Gelator, injectable gel, gel composition, cell scaffold material, and medical material

Abstract

To provide a gelator capable of forming a gel composition having biocompatibility, biodegradability, and cell adhesion.SOLUTION: A gelator comprises a first polymer, which is a derivative of a first biopolymer, and a second polymer, which is at least one selected from the group consisting of a second biopolymer and derivatives thereof. Out of the first and second polymers, only the first polymer has a ureidopyrimidinone group, and only one of the first polymer or the second polymer has a cross-linkable group.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a gelling agent, an injectable gel, a gel composition, a cell scaffold material, and a medical material. [Background technology]

[0002] Hydrogels are substances with a network structure in which polymers are crosslinked three-dimensionally, and are characterized by the inclusion of a large amount of water inside. Hydrogels are expected to have various applications, such as cell scaffolding materials, drug delivery systems, and medical adhesives. In particular, injectable gels have attracted attention in the medical field. An injectable gel is, for example, a solution (sol) of a gelling agent that is injected into the body in liquid form using a syringe or the like and gels in the body after injection. Compared to implanting an already gelled substance in the body, it has the advantage of reducing the physical burden on the patient. In addition, by encapsulating cells in the injectable gel, cells can be easily delivered locally to the target tissue.

[0003] Hydrogels are further classified into chemical gels, which form cross-linked structures through chemical reactions, and physical gels, which are formed by physical interactions between polymers. Generally, physical gels have low mechanical strength, so chemical reactions are necessary to create highly stable hydrogels.

[0004] However, chemical gels have a problem in that the polymer network is very dense, which hinders the diffusion of nutrients and oxygen and the migration of cells. To solve such problems, research on gels with a porous structure is being conducted. For example, Non-Patent Document 1 reports a porous gel that uses fine ice particles as a porogen. In addition, Non-Patent Document 2 reports a porous injectable gel. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Q. Zhang et al., Pore size effect of collagen scaffolds on cartilage regeneration. Acta Biomaterialia 10 (2014) 2005-2013. [Non-Patent Document 2] N. Huebsch et al., Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation. Nat. Mater. 14 (2015) 1269-1277. Summary of the Invention [Problem to be solved by the invention]

[0006] However, the method reported in Non-Patent Document 1 is not injectable, so the operation is complicated, and a cell culture facility is required, which is costly. In addition, since the cells are seeded after the gel is produced, the cells cannot be encapsulated inside the gel. The injectable porous gel reported in Non-Patent Document 2 can encapsulate cells inside the gel, but it has independent pores rather than continuous pores. For this reason, it is presumed that material diffusion to the cells contained inside the gel and cell migration are unlikely to occur. In addition, when a porous structure is formed using solid particles as porogens, there is a concern that the mechanical strength may decrease.

[0007] The present invention solves the above-mentioned problems by providing a gelling agent that can be used as an injectable agent and that can form a porous gel composition having high mechanical strength by utilizing the liquid-liquid phase separation phenomenon. [Means for solving the problem]

[0008] As a result of extensive investigations aimed at achieving the above object, the present inventors have found that the above object can be achieved by the following configuration.

[0009] [1] A gelling agent comprising: a first polymer that is a derivative of a first biopolymer; A second polymer, which is at least one selected from the group consisting of a second biopolymer and a derivative thereof; Of the first polymer and the second polymer, only the first polymer has a ureidopyrimidinone group; A gelling agent, wherein only one of the first polymer and the second polymer has a crosslinkable group. [2] The gelling agent according to [1], wherein the first biopolymer is gelatin. [3] The gelling agent according to [1] or [2], wherein the second biopolymer is gelatin. [4] The gelling agent according to any one of [1] to [3], wherein the crosslinkable group contains a thiol group and a vinyl sulfone group, or contains an ethylenically unsaturated group. [5] The gelling agent according to [4], wherein the crosslinkable group contains a thiol group and a vinyl sulfone group. [6] The gelling agent according to any one of [1] to [5], wherein the second polymer has the crosslinkable group. [7] the second polymer, a polymer having a thiol group introduced therein as a second biopolymer; The gelling agent according to [6], wherein the second biopolymer contains a polymer having a vinyl sulfone group introduced therein. [8] The gelling agent according to any one of [1] to [5], wherein the first polymer has the crosslinkable group. [9] The first polymer, a polymer having a ureidopyrimidinone group and a thiol group introduced into a first biopolymer; The gelling agent according to [8], comprising a polymer having a ureidopyrimidinone group and a vinyl sulfone group introduced into the first biopolymer.

[10] The gelling agent according to any one of [1] to [9], which is capable of forming a gel composition having a phase-separated structure.

[11] The gelling agent according to

[10] , wherein the phase-separated structure includes a fiber structure or a gyroid structure.

[12] The gelling agent according to

[10] or

[11] , wherein the gel composition becomes porous in an aqueous solvent.

[13] An injectable gel comprising the gelling agent according to any one of [1] to

[12] .

[14] A gel composition comprising the gelling agent according to any one of [1] to

[12] .

[15] A first phase comprising a first polymer; a second phase comprising a second polymer, the second phase being phase-separated from the first phase; The gel composition according to

[14] , wherein only one of the first phase and the second phase has a crosslinked structure.

[16] In aqueous solvents, The gel composition according to

[15] , wherein the other of the first and second phases, which does not have a cross-linked structure, is dissolved and removed, and a porous structure composed of the one of the phases is formed.

[17] A cell scaffold material comprising a gel composition according to any one of

[14] to

[16] .

[18] A medical material comprising the gel composition according to any one of

[14] to

[16] . Effect of the Invention

[0010] The gelling agent of the present invention can be used as an injectable agent, and by utilizing the liquid-liquid phase separation phenomenon, a porous gel composition having high mechanical strength can be formed. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a diagram illustrating a method for producing (preparing) a phase-separated gel composition according to an embodiment. [Diagram 2] FIG. 2 is a diagram illustrating the interaction (hydrogen bond) between ureidopyrimidinone groups. [Diagram 3] FIG. 1 is a diagram illustrating a method for synthesizing ureidopyrimidinone-modified (UPy-modified) gelatin in an embodiment. [Figure 4] FIG. 2 is a diagram showing a click reaction between thiolated (SH) gelatin and vinyl sulfonated (VS) gelatin in an embodiment. [Diagram 5] FIG. 2 is a diagram illustrating a method for synthesizing thiolated gelatin in an embodiment. [Figure 6] FIG. 1 is a diagram illustrating a method for synthesizing vinylsulfonated gelatin in an embodiment. [Figure 7] 7 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) of the gel compositions prepared in the examples. Fig. 7(a) shows gel composition A, Fig. 7(b) shows gel composition B, and Fig. 7(c) shows gel composition 1. [Figure 8] FIG. 2 is a diagram showing the relationship between the concentration of a gelatin solution used in preparing a gel composition and the phase-separated structure of the gel composition in the Examples. [Figure 9] 1 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) of gel compositions 1 to 9 prepared in the examples. [Figure 10] 1 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) of gel compositions 1 and 1-1 to 1-4 prepared in the examples. [Figure 11] 1 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) of gel compositions 1 and 1-5 to 1-6 prepared in the examples. [Figure 12] 1 is a graph showing the change over time in the shear modulus of gel composition 10 prepared in an example. [Figure 13] 1 is a graph showing the mechanical strength in a tensile test of gel compositions 10 and 11 prepared in the examples. [Figure 14] 1 is a graph showing the adhesive strength of gel compositions 10 and 11 prepared in the examples. [Figure 15] 15A and 15B are confocal laser scanning microscope (CLSM) photographs (grayscale converted) of the gel composition 10 prepared in the example, in which Fig. 15(a) is a photograph before being made porous, and Fig. 15(b) is a photograph after being made porous. [Figure 16] FIG. 2 is a graph showing the albumin permeability of gel composition 10 prepared in the example. [Figure 17]17(a) and 17(b) are confocal laser scanning microscope (CLSM) photographs (grayscale converted) of gel compositions after protein (albumin) permeability evaluation in the Examples. FIG. 17(a) shows gel composition 11, and FIG. 17(b) shows gel composition 10. [Figure 18] 18 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) of gel compositions after cell entrapment test 1 in the examples. Figure 18(a) shows gel composition 11, and Figure 18(b) shows gel composition 10, with the light-colored areas being human mesenchymal stem cells (stained actin and nuclei). [Figure 19] FIG. 18 is a graph showing the ratio of the area occupied by human mesenchymal stem cells (areas where actin and nuclei are stained) to the total area of ​​the photograph for (a) gel composition 11 and (b) gel composition 10. [Figure 20] 1 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) of gel compositions 10, 30, 40 and 50 after cell encapsulation test 2 in an example. [Figure 21] 1 is a graph showing the results of a cell proliferation test using gel compositions 10 and 11 in an example. [Figure 22] 1 is a graph showing the results of angiogenesis evaluation using gel compositions 10 and 11 in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment. In this specification, a range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0013] In the description of groups (atomic groups) in this specification, the description without indicating whether substituted or unsubstituted includes both unsubstituted and substituted groups, as long as it does not impair the effects of the present invention. For example, "alkyl group" includes not only alkyl groups without substituents (unsubstituted alkyl groups), but also alkyl groups with substituents (substituted alkyl groups). This also applies to each compound. In addition, in this specification, "(meth)acryloyl" means either one or both of acryloyl and methacryloyl.

[0014] [First embodiment] The gelling agent of this embodiment includes a first polymer having a ureidopyrimidinone group (UPy group) and a second polymer having a crosslinkable group. When the gelling agent of this embodiment is used, a gel composition 100 is obtained in which a first phase 21 containing the first polymer and a second phase 22 containing the second polymer are phase-separated on a fine scale (micro-order scale) as shown in FIG. 1.

[0015] 1. First polymer The first polymer is a derivative of a first biopolymer into which a ureidopyrimidinone group (UPy group) has been introduced. Here, the biopolymer means a natural polymer produced by cells of an organism. The gelling agent of this embodiment uses a material derived from a biopolymer for both the first polymer and the second polymer described below, thereby obtaining a gel composition having excellent biocompatibility, biodegradability, and cell adhesiveness. Examples of the first biopolymer include proteins such as gelatin, elastin, and albumin, and polysaccharides such as hyaluronic acid and chondroitin. Among them, gelatin is preferred from the viewpoints of cell adhesiveness and solubility.

[0016] Gelatin (raw gelatin) used as the first biopolymer can be any gelatin obtained by natural origin, chemical synthesis, fermentation, genetic recombination, or the like, without any particular limitation. Among them, gelatin of natural origin is preferred. Examples of naturally derived gelatin include gelatin derived from mammals such as cows, pigs, and humans, and gelatin derived from fish such as sea bream, sturgeon, salmon, and cod. Among them, porcine gelatin is preferred, and porcine skin gelatin and porcine tendon gelatin are more preferred.

[0017] The molecular weight of the raw material gelatin is not particularly limited, and generally, the weight average molecular weight is preferably 10,000 to 500,000. The upper limit is not particularly limited, but from the viewpoint of solubility, it is more preferably 350,000 or less. The lower limit is not particularly limited, but from the viewpoint of the obtained gel having superior mechanical strength, it is more preferably 50,000 or more.

[0018] In the present specification, the term "ureidopyrimidinone group" (UPy group) refers to a monovalent group obtained by removing one hydrogen atom from ureidopyrimidinone or a derivative thereof, for example, a group represented by the following formula (11).

[0019] [ka]

[0020] In formula (11), R 1 is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and * indicates the linking position. 1 is preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group.

[0021] The ureidopyrimidinone group is preferably introduced indirectly (i.e., via a spacer that is a divalent group) into the first biopolymer. For example, a group represented by the following formula (12) may be introduced into an amino group of the first biopolymer (e.g., an amino group of raw gelatin). In the case of formula (12), L having an isocyanate group at its terminal may be used as a spacer. 6A ureidopyrimidinone group is introduced into the first biopolymer via (see FIG. 3).

[0022] [ka]

[0023] In formula (12), R 1 is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and L 6 is a single bond or a divalent group, and * represents the linking position.

[0024] R 1 is preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group. 6 The divalent group as includes, for example, a hydrocarbon chain having 1 to 20 carbon atoms. The hydrocarbon chain may be, for example, an alkyl group or an ethylene oxide chain, and preferably has 2 to 12 carbon atoms.

[0025] Here, when gelatin is used as the first biopolymer, the ratio of the content (molar concentration) of ureidopyrimidinone groups in the first polymer to the content (molar concentration) of amino groups (primary amino groups, -NH2) in gelatin (raw gelatin) is defined as "ureidopyrimidinone group introduction rate (UPy group introduction rate, UPy group substitution rate) (mol%)". The method of determining the UPy group introduction rate is not particularly limited, and may be calculated from a value determined directly or indirectly using, for example, the TNBS method (2,4,6-trinitrobenzenesulfonic acid method, amino group quantification), Ellman's method (thiol group quantification), or the like. For example, it may be determined by the method described in the examples below.

[0026] The UPy group introduction rate is not particularly limited, but may be, for example, 30 mol% to 70 mol%, or 40 mol% to 60 mol%, from the viewpoint of efficiently causing phase separation in the gel composition and from the viewpoint of increasing the gel strength and / or adhesive strength to biological tissue.

[0027] The molecular weight of the first polymer is not particularly limited and is determined by the molecular weight of the first biopolymer and the type and amount (number) of the introduced group. Therefore, the range of the weight average molecular weight (Mw) of the first polymer is the same as the range of the weight average molecular weight (Mw) of the first biopolymer described above.

[0028] The first polymer may be a single type of polymer or a mixture of two or more types of polymers.

[0029] The synthesis method of the first polymer is not particularly limited, and a known method can be used, for example, the method described in JP 2023-027762 A, the contents of which are incorporated herein by reference. When gelatin is used as the first biopolymer, it may be synthesized, for example, by the method described below (see FIG. 3). First, gelatin (raw gelatin) is dissolved in an organic solvent such as dimethyl sulfoxide. Next, a ureidopyrimidinone derivative having an isocyanate group at its terminal is prepared and dissolved in an organic solvent such as dimethyl sulfoxide. The ureidopyrimidinone derivative may be a commercially available product or may be synthesized by a known method. A gelatin solution and a ureidopyrimidinone derivative solution are mixed and stirred so that the ureidopyrimidinone group is 30 to 200 mol% when the amount of amino groups (n) in the gelatin is 100 mol%. Ureidopyrimidinone gelatin (UPy gelatin) can be synthesized by stirring for 16 to 24 hours at 20 to 30°C. After the reaction is complete, it is reprecipitated with a mixed solvent of cold ethanol and ethyl acetate, washed with cold chloroform and cold ethanol, and then dried under reduced pressure to obtain a powder of UPy gelatin (first polymer).

[0030] The first polymer may be a derivative in which only a UPy group is introduced into the first biopolymer, or may be a derivative in which a functional group different from the UPy group is further introduced, as long as the effect of this embodiment is achieved. However, the first polymer does not have a crosslinkable group that the second polymer described below has. As a result, in the gel composition 100 generated by the gelling agent, the first phase 21 containing the first polymer does not have a crosslinked structure and can maintain water solubility. By placing the gel composition 100 under a wet condition (for example, in a living body), the first phase 21 is dissolved and removed, and a porous gel can be obtained.

[0031] 2. Second polymer The second polymer is a derivative of the second biopolymer in which a crosslinkable group is introduced into the second biopolymer. The second biopolymer may be the same as the first biopolymer described above, and the preferred embodiments are also the same. The second biopolymer may be a different type of biopolymer from the first biopolymer, or may be the same type of biopolymer. Usually, a solution of two types of polymers having a common backbone tends to form a homogeneous phase. However, in the gelling agent of this embodiment, even if the first polymer and the second polymer have a common backbone, due to the difference in the presence or absence of a UPy group, a homogeneous phase is not formed, and a phase-separated gel is obtained.

[0032] The crosslinkable group is not particularly limited, but examples thereof include functional groups capable of click reaction, such as thiol groups (SH groups) and vinyl sulfone groups (VS groups), and ethylenically unsaturated groups capable of radical polymerization, such as (meth)acryloyl groups, styryl groups, and allyl groups. Among these, thiol groups and vinyl sulfone groups capable of click reaction are preferred as crosslinkable groups (see FIG. 4). Click reactions do not require a catalyst or external energy, proceed rapidly in an aqueous environment, and are highly bioorthogonal and biocompatible.

[0033] When the crosslinkable group is a thiol group and a vinylsulfone group, the second polymer preferably contains two types of polymers: an SH polymer in which a thiol group is introduced into the second biopolymer and a VS polymer in which a vinylsulfone group is introduced into the second biopolymer. The click reaction between the thiol group and the vinylsulfone group is highly reactive. By introducing the thiol group and the vinylsulfone group into different polymers (different polymer chains) to form two types of modified polymers, for example, until the crosslinking agent is used, the two types of polymers can be stored and managed in separate containers, thereby suppressing the curing reaction (click reaction) before use. The SH polymer and the VS polymer before use may be stored separately in containers, for example, together with a desiccant such as silica gel, or preferably stored under reduced pressure (under vacuum) at a low temperature (frozen state).

[0034] The content ratio of the SH polymer and the VS polymer in the gelling agent is not particularly limited. From the viewpoint of increasing the gel strength, for example, it is preferable that the vinylsulfone group of the VS polymer contains 0.2 to 5 molar equivalents, more preferably 0.5 to 2 molar equivalents, relative to 1 molar equivalent of the thiol group of the SH polymer. The details of the SH polymer and the VS polymer will be described below.

[0035] <SH polymer> When the second biopolymer is gelatin, the SH polymer (thiolated gelatin) has a structure in which, for example, a thiol group is introduced into the amino group of the raw gelatin via a first spacer. For example, thiolated gelatin (SH gelatin) has a structure represented by the following formula (1).

[0036]

Chemical formula

[0037] In formula (1), Gltn 1 is a gelatin residue, and L 1is a first spacer which is a divalent group. The first spacer L 1 preferably contains at least one selected from the group consisting of -C(O)-, -S-, -O-, and an alkylene group having 2 to 18 carbon atoms. The alkylene group may be linear or branched, and is preferably an alkylene group having 2 to 5 carbon atoms.

[0038] From the viewpoint of efficiently synthesizing thiolated gelatin, the thiolated gelatin may have a structure represented by the following formula (3): That is, the thiolated gelatin may have an amide group (-NHC(O)-).

[0039] [ka]

[0040] In formula (3), Gltn 1 is a gelatin residue, and L 3 is an alkylene group having 2 to 18 carbon atoms, which may be linear or branched, and is preferably an alkylene group having 2 to 5 carbon atoms.

[0041] In the above formulas (1) and (3), the gelatin residue Gltn 1 The nitrogen atom (N) directly bonded to is derived mainly from the ε-amino group of lysine (Lys) in gelatin.

[0042] Here, the ratio of the content (molar concentration) of thiol groups in thiolated gelatin to the content (molar concentration) of amino groups (primary amino groups, -NH2) in raw gelatin is defined as "thiol group introduction rate (SH group introduction rate, SH group substitution rate) (mol%)". There is no particular limitation on the method for determining the thiol group introduction rate, and it may be calculated from a value determined directly or indirectly using, for example, the TNBS method (2,4,6-trinitrobenzenesulfonic acid method, amino group quantification), Ellman's method (thiol group quantification), etc. For example, it may be determined by the method described in the examples below.

[0043] Although the SH group introduction rate is not particularly limited, the mechanical properties of the resulting gel can be controlled by changing the SH group introduction rate. If the thiol group introduction rate is too high, the water solubility decreases and it becomes difficult to form a gel (hydrogel). From the viewpoint of facilitating the formation of a gel and increasing the gel strength and / or adhesive strength to biological tissue, the thiol group introduction rate may be, for example, 20 mol% to 80 mol%, 40 mol% to 80 mol%, or 50 mol% to 70 mol%.

[0044] The molecular weight of thiolated gelatin is not particularly limited, and is determined by the molecular weight of the second biopolymer (raw gelatin) and the type and amount (number) of the introduced group. Therefore, the range of the weight average molecular weight (Mw) of thiolated gelatin is the same as the range of the weight average molecular weight (Mw) of the second biopolymer.

[0045] The gelling agent of the present embodiment may contain only one type of thiolated gelatin, or may contain two or more types of thiolated gelatin.

[0046] The method for producing thiolated gelatin (synthesis method) is not particularly limited, and it can be synthesized by a known method. Examples of the method for binding a thiol group to an amino group of raw gelatin, preferably an amino group of lysine, via a first spacer include the so-called reductive amination reaction (method using an aldehyde or ketone), the Schotten-Baumann reaction (method using an acid chloride), and a reaction using N-hydroxysuccinimide. For example, as shown in FIG. 5, a thiol group may be introduced by reacting a thiolactone compound (e.g., γ-thiobutyrolactone) with a primary amine of raw gelatin. The number of carbon atoms of the lactone compound used in the synthesis may be appropriately selected depending on the type of the first spacer of the thiolated gelatin to be synthesized.

[0047] The method for producing thiolated gelatin is not limited to the above method. For example, instead of the thiolactone compound, it may be synthesized using a carboxylic acid compound having a thiol group and / or an aldehyde compound having a thiol group (see Fig. 5).

[0048] In addition, in thiolated gelatin, only a thiol group (-L 1 -SH) may be introduced to the amino group of the raw material gelatin, or other functional groups may be introduced as long as the effects of the present invention are achieved. However, the thiolated gelatin (SH polymer) of the present embodiment does not have a ureidopyrimidinone group (UPy group). Thereby, in the gel composition 100, the phase separation between the first phase 21 and the second phase 22 can be promoted (see Fig. 1).

[0049] <VS polymer> When the second biopolymer is gelatin, the VS polymer (vinylsulfonated gelatin) has, for example, a structure in which a vinylsulfone group is introduced to the amino group of the raw material gelatin via a second spacer. For example, vinylsulfonated gelatin (VS gelatin) has a structure represented by the following formula (2).

[0050]

Chemical formula

[0051] In formula (2), Gltn 2 is a gelatin residue, and L 2 is a second spacer which is a divalent group. The second spacer L 2 preferably contains at least one selected from the group consisting of -C(O)-, -S-, -O- and an alkylene group having 2 to 18 carbon atoms. The alkylene group may be linear or branched, and is preferably an alkylene group having 2 to 5 carbon atoms.

[0052] From the viewpoint of efficiently synthesizing vinylsulfonated gelatin, the vinylsulfonated gelatin may have a structure represented by the following formula (4): That is, the vinylsulfonated gelatin may have an amide group (-NHC(O)-).

[0053] [ka]

[0054] In formula (4), Gltn 2 is a gelatin residue, and L 4 is a divalent group. As the divalent group, the second spacer L 2 From the viewpoint of more efficient synthesis of vinylsulfonated gelatin, the functional groups L 4 may be a group represented by the following formula (5): 51 is an alkylene group having 2 to 18 carbon atoms, X is a sulfur atom (S) or an oxygen atom (O), and R 52 is an ethylene group. More preferably, in formula (5), X is a sulfur atom (S) and R 51 is an alkylene group having 2 to 5 carbon atoms.

[0055] [ka]

[0056] In the above formulas (2) and (4), the gelatin residue Gltn 2 The nitrogen atom (N) directly bonded to is derived from the ε-amino group of lysine (Lys) in the raw gelatin.

[0057] Here, the ratio of the content (molar concentration) of vinyl sulfone groups in vinyl sulfonated gelatin to the content (molar concentration) of amino groups (primary amino groups, -NH2) in raw gelatin is defined as the "vinyl sulfone group introduction rate (VS group introduction rate, VS group substitution rate) (mol%)". There is no particular limitation on the method for determining the vinyl sulfone group introduction rate, and it may be calculated from a value determined directly or indirectly using, for example, the TNBS method (2,4,6-trinitrobenzenesulfonic acid method, amino group quantification), the Ellman method (thiol group quantification), or the like. For example, it may be determined by the method described in the examples below.

[0058] The VS group introduction rate is not particularly limited, and the mechanical properties of the resulting gel can be controlled by changing the VS group introduction rate. If the VS group introduction rate is too high, the water solubility decreases, making it difficult to form a gel (hydrogel). From the viewpoint of facilitating the formation of a gel and increasing the gel strength and / or adhesive strength to biological tissue, the VS group introduction rate may be, for example, 20 mol% to 80 mol%, 40 mol% to 80 mol%, or 50 mol% to 70 mol%.

[0059] The molecular weight of vinylsulfonated gelatin is not particularly limited, and is determined by the molecular weight of the second biopolymer (raw gelatin) and the type and amount (number) of the introduced group. Therefore, the range of the weight average molecular weight (Mw) of vinylsulfonated gelatin is almost the same as the range of the weight average molecular weight (Mw) of the second biopolymer (raw gelatin).

[0060] The raw gelatin for the vinyl sulfonated gelatin may be the same type as the raw gelatin for the sulfonated gelatin, or may be a different type. From the viewpoints of raw material management and efficient synthesis of modified gelatin, it is preferable that the raw gelatin for the sulfonated gelatin and the raw gelatin for the vinyl sulfonated gelatin are the same type.

[0061] The gelling agent of the present embodiment may contain only one type of vinyl sulfonated gelatin, or may contain two or more types of vinyl sulfonated gelatin.

[0062] The method for producing vinylsulfonated gelatin (synthesis method) is not particularly limited, and it can be synthesized by a known method. For example, as shown in FIG. 6, thiolated gelatin may be synthesized first, and then vinylsulfone groups may be introduced by reacting divinylsulfone with the thiol groups of the thiolated gelatin. The thiolated gelatin may be synthesized by the above-mentioned method. The method for introducing vinylsulfone groups to thiol groups is not particularly limited, but for example, 150 mol% to 300 mol%, preferably 180 mol% to 250 mol% of divinylsulfone may be mixed with respect to 100 mol% of the thiol groups of the thiolated gelatin in a solvent, and reacted at 20°C to 70°C for 10 hours to 30 hours. After the reaction, reduction, reprecipitation, filtration, dialysis, drying under reduced pressure, etc. may be performed as necessary to obtain a powder of vinylsulfonated gelatin.

[0063] The method for producing vinylsulfonated gelatin is not limited to the above-mentioned method. For example, raw gelatin may be used instead of thiolated gelatin, and vinylsulfone groups may be introduced by reacting divinylsulfone with hydroxyl groups contained in the raw gelatin (see FIG. 6).

[0064] In the synthesis of vinylsulfonated gelatin, as described above, it is preferable to mix and react an excess equivalent (150 mol% or more, preferably 180 mol% or more) of divinylsulfone with the thiol group of thiolated gelatin (or the hydroxyl group of raw gelatin). This can suppress the side reaction of the reaction of both of the two vinyl groups of divinylsulfone with the thiol group (or the hydroxyl group), and can efficiently introduce the vinylsulfone group into the thiol group. On the other hand, since divinylsulfone has cytotoxicity, it is preferable to use a small amount. By setting the mixing ratio of divinylsulfone to the thiol group (or the hydroxyl group) to 300 mol% or less, preferably 250 mol% or less, the divinylsulfone concentration in the obtained gel can be kept low. In order to further suppress the divinylsulfone concentration in the gel, it is preferable to purify the vinylsulfonated gelatin by dialysis after synthesis.

[0065] When synthesizing vinylsulfonated gelatin via thiolated gelatin, vinylsulfone groups may be introduced to all thiol groups, or vinylsulfone groups may be introduced to only some of the thiol groups. However, when thiol groups remain in the vinylsulfonated gelatin, the thiol group introduction rate is preferably, for example, 10 mol% or less, or 5 mol% or less. By suppressing the thiol group introduction rate low, unnecessary reactions (intramolecular crosslinking) before using a gelling agent can be suppressed.

[0066] In vinylsulfonated gelatin, only vinylsulfone groups (VS) may be introduced to the amino groups of raw gelatin, or thiol groups (SH) may be introduced as described above. Furthermore, other functional groups may be introduced within the range in which the effects of the present invention are achieved. However, the vinylsulfonated gelatin (VS polymer) of this embodiment does not have a ureidopyrimidinone group (UPy group). This can promote phase separation between the first phase 21 and the second phase 22 in the gel composition 100 (see FIG. 1).

[0067] 3. Gelling agent and gel composition The gelling agent of the present embodiment includes two types of polymers, a first polymer having the above-mentioned ureidopyrimidinone group (UPy group) and a second polymer having no UPy group. The inventors of the present application found that when a first polymer having a UPy group and a second polymer having no UPy group are mixed in an aqueous solvent (e.g., a buffer solution), a homogeneous phase is not formed, and fine liquid-liquid phase separation occurs between a phase containing the first polymer and a phase containing the second polymer, and the present invention was achieved by applying this phenomenon. By gelling the sol solution that has undergone liquid-liquid phase separation, as shown in FIG. 1, a gel composition 100 having a fine phase-separated structure of a first phase 21 containing the first polymer and a second phase 22 containing the second polymer is obtained. The mechanism by which the fine phase-separated structure is formed is unclear, but it is presumed to be due to hydrogen bonding between the UPy groups of the first polymer as shown in FIG. 2. In the gelling agent aqueous solution, the first polymers strongly interact with each other, and therefore the compatibility with the second polymer decreases. It is believed that the polymers that do not mix with each other form micro regions and undergo phase separation. In the gelling agent of this embodiment, only the second polymer has a crosslinkable group, so only the second phase 22 has a crosslinked structure. The first phase 21 that does not have a crosslinked structure is water-soluble, and is dissolved and removed when placed under a moist condition (for example, in a living body), forming voids. In this way, by using the gelling agent of this embodiment, a porous gel composed of the second gel 22 having a crosslinked structure can be produced.

[0068] The second phase 22 has a chemically crosslinked structure and is a gel (may be referred to as "second gel") at temperatures near human body temperature (e.g., 37°C). On the other hand, the first phase 21 may be a gel (may be referred to as "first gel") or in a sol state at temperatures near human body temperature (e.g., 37°C).

[0069] The gel composition obtained has high mechanical strength and strong adhesiveness to a living body, compared with a gel composition that does not have a phase separation structure (i.e., a homogeneous phase). The mechanism behind this is unclear, but is speculated as follows. If the gel composition were a homogeneous phase, the entire gel composition would be chemically crosslinked only by the second polymer having a crosslinking group, and the crosslink density would decrease. It is speculated that the mechanical strength and adhesiveness would also decrease accordingly. In contrast, since the gel composition 100 of this embodiment has a phase separation structure, the crosslinked structure is concentrated in the second gel 22, and the crosslink density is high. Since the phase separation occurs throughout the gel composition 100 on a fine scale (micro-order scale), the second gel 22 having a high crosslink density is arranged throughout the gel composition 100. As a result, it is speculated that the mechanical strength and adhesiveness of the entire gel composition 100 are improved.

[0070] In addition, when the gel composition is made porous, the uncrosslinked first phase is removed, and the second phase having a high crosslink density remains. Therefore, it is presumed that the porous gel composition also has high mechanical strength and adhesiveness. In addition, although the mechanical strength of a porous gel generally tends to decrease when a solid porogen is used, the gel composition 100 of the present embodiment is made porous by utilizing liquid-liquid phase separation, and therefore can maintain high mechanical strength.

[0071] The fine phase separation structure of the gel composition 100 is not particularly limited, and examples thereof include a fiber (fibrous) structure, a gyroid structure, a droplet structure, a cylinder (columnar) structure, and a lamellar (plate) structure. Here, the fiber structure means a structure in which either one or both of the first and second phases extend in a fibrous shape with a diameter of about several μm (diameter less than 10 μm) to form a network. The gyroid structure means a structure in which either one or both of the first and second phases extend in a branch-like (bone-like) shape thicker than a fiber shape (diameter 10 μm or more) to form a network. The first and second phases of the fiber structure and the gyroid structure are both continuous phases. On the other hand, the droplet structure is a so-called sea-island structure in which one of the first and second phases (sea, continuous phase) is dispersed in the other phase (island, discontinuous phase).

[0072] When the gel composition 100 is used as a cell scaffold material, the phase-separated structure preferably includes a fiber (fibrous) structure or a gyroid structure, and more preferably includes a fiber (fibrous) structure. The fiber (fibrous) structure and the gyroid structure can form connecting holes when the gel composition is made porous, and can promote the diffusion of nutrients and oxygen and the migration of cells through the connecting holes. In particular, the fiber structure can form many connecting holes of a moderate size (narrowness) (diameter: about several micrometers, about 1 μm to 9 μm), so that excessive outflow of the medium can be prevented compared to the gyroid structure (diameter of connecting hole: about several tens of micrometers, 10 μm to 99 μm), and cell culture can be further promoted.

[0073] What type of phase-separated structure is formed in the gel composition is determined by the types of the first polymer and the second polymer (types of biopolymers used as raw materials, introduction rates of each functional group, types of crosslinking groups, molecular weight, etc.), the composition of the gel composition (ratio of the first polymer to the second polymer, gelling agent concentration, etc.), etc. Those skilled in the art can select a gelling agent that forms a desired phase-separated structure based on the phase diagram shown in Figure 8, for example, as described in the Examples below.

[0074] In the gelling agent of this embodiment and the resulting gel composition, the ratio of the first polymer to the second polymer is not particularly limited, and for example, the ratio (PM1 / PM2) of the mass of the first polymer (PM1) to the mass of the second polymer (PM2) can be 1 / 10 to 100 / 10, or 5 / 10 to 20 / 10.

[0075] The gelling agent of the present embodiment may be composed of only the first polymer and the second polymer. The gelling agent of the present embodiment and the resulting gel composition may contain other components as necessary. For example, the other components may contain a solvent containing water (hereinafter, may be referred to as "aqueous solvent" as appropriate). By dissolving the first polymer and the second polymer in an aqueous solvent, the gelling agent of the present embodiment can be used as an injectable gel. In addition, when the second polymer contains an ethylenically unsaturated group as a crosslinkable group, it is preferable to contain a polymerization initiator to promote the crosslinking reaction.

[0076] Depending on the application, the gelling agent of the present embodiment and the resulting gel composition may contain various additives as other components. For example, when the gel composition is used as a medical material such as a drug delivery carrier (local delivery carrier, sustained release delivery carrier) or a vaccine carrier, the gelling agent may contain a drug or a protein. Examples of the drug and protein include anticancer drugs, anti-inflammatory drugs, antithrombotic drugs, antibiotics, biological preparations, growth factors such as fibroblast growth factor, vascular endothelial growth factor, and hepatocyte growth factor, and antigen proteins (vaccines) for viruses and cancers.

[0077] Furthermore, the gelling agent of the present embodiment and the resulting gel composition may contain a decellularized matrix, which is a matrix structure of biological tissue, as another component (additive). By containing extracellular matrix components remaining after removing cellular components from organs or tissues of animals such as pigs and cows, biological functions can be imparted to the resulting gel composition. As the decellularized matrix, for example, a decellularized matrix prepared from an organ such as the bladder, heart, liver, pancreas, or small intestine can be used. A gel composition containing such an additive such as a decellularized matrix can be used as a cell scaffold material, a medical material, or the like.

[0078] Furthermore, the gelling agent of the present embodiment and the resulting gel composition may contain other general-purpose additives, such as colorants, pH adjusters, and preservatives, within the scope of the effects of the present invention.

[0079] The gel composition (typically, a hydrogel) obtained in this embodiment contains a gelling agent, an aqueous solvent, and, if necessary, other additives, as described above. The type of aqueous solvent will be described later. The total content of the first polymer and the second polymer in the gel composition is not particularly limited, but may be, for example, 1% by mass to 20% by mass, and preferably 5% by mass to 10% by mass, from the viewpoint of increasing the strength of the gel composition.

[0080] The method of using the gelling agent of this embodiment (i.e., the method of producing a gel composition) is not particularly limited, and may involve mixing a first polymer having a UPy group, a second polymer having a crosslinkable group, and an aqueous solvent. When the crosslinkable group is an SH group or a VS group, a click reaction (crosslinking reaction) occurs in the second polymer by mixing, and a gel composition (typically, a hydrogel) is produced. When the crosslinkable group is an ethylenically unsaturated group capable of radical polymerization, the mixture (for example, a sol solution) may be irradiated with UV light to promote gelation (polymerization). The gelling agent of the present embodiment can also be used as an injectable agent, as described below.

[0081] <Injectable gel> The gelling agent of the present embodiment can be dissolved in an aqueous solvent and used as an injectable gel (gelling agent solution). The following describes a case where the second polymer is composed of an SH polymer having a thiol group introduced therein and a VS polymer having a vinyl sulfone group introduced therein.

[0082] In this case, the injectable gel is preferably composed of three types of agents (compositions, solutions): a first agent in which a first polymer is dissolved in an aqueous solvent, a second agent SH in which an SH polymer is dissolved in an aqueous solvent, and a second agent VS in which a VS polymer is dissolved in an aqueous solvent. By making each polymer into a separate composition (solution), it is possible to suppress the gelation reaction (crosslinking reaction) outside the body before use.

[0083] The aqueous solvent used in the first agent, the second agent SH, and the second agent VS is not particularly limited as long as it is a solvent containing water, and examples thereof include ultrapure water, physiological saline, buffer solutions of various inorganic salts such as boric acid, phosphoric acid, and carbonate, or mixtures thereof. When it is assumed that the obtained gel will be used for cell culture, etc., the aqueous solvent is preferably a buffer solution. In addition, the aqueous solvents used in the first agent, the second agent SH, and the second agent VS may be different types of solvents, but are preferably the same type of solvent.

[0084] The first agent, the second agent SH, and the second agent VS may each be composed only of the respective polymers and an aqueous solvent, or may contain other additives that may be contained in the gel composition described above, if necessary.

[0085] The concentration of the UPy polymer in the first agent may be, for example, 1% by mass to 20% by mass, or 5% by mass to 10% by mass. When the concentration of the UPy polymer in the first agent is 5% by mass or more, or 7% by mass or more, a gel phase-separated structure having a fiber structure is easily obtained.

[0086] The concentration of the SH polymer in the second part SH may be, for example, 1 to 20 mass %, or 5 to 10 mass %. The concentration of the VS polymer in the second part VS may be, for example, 1 to 20 mass %, or 5 to 10 mass %.

[0087] From the viewpoint of facilitating the formation of a uniform gel, it is preferable that the concentration of the UPy polymer in the first agent (C1) and the sum (C2) of the concentration of the SH polymer in the second agent SH and the concentration of the VS polymer in the second agent VS are close to each other, for example, the ratio (C1) / (C2) may be 0.8 to 1.2.

[0088] In the injectable gel, the volume ratios of the first agent, the second agent SH, and the second agent VS may be appropriately adjusted depending on the introduction rate of the functional groups (Upy group, SH group, VS group) of each polymer, etc., but from the viewpoint of facilitating the formation of a uniform gel, it is preferable that the volume (V1) of the first agent and the total volume (V2) of the second agent SH and the second agent VS are close to each other. For example, the ratio (V1) / (V2) may be 0.8 to 1.2.

[0089] The injectable gel may be used, for example, as follows. First, immediately before use, each polymer is dissolved in an aqueous solvent to prepare a first agent, a second agent SH, and a second agent VS. The preparation method is not particularly limited, and they can be prepared by mixing them by a known method. At this time, the pH and temperature of the first agent, the second agent SH, and the second agent VS may be, for example, pH=6.0 to 8.5, and temperature: 30°C to 60°C. Next, these three types of drugs (solutions) are applied (injected) to a target part (affected part) in a living body using a syringe or the like. As a result, the three types of drugs come into contact with each other at the target part, and a crosslinking reaction (click reaction between the SH group and the VS group) immediately occurs to produce a gel composition (see Figures 1 and 4).

[0090] The injectable gel may be applied to the target portion using, for example, a triple syringe type dispenser or sprayer capable of mixing three types of drugs at the tip. Alternatively, the three liquids may be mixed and then injected using a syringe. After application of the injectable gel, it is preferable to leave it for, for example, 1 to 30 minutes to allow it to gel sufficiently. The higher the pH, the shorter the curing time tends to be, so the curing time can be adjusted by the pH depending on the application of the resulting gel.

[0091] In the above, the injectable gel composed of three kinds of drugs in the case where the crosslinkable group is a functional group capable of a click reaction (SH group, VS group) has been described, but the present embodiment is not limited to this. For example, when the crosslinkable group is an ethylenically unsaturated group, a sudden reaction such as a click reaction does not occur. Therefore, the injectable gel can be used as a one-liquid composition (solution) in which all the constituent materials are mixed.

[0092] 4. Uses of the gel composition The gel composition obtained by using the gelling agent of the present embodiment is derived from a biopolymer, and therefore has high biocompatibility, biodegradability, and cell adhesiveness. In addition, since the gel composition has a phase separation structure, it has high mechanical strength and adhesiveness, and furthermore, when placed under a wet condition (for example, in a living body), it becomes porous. Therefore, the gel composition of the present embodiment is excellent, for example, as a cell scaffold material. For example, by applying the gel of the present embodiment containing cells to a diseased site or a tissue defect site, tissue regeneration of the defect in the affected area can be promoted. Therefore, the gel composition (cell scaffold material) of the present embodiment can be used as a medical material for wound covering, muscle tissue regeneration, bone regeneration, nerve regeneration, ischemia treatment, diabetes treatment, heart disease treatment, and the like. Furthermore, by culturing cancer cells using the gel composition of the present embodiment, it becomes possible to construct a cancer tissue model, and application to drug discovery screening can also be expected.

[0093] [Second embodiment] In the first embodiment, the first polymer has a UPy group and the second polymer has a crosslinkable group, but the present invention is not limited to this form. In the present embodiment, the first polymer has both a UPy group and a crosslinkable group, and the second polymer has neither a UPy group nor a crosslinkable group. The following mainly describes the differences between this embodiment and the first embodiment. Therefore, the points that are not described in this embodiment are the same as those in the first embodiment.

[0094] 1. First polymer The first polymer of this embodiment is a polymer in which a UPy group and a crosslinkable group are introduced into a first biopolymer. The first biopolymer, the Upy group, and the crosslinkable group are the same as those in the first embodiment, and preferred embodiments are also the same.

[0095] The first polymer may be composed of only one type of polymer, or may contain two or more types of polymers. When the crosslinkable groups are SH and VS groups, the first polymer preferably contains two types of polymers: a UPy-SH polymer in which a UPy group and an SH group are introduced into the first biopolymer, and a UPy-VS polymer in which a UPy group and a VS group are introduced into the first biopolymer.

[0096] In the UPy-SH polymer, from the viewpoint of increasing the strength of the gel composition, the molar ratio of the thiol group (SH), the ureidopyrimidinone group (UPy), and the primary amino group (A) may be, for example, (SH) / (UPy) / (A)=(40-60) / (35-55) / (5-20).

[0097] In the UPy-VS polymer, from the viewpoint of increasing the strength of the gel composition, the molar ratio of the vinyl sulfone group (VS), the ureidopyrimidinone group (UPy), and the primary amino group (A) may be, for example, (VS) / (UPy) / (A)=(40-60) / (35-55) / (5-20).

[0098] The concentration (introduction rate of functional groups) of the functional groups (UPy groups, SH groups, VS groups) in each polymer can be obtained by the method described in the first embodiment, and the molar ratio of the functional groups in each of the above-mentioned polymers can be calculated therefrom.

[0099] The manufacturing method (synthesis method) of the UPy-SH polymer and the UPy-VS polymer is not particularly limited, and they can be synthesized by a known method. For example, first, a ureidopyrimidinone group is introduced into the amino group of a first biopolymer (e.g., gelatin) by the method described in the first embodiment to synthesize a UPy-polymer (see FIG. 3). A UPy-SH polymer is obtained by introducing a thiol group into the UPy-polymer by the method described in the first embodiment. Also, a UPy-VS polymer is obtained by introducing a vinyl sulfone group into the UPy-polymer by the method described in the first embodiment.

[0100] 2. Second polymer The second polymer of this embodiment can be the second biopolymer described in the first embodiment. The second polymer may be a derivative of the second biopolymer to which the UPy group and the crosslinkable group are not introduced. The second polymer may be composed of only one type of polymer, or may contain two or more types of polymers.

[0101] 3. Gelling agent and gel composition The gelling agent of this embodiment contains a first polymer having both a UPy group and a crosslinkable group, a second polymer having neither a UPy group nor a crosslinkable group, and, if necessary, other additives. The other additives include the same ones as in the first embodiment, and the preferred embodiments are also the same. The gelling agent of this embodiment can also be used as an injectable gel, like the first embodiment.

[0102] When the gelling agent of this embodiment is mixed with an aqueous solvent, it does not become a homogeneous phase, and fine liquid-liquid phase separation occurs between a phase containing a first polymer and a phase containing a second polymer. By gelling this liquid-liquid phase-separated sol solution, a gel composition having a fine phase-separated structure of a first phase containing a first polymer and a second phase containing a second polymer is obtained. In this embodiment, the first phase having UPy groups has a crosslinked structure. By placing it under a wet condition (for example, in a living body), the second phase not having a crosslinked structure is dissolved and removed to form voids. In this way, by using the gelling agent of this embodiment, a porous gel consisting of a first phase (first gel having UPy groups) having a crosslinked structure can be produced.

[0103] The gel composition of this embodiment can also take various forms such as a fiber (fibrous) structure, a gyroid structure, etc., as in the first embodiment, and as will be described in the examples below, those skilled in the art can select a gelling agent capable of forming a desired phase separation structure based on a phase diagram (FIG. 8) represented by the ratio of the first polymer and the second polymer, etc. The gel composition of this embodiment has the same effects as the gel composition of the first embodiment, and can be used for the same purposes. EXAMPLES

[0104] The present invention will be described below with reference to examples, but the present invention is not limited to these.

[0105] [Experiment 1] In experiment 1, two types of polymers, gelatin with UPy groups introduced (UPy-modified pig tendon gelatin or UPy-modified pig skin gelatin) and a biopolymer without UPy groups introduced, were mixed in an aqueous solvent and gelled, and the phase separation phenomenon of the gel composition was observed. In addition, no crosslinkable group was introduced into any of the gelatins used in this experiment. However, the presence or absence of a crosslinkable group does not have a significant effect on the phase separation of the gel composition. Therefore, it is assumed that the same results as those of this experiment shown below will be obtained even if either one of the two types of polymers to be mixed has a crosslinkable group.

[0106] 1. Synthesis of UPylated gelatin <UPy-functionalized porcine tendon gelatin> First, a ureidopyrimidinone derivative having an isocyanate group at the end, as shown in Fig. 3, was synthesized. 2-Amino-4-hydroxy-6-methylpyrimidine (17.4 mmol, 2.178 g, Sigma-Aldrich) was dispersed in 1,6-diisocyanatohexane (78.3 mmol, 13.163 g, Tokyo Chemical Industry Co., Ltd.), heated to 100 °C for reaction, and continuously stirred for 16 hours. After cooling to 25 °C, 10 volumes of n-hexane were added to precipitate the product. The obtained precipitate was filtered and collected, and washed three times with n-hexane. The product was dried under reduced pressure at 50 °C to obtain a ureidopyrimidinone derivative (molecular weight 293) having an isocyanate group at the end as a white powder.

[0107] Next, the raw material gelatin (porcine tendon gelatin, Tendon gelatin, TG, weight average molecular weight: 340,000, Nitta Gelatin Inc.) was dissolved in dimethyl sulfoxide at 50 °C and cooled to room temperature. Also, the synthesized ureidopyrimidinone derivative was dissolved in dimethyl sulfoxide. A predetermined amount of the ureidopyrimidinone derivative solution (0.45 molar equivalent of UPy groups relative to 1 molar equivalent of the amino group amount of the raw material gelatin (293 μmol / g, measured by the TNBS method)) was added to the gelatin solution, mixed, and stirred. UPy-functionalized gelatin was synthesized by stirring at 20 - 30 °C for 16 - 24 hours (see Fig. 3). After completion of the reaction, reprecipitation treatment was performed with a mixed solvent of ethanol and ethyl acetate, washed with cold chloroform and cold ethanol, and then dried under reduced pressure to obtain the target powder.

[0108] <Measurement of UPy group introduction rate> The UPy group introduction rate was calculated by the following formula from the values obtained by quantifying the primary amino group concentration (mol / g) of the raw material gelatin and the primary amino group (remaining amino group) concentration (mol / g) of the UPy-functionalized porcine tendon gelatin by the TNBS method. The UPy group introduction rate was 42 mol%.

[0109] UPy group introduction rate (mol%) =[(Primary amino group concentration of raw gelatin) - (Primary amino group concentration of UPy - modified porcine tendon gelatin)] / (Primary amino group concentration of raw gelatin) × 100

[0110] <UPy - modified porcine skin gelatin> As the raw gelatin, except that porcine skin gelatin (Skin - derived gelatin, SG, weight - average molecular weight: 180,000, Nitta Gelatin Inc.) was used instead of porcine tendon gelatin, UPy - modified porcine skin gelatin was synthesized by the same method as the above - mentioned UPy - modified porcine tendon gelatin. When the UPy group introduction rate was calculated by the above - mentioned method, the UPy group introduction rate was 42 mol%.

[0111] 2. Observation of phase - separation phenomenon of gel composition Two kinds of polymers, UPy - modified gelatin (UPy - modified porcine tendon gelatin or UPy - modified porcine skin gelatin) and porcine skin gelatin, were mixed and gelled in the presence of an aqueous solvent, and the phase - separation phenomenon of the gel composition was observed by a confocal laser scanning microscope (CLSM). The UPy - modified porcine tendon gelatin was fluorescently labeled with Cy5.5, and the UPy - modified porcine skin gelatin was fluorescently labeled with fluorescein.

[0112] <Observation 1> The gel composition was prepared by the following procedure. The synthesized UPy - modified porcine tendon gelatin was dissolved in phosphate - buffered saline (PBS, pH = 7.4) at 100 mg / mL (= 10 wt%), heated at 50 °C, and then the pH was adjusted to 7.0 - 8.0. Next, porcine skin gelatin was dissolved in PBS (pH = 7.4) at 100 mg / mL (= 10 wt%), heated at 50 °C, and then the pH was adjusted to 7.0 - 8.0. Each gelatin solution was heated at 37 °C for 30 minutes and mixed in equal amounts. The mixed solution was dropped onto a substrate and allowed to stand for 1 - 30 minutes and cooled to below the sol - gel transition temperature (30 - 35 °C) to obtain Gel Composition 1. The porcine skin gelatin used for the preparation of Gel Composition 1 was the same as the raw gelatin used for the synthesis of UPy - modified porcine skin gelatin.

[0113] In addition, gel composition A was prepared using only pig skin gelatin, and gel composition B was prepared using only UPy pig tendon gelatin by the same method. The observation results of gel compositions A, B, and 1 are shown in Figures 7(a) to (c).

[0114] In Fig. 7(c), the dark colored portion is the phase containing UPy-modified porcine tendon gelatin. As shown in Fig. 7(c), in gel composition 1, which was prepared by mixing UPy-modified porcine tendon gelatin and pig skin gelatin, the phase containing UPy-modified porcine tendon gelatin extended in a fibrous form, forming a phase-separated structure (fiber structure). On the other hand, as shown in Figs. 7(a) and (b), in gel compositions A and B, uniform fluorescence was observed throughout, and no phase-separated structure was observed.

[0115] <Observation 2> The concentration of UPylated porcine tendon gelatin in the PBS solution was varied in the range of 1 wt% to 10 wt%, and the concentration of pig skin gelatin in the PBS solution was varied in the range of 1 wt% to 10 wt%, and multiple gel compositions were prepared in the same manner as for the above-mentioned gel composition 1, and the phase separation phenomenon of these gel compositions was observed. The relationship between the gelatin solution concentration in each sample and the observation results is shown in Figure 8.

[0116] As shown in Figure 8, by changing the concentration of each gelatin solution, the phase separation structure of the resulting gel composition changed (fiber structure, gyroid structure, droplet structure). This confirmed that the phase separation structure can be controlled by changing the concentration of each gelatin solution and / or adjusting the composition of the gel composition.

[0117] <Observation 3> As shown in Table 1, two types of polymers, UPy-modified gelatin and a biopolymer having no UPy group, were combined to prepare gel compositions 2 to 9 in the same manner as gel composition 1, and the phase separation phenomenon of each gel composition was observed. The observation results of gel compositions 1 to 9 are shown in Figure 9. As shown in Figure 9, gel compositions with various phase separation structures were obtained by changing the combination of UPy-modified gelatin and biopolymer used.

[0118] [Table 1]

[0119] In gel compositions 1 to 7 (No. 1 to No. 7) in FIG. 9, the light-colored parts are phases containing UPy-modified gelatin. In gelatin composition 2, which is a combination of pig tendon gelatin and UPy-modified pig tendon gelatin, both of which have the same origin, and gelatin 5, which uses cod skin gelatin, a phase-separated structure (fiber structure) with very small domains was formed. In contrast, in gel compositions 3 and 4, which used UPy-modified pig skin gelatin, a phase-separated structure (gyroid structure) with large domains was formed. In addition, in gel composition 6, which used bovine bone gelatin, a fiber structure with a domain size similar to that of gel composition 1, which used pig skin gelatin, was obtained. In addition, in gel composition 7, which used bovine serum albumin instead of gelatin as a biopolymer, a fiber structure was also formed.

[0120] In addition, in gel composition 8, which used hyaluronic acid instead of gelatin as a biopolymer, the gel containing hyaluronic acid and the gel containing UPy pig tendon gelatin each formed a fibrous phase (fiber) and separated, forming a network structure in which these two types of fibers were mixed. Gel composition 9, which used chondroitin sulfate instead of gelatin as a biopolymer, also formed a network structure in which two types of fibers were mixed. In gel compositions 8 to 9 (No. 8 to No. 9) in Figure 9, a network structure of two types of fibrous phases can be confirmed in the light-colored areas.

[0121] <Observation 4> Gel compositions 1-1, 1-2, and 1-3 were prepared under the same conditions as gel composition 1 (weight average molecular weight: 180,000), except that the weight average molecular weight of the pig skin gelatin was set to 20,000, 60,000, and 120,000, and the phase separation phenomenon of each gel composition was observed. The observation results of gel compositions 1 and 1-1 to 1-3 are shown in Figure 10. In gel compositions 1 and 1-1 to 1-3 (No. 1 and No. 1-1 to 1-3) in Figure 10, the light-colored parts are phases containing UPy-modified gelatin.

[0122] As shown in Figure 10, a phase separation structure was confirmed in gel compositions 1-2, 1-3, and 1, in which the weight-average molecular weight of the pig skin gelatin was 60,000 or more. This is presumably because the larger the molecular weight of the gelatin, the lower the solubility and the easier it is to undergo phase separation. From this result, it is presumed that a relatively large weight-average molecular weight of the polymer can efficiently form a phase separation structure.

[0123] <Observation 5> Gel compositions 1-4 to 1-6 were prepared under the same conditions as gel composition 1 (UPy group introduction rate: 42%), except that the UPy group introduction rate in the UPy-modified porcine tendon gelatin was set to 0% (i.e., porcine tendon gelatin), 26%, and 53%, and the phase separation phenomenon of each gel composition was observed. The observation results of gel compositions 1 and 1-4 to 1-6 are shown in Figure 11. In gel compositions 1 and 1-4 to 1-6 (No. 1 and No. 1-4 to 1-6) in Figure 11, the dark colored parts are phases containing UPy-modified gelatin.

[0124] As shown in Figure 11, a phase-separated structure was confirmed in gel compositions 1 and 1-6, in which the UPy group introduction rate of the UPy-modified porcine tendon gelatin was 40% or more. This is presumed to be because the higher the UPy group introduction rate, the stronger the cohesive force of the UPy-modified porcine tendon gelatin becomes, making it easier to separate into phases. From this result, it is presumed that a relatively high UPy group introduction rate can efficiently form a phase-separated structure.

[0125] From the results of Observations 1 to 5 described above, it was confirmed that the phase separation structure in the gel composition is affected by the concentration of the gelatin solution, the polymer composition in the gel composition, the type of biopolymer from which the modified polymer is derived, the molecular weight, the introduction rate of the UPy group, etc. It is presumed that the phase separation structure can be controlled by adjusting these parameters.

[0126] [Experiment 2] In experiment 2, a gel composition was prepared using a gelling agent consisting of a first polymer having a UPy group and a second polymer not having a UPy group, in which only one of the first polymer and the second polymer had a crosslinkable group.

[0127] 1.Synthesis of SH-gelatin Raw gelatin (pig skin-derived gelatin, SG, weight-average molecular weight: 180,000, Nitta Gelatin Co., Ltd.) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 60 mg / mL and heated at 50°C. A predetermined amount of γ-thiobutyrolactone (Sigma-Aldrich) was added to the solution, mixed, and reacted by stirring at 50°C for 16 to 24 hours (see Figure 5). The mixing ratio of raw gelatin and γ-thiobutyrolactone was adjusted so that γ-thiobutyrolactone was 2 molar equivalents per molar equivalent of the amino group amount of raw gelatin (350 μmol / g, quantified by TNBS method).

[0128] The solution after the reaction was cooled to room temperature, and tris(2-carboxyethyl)phosphine was added at a concentration of 0.2 mg / mL and reduced by stirring for 30 minutes. The solution was then slowly dropped into a 20-fold amount of a mixed solvent of cold ethanol / cold ethyl acetate (volume ratio 1:1) while stirring to cause reprecipitation. The solution was filtered using a glass filter, washed with chloroform and ethanol, and dried under reduced pressure to recover a white powder of thiolated gelatin.

[0129] <Measurement of thiol group introduction rate> The primary amino group concentration (mol / g) of the raw gelatin was quantified by the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method), and the thiol group concentration (mol / g) of the thiolated gelatin was quantified by the Ellman method. The thiol group introduction rate was calculated from the obtained values ​​using the following formula. The thiol group introduction rate was 64 mol%. Thiol group introduction rate of thiolated gelatin (mol%) = (thiol group concentration of thiolated gelatin) / (primary amino group concentration of raw gelatin) x 100

[0130] 2. Synthesis of VS-gelatin The synthesized thiolated gelatin was dissolved in ultrapure water at a concentration of 10 mg / mL and heated at 50°C. A predetermined amount of divinyl sulfone (Tokyo Chemical Industry Co., Ltd.) was added to the solution (2 molar equivalents of divinyl sulfone per 1 molar equivalent of thiol group), mixed, and reacted at 50°C for 16 to 24 hours by stirring (see Figure 6). The solution after the reaction was cooled to room temperature, and tris(2-carboxyethyl)phosphine was added at a concentration of 0.2 mg / mL, and reduced by stirring for 30 minutes. The solution was then placed in a dialysis membrane (molecular weight cutoff 12,000 to 15,000), dialyzed for 3 days, and freeze-dried to recover a white powder of vinylsulfonated gelatin.

[0131] <Measurement of vinyl sulfone group introduction rate> The primary amino group concentration (mol / g) of raw gelatin was quantified by the TNBS method, and the thiol group concentration (mol / g) of thiolated gelatin and the thiol group concentration (mol / g) of vinylsulfonated gelatin were quantified by the Ellman method, and the vinylsulfone group introduction rate was calculated from the obtained values ​​using the following formula. The vinylsulfone group introduction rate was 60 mol%. Vinyl sulfonate group introduction rate (mol%) of vinyl sulfonated gelatin = [(thiol group concentration of SH gelatin) - (thiol group concentration of VS gelatin)] / (primary amino group concentration of raw gelatin) x 100

[0132] 3.Synthesis of UPy-SH-modified gelatin A thiol group was further introduced to the primary amino group of the previously synthesized UPy-modified porcine tendon gelatin by the same method as the above-mentioned SH-modified gelatin, to synthesize UPy-SH-modified gelatin. The mixing ratio of the Upy-modified gelatin and γ-thiobutyrolactone was adjusted so that γ-thiobutyrolactone was 2 molar equivalents per 1 molar equivalent of the amino group of the Upy-modified gelatin. The thiol group introduction rate was measured by the above-mentioned method. The thiol group introduction rate was 32 mol%.

[0133] 4. Synthesis of UPy-VS-modified gelatin Instead of SH-gelatin, the synthesized UPy-SH-gelatin was used to introduce VS groups in the same manner as the above-mentioned VS-gelatin, to synthesize UPy-VS-gelatin. The vinyl sulfone group introduction rate was measured by the above-mentioned method. The vinyl sulfone group introduction rate was 30 mol%.

[0134] 5. Preparation and evaluation of gel composition (1) Evaluation of time dependence of viscoelasticity The synthesized gelatins were combined as shown in Table 2 to prepare a gelling agent 10. As shown in Fig. 15(a), in the gel composition 10 formed by the gelling agent 10, a structure (fiber structure) was confirmed in which the first gel 21 containing UPy-gelatin (first polymer) extended in a fibrous form from the second gel 22 containing a crosslinked product of SH-gelatin and VS-gelatin (second polymer) and was phase-separated.

[0135] [Table 2]

[0136] The viscoelasticity of the gel composition 10 prepared using the gelling agent 10 was measured by the following procedure. First, UPy-modified porcine tendon gelatin was dissolved in phosphate buffer (PBS, pH=7.4) at 100 mg / mL, heated at 50°C, and then the pH was adjusted to 7.0-8.0 (UPy-modified gelatin solution). Thiol-modified gelatin was dissolved in PBS (pH=7.4) at 100 mg / mL, heated at 50°C, and then the pH was adjusted to 7.5-8.2 (SH-modified gelatin solution). Vinyl sulfonated gelatin was dissolved in PBS (pH=7.4) at 100 mg / mL, heated at 50°C, and then the pH was adjusted to 6.0-7.0 (VS-modified gelatin solution). Each solution was heated at 37° C. for 30 minutes and mixed in a ratio of (UPy-modified gelatin solution):(SH-modified gelatin solution):(VS-modified gelatin solution)=2:1:1 (volume ratio) to prepare a mixed solution (pre-gel solution).

[0137] 100 μL of the prepared mixture (pre-gel solution) was dropped onto a substrate, which was then placed on the stage of a viscoelasticity measuring device (Rheoplus, Anton Paar) and clamped with a jig having a diameter of 10 mm. The time dependence (1 to 30 minutes) of the elastic modulus of the gel composition formed from the pre-gel was measured under the conditions of 37°C, 1% strain, and 10 rad / s. The results are shown in Figure 12.

[0138] As shown in FIG. 12, the storage modulus (G') exceeded the loss modulus (G"), and it was confirmed that a stable gel (gel composition 10) was formed after 20 minutes.

[0139] (2) Mechanical strength measurement by tensile test First, a mixture (pre-gel solution) was prepared by mixing (UPy-modified gelatin solution):(SH-modified gelatin solution):(VS-modified gelatin solution) at a volume ratio of 2:1:1 using gelling agent 10 in the same manner as described above, poured into a silicon mold of ISO37-2 size, and gelled at 37° C. for 60 minutes to obtain gel composition 10. A tensile test of gel composition 10 was performed at 25° C. using a texture analyzer. The results are shown in FIG. 13.

[0140] Next, porcine tendon gelatin (without UPy groups), the synthesized SH-modified gelatin, and VS-modified gelatin were combined as shown in Table 3 to prepare gelling agent 11. Using gelling agent 11 instead of gelling agent 10, a tensile test was carried out on gel composition 11 formed from gelling agent 11 in the same manner. The results are shown in Figure 13. It was confirmed that gel composition 11 formed a homogeneous phase and did not have a phase-separated structure.

[0141] [Table 3]

[0142] As shown in FIG. 13, Gel Composition 10 (phase separation) exhibited higher breaking strain and breaking stress (strain: 345%, stress: 70.1 kPa) compared to Gel Composition 11 (homogeneous phase).

[0143] (3) Adhesion test For Gel Compositions 10 and 11, an adhesiveness evaluation was carried out in accordance with ASTM-F2392-04R using a collagen casing as a model tissue for evaluating tissue adhesiveness.

[0144] First, a pinhole with a diameter of 3 mm was formed in a collagen casing with a diameter of 35 mm. Pre-gel solutions of gel compositions 10 and 11 were prepared in the same manner as described above, and 300 μL of the pre-gel solution was dropped onto the collagen casing. This was left to stand at 37° C. for 60 minutes, and then the pressure resistance strength was measured. The results are shown in FIG. 14. As shown in FIG. 14, gel composition 10 (phase separation) showed higher adhesive strength than gel composition 11 (homogeneous phase).

[0145] (4) Formation and observation of porous structures A porous structure was formed in gel composition 10 and observed by the following procedure. First, a pre-gel solution was prepared using gelling agent 10 by the same method as described above, and 20 μL of the pre-gel solution was added to a glass substrate and allowed to stand at 37° C. for 30 minutes. As a result, a gel composition 10 having a phase-separated structure was formed, as shown in FIG. 15(a). In FIG. 15(a), the dark colored portion is the first phase 21 containing the first polymer (UPy-modified porcine tendon gelatin).

[0146] Next, 5 mL of PBS was added to the gel composition 10, and the mixture was allowed to stand for 24 hours. After standing, as shown in Fig. 15(b), the first gel 21 containing the UPy-modified gelatin was dissolved in PBS and removed, and a porous gel consisting of the second gel 22 containing a crosslinked product of SH-modified gelatin and VS-modified gelatin was obtained. The part (dark part) indicated by the white arrow in Fig. 15(b) is the part from which the first gel 21 was removed.

[0147] (5) Evaluation of protein permeability The protein permeability of gel compositions 10 and 11 was evaluated by the following procedure. First, pre-gel solutions were prepared using gelling agents 10 and 11 in the same manner as described above, and 20 μL of the pre-gel solutions were added to a glass substrate and left to stand at 37° C. for 30 minutes to obtain gel compositions 10 and 11. 5 mL of PBS (fluorescent reagent) containing fluorescently labeled bovine serum albumin at a concentration of 1 mg / mL was added to gel compositions 10 and 11, and line scan observation was performed over time to calculate the albumin permeability of gel compositions 10 and 11. The results are shown in FIG. 16. In FIG. 16, the "albumin permeability" on the vertical axis is the relative value (%) of the fluorescence intensity of the fluorescent reagent that has permeated into the gel composition when the initial fluorescence intensity of the fluorescent reagent is taken as 100%. CLSM photographs of gel compositions 10 and 11 24 hours after the addition of the fluorescent reagent are shown in FIG. 17(a) and (b).

[0148] As shown in FIG. 16, the gel composition 10 (phase separation) had a higher albumin permeability than the gel composition 11 (homogeneous phase). This is presumably because the first gel 21 of the gel composition 10 was dissolved in PBS, causing the gel composition 10 to become porous. As shown in FIG. 17(b), fibrous connecting holes were formed in the gel composition 10 24 hours after the addition of the fluorescent reagent, and albumin 40 was confirmed therein. It is presumed that the albumin 40 diffused through these connecting holes. On the other hand, as shown in FIG. 17(a), the gel composition 11 was in a homogeneous phase even 24 hours after the addition of the fluorescent reagent, and no connecting holes were present that could serve as a passageway for albumin. From the above results, the gel composition 10 can obtain a porous structure under a wet condition (e.g., in a living body), and nutrients and oxygen can be diffused into the gel through the holes, which makes it considered to be a gel suitable for cell culture.

[0149] (6) Cellular Encapsulation Test 1 A cell encapsulation test was carried out by the method described below using gel compositions 10 and 11. Human mesenchymal stem cells were used as the cells to be evaluated.

[0150] First, a pregel solution was prepared using gelling agents 10 and 11 in the same manner as described above. 4 20 μL of pregel solution was added to each human mesenchymal stem cell, and the cells were seeded on a 24-well plate. After 20 minutes, 500 μL of DMEM medium (15% fetal bovine serum, 1% penicillin-streptomycin) was added, and the cells were cultured for 24 hours in an incubator at 37°C and 5% CO2. After culture, the samples were fixed with formalin, stained with actin and nuclei, and observed with a confocal laser scanning microscope (CLSM). Figure 18 shows a confocal laser scanning microscope (CLSM) photograph. Figure 19 shows the percentage (%) of the area of ​​the stained portion relative to the total area of ​​the fluorescent microscope photograph shown in Figure 18.

[0151] As shown in Figures 18(a) and (b), it was confirmed that human mesenchymal stem cells (stained actin and nuclei) were more adherent and spread out in gel composition 10 (phase separation) compared to gel composition 11 (homogeneous phase). As can be seen from Figure 19, gel composition 10 (phase separation) had a larger area of ​​stained parts, i.e., the area occupied by human mesenchymal stem cells, compared to gel composition 11 (homogeneous phase). This is presumably because gel composition 10 was made porous by PBS. From these results, it was confirmed that gel composition 10 is capable of encapsulating cells while maintaining a high cell viability, and also has cell adhesive properties.

[0152] (7) Cellular encapsulation test 2 Gel compositions 10, 30, 40, and 50 having various phase separation structures were prepared using gelators 10, 30, 40, and 50 shown in Table 4, and a cell encapsulation test was performed in the same manner as in the cell encapsulation test 1 described above. In addition, in all gelators shown in Table 4, only the first polymer has a UPy group. Of these, in gelators 10, 30, and 40, only the second polymer has a crosslinkable group (SH group and VS group), and in gelator 50, only the first polymer has a crosslinkable group (SH group and VS group). Fluorescence microscope photographs of each gel composition are shown in FIG. 20, and the phase separation structure of each gel composition is summarized in Table 4. In gels 10, 30 and 40 (No. 10, 30, 40) in Figure 20, the light-colored parts are porous structures formed by the second phase (cross-linked product of the second polymer, and cross-linked product of SH- and VS-modified pig skin gelatin), and in gel 50 (No. 50), the light-colored parts are porous structures formed by the first phase (cross-linked product of the first polymer, and cross-linked product of UPy-SH- and UPy-VS-modified pig tendon gelatin).

[0153] [Table 4]

[0154] When the morphology of the encapsulated human mesenchymal stem cells was observed under a fluorescent microscope, the cells were spread out and good cell adhesion was observed in gel composition 10 (fiber structure) and gel composition 50 (fiber structure). This is presumably because connected pores were formed in gel compositions 10 and 50 by making the gel porous. Note that gel composition 10 before being made porous had a phase-separated structure in which only the first phase (first polymer) extended in a fibrous form, and gel composition 50 before being made porous had a phase-separated structure in which both the first phase (first polymer) and the second phase (second polymer) extended in a fibrous form.

[0155] On the other hand, in gel composition 30 (gyroid) and gel composition 40 (droplet structure), cell spreading was less compared to gel compositions 10 and 50. It is presumed that in gel composition 30 (gyroid), the size of the connecting pores formed by porosity is large, causing the cells to flow out of the gel. It is presumed that in gel composition 40 (droplet structure), the pores formed by porosity are independent pores, and therefore the effect of promoting cell spreading was lower compared to gel compositions 10 and 50 in which connecting pores are formed.

[0156] (8) Cellular Encapsulation Test 3 Except for using cells other than human mesenchymal stem cells as evaluation cells, cell encapsulation tests were performed using gelators 10 and 11 in the same manner as in the above-mentioned cell encapsulation test 1. Four types of evaluation cells were used: fibroblasts, myoblasts, cardiomyocytes, and endothelial cells.

[0157] Confocal laser scanning microscope (CLSM) observations confirmed that, in all four types of cells, cells adhered and spread more in gel composition 10 (phase-separated) than in gel composition 11 (homogeneous phase), as in cell encapsulation test 1.

[0158] (9) Cell proliferation test First, a pregel solution was prepared using gelling agents 10 and 11 in the same manner as described above. The pregel solution was diluted to 20 μL and diluted with 1x10 4Mouse myoblasts were dispersed and seeded on a 24-well plate. After 20 minutes, 500 μL of DMEM medium (15% fetal bovine serum, 1% penicillin-streptomycin) was added, and the cells were cultured in an incubator at 37°C and 5% CO2. After 24 hours of culture, 48 hours of culture, and 72 hours of culture, the samples (gel composition) were dissolved in collagenase, and the cells were collected by centrifugation and counted using a hemocytometer. The results are shown in Figure 21.

[0159] 21, cell proliferation was more promoted in gel composition 10 (phase separation) than in gel composition 11 (homogeneous phase). From this result, it was confirmed that gel composition 10 (phase separation) promotes not only cell adhesion but also cell proliferation.

[0160] (10) Angiogenesis evaluation Gel compositions 10 and 11 and human mesenchymal stem cells were implanted into C57BL / 6J mice (6 to 8 weeks old, female) to evaluate the angiogenic function.

[0161] First, mice were anesthetized with isoflurane inhalation, disinfected with 70% ethanol, and then the skin was incised with a scalpel, the femoral artery was ligated in two places, and the blood vessels between them were cut to create an ischemia model. Pregel solutions were prepared using gelling agents 10 and 11, respectively, in the same manner as described above, and human mesenchymal stem cells (5x10 5 The gelling agent was removed and the human mesenchymal stem cells were added to the affected area of ​​the mouse, and the wound was closed by suturing the skin. The blood flow in the lower limbs of the mouse was then measured over time. The results are shown in Figure 22. For comparison, the blood flow in the lower limbs of an untreated mouse (ischemia model) and a mouse to which only the human mesenchymal stem cells were added without the gelling agent were also measured over time. The results are also shown in Figure 22.

[0162] As shown in Figure 22, when gel composition 10 (phase separation) was used, a significant improvement in blood flow was confirmed. On the other hand, when gel composition 11 (homogeneous phase) was used, blood flow improved compared to the untreated case, but the degree of improvement was equivalent to that when only human mesenchymal stem cells were added. [Industrial Applicability]

[0163] The gel composition obtained from the gelling agent of the present embodiment described above has high biocompatibility, biodegradability, and cell adhesiveness, and is very useful, for example, for medical applications as a medical material, a cell scaffold material, etc.

Claims

1. It is a gelling agent, The first polymer is a derivative of the first biopolymer, The present invention comprises a second polymer, which is at least one selected from the group consisting of a second biopolymer and its derivatives, Of the first polymer and the second polymer, only the first polymer has a ureidopyrimidinone group. A gelling agent wherein only one of the first polymer and the second polymer has a crosslinkable group.

2. The gelling agent according to claim 1, wherein the first biopolymer is gelatin.

3. The gelling agent according to claim 1, wherein the second biopolymer is gelatin.

4. The gelling agent according to claim 1, wherein the crosslinkable group contains a thiol group and a vinyl sulfone group, or contains an ethylenically unsaturated group.

5. The gelling agent according to claim 4, wherein the crosslinkable group contains a thiol group and a vinyl sulfone group.

6. The gelling agent according to claim 1, wherein the second polymer has the crosslinkable group.

7. The second polymer is A polymer in which a thiol group is introduced into the second biopolymer, The gelling agent according to claim 6, comprising a polymer in which a vinyl sulfone group is introduced as the second biopolymer.

8. The gelling agent according to claim 1, wherein the first polymer has the crosslinkable group.

9. The first polymer is A polymer in which a ureidopyrimidinone group and a thiol group are introduced into the first biopolymer, The gelling agent according to claim 8, comprising a polymer in which a ureidopyrimidinone group and a vinyl sulfone group are introduced into the first biopolymer.

10. The gelling agent according to claim 1, capable of forming a gel composition having a phase separation structure.

11. The gelling agent according to claim 10, wherein the phase separation structure includes a fiber structure or a gyroid structure.

12. The gelling agent according to claim 10, wherein the gel composition becomes porous under wet conditions.

13. An injectable gel containing the gelling agent described in any one of claims 1 to 12.

14. A gel composition comprising the gelling agent according to any one of claims 1 to 12.

15. Phase 1 containing the first polymer, It comprises a second phase containing a second polymer that is phase-separated from the first phase, The gel composition according to claim 14, wherein only one of the first phase and the second phase has a crosslinked structure.

16. In a wet state, The gel composition according to claim 15, wherein the other phase of the first and second phases that does not have a crosslinking structure is dissolved and removed, and a porous structure composed of the one of the phases is formed.

17. A cell scaffold material comprising the gel composition described in claim 14.

18. A medical material comprising the gel composition described in claim 14.

19. A method for producing a gel composition using the gelling agent described in any one of claims 1 to 12, A method for producing a gel composition, comprising mixing a first polymer, a second polymer, and an aqueous solvent to form a gel.

20. The method for producing a gel composition according to claim 19, further comprising placing the gelled mixture under a wet state to make it porous.

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