 18. (A) shows HE-stained, SOFG-stained, and MTC-stained images 12 weeks after implantation of high-endotoxin AL20. (B) shows HE-stained, SOFG-stained, and MTC-stained images 12 weeks after implantation of low-endotoxin AL20. [Figure 19] Figure 19 shows the results of Example 19. (A) shows HE-stained, SOFG-stained, and MTC-stained images 12 weeks after implantation of high-endotoxin AL500. (B) shows HE-stained, SOFG-stained, and MTC-stained images 12 weeks after implantation of low-endotoxin AL500. [Figure 20] 20 shows the results of Comparative Example 1. (A) shows HE-stained, SOFG-stained, and MTC-stained images one week after hyaluronic acid implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images one week after collagen implantation. [Figure 21]21 shows the results of Comparative Example 2. (A) shows HE-stained, SOFG-stained, and MTC-stained images 3 weeks after hyaluronic acid implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images 3 weeks after collagen implantation. [Figure 22] 22 shows the results of Comparative Example 3. (A) shows HE-stained, SOFG-stained, and MTC-stained images 6 weeks after hyaluronic acid implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images 6 weeks after collagen implantation. [Figure 23] 23 shows the results of Comparative Example 4. (A) shows HE-stained, SOFG-stained, and MTC-stained images 12 weeks after hyaluronic acid implantation. (B) shows HE-stained, SOFG-stained, and MTC-stained images 12 weeks after collagen implantation. [Figure 24] Figure 24 shows the results of Example 20. (A) shows HE-stained and SOFG-stained images of calcium-crosslinked AL10 12 weeks after implantation. (B) shows HE-stained and SOFG-stained images of hyaluronic acid 12 weeks after implantation. [Figure 25] Figure 25 shows the results of Example 21. (A) shows HE-stained and SOFG-stained images of calcium-crosslinked AL10 24 weeks after implantation. (B) shows HE-stained and SOFG-stained images of hyaluronic acid 24 weeks after implantation. [Figure 26] Figure 26 shows the results of Example 22. (A) shows an HE-stained image one week after implantation of sulfated sodium alginate. (B) shows an HE-stained image one week after implantation of sodium alginate (AL100). [Figure 27] Figure 27 shows the results of Example 23. (A) shows an HE-stained image of a mixture of sodium alginate (AL10) and sulfated dextran two weeks after implantation. (B) shows an HE-stained image of sodium alginate (AL10) two weeks after implantation. [Figure 28]Figure 28 shows the results of Example 24. (A) shows an HE-stained image of calcium-crosslinked sodium alginate (AL10) one week after implantation. (B) shows an HE-stained image of sodium alginate (AL10) one week after implantation. [Figure 29] Figure 29 shows the results of Example 25. (A) shows an HE staining image 4 weeks after the combined implantation of calcium-crosslinked sodium alginate (AL10) and PGA nonwoven fabric into the rat peritoneum. (B) shows an HE staining image 4 weeks after the combined implantation of poly-L-lysine-crosslinked sodium alginate (AL10) and PGA nonwoven fabric into the rat peritoneum. (C) shows an HE staining image 4 weeks after the implantation of PGA nonwoven fabric alone into the rat peritoneum. (D) shows an HE staining image 4 weeks after the implantation of calcium-crosslinked sodium alginate (AL10) alone into the rat peritoneum. [Figure 30] Figure 30 shows the results of Example 26. (A) shows an HE-stained image from a control experiment. (B) shows an HE-stained image from a site injected with relatively small molecules of sodium alginate. (C) shows an HE-stained image from a site injected with AL10. (D) shows an HE-stained image from a site injected with relatively small molecules of alginate and sodium polyglutamate solution at the same site. (E) shows an HE-stained image from a site injected with AL10 and sodium polyglutamate solution at the same site. DETAILED DESCRIPTION OF THE INVENTION

[0028] Each embodiment of the present invention will be described in further detail below.

[0029] Tissue-forming agents The tissue-forming agent of the present invention contains alginic acid or a salt thereof. The above-mentioned alginic acid salt is not particularly limited, but is limited to the range in which the effects of the present invention are exhibited. Specifically, it refers to a salt in which the hydrogen ion of the carboxyl group of alginic acid is liberated and bound to a cation. Examples of such cations include monovalent cations such as sodium ion, potassium ion, and ammonium ion, inorganic polyvalent ions such as calcium ion, magnesium ion, iron ion, and ammonium ion, and polyvalent cations such as organic polyvalent ions such as polylysine. Among these salts, sodium salts are preferred due to their water solubility.

[0030] The tissue in the tissue-forming agent of the present invention is not particularly limited, and specific examples include epithelial tissue, tissue lining the epithelial tissue layer, and tissue located deeper than these tissues.

[0031] The epithelial tissue is not particularly limited as long as it exhibits the effects of the present invention, and specific examples include mesothelial tissues such as peritoneum, pleura, and pericardium, and endothelial tissues such as blood vessels, lymphatic vessels, and meninges, as well as epithelial tissues in the narrow sense such as skin, urinary organs, reproductive organs, digestive organs, and respiratory organs. Among these, mucosal tissues of the skin, digestive organs, uterus, bladder, and trachea, kidney, and liver tissues are preferred.

[0032] The tissue lining the epithelial tissue layer is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include dermal tissue in the skin, and the muscularis mucosae and lamina propria in the mucosa of the digestive, respiratory, urinary, or genital organs.

[0033] The tissue located deeper than the epithelial tissue and the tissue lining the epithelial tissue is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include subcutaneous tissue in the skin, submucosal tissue in the mucosa, the peritoneal submesothelial layer and the pleural submesothelial layer in mesothelial tissue, and the subendothelial layer of the vascular endothelium in endothelium.

[0034] The amount of endotoxin contained in the tissue-forming agent of the present invention is not particularly limited, as long as it is within the range in which the effects of the present invention are exhibited. Specifically, it can be about 1500 EU / mL or mg or less, preferably about 500 EU / mL or mg or less, more preferably about 100 EU / mL or mg or less, and most preferably about 50 EU / mL or mg or less. Note that such endotoxin is measured by the method set forth in the endotoxin test method in the 17th edition of the Japanese Pharmacopoeia.

[0035] The viscosity of the alginic acid or salt thereof contained in the tissue-forming agent of the present invention is not particularly limited, as long as it is within a range in which the effects of the present invention are exhibited. Specifically, it can be about 0.1 to 1500 mPa / s. Preferably, it is about 2 to 500 mPa, more preferably about 50 to 100 mPa, and most preferably about 10 to 20 mPa.

[0036] Such viscosity was measured in an aqueous solution with a concentration of 1% (by mass) under measurement conditions of 20°C.

[0037] The M / G ratio of alginic acid or a salt thereof contained in the tissue-forming agent of the present invention is not particularly limited as long as it is within a range in which the effects of the present invention are exhibited, and specifically, can be about 0.001 to 0.999.

[0038] The alginic acid or a salt thereof contained in the tissue-forming agent of the present invention may be crosslinked with a cation such as calcium, a polyvalent cation such as polylysine or acid-treated collagen, or a compound in which a polysaccharide such as chitosan has become cationic. The specific crosslinking method is not particularly limited as long as it can exert the effects of the present invention. For example, the alginic acid or a salt thereof may be obtained by mixing calcium, a compound in which a polysaccharide such as chitosan has become cationic, or polylysine.

[0039] The calcium is not limited to any particular form as long as it can achieve the effects of the present invention. Specific examples include calcium in the form of a salt. Such calcium salts are not particularly limited as long as they can achieve the effects of the present invention. Examples include calcium salts of organic acids and calcium salts of inorganic acids, and more specifically, calcium salts of gluconic acid.

[0040] The polylysine is not limited to any form of polylysine as long as it can exhibit the effects of the present invention. Specific examples include polylysine in the form of a salt. Such polylysine salts are not particularly limited as long as they can exhibit the effects of the present invention. Examples include inorganic acid salts such as hydrochloride, hydrobromide, and phosphate, and organic acid salts such as propionate, acetate, fumarate, malate, and citrate. The polylysine may be D-lysine, L-lysine, or a mixture of D-lysine and L-lysine, but is preferably composed entirely of L-lysine.

[0041] The alginic acid or salt thereof contained in the tissue-forming agent of the present invention may be cross-linked with sulfuric acid. The specific sulfation method is not particularly limited, as long as it achieves the effects of the present invention. For example, sulfated alginic acid may be prepared according to the method described in "Whistler R L., et al., Sulfates. Edited by Whistler R L., et al., Methods in Carbohydrate Chemistry. Vol. II, 298-303. Academic Press (New York) (1963)."

[0042] More specifically, sodium alginate and sulfur trioxide pyridine complex were stirred in dimethylformamide (40°C for 12 hours) to introduce sulfate groups into alginic acid, and then sulfated alginic acid was precipitated. This was then neutralized and dissolved in dilute aqueous sodium hydroxide solution, dialyzed for 72 hours, and freeze-dried to prepare sulfated alginic acid.

[0043] The tissue-forming agent of the present invention may contain substantially only the above-mentioned alginic acid or a salt thereof, or may contain other components in addition to alginic acid or a salt thereof.

[0044] The above-mentioned other components are not particularly limited as long as they can exert the effects of the present invention. Examples include pharmaceutically acceptable bases, carriers, and additives (e.g., excipients, solvents, surfactants, preservatives, pH adjusters, thickeners, etc.). Such bases, carriers, and additives are specifically described in, for example, a dictionary of pharmaceutical additives, and those described therein can be used as appropriate.

[0045] A sulfate can be used together with the alginic acid or a salt thereof. Such a sulfate can be selected from those widely known as pharmaceutically acceptable sulfates, such as sulfated dextran, heparan sulfate (including heparin and heparinoid), chondroitin sulfate, keratan sulfate, carrageenan, funoran, dermatan sulfate, porphyrin, and fucoidan. Among these, sulfated dextran or heparan sulfate is preferred.

[0046] The above-mentioned alginic acid and its salts exhibit a more excellent tissue-forming effect when used together with a biocompatible substance that exhibits strong adhesion to the surroundings. The biocompatible substance that exhibits strong adhesion to the surroundings is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include pullulan and polyglutamates. Other substances that exhibit a more excellent tissue-forming effect when used together include thickeners (carrageenan, pectin, gelatin, etc.), compounds in which polysaccharides such as chitosan have been converted into cationic forms, and polyvinylbenzyltrimethylammonium. Among these, pullulan and polyglutamates are preferred, with polyglutamates being most preferred.

[0047] The formulation of the tissue-forming agent of the present invention is not particularly limited as long as it can exert the effects of the present invention. For example, alginic acid or a salt thereof and other components can be mixed in a conventional manner and then appropriately prepared into a formulation such as an injection or to be placed inside a wound or tissue, depending on the administration form of the tissue-forming agent of the present invention.

[0048] The method of administration or placement of the tissue-forming agent of the present invention is not particularly limited, as long as it is a known administration method optimized for each of the above-mentioned dosage forms within the scope of the effect of the present invention. Specific examples include administration and placement into epithelium and its adjacent tissues (epithelial tissue, mesothelial tissue, endothelial tissue, tissues lining these epithelial tissues, and tissues located deeper than these, which are included in epithelium in the broad sense), or into open wounds, closed wounds, adipose tissue, etc., in areas adjacent to these tissues.

[0049] The site of administration or placement of the tissue-forming agent of the present invention is not particularly limited as long as the effects of the present invention can be achieved, and examples thereof include administration or placement at a site of skin damage caused after surgery or the like, a wounded site, epithelium and its adjacent tissue, or other open wounds, closed wounds, intramuscular tissue, adipose tissue, intraarticular tissue, bone tissue, organs, and body cavities.

[0050] The dosage of the tissue-forming agent of the present invention cannot be specified, as it is determined depending on the desired site of formation, the extent of formation, and the degree of damage. For example, the tissue-forming agent of the present invention may be administered at approximately 10% to 300% of its volume per administration. Preferably, the amount administered per administration is approximately 1000 ml or less. This dosage can be administered once a day, or in divided doses several times a day. It can also be administered several times every six months to a month, several times every two weeks, or several times a week, or once every six, five, four, three, or two days.

[0051] tissue fillers The tissue filler of the present invention contains alginic acid or a salt thereof.

[0052] The alginic acid or a salt thereof contained in the tissue filler of the present invention, the dosage of the filler, the administration method, etc. can be the same as those of the tissue-forming agent of the present invention described above. Furthermore, the tissue targeted by the tissue filler can also be the same as those of the tissue-forming agent of the present invention described above.

[0053] The alginic acid or salt thereof contained in the tissue filler of the present invention tends to be crosslinked to a higher degree than the alginic acid or salt thereof contained in the tissue-forming agent of the present invention described above.

[0054] Tissue scaffolding agents The tissue scaffolding agent of the present invention contains alginic acid or a salt thereof.

[0055] The alginic acid or a salt thereof contained in the tissue scaffold agent of the present invention, the dosage of the scaffold agent, the administration method, etc. may be the same as those of the tissue-forming agent of the present invention described above. Furthermore, the tissue targeted by the tissue scaffold agent may also be the same as those of the tissue-forming agent of the present invention described above.

[0056] The tissue scaffolding agent of the present invention can be used in combination with the above-mentioned alginic acid or its salt, as well as conventionally known tissue reinforcement materials (also called organ defect reinforcement materials). Such tissue reinforcement materials can be selected from those widely known as pharmaceutically acceptable tissue reinforcement materials, such as polyglycolic acid nonwoven fabric (PGA nonwoven fabric), lactic acid / caprolactone copolymer nonwoven fabric (LA / CL nonwoven fabric), and other nonwoven fabrics containing polyglycolic acid, polylactic acid or a portion thereof, and nonwoven fabrics containing any biologically derived component such as a polyvinyl component, a polyurethane component, a cellulose component, collagen, or gelatin. Among these tissue reinforcement materials, PGA nonwoven fabric and LA / CL nonwoven fabric are preferred.

[0057] In this specification, the expression "comprises" or "contains" a certain component means that the component in question is included and may further include other components, and also encompasses the concepts of "consisting only of" meaning that the component is included only, and "consisting essentially of" meaning that the component is included as an essential component.

[0058] The contents of the publications and web pages cited herein are hereby incorporated by reference.

[0059] Furthermore, the various characteristics, such as properties, structures, and functions, described for each embodiment of the present invention above can be combined as appropriate to specify aspects included in the present invention. In other words, the present invention can include all inventions of aspects of each characteristic that can be combined as disclosed in this specification. [Example]

[0060] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

[0061] The reagents used in the following examples will be described.

[0062] Saline solution: Otsuka saline injection 20mL plastic ampule, product number (YJ code): 3311401A2026 Various sodium alginates (Kimika Co., Ltd.) Low-endotoxin sodium alginate AL2 (Weight average molecular weight: 7,700kDa, viscosity 1% aqueous solution) Low-endotoxin sodium alginate AL10 (Weight average molecular weight: 57kDa, viscosity 1% aqueous solution: 11mPa / s) Low-endotoxin sodium alginate AL20 (Weight average molecular weight: 91kDa, viscosity 1% aqueous solution: 20-25mPa / s) Low-endotoxin sodium alginate AL100 (Weight average molecular weight: 170-180kDa viscosity 1% aqueous solution: 100-120mPa / s) Low-endotoxin sodium alginate AL500 (Weight average molecular weight: 280-300kDa, viscosity 1% aqueous solution: 400-600mPa / s) Low-endotoxin sodium alginate ALG2 (Weight average molecular weight: 7.7kDa, viscosity 1% aqueous solution) Low-endotoxin sodium alginate ALG20 (Weight average molecular weight: 91kDa, viscosity 1% aqueous solution: 20-25mPa / s) Low-endotoxin sodium alginate ALG500 (Weight average molecular weight: 280-300kDa, viscosity 1% aqueous solution: 400-600mPa / s) The endotoxin content of the low-endotoxin sodium alginate is ≦EU50.

[0063] High-endotoxin sodium alginate AL20 (Weight average molecular weight: 91kDa, viscosity 1% aqueous solution: 30mPa / s) High-endotoxin sodium alginate AL500 (Weight average molecular weight: 280-300kDa, viscosity 1% aqueous solution: 400mPa / s) The amount of endotoxin contained in the high-endotoxin sodium alginate is ≧10,000 EU or more.

[0064] Koken Atelocollagen Implant (Koken Co., Ltd.: Product Number: #1332) Hyaluronic acid (for joint injection) Product code (brand code): 3999408G1417 Sodium hyaluronate for joint injection 25 mg syringe "Nichiiko" 25 mg / 2.5 ml TEOSYAL RHA1 (hyaluronic acid) TEOXANE product ID: 1208 crosslinker rate: 1.9%

[0065] Example 1 Six rats (Wistar / ST female) were anesthetized with intraperitoneal administration of pentobarbital and inhaled isoflurane, and each solution (physiological saline and low-endotoxin sodium alginate) was injected into eight sites from the upper to lower back. AL2Using a 1 ml syringe and a 23G needle, 1 ml of each solution was injected (implanted) into the tissue between the subcutaneous fascia at a depth of approximately 1 cm. The injection of each solution was randomized. The needle insertion site was sutured with a single stitch using 5-0 proline thread.

[0066] Three weeks later, each rat was sacrificed by pentobarbital overdose, and the implantation site was excised from the back and immersion-fixed in 10% formaldehyde solution. The fixed tissue was stained with hematoxylin and eosin (HE staining) and safranin-o (SOFG staining) according to standard methods. The results are shown in Figure 1.

[0067] The results shown in Figure 1 indicate that AL2 disappeared and no inflammatory changes were observed.

[0068] Example 2 Low Endotoxin Sodium Alginate ALG20 and low-endotoxin sodium alginate AL20 1 ml of each of the 2% aqueous solutions was implanted into 6 rats in the same manner as in Example 1. After 12 weeks, HE staining and SOFG staining were carried out in the same manner as in Example 1, and the results are shown in FIG.

[0069] The results shown in Figure 2 indicate that implantation of ALG20 and AL20 resulted in little retention, and both were thought to have undergone changes in orientation. Furthermore, although inflammatory changes were alleviated, some cases persisted. Furthermore, no significant differences were observed between ALG20 and AL20.

[0070] Example 3 Low Endotoxin Sodium Alginate ALG500 and low-endotoxin sodium alginate AL500 1 ml of each 2% aqueous solution was implanted into 6 rats in the same manner as in Example 1. After 12 weeks, HE staining and SOFG staining were carried out in the same manner as in Example 1, and the results are shown in FIG.

[0071] As shown in Figure 3, implantation of ALG500 and AL500 resulted in significant retention. Inflammatory changes were alleviated, but some cases persisted. No significant differences were observed between ALG500 and AL500.

[0072] Example 4 Low Endotoxin Sodium Alginate ALG20 and low-endotoxin sodium alginate AL20 1 ml of each 2% aqueous solution was implanted into 6 rats in the same manner as in Example 1. 24 weeks later, HE staining and SOFG staining were carried out in the same manner as in Example 1, and the results are shown in FIG.

[0073] The results shown in Figure 4 indicate that implantation of ALG20 and AL20 resulted in little retention, and both showed significant changes in orientation. Furthermore, although inflammatory changes were alleviated, some cases persisted. Furthermore, no significant differences were observed between ALG20 and AL20.

[0074] Example 5 Low Endotoxin Sodium Alginate ALG500 and low-endotoxin sodium alginate AL500 1 ml of each 2% aqueous solution was implanted into 6 rats in the same manner as in Example 1. 24 weeks later, HE staining and SOFG staining were carried out in the same manner as in Example 1, and the results are shown in FIG.

[0075] The results shown in Figure 5 indicate that implantation of ALG500 and AL500 resulted in retention. Although inflammatory changes were alleviated, some cases persisted. No significant differences were observed between ALG500 and AL500.

[0076] Example 6 In addition to HE staining and SOFG staining, MTC staining and type I collagen staining were also performed on the implanted rat tissue in the same manner as in Example 4. The results are shown in Figure 6. MTC staining and type I collagen staining were performed according to standard methods.

[0077] From the results shown in Figure 6, in both implanted ALG20 and AL20, SOFG staining, MTC staining, and type I collagen staining were positive in the areas where changes in orientation were observed, suggesting that collagen tissue, etc. was formed and that there was a significant orientation according to the collagen tissue tension.

[0078] Example 7 In addition to HE staining, Col-I staining, vWF staining, and alfa-SMA staining were also performed on the implanted rat tissue in the same manner as in Example 4. The results are shown in Figure 7. Note that vWF staining and alfa-SMA staining were performed according to standard methods.

[0079] The results shown in Figure 7 indicate that in both ALG20 and AL20 implants, Col-I, vWF, and alfa-SMA staining were positive at the site where changes in orientation were observed, suggesting that adipose tissue and vascular tissue had formed in the subcutaneous tissue. No scarring was observed.

[0080] Example 8 Low Endotoxin Sodium Alginate AL10 and low-endotoxin sodium alginate AL100 1 ml of each 2% aqueous solution was implanted into 6 rats in the same manner as in Example 1. One week later, HE staining and SOFG staining were carried out in the same manner as in Example 1, and further MTC staining was carried out, and the results are shown in Figure 8.

[0081] From the results shown in FIG. 8, it was found that the injected sodium alginate remained in both the AL10 and AL100 implants.

[0082] Example 9 Six rats were implanted in the same manner as in Example 8, and 3 weeks later, HE staining, SOFG staining, and MTC staining were performed in the same manner as in Example 8. The results are shown in FIG.

[0083] From the results shown in FIG. 9, it was found that the injected sodium alginate remained in both the AL10 and AL100 implants.

[0084] Example 10 Six weeks after implantation in six rats in the same manner as in Example 8, HE staining, SOFG staining, and MTC staining were carried out in the same manner as in Example 8. The results are shown in FIG.

[0085] The results shown in Figure 10 indicate that the injected sodium alginate remained in the AL100 implant, but the amount remaining was significantly reduced in the AL10 implant. In addition, some changes in orientation were observed in the AL10 implant.

[0086] Example 11 Six rats were implanted in the same manner as in Example 8, and 12 weeks later, HE staining, SOFG staining, and MTC staining were performed in the same manner as in Example 8. The results are shown in FIG.

[0087] The results shown in Figure 11 indicate that the injected sodium alginate remained in the AL100 implant, but not in the AL10 implant. In addition, some changes in orientation were observed in the AL100 implant, but not in the AL10 implant.

[0088] Example 12 High-Endotoxin Sodium Alginate AL20 (High Endo AL20) and Low Endotoxin Sodium Alginate AL20 1 ml of a 2% aqueous solution of (Low End AL20) was implanted into six rats in the same manner as in Example 1. One week later, HE staining, SOFG staining, and MTC staining were performed in the same manner as in Example 1, and the results are shown in Figure 12.

[0089] The results shown in Figure 12 show that in the case of high-end AL20 implants, inflammatory cells infiltrated not only around the implant site but also into the implant site. In the case of low-end AL20 implants, inflammatory cells were only observed around the implant site.

[0090] Example 13 High-Endotoxin Sodium Alginate AL500 (High Endo AL500) and Low Endotoxin Sodium Alginate AL500 1 ml of a 2% aqueous solution of (Low End AL500) was implanted into six rats in the same manner as in Example 1. One week later, HE staining, SOFG staining, and MTC staining were performed in the same manner as in Example 1, and the results are shown in Figure 13.

[0091] The results shown in Figure 13 indicate that in the case of high-end AL500 implants, inflammatory cells infiltrated not only around the implant site but also into the implant site. In the case of low-end AL500 implants, inflammatory cells were only observed around the implant site.

[0092] Example 14 High End AL20 and Low End AL20 were implanted into rats in the same manner as in Example 12, and three weeks later, staining was carried out in the same manner as in Example 12. The results are shown in FIG.

[0093] From the results shown in FIG. 14, more inflammatory cells were observed in the implants of high-end AL20 than in the implants of low-end AL20.

[0094] Example 15 As in Example 13, High End AL500 and Low End AL500 were implanted into rats, and three weeks later, staining was carried out in the same manner as in Example 13. The results are shown in FIG.

[0095] From the results shown in Figure 15, when High End AL500 was implanted, infiltration of inflammatory cells was observed not only around the implant site but also inside the site. When Low End AL500 was implanted, inflammatory cells were observed only around the implant site, as in Figure 13 above, one week after implantation.

[0096] Example 16 High End AL20 and Low End AL20 were implanted into rats in the same manner as in Example 12, and 6 weeks later, staining was carried out in the same manner as in Example 12. The results are shown in FIG.

[0097] From the results shown in Figure 16, implantation of high-end AL20 suppressed the infiltration of inflammatory cells more than 3 weeks after implantation in Figure 14 above, and was slightly more suppressed than implantation of low-end AL20.

[0098] Example 17 High End AL500 and Low End AL500 were implanted into rats in the same manner as in Example 13, and 6 weeks later, staining was carried out in the same manner as in Example 13. The results are shown in FIG.

[0099] From the results shown in Figure 17, when High End AL500 was implanted, infiltration of inflammatory cells was observed not only around the implant but also inside the implant, as in the case of the implant site 3 weeks after implantation in Figure 15. When Low End AL500 was implanted, inflammatory cells were observed only around the implant site, as in the case of the implant site 1 week and 3 weeks after implantation in Figures 11 and 13 above.

[0100] Example 18 High End AL20 and Low End AL20 were implanted into rats in the same manner as in Example 12, and 12 weeks later, staining was carried out in the same manner as in Example 12. The results are shown in FIG.

[0101] The results shown in Figure 18 indicate that the implantation of high-end AL20 resulted in the same level of inflammatory cell infiltration as the implantation of low-end AL20. With low-end AL20, inflammatory cells were observed only around the implant site, as shown in Figures 14 and 16 above, at 1 week and 3 weeks after implantation. A significant change in orientation was also observed. This significant change in orientation was not observed with high-end AL20.

[0102] Example 19 As in Example 13, High End AL500 and Low End AL500 were implanted into rats, and 12 weeks later, staining was carried out in the same manner as in Example 13. The results are shown in FIG.

[0103] From the results shown in Figure 19, when high-end AL500 was implanted, inflammatory cell infiltration was observed not only around the implant but also inside the implant, as in the case of the implants in Figures 15 and 17 at 3 and 6 weeks after implantation. When low-end AL500 was implanted, inflammatory cells were observed only around the implant, as in the case of the implants in Figures 11 and 13 at 1 and 3 weeks after implantation. Furthermore, significant changes in orientation were also observed depending on the site. This significant change in orientation was not observed with high-end AL500.

[0104] (Comparative Example 1) Hyaluronic acid manufactured by Nichi-Iko Pharmaceuticals, Inc., at a concentration of 25 mg / 2.5 ml and collagen at a concentration of 2% were implanted in 1 ml each into six rats in the same manner as in Example 1. One week later, HE staining and SOFG staining were performed in the same manner as in Example 1, and further MTC staining was performed, and the results are shown in Figure 20.

[0105] From the results shown in Figure 20, hyaluronic acid disappeared from the injection site. Collagen was present in an unstructured form at the injection site, and no cell infiltration was confirmed.

[0106] (Comparative Example 2) As in Comparative Example 1, hyaluronic acid and collagen were implanted at 2% into six rats, and three weeks later, HE staining, SOFG staining, and MTC staining were performed. The results are shown in FIG.

[0107] From the results shown in Figure 21, similar to the results one week after implantation shown in Figure 20, hyaluronic acid had disappeared from the injection site, and collagen was also present in an unstructured form at the injection site, with no cell infiltration confirmed.

[0108] (Comparative Example 3) As in Comparative Example 1, hyaluronic acid and collagen were implanted at 2% into six rats, and six weeks later, HE staining, SOFG staining, and MTC staining were performed. The results are shown in FIG.

[0109] From the results shown in Figure 22, similar to the results shown in Figures 20 and 21, 1 week and 3 weeks after implantation, hyaluronic acid had disappeared from the injection site, and collagen was also present in an unstructured form at the injection site, with no cell infiltration confirmed.

[0110] Comparative Example 4 As in Comparative Example 1, hyaluronic acid and collagen were implanted at 2% into six rats, and 12 weeks later, HE staining, SOFG staining, and MTC staining were performed. The results are shown in FIG.

[0111] The results shown in Figure 23 indicate that hyaluronic acid disappeared from the injection site, similar to the results shown in Figures 20, 21, and 22, 1 to 6 weeks after implantation. Similarly, collagen was present in an unstructured manner at the injection site, and no cell infiltration was observed. Although fat cells were observed around the collagen injection site, no change in orientation was observed.

[0112] Example 20 1 ml each of calcium-crosslinked hyaluronic acid AL10 and TEOSYAL RHA1 (manufactured by TEOXANE) was implanted into six rats in the same manner as in Example 1. Twelve weeks later, HE staining and SOFG staining were performed in the same manner as in Example 1, and the results are shown in Figure 24.

[0113] The calcium-crosslinked AL10 was prepared by injecting a total of 1 ml of 4% AL10 solution and 0.5 ml of calcichol solution diluted 1 / 8 with water for injection simultaneously using a double syringe that formed a gel at the nozzle. The procedure is as follows. In this case, the calcium crosslinking was mild.

[0114] As shown in Figure 24, the formation of oriented tissue and angiogenesis were observed in the mildly calcium-crosslinked AL10. On the other hand, in the case of hyaluronic acid, hyaluronic acid remained throughout the entire layer, and no cell infiltration was observed, resulting in no formation of oriented tissue or angiogenesis.

[0115] Example 21 As in Example 20, calcium-crosslinked hyaluronic acid AL10 and TEOSYAL RHA1 manufactured by TEOXANE were implanted into six rats, and the results of HE staining and SOFG staining 24 weeks later are shown in Figure 25.

[0116] As shown in Figure 25, in the case of the mildly calcium-crosslinked AL10, the formation of oriented tissue and angiogenesis were clearly observed compared to the results after 12 weeks shown in Figure 20. On the other hand, in the case of hyaluronic acid, even after 24 weeks, hyaluronic acid remained throughout the entire layer, no cell infiltration was observed, and no oriented tissue formation or angiogenesis was observed, similar to the results after 12 weeks shown in Figure 20.

[0117] Example 22 Regarding the tissue-forming effect when a sulfate group component is used in combination with alginic acid, the following study was carried out on sulfated alginic acid, in which sulfate groups have been introduced into alginic acid itself, or its salts.

[0118] Sodium sulfated alginate at a concentration of 0.5% was injected and implanted subcutaneously into the dorsal skin of rats, and then histopathologically evaluated in the same manner as in Example 1. The sulfated alginate used was synthesized by Tokyo Chemical Industry Co., Ltd. and was sodium alginate with a viscosity of 100 mP / s (concentration 10 g / L, 20°C) and 0.2 sulfate groups per monosaccharide structural moiety.

[0119] The results of HE staining of the implantation site of sulfated sodium alginate one week after subcutaneous implantation are shown in Figure 26. For comparison, a similar study was conducted using the same sodium alginate but without sulfate groups, and the results of HE staining of the implantation site one week after implantation are also shown in Figure 26.

[0120] The results shown in Figure 26 indicate that with sulfated alginate, tissue consisting of a distinct collagen layer structure was formed, and that a vascular network and interstitial tissue had already formed along this collagen layer structure (Figure 26A). On the other hand, with the comparison alginate without sulfate groups, neither the collagen layer structure nor the interstitial tissue with its vascular network were clear (Figure 26B). Thus, it became clear that tissue formation with sulfated alginate was more promoted than with the injection and implantation of regular sodium alginate.

[0121] Example 23 The following study was carried out to examine the tissue formation effect when alginic acid and a sulfate group component were used in combination.

[0122] As in Example 22, 2% AL10 sodium alginate mixed with sulfated dextran (Sigma-Aldrich) at a concentration of 0.01 mg / mL was implanted subcutaneously in rats, and the implantation site was subjected to HE staining two weeks later. The results are shown in Figure 27. For comparison, a similar study was performed using the same sodium alginate alone but without sulfated dextran. The results of HE staining of the implantation site two weeks later are shown in Figure 27.

[0123] The results shown in Figure 27 indicate that when sulfated alginate was mixed, tissue consisting of a collagen layer structure was already formed, and that in addition to a vascular network and interstitial tissue along this collagen layer structure, subcutaneous fat tissue had already formed (Figure 27A). On the other hand, when the comparison alginate was mixed without sulfated dextran, neither the collagen layer structure nor the interstitial tissue with its vascular network was clear (Figure 27B). Thus, tissue formation using sodium alginate mixed with dextran sulfate was shown to be more accelerated than when regular sodium alginate was injected and implanted.

[0124] Example 24 In order to confirm the tissue filling effect of alginate and the like, the following study was carried out.

[0125] A sample of highly calcium-crosslinked alginate, prepared by mixing 1% sodium alginate (AL10) with an equal volume of stock calcicol solution, was injected and implanted into the subcutaneous tissue of a rat in the same manner as in Example 22, and the implantation site was subjected to HE staining one week later. The results are shown in Figure 28. For comparison, a similar study was conducted using the same sodium alginate alone but at the same concentration (i.e., 0.5%), and the results of HE staining of the implantation site one week later are shown in Figure 28.

[0126] The results shown in Figure 28 indicate that when highly calcium-crosslinked alginate was injected, crosslinked alginate masses were present at the injection site, but there was little cell or vascular infiltration within them (Figure 28A), as seen with conventional tissue fillers such as comparative collagen (Figure 23B) and comparative hyaluronic acid (Figure 24B). This indicates that highly crosslinked alginate acts as a tissue filler, not a tissue-forming agent. On the other hand, when the same sodium alginate was implanted at the same concentration as a control, significant cell infiltration was observed at the alginate site, and the formation of interstitial tissue with a collagen layer structure and a vascular network was initiated (Figure 28B).

[0127] Example 25 In order to confirm the tissue scaffolding effect of sulfate group components in alginate, the following study was carried out.

[0128] The tissue reinforcement material used was a PGA nonwoven fabric (Neoveil (trademark) sheet, Gunze Medical Co., Ltd.), and the alginic acid used was the same as that described in Example 20 above, a gel of sodium alginate (AL10) gently cross-linked with calcium (i.e., a gel prepared by mixing equal volumes of 4% AL10 solution and calcichol solution diluted 1 / 8 with water for injection).

[0129] The PGA nonwoven fabric and the gel were combined and implanted into rat peritoneal submesothelial tissue. Instead of calcium (calcicol solution), a 1% poly-L-lysine solution was used as a crosslinker. Specifically, a gel prepared by mixing equal volumes of 4% AL10 solution and a 1% solution of poly-L-lysine dissolved in distilled water for injection was combined with PGA nonwoven fabric and implanted into rat peritoneal submesothelial tissue. For comparison, a gel prepared by mixing equal volumes of 4% AL10 solution and calcicol solution diluted 1 / 8 with water for injection without the nonwoven fabric was implanted submesothelially, and the nonwoven fabric alone was also implanted. HE staining of the implanted sites after 4 weeks is shown in Figure 29.

[0130] As shown in Figures 29A and 29B, when alginate was used, a sufficient thickness of peritoneal submesothelial tissue was formed, with residual alginate around the fibers of the nonwoven fabric, and a rich vascular network and infiltration and proliferation of interstitial cells were observed within the remaining alginate. On the other hand, when nonwoven fabric was implanted alone without alginate, the thickness of the formed peritoneal submesothelial tissue was very thin, with only slight tissue formation observed around the fibers of the nonwoven fabric, such as PGA (Figure 29C). When calcium-crosslinked alginate alone was implanted without nonwoven fabric, only sparse peritoneal tissue regeneration was observed (Figure 29D).

[0131] The mechanical strength of a gel scaffold made by calcium cross-linking only alginate was compared with that of a PGA nonwoven fabric or an LA / CL nonwoven fabric combined with the above gel. To measure the mechanical strength of the scaffold, a gel scaffold made by calcium cross-linking only alginate was prepared by simultaneously ejecting 0.5 ml of a 4% AL10 solution and 0.5 ml of a calcichol solution diluted 1 / 8 with water for injection using a double syringe, which formed a gel at the nozzle, and then injecting the entire volume (1 ml) into a 10 mm wide x 30 mm long silicone container. To prepare scaffolds for measuring the mechanical strength of scaffolds, PGA or LA / CL nonwoven fabrics were combined with this gel. The doubled PGA or LA / CL nonwoven fabric was placed in the bottom of a silicon container, and 0.5 ml of 4% AL10 solution and 0.5 ml of calcicol solution diluted 1 / 8 with water for injection were simultaneously sprayed onto the doubled PGA or LA / CL nonwoven fabric. The specimens were then clamped at 10 mm width on both ends of the jig and pulled in both directions, and the breaking strength was measured using the following equipment. Load measurement equipment: Desktop load measurement instrument MODEL-1356R (Aiko Engineering Co., Ltd.) Force gauge: MODEL-RX-10 (Aiko Engineering Co., Ltd.) Hardware: Powerlab2 / 26 (AD Instruments) Software: Labchart (AD Instruments)

[0132] The breaking strength of 10 mm wide scaffolds when PGA nonwoven fabric or LA / CL nonwoven fabric was combined with cross-linked alginate gel was 2 N or more. On the other hand, the breaking strength of cross-linked alginate gel alone was 0.1 N or less. Thus, it became clear that when low-endotoxin alginate components are combined with conventional tissue reinforcement materials, excellent scaffolds can be produced that have sufficient tensile strength and also have the function of actively forming tissue of sufficient thickness.

[0133] Example 26 Two-year-old male or female beagle dogs weighing approximately 10 kg underwent laparotomy under general anesthesia, and the stomach was incised to expose the mucosal surface. A total of 4 ml of each alginate preparation (including those containing sodium polyglutamate) was injected into the submucosal layer. The stomach incision was sutured closed, and the abdominal laparotomy wound was sutured closed. Four weeks later, the stomach was removed from the beagle dog, and the stomach wall at the alginate-injected site was fixed in formalin. Histopathological specimens were prepared using standard methods under a microscope and evaluated using HE staining. A control experiment was also conducted in which no alginate preparation was injected. The results are shown in Figure 30.

[0134] Figure 30A shows an HE-stained image of the control. Figure 30B shows an HE-stained image of a site injected with relatively small molecular weight sodium alginate (molecular weight 10,000 or less). Compared to the control in Figure 30A, slight tissue regeneration in the submucosal layer was observed. Figure 30C shows an HE-stained image of a site injected with 0.5% AL10. Significant tissue regeneration in the submucosal layer was observed. Figure 30D shows an HE-stained image of a site injected with 2 ml of relatively small molecular weight alginate (molecular weight 10,000 or less) and 2 ml of 0.5% sodium polyglutamate solution at the same site. More significant submucosal regeneration was observed compared to the sodium alginate injection shown in Figure 30B. Figure 30E shows an HE-stained image of a site injected with 2 ml of 0.5% AL10 and 2 ml of 0.5% sodium polyglutamate solution at the same site. Even more significant submucosal tissue regeneration was observed compared to the AL10 alone shown in Figure 30C.

Claims

1. A tissue scaffold comprising alginic acid or a salt thereof and a nonwoven fabric, A scaffolding agent, wherein the tissue is any one of epithelial tissue, tissue lining an epithelial tissue layer, or tissue located deeper than these tissues (excluding brain, nerve, heart, or hard tissue).

2. A tissue scaffold agent used together with a nonwoven fabric, comprising alginic acid or a salt thereof, A scaffolding agent, wherein the tissue is any one of epithelial tissue, tissue lining an epithelial tissue layer, or tissue located deeper than these tissues (excluding brain, nerve, heart, or hard tissue).

3. A wound healing agent containing alginic acid or a salt thereof, A wound healing agent, wherein the wound tissue is any tissue selected from epithelial tissue, tissue lining an epithelial tissue layer, and tissue located deeper than these tissues (excluding the brain, nerves, heart, or hard tissue).

4. The wound healing agent according to claim 3, wherein the alginic acid or salt thereof is sulfated.

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