JP6055466B2 - Gel material crosslinked with oxidized oligosaccharide - Google Patents

Description

  The present invention relates to a gel material cross-linked with an oligosaccharide oxide, a cross-linking agent comprising an oligosaccharide oxide used for the preparation of the gel material, a cell culture system and a transplant system using the gel material.

  In recent years, research on three-dimensional culture of cells to construct a large regenerative tissue has been actively conducted, and a technique for culturing and stacking cells on a cell sheet has been proposed (Non-patent Document 1). ~ 3). Here, when constructing a large regenerative tissue, it is necessary to examine how to introduce the vascular system. That is, since the exchange of oxygen and substances by passive diffusion is limited to several hundred microns, even if a large regenerated tissue can be created, if the vascular system is not introduced, the regenerated tissue Even if transplanted to the host, the center is necrotic.

  Although it is ideal to create a regenerative tissue containing the vascular system in terms of tissue engineering as a method for introducing the vascular system into a large regenerative tissue, it is necessary to establish a vascular system construction technology for this purpose. The technology is not established at this time. As another method, it is conceivable to control the angiogenesis mechanism in the host transplantation bed. This is a technique in which regenerative tissue is transplanted into a host, angiogenesis is induced from the host mother bed, and nutrient blood vessels are drawn into the regenerated tissue.

In recent years, spheroids have attracted attention as microregenerative tissue units. Spheroids are those in which a large number of cells aggregate to form a spherical mass, and spheroid culture can maintain the function of cells for a long period of time compared to conventional monolayer culture, and can be said to be a culture method closer to a living body. Therefore, a technique for transplanting a spheroid to a host, inducing (organizing) a blood vessel to the spheroid, and constructing the organized spheroid three-dimensionally (Patent Document 1, Non-Patent Documents 4 to 5) has been studied.
However, even if only the spheroids are transplanted into the host mother bed, they are quickly dispersed and are difficult to keep in place. Furthermore, angiogenesis cannot be successfully induced because there is no support for maintaining the stretch and structure of blood vessels around the spheroids. Therefore, a scaffold material that induces angiogenesis is required.

  As such a scaffold material, collagen has been studied so far because it is excellent in cell adhesion and biodegradability (Patent Document 2). In recent years, in-gel cell culture systems based on collagen are commercially available. However, since the gelation mechanism of collagen is due to the aggregation of collagen molecules, the strength and transparency of the gel obtained depend greatly on the collagen concentration, ionic strength, and pH. Therefore, it is difficult to say that a gel formed of such aggregates is stable in vivo.

JP 2010-065082 A JP 2011-160817 A

Haraguchi Y. Shimizu T. Yamato M .; , Okano T .; , J .; Tissue Eng Regen. Med. , 4 (4), 291-299 (2010). Sekiya S. et al. Shimizu T. Yamato M .; , Okano T .; , J .; Artif. Organs, 14 (1), 43-51 (2011). Ohashi K.K. , Yokoyama T .; Yamato M .; , Kuge H., et al. Kanehiro H .; Tsutsumi M., et al. , Amanuma T .; Iwata, H .; Yang J. et al. , Okano T .; Nakajima Y .; Nat. Med. 13, 880-885 (2007). Lazar A.I. Mann HJ. , Remmel RP. , Chatter R-A. , Cerra FB. , Hu W-S. In Vitro Cell Dev. Biol. Anim. , 31 (5): 340-6 (1995). Toshio Takayama, Satoshi Taguchi, Hiroyuki Koyama, Naotoshi Kobayashi, Tetsuro Miyata, Koichi Nakawa, Angiology 48, S100 (2008)

  The present invention is a scaffold for constructing a large regenerative tissue. (1) It has a high cell affinity, (2) it has a stable structure even in a physiological environment, and (3) it is rapidly produced after transplantation. (4) can be stably carried inside without leaking the blood vessel inducing factor, and (5) has transparency enough to observe the state of cells cultured in the gel over time, ( 6) It aims at providing the material which has the characteristics which can adjust the intensity | strength of the gel obtained easily.

The present inventors have conceived that collagen is prevented from forming an aggregate by forming a chemical crosslink in order to stably maintain a collagen gel under conditions suitable for cell culture. Here, water-soluble carbodiimide, glutaraldehyde, and the like are known as water-soluble cross-linking agents that form chemical cross-links. However, it is not appropriate to use these because of problems of irritation to cells and toxicity. As a result of extensive studies, it was found that a colorless and transparent gel can be obtained when an oligosaccharide oxide is used as a crosslinking agent.
As the oxidation reaction, an oxidation reaction in an aqueous system is preferable because the solubility of oligosaccharides in an organic solvent is low, and in the oxidation reaction using lead tetraacetate, the toxicity of residual lead has been a concern. Accordingly, the present inventors have established a technique that allows an iodine compound to be easily removed by selecting an oxidation reaction with periodic acid and adding an alcohol after the oxidation reaction.

That is, the present invention
(1) a crosslinkable composition comprising an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain;
(2) The crosslinkable composition according to (1), wherein the oligosaccharide oxide is generated from periodate oxidation of an oligosaccharide,
(3) The crosslinkable composition according to (1) or (2), wherein the polypeptide is selected from collagen, atelocollagen, gelatin, derivatives thereof or mixtures thereof,
(4) The polysaccharide is deacetylated chitin or chitosan, succinylated chitosan, glycerylated chitosan, carboxymethylated chitosan, hydroxybutyl-hydroxypropylated chitosan, aminated dextran, aminated starch, aminoalkylated pullulan, amino The crosslinkable composition according to (1) or (2), which is selected from hydrogenated hyaluronic acid, aminated chondroitin sulfate, aminated cellulose, derivatives thereof or mixtures thereof,
(5) The crosslinkable composition according to any one of (1) to (4), wherein the oligosaccharide is selected from the group consisting of sucrose, maltose, raffinose, derivatives thereof and a mixture of two or more thereof. ,
(6) A crosslinked product obtained from the crosslinkable composition according to any one of (1) to (5),
(7) The crosslinked product according to (6), wherein an aldehyde group of the oligosaccharide oxide and an amino group of the polypeptide or polysaccharide are covalently bonded,
(8) The crosslinked product according to (6) or (7), which is in a gelled state,
(9) The crosslinked product according to any one of (6) to (8), which contains an angiogenic factor,
(10) The crosslinked body according to any one of (6) to (9), which contains cells or cell tissues,
(11) The crosslinked product according to (10), which contains a cell culture medium,
(12) A cell culture system using the crosslinked product according to any one of (6) to (11),
(13) A system for transplanting the crosslinked body according to any one of (6) to (11) into the body of a subject,
(14) A system for transplanting cultured cells obtained by the cell culture system according to (12) into the body of a subject,
(15) (a) a step of preparing the crosslinkable composition according to any one of (1) to (5),
(B) a method of preparing a gel of the crosslinkable composition, comprising a step of adjusting the pH of the crosslinkable composition to 7.0 or more,
(16) (a) a step of preparing the crosslinkable composition according to any one of (1) to (5),
(B) adjusting the pH of the crosslinkable composition to 7.0 or higher;
(C) including a step of gelling the crosslinkable composition, and adding cells or cell tissues to the crosslinkable composition at a time before, during or after the step of (c), culturing cells Method,
(17) The method for culturing cells according to (16), wherein a culture solution is added to the crosslinkable composition before, during or after the step of (c),
(18) A method of transplanting a gel obtained by the method according to (15) into the body of a subject,
(19) A method for transplanting cultured cells obtained by the method according to (16) or (17) into the body of a subject,
(20) Cells or cell tissues are embedded in a gel formed from a crosslinkable composition containing an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain, Transplant material for constructing regenerative tissue, used to be implanted in the body,
(21) A crosslinking agent comprising an oligosaccharide oxide having an aldehyde group and used for a crosslinking reaction with a polypeptide or polysaccharide having an amino group in the side chain,
(22) The crosslinking agent according to (21), wherein the oligosaccharide oxide is generated from periodate oxidation of an oligosaccharide,
(23) The cross-linking agent according to (21) or (22), wherein the oligosaccharide is selected from the group consisting of sucrose, maltose, raffinose, derivatives thereof and a mixture of two or more thereof.
(24) The cross-linking agent according to any one of (21) to (23), wherein the polypeptide having an amino group in the side chain is selected from collagen, atelocollagen, gelatin, derivatives thereof, or a mixture thereof, and (25) A polysaccharide having an amino group in the side chain is deacetylated chitin or chitosan, succinylated chitosan, glycerylated chitosan, carboxymethylated chitosan, hydroxybutyl-hydroxypropylated chitosan, aminated dextran, aminated starch, The crosslinking agent according to any one of (21) to (23), which is selected from aminoalkylated pullulan, aminated hyaluronic acid, aminated chondroitin sulfate, aminated cellulose, a derivative thereof, or a mixture thereof. To do.

Polysaccharides with amino groups in the side chains such as collagen or polysaccharides with amino groups such as chitosan derivatives are chemically cross-linked with oligosaccharide oxides to suppress the formation of aggregates and prepare transparent and uniform gels It became possible to do. A transparent and uniform gel allows easy observation of cells and progresses promptly with no degradation in vivo. In addition, chemical cross-linking limits and suppresses structural changes such as gel shrinkage, swelling, and dissolution under physiological conditions, and maintains an appropriate cross-link density that provides sufficient strength and decomposition rate as a cell scaffolding material. be able to.
In addition, oligosaccharide oxides are less irritating and toxic than lower aldehydes, and are safe crosslinking agents for cells and as a transplant material.
Furthermore, the aldehyde group of the oligosaccharide oxide can carry angiogenic factors and the like by covalent bonds. In addition, a carboxylic acid generated by oxidation of an aldehyde can also carry a basic factor such as bFGF by electrostatic interaction. Therefore, since the gel material of the present invention hardly releases angiogenic factors or the like outside in a living body, it becomes possible to induce angiogenesis efficiently.

Microscopic observation results of normal human umbilical vein endothelial cells (HUVEC) cultured using the gel of the present invention (5th day of culture) Results of microscopic observation of spheroids of hepatocytes cultured using the gel of the present invention (3rd day of culture) Results of microscopic observation of spheroids of cardiomyocytes cultured using the gel of the present invention (3rd culture day) Schematic flow diagram of rat transplantation experiment Results of optical microscope observation of the gel cross section taken 5 days after transplantation

Crosslinking agent One form of this invention is a crosslinking agent which consists of an oligosaccharide oxide which has an aldehyde group.

An oligosaccharide refers to a compound having a molecular weight of about 300 to 3000 among compounds in which monosaccharides are bonded by a glycosidic bond. Oligosaccharides that can be used in the present invention include disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose; trisaccharides such as raffinose, panose, maltotriose, melezitose, and gentianose; Cyclic dextrins such as cyclodextrin and cycloawaodoline, etc .; fructooligosaccharides and galactooligosaccharides that are monosaccharide polymers; mannan oligosaccharides and xylooligosaccharides as degradation products of polysaccharides; Extract; selected from the group consisting of these derivatives and mixtures of two or more thereof.
In the present invention, among these, sucrose, maltose, raffinose and cyclodextrin are preferable, and sucrose, maltose and raffinose are particularly preferable.

  The oligosaccharide oxide having an aldehyde group in the present invention refers to one in which the vicinal diol group of the oligosaccharide is partially dissociated and oxidized to an aldehyde group. Here, an oligosaccharide having a vicinal diol group oxidized to an aldehyde group can be obtained by various methods. In the present invention, as an oligosaccharide oxide having an aldehyde group, an oligosaccharide periodate is used. Particularly preferred is an oxide generated from oxidation.

Regarding the method for producing a crosslinking agent of the present invention, a method for producing oligosaccharide by periodate oxidation will be described.
Oligosaccharide is dissolved in a basic aqueous solution such as water or sodium hydroxide solution, and periodate oxidation reaction of oligosaccharide is performed by adding sodium periodate under ice cooling. The pH of the reaction solution is preferably 7 or less, particularly preferably around 5-6.
In addition, excessive oxidation degrades the sugar, and there are concerns that by-products such as formic acid and formaldehyde are generated. Therefore, the ratio of sodium periodate to oligosaccharide sugar units is preferably 1: 2 or less. As the reaction solvent, for example, various solvents such as water, water / ethanol mixed solvent, water / DMF mixed solvent and the like can be used, but water is preferably used.

  While the reaction time is appropriately determined depending on the type of sugar, it can usually be carried out in 5 minutes to 96 hours, particularly preferably 1 to 60 hours. After completion of the reaction, in order to remove the remaining iodine compound by periodate oxidation, a polar solvent is added to precipitate and remove the iodine compound. The polar solvent is preferably a low boiling point solvent because it must be removed under reduced pressure during the preparation of the oligosaccharide oxide. Moreover, it is preferable that it is a solvent with high safety | security with respect to a biological body. From these points, it is preferable to use alcohols such as ethanol and isopropanol, acetone, tetrahydrofuran and the like as the polar solvent, and ethanol is particularly preferable.

  The cross-linking agent comprising an oligosaccharide oxide having an aldehyde group of the present invention can be suitably used for a cross-linking reaction with a polypeptide or polysaccharide having an amino group in the side chain, as will be described later.

Crosslinkable composition Another form of the present invention is a crosslinkable composition comprising a crosslinking agent comprising an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain.

  Non-limiting examples of the polypeptide having an amino group in the side chain include naturally occurring polypeptides, artificially synthesized polypeptides that mimic or improve their functions, genetically modified proteins, and the like.

  Naturally-derived polypeptides include silkworm-derived fibroin, sericin, and the like, and naturally-derived extracellular matrix components such as collagen, atelocollagen, gelatin, proteoglycan, fibronectin, laminin, elastin, entactin and their derivatives. It is done.

  Artificially synthesized polypeptides and genetically engineered proteins include, but are not limited to, dehydrated condensates of oligopeptides such as Poly (PHG), Poly (PHG / QGIA), Poly (PHG / RGD), Poly (PHG / YIGSR / IKVAV) ) And the like (Japanese Unexamined Patent Publication No. 2003-321500), “Pronectin F” and “Pronectin L” manufactured by Sanyo Chemical Industries, “RetroNectin” manufactured by Takara Shuzo, and the like.

  In the present invention, the polypeptide having an amino group in the side chain is preferably collagen, atelocollagen, gelatin, fibronectin, Poly (PHG / RGD), derivatives thereof, or a mixture thereof, more preferably collagen, atelocollagen. Gelatin, fibronectin, derivatives thereof and mixtures thereof. Particularly preferred are mammalian or fish-derived collagen, atelocollagen, gelatin, derivatives thereof and mixtures thereof.

  Collagen is one of the proteins that constitute the dermis, ligaments, tendons, bones, cartilage, and the like, and is an element that constitutes the extracellular matrix. The peptide chain of the collagen protein has a primary structure in which “-(glycine)-(amino acid X)-(amino acid Y)-” and glycine repeat every 3 residues. In many types of collagen, three of these peptide chains gather to form a helical structure and are called tropocollagen. For example, one peptide chain of type I collagen has a sequence that repeats 1014 amino acid residues and has a molecular weight of about 100,000. It is known that there are more than 30 types of human collagen. For example, type I collagen is the main component in dermis, ligaments, tendons, bones, etc., and type II collagen is the main component in articular cartilage. In addition, type IV collagen is mainly contained in the basement membrane which is the lining structure of all epithelial tissues. The most abundant in the body is type I collagen.

  Although it will not be limited if it is collagen, Preferably it is soluble collagen. More preferred is collagen that is soluble in water. For soluble collagen, neutral salt soluble collagen, acid (eg, 0.5M acetic acid) solubilized in a neutral salt (eg, 0.15-0.5M NaCl in 0.05M Tris = HCl, pH 7.5) solution. Or 0.1-0.3 M citrate buffer, pH 3.5-3.7) acid-soluble collagen solubilized in solution, enzyme-soluble collagen solubilized with enzyme (for example, pepsin), and the like. The collagen may be peptided collagen (collagen fragment).

  Atelocollagen is collagen obtained by removing telopeptides present at both ends of a collagen molecule by enzymatic treatment, and is soluble in water. Atelocollagen may be peptideized.

  Gelatin means the collagen extracted into water by heating with water for a long time. The gelatin may be peptideized. The peptide of gelatin is the same as that of collagen.

  Since gelatin is a heat-denaturing substance of collagen, the amino acid composition is almost the same as that of collagen. Although it depends on the origin, there are about 12% acidic groups, and about one third of them are amidated. Gelatin includes acid-treated gelatin, alkali-treated gelatin, and the like depending on the pretreatment step of water extraction. Acid-treated gelatin is treated with an inorganic acid such as hydrochloric acid or sulfuric acid for several tens of hours to several days, and the deamidation rate is low, whereas alkali-treated gelatin is almost 100% deamidated in the liming process of 2 to 3 months. Has been.

  In the present invention, the polypeptide having an amino group in the side chain is preferably selected from collagen, atelocollagen, gelatin, derivatives thereof, or a mixture thereof.

  In the present invention, a polysaccharide is a sugar in which a number of monosaccharide molecules are polymerized by glycosidic bonds. Examples of the polysaccharide having an amino group in the side chain in the present invention include deacetylated chitin and chitosan (eg, Daichitosan VL manufactured by Dainichi Seika), succinylated chitosan, glycerylated chitosan, carboxymethylated chitosan, hydroxybutyl-hydroxy Examples include chitosan derivatives such as propylated chitosan, derivatives of natural and semi-artificial polysaccharides such as aminated dextran, aminated starch, aminoalkylated pullulan, aminated hyaluronic acid, aminated chondroitin sulfate, and aminated cellulose.

  In the crosslinkable composition of the present invention, the concentration of the polypeptide or polysaccharide or oligosaccharide oxide having an amino group in the side chain depends on the type thereof, taking into account the reactivity or viscosity of the composition solution. It can be set appropriately. For example, when the polypeptide having an amino group in the side chain is collagen, the concentration is preferably 0.3% or more, and in the case of gelatin, the concentration is preferably 5% or more. For example, when the polysaccharide having an amino group in the side chain is chitosan, the concentration is preferably 1% or more. The concentration of the oligosaccharide oxide is usually 0.01% or more, preferably 0.05% or more in the case of raffinose oxide, for example.

  The crosslinkable composition of the present invention includes other additives such as a crosslinking agent, a reaction accelerator, a retarder, a dispersant (anti-aggregation agent), and a reinforcing material (fiber, filament, whisker, particle, microgel). , Droplets and the like can be contained.

Crosslinked body The further form of this invention is a crosslinked body obtained from the said crosslinkable composition.
The cross-linked product of the present invention refers to an oligosaccharide oxide-polypeptide complex in which an aldehyde group of an oligosaccharide oxide and an amino group of a polypeptide or polysaccharide are covalently bonded (-polypeptide-oligosaccharide oxide-poly Peptide-) and oligosaccharide oxide-polysaccharide complex (polysaccharide-oligosaccharide oxide-polysaccharide). Here, the covalent bond between the oligosaccharide oxide and the polypeptide is usually a covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the side chain of the polypeptide, and the amino group of the oligosaccharide oxide and the polypeptide. It consists of a mixture of covalent bonds of the terminal amino group, but it may consist only of a covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the side chain of the polypeptide, or it may consist of the aldehyde group and the polysaccharide of the oligosaccharide oxide. It may consist only of a covalent bond of the amino group at the amino terminus of the peptide.

  The covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide means that the covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide is present in the crosslinked product. It means that there is at least one.

  The cross-linked product preferably contains an aldehyde group of an oligosaccharide oxide that does not participate in a covalent bond and an amino group of a polypeptide or polysaccharide. In order to carry an angiogenic factor for inducing vascular elongation, a functional group for interacting with these physiologically active substances may be required. For example, bFGF whose charge is positively biased has an electrostatic interaction with the aldehyde group of the oligosaccharide oxide. In addition, in order to maintain good cell adhesion and enzymatic degradability, the side chain structure of the polypeptide is destroyed by the formation of a covalent bond between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide. It is desirable to minimize structural changes.

  A preferred embodiment of the present invention is a state in which the crosslinked product is gelled (hereinafter also simply referred to as “gel”). Here, the gel means that the fluidity is lost in the crosslinked body (crosslinkable composition). As described above, the cross-linked product in the present invention refers to an oligosaccharide oxide-polypeptide complex, or an aldehyde group of an oligosaccharide oxide and an aldehyde group of an oligosaccharide oxide. It refers to those in which the amino group of the saccharide is covalently bonded to form an oligosaccharide oxide-polysaccharide complex, and the crosslinked product of the present invention includes cases where it is not gelled. By making the crosslinkable composition into a gel, it is possible to obtain a large regenerative tissue scaffolding material having a stable structure, which is particularly preferable.

The crosslinked body of the present invention can contain a physiologically active substance.
Non-limiting examples of physiologically active substances include growth factors and cytokines, antibodies and the like. Examples of growth factors and cytokines include, but are not limited to, vascular endothelial growth factor (VEGF), acidic fibroblast growth factor (a-FGF), basic fibroblast growth factor. (Basic fibroblast growth factor, b-FGF), platelet-derived endothelial cell growth factor (PD-ECGF), transforming growth factor (transforming growth factor, TGF-α, TGF-β), tumor Necrosis factor (angiogenin tumor necrosis factor-α, TNF-α), hepatocyte growth factor (HGF), granulocyte / macrophage-colony stimulating factor (GM-CSF), insulin, Insulin-like growth factor (IGF-I, IGF-II), Ellis Lopoietin (EPO), thrombopoietin (TPO), epidermal growth factor (EGF), heparin binding growth factor (hbgf), nerve growth factor (NGF), muscle formation factor (Muscle morphogenic factor: MMF), bone morphogenetic protein (BMP) and the like. Non-limiting examples of antibodies include antibodies that can stimulate cells such as anti-CD3 antibodies and anti-CD28 antibodies. Preferably, basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor, PD-ECGF), hepatocyte growth factor (HGF), granulocyte / macrophage-colony stimulating factor (GM-CSF), more preferably basic fibroblast growth factor (Basic fibroblast growth factor, b-FGF), vascular endothelial growth factor (VEGF). The physiologically active substance may be a cell that secretes a physiologically active substance.

Moreover, the crosslinked body of this invention can contain a cell.
The cell type is not limited, but preferably chondrocytes; fibrochondrocytes; bone cells; osteoblasts; osteoclasts; synovial cells; bone marrow cells; mesenchymal cells; Peripheral blood progenitor cells; insulin-producing cells; antibody-producing cells such as B cells or hybridomas; hormone-producing cells; mast cells; chemotransmitter-producing cells (immune cells); genetically modified cells; stem cells (ES cells, EG cells) , IPS cells, and adult (tissue) stem cells); cells that produce bioactive factors of the liver, nerves, thyroid, thymus, adrenal medulla, adrenal cortex, kidney or gastrointestinal tract; and combinations of these cells with other cells Is mentioned. The cells may be in aggregated form (spheroids etc.) or part of tissue. The origin of the cells is not limited, but preferably mammals, more preferably pets such as dogs and cats; humans; livestock animals such as cows, pigs, horses, sheep, goats; monkeys, rabbits, mice, rats And the like, particularly preferably humans. Moreover, in order to avoid rejection by immunity, the cells are preferably derived from a transplant subject.

Moreover, the crosslinked body of this invention can contain a culture solution further.
The type of the culture solution is variously prepared depending on the type, state, and purpose of the cell. Examples include, but are not limited to, pure water, physiological saline, and body fluid of the host.

The crosslinked product of the present invention can be produced by preparing a crosslinkable composition containing an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain, and allowing the crosslinking reaction to proceed. it can. Specifically, a phosphate buffer solution of a polypeptide or polysaccharide having an amino group in the side chain is prepared, pH is adjusted, and an oligosaccharide oxide having an aldehyde group is added thereto to obtain a crosslinkable composition. The reaction is allowed to proceed with stirring for a predetermined time. If the stirring is continued, the solution thickens, and if the stirring is further performed, the fluidity is lost and a gel can be formed.
In the present invention, since the chemical bonding reaction between the aldehyde group of the oligosaccharide oxide and the amino group of the polypeptide or polysaccharide is considered to be significantly accelerated under alkaline conditions, the pH of the crosslinkable composition is preferably It is adjusted to 7.0 or more, more preferably 7.6 or more.

  In the method for producing a crosslinked product of the present invention, the reaction temperature can be set according to the type of polypeptide to be used, but the higher the reaction temperature, the shorter the gelation time. In addition, the concentration of the oligosaccharide oxide can be set according to the type of polypeptide used, etc., but the gelation time can be shortened as the concentration of the oligosaccharide oxide increases.

Cell culture system The further form of this invention is related with the cell culture system using the bridge | crosslinking body of this invention, and the method of culture | cultivating a cell.
In the cell culture method of the present invention, an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain are mixed to prepare a crosslinkable composition, its pH is adjusted, and a gel is prepared. Progress. Then, before the gelation progresses, during the progress, and at any time after the completion, the cells and the culture solution are added to culture the cells.
As one aspect of the present invention, when the crosslinkable composition is thickened, the cells are added to and mixed with this, and the cell mixture is injected into a petri dish or the like, and left to stand for a predetermined temperature and time for gelation. The cells can be cultured by coating a disc-shaped gel having a predetermined thickness and diameter with a culture solution.
As another aspect, when the crosslinkable composition has thickened, spheroids obtained by seeding cells in a separate step and culturing for a predetermined period are added and mixed, and allowed to stand at a predetermined temperature and time. The cells can be cultured by coating a disc-shaped gel having a predetermined thickness and diameter with a culture solution.

  Examples of the culture solution include pure water, physiological saline, phosphate buffer, Eagle basal medium (BME), Dulbecco's Modified eagle's Medium (DMEM), DMEM: F12 medium, Glasgow Minimum Essential Medium (GMEM), Grace's Insect Medium, Ham's Medium, Iscove's Dumbecco Medium : IMDM), L-15 (Leibovitz) medium (L-15 (Leibovitz) Medium), McCoy's 5A medium (McCoy's 5 A Medium), Minimum Essential Medium-Eagle (MEM Eagle / E-MEM), 199 Medium (Medium 199), NCTC-109 Medium (NCTC-109 Medium), Richter CM (Richter's CM), RPMI 1640 Medium (RPMI 1640) Medium), insect medium, Weymouth's Medium, William's Medium, body fluid, and the like, but are not limited thereto.

In the cell culture system and the cell culture method of the present invention, the cells, spheroids, and cell culture solution described above can be used. In the cell culture system of the present invention, the aforementioned physiologically active substance can be appropriately added to the gel. Moreover, in order to raise the mechanical strength of a gel, a reinforcing material can be contained in a gel.
In addition, a flow system can be incorporated that injects the culture medium into the gel as described in the implantation system below.

  In addition, the cell culture system of the present invention is highly transparent and suitable for cell observation. Therefore, in addition to the components and factors necessary for cell growth assuming transplantation, it is also possible to add other drugs that affect the behavior of cells (good or bad influence) into the gel. In this case, the gel of the present invention can exhibit a function as a drug carrier.

Transplantation system A further aspect of the present invention is a system and method for transplanting the crosslinked body of the present invention or the cultured cells obtained by the cell culture system of the present invention into the body. In another aspect of the present invention, cells or tissue are encapsulated in a gel formed from a crosslinkable composition containing an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having an amino group in the side chain. It is a transplant material for constructing a regenerative tissue that is buried and used to be transplanted into the body of a subject.

In the transplant system of the present invention, a cross-linked body containing cells, a cross-linked body containing cell tissues such as spheroids (for example, a spheroid surrounded by a cross-linked body that is a scaffold material), a plurality of cell tissues A cross-linked body encapsulated (for example, a plurality of cellular tissues covered with a cross-linked body in a two-dimensional or three-dimensional manner) or a cross-linked body not containing cells can be transplanted into a host mother bed.
The cross-linked body used in the transplantation system of the present invention has a matrix structure with appropriate gaps, and can maintain a stable structure in vivo because of its good adhesion to vascular wall constituent cells, Since it is decomposed by angiogenic protease, a space for blood vessel growth can be secured, and angiogenesis can be efficiently induced.

  In the transplantation system of the present invention, the cross-linked body preferably contains an angiogenic factor, and particularly preferably bound to a polypeptide in the cross-linked body. Thereby, the blood vessel can be induced inside the crosslinked body without releasing the angiogenic factor outside the crosslinked body.

Moreover, the transplant system of this invention can also be equipped with the means to supply a culture solution from the exterior to the bridge | crosslinking body or culture cell after transplant. It takes several days to induce angiogenesis from the host vascular bed and draw it into the cross-linked body to induce vascular-rich granulation tissue in the transplanted cells, while the number of cells tends to gradually decrease due to central necrosis. By supplying the culture solution from the outside, it is possible to suppress the central necrosis and allow the cells to survive for several days until the granulation tissue is induced.
In order to supply the culture solution from the outside, the culture solution can be injected under pressure into the center (in some cases, a plurality of points) of the gel by a pump or the like.

  If the transplantation system of the present invention is used for humans, the transplanted cultured cells will express their functions in the human body for a long period of time, and become artificial organs as they are. The expression level of the function can be controlled by the size and shape of the cultured cells, or both. If hepatocytes are used as cultured cells, it shows a role as an artificial liver, such as liver enzyme deficiency, hemophilia, coagulopathy, liver failure, fulminant hepatitis, chronic hepatitis, cirrhosis, treatment of patients with hepatectomy, It is used for the essential treatment of each disease mentioned in infectious diseases and the like or for the purpose of assisting liver function. The transplanted tissues (organs) include liver, heart, vascular tissue, kidney, lung, pancreas (islet), gastrointestinal tissue, eye (cornea), brain, nerve tissue, bone marrow, bone, tooth, cartilage, and skin. (Hair) and muscles.

  When the transplant system of the present invention is used for animals, it becomes a cultured cell transplant animal. The expression level of the function can be controlled by the size and shape of the cultured cells. Examples of animals used herein include rats, mice, guinea pigs, marmosets, rabbits, dogs, pigs, chimpanzees, and immunodeficient animals thereof, but are not particularly limited. Such an islet transplanted animal is used for the purpose of, for example, a pancreatic function evaluation system in which a test substance is administered to the pancreatic islet transplanted animal and the influence of the test substance on pancreatic function is determined. Is not to be done.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Preparation of oligosaccharide oxide having aldehyde group [Synthesis Example 1]
Raffinose pentahydrate (Wako Pure Chemicals, Mw: 594.5) 800 mg (1.4 mmol, 4 mmol for sugar units) (1) 0.1N aqueous sodium hydroxide (10 mL) or (2) distilled water (10 mL) Then, 640 mg (3 mmol) of sodium periodate was added under ice cooling. This mixed solution was stirred at 24 ° C. for 60 hours under light shielding to carry out an oxidation reaction. Ethanol (40 mL) was added to the reaction solution and allowed to stand for 12 hours, and then the deposited iodine oxide precipitate was removed by filtration. The filtrate was concentrated to dryness under reduced pressure to obtain raffinose oxide (yield: 580 mg).

[Synthesis Example 2]
690 mg (2 mmol, 4 mmol for sugar units) of sucrose (Wako Pure Chemical, Mw: 342.3) was dissolved in (1) 0.1N aqueous sodium hydroxide (10 mL) or (2) distilled water (10 mL), and iced 640 mg (3 mmol) of sodium periodate was added under cooling. This mixed solution was stirred at 24 ° C. for 60 hours under light shielding to carry out an oxidation reaction. Ethanol (40 mL) was added to the reaction solution and allowed to stand for 12 hours, and then a white precipitate of precipitated iodine oxide was removed by filtration. The hydrated ethanol in the filtrate was evaporated azeotropically under reduced pressure to obtain sucrose oxide as a concentrated dry solid (yield: 640 mg).

[Synthesis Example 3]
Glucose (Wako Pure Chemicals, Mw: 180.2) 730 mg (4 mmol) was dissolved in (1) 0.1N aqueous sodium hydroxide (10 mL) or (2) distilled water (10 mL), and periodate was added under ice cooling. 640 mg (3 mmol) of sodium acid was added. This mixed solution was stirred at 24 ° C. for 60 hours under light shielding to carry out an oxidation reaction. Ethanol (40 mL) was added to the reaction solution and allowed to stand for 12 hours, and then a white precipitate of precipitated iodine oxide was removed by filtration. The hydrous ethanol in the filtrate was evaporated by azeotropic distillation under reduced pressure to obtain glucose oxide as a concentrated dry solid (yield: 730 mg).

[Synthesis Example 4]
Maltose monohydrate (Wako Pure Chemicals, Mw: 360.3) 730 mg (2 mmol, 4 mmol for sugar units) is dissolved in (1) 0.1N aqueous sodium hydroxide solution (10 mL) or (2) distilled water (10 mL) Further, 640 mg (3 mmol) of sodium periodate was added under ice cooling. This mixed solution was stirred at 24 ° C. for 60 hours under light shielding to carry out an oxidation reaction. Ethanol (40 mL) was added to the reaction solution and allowed to stand for 12 hours, and then a white precipitate of precipitated iodine oxide was removed by filtration. The hydrous ethanol in the filtrate was evaporated azeotropically under reduced pressure to obtain maltose oxide as a concentrated dry solid (yield: 610 mg).

[Synthesis Example 5]
730 mg (4 mmol) of fructose (Wako Pure Chemical, Mw: 180.2) is dissolved in (1) 0.1N aqueous sodium hydroxide solution (10 mL) or (2) distilled water (10 mL), and periodate is added under ice cooling. 640 mg (3 mmol) of sodium acid was added. This mixed solution was stirred at 24 ° C. for 60 hours under light shielding to carry out an oxidation reaction. Ethanol (40 mL) was added to the reaction solution and allowed to stand for 12 hours, and then a white precipitate of precipitated iodine oxide was removed by filtration. The hydrous ethanol in the filtrate was evaporated by azeotropic distillation under reduced pressure to obtain fructose oxide as a concentrated dry solid (yield: 580 mg).

[Example 1]
(1) Gelatin of collagen solution A 0.8% atelocollagen (Nitta gelatin, collagen BM, derived from pig, Type-I) phosphate buffer solution (pH 7.6) was prepared on gelled ice (4 ° C.). This solution was dispensed into an Eppendorf tube in an amount of 0.3 mL, 0.08 mL of an aqueous solution of sugar oxide (3%) prepared in conditions (1) and (2) of Synthesis Examples 1 to 5 was added, and the mixture was stirred. Allowed to stand at ° C. Periodically, the solution was contacted with a glass rod, and the time required from the addition of oxidized sugar to the loss of fluidity was defined as the gel time. For comparison, an experiment was also performed on sugars that had not been subjected to periodate oxidation treatment.

When periodic acid is added to the aqueous sugar solution (preparation condition (2)), the pH is about 2-3. When the oxidation reaction was performed in this state, heat was generated at the initial stage of the reaction, the solution gradually became yellowish brown, and black purple iodine microcrystals were deposited. The resulting sugar oxide turned brown. When the same reaction was carried out with a 0.1N aqueous sodium hydroxide solution (preparation conditions (1)), the pH became about 5 to 6, and no browning of the solution or precipitation of iodine occurred.
When periodate oxide of sugar is added to collagen solution, gelation of sucrose oxide, raffinose oxide, maltose oxide derived from oligosaccharide compared to glucose oxide and fructose oxide which are monosaccharides I found it faster.
In addition, there was a difference in gelation depending on the preparation conditions of the sugar oxides, and there was a tendency for gelation when the sugar oxides oxidized at pH 5-6 (preparation condition (1)) were added to the collagen solution. .

(2) Effect of gel preparation temperature on gelation time 0.3 mL of 0.8% atelocollagen (Nitta gelatin, collagen BM) phosphate buffer solution (pH 7.6) is prepared on ice (4 ° C.). did. To this solution, 0.08 mL of an aqueous solution of 3% (50 mM) raffinose oxide or raffinose was added and stirred, and allowed to stand at 4 ° C., 18 ° C., 24 ° C., and 37 ° C.

  The collagen solution prepared at a neutral pH is fibrillated and gelled due to the influence of hydrophobic interaction and the like as the temperature rises. The collagen solution to which raffinose was added turned into a fibrillated and white turbid gel at about 20 minutes at 37 ° C. On the other hand, when raffinose oxide was added to the collagen solution, fibrosis was not observed even at 37 ° C., and gelled in about 1 hour while being colorless and transparent. This is probably because the cross-linking of collagen molecules with raffinose oxide limited the association / aggregation of collagens, so that fibrosis was suppressed and a colorless and transparent gel was obtained. When the preparation temperature was 4 ° C., the gelation time was as slow as 7 hours, and as the temperature increased, the gelation time tended to be shortened.

(3) Effect of gelation of collagen solution on gelation On ice (4 ° C.), 0.3 mL of 0.8% atelocollagen (Nitta gelatin, collagen BM) phosphate buffer solution (pH 7.2, pH 7.6, pH 8.0) was prepared. To this solution, 0.08 mL of 3% (50 mM) aqueous raffinose aqueous solution was added and stirred, and allowed to stand at 18 ° C.

  The reaction did not proceed sufficiently at a pH lower than neutral, and gelation proceeded quickly when the pH was 7.6 or higher. Further, even when raffinose oxide (Synthesis Example 1 (2)) oxidized with perchloric acid at a pH of 5 or less is used, gelation occurs when the pH of the collagen solution during gel preparation is high. It was confirmed.

[Example 2]
Gelation of gelatin solution
(1) Gelation of porcine-derived gelatin with sugar oxide An aqueous solution of gelatin (077-03155 manufactured by Wako Pure Chemical Industries, Ltd., derived from swine) is prepared at 40 ° C., and a phosphate buffer solution is added, and finally 15% A gelatin solution (pH 7.6) was prepared. To 0.3 mL of this solution, 0.06 mL of a 3% sugar oxide aqueous solution was added and stirred, and allowed to stand at 40 ° C.

  Since pig-derived gelatin forms a reversible physical gel at room temperature, the gel preparation test was performed at 40 ° C. at which the gelatin solution melts. Gelation was observed when raffinose oxide and sucrose oxide were added, but no gelation was observed with glucose oxide, maltose oxide, or flucotose oxide. At 40 ° C., it is expected that the thermal degradation of collagen and the cross-linking reaction by sugar oxide proceed at the same time. Therefore, it is considered that a quick cross-linking reaction is necessary to prepare the gel. Although all of the above sugar oxides are considered to form a crosslinked structure, raffinose oxide and sucrose oxide were recognized to have an ability to gel efficiently.

(2) Effect of gelatin solution on gelation by pH An aqueous solution of gelatin (077-03155 manufactured by Wako Pure Chemical Industries, Ltd., derived from swine) is prepared at 40 ° C., a phosphate buffer solution is added, and finally 15% A gelatin solution (pH 8, pH 9) was prepared. To 0.3 mL of this solution, 0.06 mL of an aqueous solution of 3% raffinose oxide or 3% sucrose oxide was added and stirred, and allowed to stand at 40 ° C.

  It was confirmed that the gelation did not proceed sufficiently in the vicinity of neutral pH. Moreover, when the pH of the gelatin solution was high, the tendency for gelation time to shorten was recognized.

(3) Effect of added concentration of sugar oxide on gelatinization of gelatin solution Prepare an aqueous solution of gelatin (manufactured by Wako Pure Chemicals, 077-03155, porcine) at 40 ° C, and add phosphate buffer solution. Finally, a 15% gelatin solution (pH 9) was prepared. To 0.3 mL of this solution, 0.06 mL of an aqueous solution of 3%, 6%, 9% raffinose oxide or 3%, 6%, 9% sucrose oxide was added, stirred, and allowed to stand at 40 ° C.

  There was a tendency that the gelation time of the gelatin solution was shortened as the concentration of added sugar oxide was increased.

(4) Gelation of fish-derived gelatin with sugar oxide An aqueous solution of gelatin (manufactured by SIGMA, G7041-100G, fish-derived) is prepared at 18 ° C., a phosphate buffer solution is added, and finally 15% gelatin A solution (pH 7.6) was prepared. 0.03 mL of 3% sugar oxide aqueous solution was added to 0.3 mL of this solution and stirred, and allowed to stand at 18 ° C.

  Since fish-derived gelatin forms a reversible physical gel at about 4 ° C. or lower, the gel preparation test was performed near room temperature (18 ° C.). Gelation was observed when raffinose oxide and sucrose oxide were added, but no gelation was observed with glucose oxide, maltose oxide, or flucotose oxide.

カルボキシメチルキトサンは分子中に反応性のアミノ基を有する水溶性の多糖類である。調製した水溶液は弱アルカリ性を示した。この水溶液にラフィノース酸化物を添加したところ、次第に増粘しゲル化した。 (5) Gelation of chitosan derivative with raffinose oxide A 2% aqueous solution (pH 8) of carboxymethyl chitosan (manufactured by Dainichi Seika, CM chitosan) was prepared at 24 ° C. 0.06 mL of an aqueous solution of 9% raffinose oxide was added to 0.3 mL of this solution, stirred, and allowed to stand at 24 ° C.

Carboxymethyl chitosan is a water-soluble polysaccharide having a reactive amino group in the molecule. The prepared aqueous solution showed weak alkalinity. When raffinose oxide was added to this aqueous solution, it gradually thickened and gelled.

[Example 3]
Cell culture test At 4 ° C., 0.8% atelocollagen (Nitta gelatin, collagen BM) phosphate buffer solution (pH 7.6, 1 mL) and 3% oxidized raffinose aqueous solution (oxidation conditions of Synthesis Example 1 (1)) (0. 2 mL) was added and stirred. This mixed solution was allowed to stand at 18 ° C. for 4 hours. When the viscosity was increased, 120 μL was collected, and each cell was added and mixed. A hole (φ8 mm) was made in a vinyl sheet having a thickness of 2 mm that was in close contact with the petri dish, and the cell mixture was poured into it, and left to stand at 18 ° C. for 1 hour to cause gelation. Disc-shaped gel (thickness 2 mm, φ8 mm) was coated with a culture solution and cultured.

(1) Normal human umbilical vein endothelial cells (HUVEC)
GFP-expressing HUVEC (Angio-Proteomie) was prepared to be mixed with 6 × 10 ⁶ cells in one disc-shaped gel (120 μL). As the culture solution, EGM-2 (Lonza) was used.
The observation results with a microscope on the fifth day after the start of culture are shown in FIG.

(2) Hepatocytes Hepatocytes were collected from the liver of Wistar rats (300 g) by the collagenase perfusion method. A Prime-Surfae 24-well plate (Sumitomo Bakelite) was seeded with hepatocytes (8 × 10 ⁴ cells / well), and after culturing for 4 days, the spheroids were added to 24 wells in a disc-shaped gel (120 μL). The culture solution was prepared according to the method of Yoshizato et al.
FIG. 2 shows the results of observation with a microscope on the third day after the spheroids were added to the gel.

(3) Cardiomyocytes Cardiomyocytes were collected from 1-day-old Wistar rat children. Prime-Surfae 24-well plates (Sumitomo Bakelite) were seeded with cardiomyocytes (8 × 10 ⁴ cells / well), and after culturing for 4 to 6 days, the spheroids were added to the 6-well portion to a disc-shaped gel (120 μL) . As a culture solution, RCBM (Rat Cardiac Myocyte Basal Medium, Lonza) was used.
FIG. 2 shows the results of observation with a microscope on the third day after the spheroids were added to the gel.

  In the experiments (1) to (3), the obtained gel was highly transparent, and it was possible to observe the three-dimensional growth process of cells in the gel over time. There was no problem with the cytotoxicity of the gel, and in the case of hepatocyte spheroids, a process in which new cells sprout from the spherical cell mass was observed as indicated by the arrows in FIG. In HIUVEC and cardiomyocytes, a cell branching structure was observed the day after in-gel culture, and a three-dimensional network structure was constructed after several days. In cardiomyocytes, the pulsation gradually shifted from the individual pulsation to the pulsation linked to the individual cells, and finally the entire gel pulsated (the part indicated by the arrow in FIG. 3).

[Example 4]
Implantation experiment to rat 0.3 mL of 0.8% atelocollagen (Nitta gelatin, collagen BM) phosphate buffer solution (pH 7.6) was prepared on ice (4 ° C.). To this solution, 0.06 mL of 3% (50 mM) raffinose oxide aqueous solution was added and stirred. 14 μL each was dispensed into an ePTFE artificial blood vessel (height 1 mm, inner diameter φ3 mm) cut into a ring and allowed to stand at 18 ° C. for 4 hours 30 minutes to gel. The gel was coated with a HANKS buffer to suppress the progress of gelation and allowed to stand overnight at 37 ° C. under humidity.
The obtained gel was implanted subcutaneously in the abdomen of Wister male rats (8 weeks old, weight 300-600 g), and extracted 5 days later. Hematoxylin-eosin (HE) staining was performed and observed histologically under an optical microscope. FIG. 4 shows a schematic flow diagram of the transplantation experiment.

  FIG. 5 shows the results of observation with an optical microscope. It can be seen that the transplanted gel (portion surrounded by a yellow broken line in the center of the photograph) was replaced with a granule tissue rich in blood vessels in 5 days. Moreover, the case where only the blood vessel has invaded using the gel as a scaffold is also seen, and the effect of inducing the blood vessel is also expected.

Claims (21)

A crosslinkable composition comprising an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having a plurality of amino groups in the side chain ,
The oligosaccharide oxide is produced by dissolving an oligosaccharide in a basic aqueous solution and performing periodate oxidation,
The polypeptide is selected from collagen, atelocollagen, gelatin, derivatives thereof or mixtures thereof;
The polysaccharide is a chitosan derivative.
The crosslinkable composition . Crosslinked hydroxypropylated chitosan down or mixtures thereof, according to claim 1 - wherein the chitosan derivative is deacetylated chitin, or chitosan, succinyl chitosan, glyceryl chitosan, carboxymethyl chitosan, hydroxybutyl Sex composition. The crosslinkable composition according to claim 1 or 2 , wherein the oligosaccharide is selected from the group consisting of sucrose, maltose, raffinose, derivatives thereof and a mixture of two or more thereof. The crosslinked body obtained from the crosslinkable composition of any one of Claims 1-3 . The cross-linked product according to claim 4 , wherein an aldehyde group of the oligosaccharide oxide and an amino group of the polypeptide or polysaccharide are covalently bonded. The cross-linked product according to claim 4 or 5 , which is in a gelled state. The cross-linked product according to any one of claims 4 to 6 , comprising an angiogenic factor. The crosslinked body according to any one of claims 4 to 7 , comprising a cell or a cell tissue. The crosslinked body of Claim 8 containing a cell culture solution. Cell culture system using a crosslinked body according to any one of claims 4-9. A system for implanting the crosslinked body according to the body of a subject in any one of claims 4-9. The system which transplants the cultured cell obtained by the cell culture system of Claim 10 in a test subject's body. (A) preparing the crosslinkable composition according to any one of claims 1 to 3 ,
(B) A method for preparing a gel of the crosslinkable composition, comprising a step of adjusting the pH of the crosslinkable composition to 7.0 or more. (A) preparing the crosslinkable composition according to any one of claims 1 to 3 ,
(B) adjusting the pH of the crosslinkable composition to 7.0 or higher;
(C) including a step of gelling the crosslinkable composition, and adding cells or cell tissues to the crosslinkable composition at a time before, during or after the step of (c), culturing cells Method. The method for culturing cells according to claim 14 , wherein a culture solution is added to the crosslinkable composition before, during or after the step (c). A method for transplanting a gel obtained by the method according to claim 13 into the body of a subject. A method for transplanting cultured cells obtained by the method according to claim 14 or 15 into the body of a subject. Cells or cell tissues are embedded in a gel formed from a crosslinkable composition containing an oligosaccharide oxide having an aldehyde group and a polypeptide or polysaccharide having a plurality of amino groups in the side chain. used to be implanted in, a graft material for building regenerated tissue,
The oligosaccharide oxide is produced by dissolving an oligosaccharide in a basic aqueous solution and performing periodate oxidation,
The polypeptide is selected from collagen, atelocollagen, gelatin, derivatives thereof or mixtures thereof;
The polysaccharide is a chitosan derivative.
The transplant material. A cross-linking agent comprising an oligosaccharide oxide having an aldehyde group and used for a cross-linking reaction with a polypeptide or polysaccharide having a plurality of amino groups in the side chain ,
The oligosaccharide oxide is produced by dissolving an oligosaccharide in a basic aqueous solution and performing periodate oxidation,
The polypeptide is selected from collagen, atelocollagen, gelatin, derivatives thereof or mixtures thereof;
The polysaccharide is a chitosan derivative.
The crosslinking agent . 20. The crosslinking agent according to claim 19 , wherein the oligosaccharide is selected from the group consisting of sucrose, maltose, raffinose, derivatives thereof and mixtures of two or more thereof. Crosslinked hydroxypropylated chitosan down or mixtures thereof, according to claim 19 - wherein the chitosan derivative is deacetylated chitin, or chitosan, succinyl chitosan, glyceryl chitosan, carboxymethyl chitosan, hydroxybutyl Agent.

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