Gelata, injectable gel, adhesive for biological tissue, photocurable composition for 3D printer, gel composition, cellular scaffold material, and medical material

Abstract

To provide a gelling agent capable of forming a gel composition having biocompatibility, biodegradability and cell adhesiveness.SOLUTION: The gelling agent includes a first polymer which 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, and a photopolymerization initiator. Of the first polymer and the second polymer, only the first polymer has a ureidopyrimidinone group, and only one of the first polymer and the second polymer has an ethylenically unsaturated group.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a gelling agent, an injectable gel, an adhesive for biological tissue, a photocurable composition for 3D printers, a gel composition, a cell scaffold material, and a medical material. [Background technology]

[0002] Hydrogels are materials with a three-dimensionally crosslinked network structure of polymers, characterized by the large amount of water contained within them. Hydrogels are expected to have a variety of applications, including 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 as a liquid using a syringe or other device and gels in the body after injection. Compared to implanting a pre-gel, this method offers the advantage of reducing the physical burden on the patient. Furthermore, 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, in which polymers are cross-linked through physical interactions. Generally, physical gels have low mechanical strength, so chemical reactions are necessary to create highly stable hydrogels.

[0004] However, chemical gels have the problem that the polymer network is very dense, which hinders the diffusion of nutrients and oxygen and the migration of cells. To solve this problem, research into gels with porous structures is being conducted. For example, Non-Patent Document 1 reports a porous gel that uses ice particles as a porogen. Non-Patent Document 2 reports a porous injectable gel. The present inventor also reported a porous injectable gel in Non-Patent Document 3. [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. [Non-patent document 3] Akihiro Nishiguchi., Injectable microcapillary network hydrogels engineered by liquid-liquid phase separation for stem cell transplantation. Biomaterials 305 (2024) 122451. Summary of the Invention [Problem to be solved by the invention]

[0006] However, the method reported in Non-Patent Document 1 is not injectable, making the operation complicated and costly due to the need for a cell culture facility. Furthermore, since cells are seeded after the gel is produced, it is not possible to encapsulate cells within the gel. The injectable porous gel reported in Non-Patent Document 2 can encapsulate cells within the gel, but it has closed pores rather than continuous pores. This is presumably why material diffusion to the cells encapsulated within the gel and cell migration are less likely to occur. Furthermore, when a porous structure is formed using solid particles as a porogen, there is a concern that the mechanical strength may be reduced.

[0007] In response to this, the present inventors have reported in Non-Patent Document 3 a method for making gels porous by utilizing the liquid-liquid phase separation phenomenon. In a gel that forms a fine phase-separated structure, only one phase is crosslinked and insolubilized. When this gel is placed in a humid environment (e.g., in vivo), the other uncrosslinked phase is dissolved and removed, forming voids. Crosslinking is achieved by a click reaction between thiol groups (SH groups) and vinyl sulfone groups (VS groups). This method reported in Non-Patent Document 3 makes it possible to easily prepare porous gels with interconnected pores, and the resulting porous gels have high mechanical strength. Further improvements and applications of gelators capable of producing such porous gels are anticipated.

[0008] The present invention solves the above-mentioned problems by providing a gelling agent that can be used as an injectable gel and can form a porous gel composition by utilizing the liquid-liquid phase separation phenomenon. [Means for solving the problem]

[0009] As a result of extensive research into achieving the above object, the present inventors have found that the above object can be achieved by the following configuration.

[0010] [1] A gelling agent, 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; a photopolymerization initiator; 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 an ethylenically unsaturated 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 second polymer has the ethylenically unsaturated group. [5] The gelling agent according to any one of [1] to [3], wherein the first polymer has the ureidopyrimidinone group and the ethylenically unsaturated group. [6] The gelling agent according to any one of [1] to [5], wherein the ethylenically unsaturated group is at least one selected from the group consisting of an acryloyl group and a methacryloyl group. [7] The gelling agent according to any one of [1] to [6], which is a photocurable gelling agent. [8] The gelling agent according to any one of [1] to [7], which is capable of forming a gel composition having a phase-separated structure. [9] The gelling agent according to [8], wherein the gel composition becomes porous in an aqueous solvent.

[10] An injectable gel containing the gelling agent according to any one of [1] to [9].

[11] The injectable gel according to

[10] , which is a one-component type.

[12] An adhesive for biological tissue, comprising the gelling agent according to any one of [1] to [9].

[13] A photocurable composition for 3D printers, comprising the gelling agent according to any one of [1] to [9].

[14] A gel composition comprising the gelling agent according to any one of [1] to [9].

[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 phase is formed.

[17] A cell scaffold material comprising the 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] . [Effects of the Invention]

[0011] The gelling agent of the present invention can be used as an injectable agent, and can form a porous gel composition by utilizing the liquid-liquid phase separation phenomenon. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 2 is a diagram illustrating a method for producing (preparing) a phase-separated gel composition according to an embodiment. [Figure 2] FIG. 1 is a diagram illustrating the interaction (hydrogen bond) between ureidopyrimidinone groups. [Figure 3] FIG. 1 is a diagram illustrating a method for synthesizing ureidopyrimidinone-modified (UPy-modified) gelatin in an embodiment. [Figure 4] FIG. 1 is a diagram illustrating a method for synthesizing methacryloylated gelatin in an embodiment. [Figure 5] FIG. 1 is a diagram illustrating the use of the gelling agent of the present embodiment and a gel composition prepared using the same as a medical material (adhesive for biological tissue). [Figure 6] 1 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) of gel compositions 1 to 4 prepared in the examples. [Figure 7] FIG. 1 shows the results of evaluating the cell infiltration of gel compositions 1 to 4 prepared in the examples. [Figure 8] FIG. 1 shows the results of evaluating the injectability of gel compositions 2 and 4 prepared in the examples. [Figure 9] FIG. 1 shows the results of a degradability test on gel compositions 2 and 4 prepared in the examples. [Figure 10] FIG. 1 shows the results of an adhesiveness test on gel compositions 2 and 4 prepared in the examples. [Figure 11] FIG. 1 shows the results of measuring cell viability for gel compositions 2 and 4 prepared in the examples. [Figure 12]FIG. 1 is a diagram illustrating the preparation of a gel composition using a 3D printer as carried out in an example, and a confocal laser scanning microscope (CLSM) photograph (grayscale converted) of the prepared gel composition. [Figure 13] 1 shows confocal laser scanning microscope (CLSM) photographs (grayscale converted) showing the results of a cell culture test using a gel composition produced by a 3D printer in an example. DETAILED DESCRIPTION OF THE INVENTION

[0013] 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.

[0014] In the description of groups (atomic groups) in this specification, when a notation that does not specify whether they are substituted or unsubstituted is used, it encompasses both unsubstituted and substituted groups, as long as it does not impair the effects of the present invention. For example, the term "alkyl group" encompasses not only alkyl groups that do not have a substituent (unsubstituted alkyl groups), but also alkyl groups that have a substituent (substituted alkyl groups). This also applies to each compound. Furthermore, in this specification, "(meth)acryloyl" means either or both of acryloyl and methacryloyl, and "(meth)acrylic acid" means either or both of acrylic acid and methacrylic acid.

[0015] [First embodiment] The gelling agent of this embodiment contains a first polymer having a ureidopyrimidinone group (UPy group), a second polymer having an ethylenically unsaturated group, and a photopolymerization initiator. 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.

[0016] 1. First polymer The first polymer is a derivative of a first biopolymer into which a ureidopyrimidinone group (UPy group) has been introduced. Here, biopolymer refers to a natural polymer produced by the cells of an organism. The gelling agent of this embodiment uses biopolymer-derived materials for both the first polymer and the second polymer (described below), thereby obtaining a gel composition with 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 these, gelatin is preferred from the standpoint of cell adhesiveness and solubility.

[0017] The gelatin (raw material gelatin) used as the first biopolymer can be any gelatin derived from nature, chemical synthesis, fermentation, genetic recombination, or the like, without any particular limitations. Naturally derived gelatin is preferred. Examples of naturally derived gelatin include those derived from mammals such as cows, pigs, and humans, and those derived from fish such as sea bream, sturgeon, salmon, and cod. Of these, porcine gelatin is preferred, with porcine skin gelatin and porcine tendon gelatin being more preferred.

[0018] The molecular weight of the starting gelatin is not particularly limited, but generally, a weight-average molecular weight of 10,000 to 500,000 is preferred. The upper limit is not particularly limited, but from the viewpoint of solubility, a molecular weight of 350,000 or less is more preferred. The lower limit is not particularly limited, but from the viewpoint of the resulting gel having superior mechanical strength, a molecular weight of 50,000 or more is more preferred.

[0019] In this 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, it is a group represented by the following formula (11).

[0020] [ka]

[0021] In equation (11), R 1 is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and * represents the linking position. 1 is preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group.

[0022] 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 serves as a spacer. 6 A ureidopyrimidinone group is introduced into the first biopolymer via (see Figure 3).

[0023] [ka]

[0024] In equation (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.

[0025] 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 alkylene group or an ethylene oxide chain, and preferably has 2 to 12 carbon atoms.

[0026] 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, -NH) in the gelatin (raw material gelatin) is defined as the "ureidopyrimidinone group introduction rate (UPy group introduction rate, UPy group substitution rate) (mol%)." The method for determining the UPy group introduction rate is not particularly limited, 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) or the like.

[0027] 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.

[0028] 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 groups. Therefore, the range of possible weight-average molecular weights (Mw) of the first polymer is the same as the range of possible weight-average molecular weights (Mw) of the first biopolymer described above.

[0029] The first polymer may be a single type of polymer or a mixture of two or more types of polymers.

[0030] The synthesis method for the first polymer is not particularly limited, and known methods can be used. For example, the method described in Japanese Patent Application Laid-Open No. 2023-027762 can be mentioned, 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 material 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-modified gelatin (UPy-modified gelatin) can be synthesized by stirring at 20-30°C for 16-24 hours. After the reaction is complete, the product is reprecipitated in a cold ethanol / ethyl acetate mixed solvent, washed with cold chloroform and cold ethanol, and then dried under reduced pressure to obtain UPy-modified gelatin (first polymer) powder.

[0031] The first polymer may be a derivative in which only a UPy group has been introduced into the first biopolymer, or may be a derivative in which a functional group other than a UPy group has been further introduced, as long as the effects of this embodiment are achieved. However, the first polymer does not have a crosslinkable group (e.g., an ethylenically unsaturated group contained in the second polymer described below). As a result, in the gel composition 100 produced 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 (e.g., in vivo), the first phase 21 is dissolved and removed, resulting in a porous gel.

[0032] 2. Second polymer The second polymer is a derivative of the second biopolymer in which an ethylenically unsaturated group has been introduced. In the gel 100 formed using the gelling agent of this embodiment, the second polymer forms a second phase 22 having a crosslinked structure in which the ethylenically unsaturated group has been polymerized (see FIG. 1).

[0033] As mentioned above, in Non-Patent Document 3, crosslinking of one phase of the phase-separated gel (corresponding to the second phase 22 in Figure 1) was carried out by a click reaction between a thiol group (SH group) and a vinyl sulfone group (VS group). The click reaction between SH groups and VS groups is highly reactive. For this reason, it was necessary to synthesize two types of modified polymers (SH-modified polymer and VS-modified polymer) by introducing the SH group and the VS group into different polymers (different polymer chains). Furthermore, these two types of modified polymers had to be stored in separate containers.

[0034] In contrast, the second polymer of this embodiment requires only one modified polymer with an ethylenically unsaturated group. Furthermore, the reactivity of the ethylenically unsaturated group is not as high as that of the click reaction, and the polymerization reaction does not occur until irradiation with ultraviolet light. Therefore, the crosslinkable second polymer of this embodiment eliminates the need for synthesis and management, thereby reducing costs.

[0035] Examples of the second biopolymer include those similar to the first biopolymer described above, and the preferred embodiments are also similar. The second biopolymer may be a biopolymer of a different type from the first biopolymer, or may be the same type as the first biopolymer. Normally, solutions of two polymers with a common backbone tend to form a homogeneous phase. However, with the gelling agent of this embodiment, even if the first polymer and the second polymer have a common backbone, a homogeneous phase is not formed, and a phase-separated gel is obtained due to the difference in the presence or absence of a UPy group.

[0036] The ethylenically unsaturated group is not particularly limited as long as it is a functional group that can be polymerized by irradiation with ultraviolet light, and examples thereof include a functional group represented by the following formula (21), a styryl group, and an allyl group, and among these, a (meth)acryloyl group is preferred.

[0037] [ka]

[0038] In equation (21), R 2 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and * represents the bonding position. 2 is preferably a hydrogen atom or a methyl group. When R is a hydrogen atom, formula (21) represents an acryloyl group, and when R is a methyl group, it represents a methacryloyl group.

[0039] When the second biopolymer is gelatin, the second polymer has a structure in which an ethylenically unsaturated group is introduced directly or via a spacer into an amino group of the starting gelatin, for example, the second polymer has a structure represented by the following formula (22):

[0040] [ka]

[0041] In equation (22), Gltn 1 is a gelatin residue, and L 1 is a single bond or a spacer that is a divalent group, and X is an ethylenically unsaturated group. 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. Examples of X, which is an ethylenically unsaturated group, include the same ethylenically unsaturated groups as those described above, and preferred embodiments are also the same.

[0042] From the viewpoint of efficient synthesis of the second polymer, the second polymer may have a structure represented by the following formula (23): That is, the second polymer may have an amide group (—NHC(O)—).

[0043] [ka]

[0044] In equation (23), Gltn 1 is a gelatin residue, and R 2 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. 2 is preferably a hydrogen atom or a methyl group.

[0045] In the above formulas (22) and (23), the gelatin residue Gltn 1 The nitrogen atom (N) directly bonded to is derived from the ε-amino group of lysine (Lys) in gelatin.

[0046] Here, the ratio of the content (molar concentration) of ethylenically unsaturated groups (X groups) in the second gelatin to the content (molar concentration) of amino groups (primary amino groups, -NH2) in the raw material gelatin (second biopolymer) is defined as the "ethylenically unsaturated group introduction rate (X group introduction rate, X group substitution rate) (mol%)." There are no particular limitations on the method for determining the X 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) or the like.

[0047] Although the X group introduction rate is not particularly limited, the mechanical properties of the resulting gel can be controlled by changing the X group introduction rate. Moreover, if the X group introduction rate is too high, the water solubility decreases, making it difficult to form a gel (hydrogel). From the viewpoint of facilitating gel formation and increasing gel strength and / or adhesive strength to biological tissue, the X group introduction rate may be, for example, 20 mol% to 80 mol%, 40 mol% to 80 mol%, or 50 mol% to 70 mol%.

[0048] The molecular weight of the second polymer is not particularly limited and is determined by the molecular weight of the second biopolymer (raw gelatin) and the type and amount (number) of introduced groups. Therefore, the range of possible weight-average molecular weights (Mw) of thiolated gelatin is the same as the range of possible weight-average molecular weights (Mw) of the second biopolymer.

[0049] The gelling agent of the present embodiment may contain only one type of second polymer, or may contain two or more types of second polymers.

[0050] The method for producing (synthesizing) the second polymer is not particularly limited, and the second polymer can be synthesized by a known method. For example, as shown in Figure 4, the second polymer can be synthesized by reacting an amino group of raw gelatin (second biopolymer), preferably an amino group of lysine, with (meth)acrylic anhydride to introduce a (meth)acryloyl group into the raw gelatin.

[0051] In the second polymer, only ethylenically unsaturated groups may be introduced into the second biopolymer, or other functional groups may be introduced as long as the effects of the present invention are achieved. However, the second polymer of this embodiment does not have a ureidopyrimidinone group (UPy group). This can promote phase separation into the first phase 21 and the second phase 22 in the gel composition 100 (see FIG. 1).

[0052] 3. Photopolymerization initiator The photopolymerization initiator is not particularly limited, and known compounds that absorb ultraviolet light and generate active species (radicals, cations, anions, etc.) can be used. Examples of photopolymerization initiators that can be used include known photoradical polymerization agents such as benzophenones, thioxanthones, acetophenones, and acylphosphine-based agents, as well as photoanionic and photocationic polymerization agents. Furthermore, when an aqueous solvent (described in detail below) is used for gelation, the photopolymerization agent is preferably water-soluble in order to efficiently carry out the crosslinking reaction. An example of a water-soluble photopolymerization initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0053] 4. Gelling agent and gel composition The gelling agent of this embodiment contains two types of polymers: a first polymer having the above-mentioned ureidopyrimidinone group (UPy group) and a second polymer having no UPy group, and further contains a photopolymerization initiator. The gelling agent of this embodiment is a photocurable gelling agent that undergoes a crosslinking reaction (photocrosslinking) upon irradiation with ultraviolet light to form a gel.

[0054] The present inventors discovered that mixing a first polymer containing UPy groups with a second polymer lacking UPy groups in an aqueous solvent (e.g., a buffer solution) does not result in a homogeneous phase, but rather results in a fine liquid-liquid phase separation between a phase containing the first polymer and a phase containing the second polymer. By applying this phenomenon, the present invention was achieved. By gelling the liquid-liquid phase-separated sol solution, a gel composition 100 is obtained, which has a fine phase-separated structure consisting of a first phase 21 containing the first polymer and a second phase 22 containing the second polymer, as shown in Figure 1. The mechanism by which this 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 Figure 2. In an aqueous gelling agent solution, the first polymers strongly interact with each other, reducing their compatibility with the second polymer. It is believed that the immiscible polymers form microdomains, resulting in phase separation.

[0055] Only one of the first polymer and the second polymer (only the second polymer in this embodiment) has a photopolymerizable ethylenically unsaturated group. By irradiating with ultraviolet light, a crosslinked structure is formed only in the second phase 22, resulting in gelation. The first phase 21, which does not have a crosslinked structure, is water-soluble and is dissolved and removed when placed under a moist condition (for example, in vivo), forming voids. In this way, by using the gelling agent of this embodiment, a porous gel composed of the second phase 22 having a crosslinked structure can be produced.

[0056] The second phase 22 has a chemically crosslinked structure and is in the form of a gel (sometimes referred to as a "second gel") at temperatures near human body temperature (e.g., 37°C). On the other hand, the first phase 21 may be in the form of a gel (sometimes referred to as a "first gel") or a sol at temperatures near human body temperature (e.g., 37°C).

[0057] The gel composition 100 of this embodiment has higher mechanical strength and stronger adhesiveness to living organisms than gel compositions without a phase-separated structure (i.e., a homogeneous phase). While the mechanism behind this is unclear, it is speculated as follows. If the gel composition were a homogeneous phase, the entire gel composition would be chemically crosslinked solely by the second polymer having crosslinking groups, resulting in a decrease in crosslink density. This is likely to result in a decrease in mechanical strength and adhesiveness. In contrast, the gel composition 100 of this embodiment has a phase-separated structure, so the crosslinked structure is concentrated in the second phase (second gel) 22, resulting in a high crosslink density. Because phase separation occurs on a fine scale (micron-order scale) throughout the gel composition 100, the second phase (second gel) 22, which has a high crosslink density, is distributed 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.

[0058] Furthermore, when the gel composition 100 is made porous, it is the uncrosslinked first phase 21 that is removed, and the second phase 22, which has a high crosslink density, remains. Therefore, it is presumed that the porous gel composition 100 also has high mechanical strength and adhesiveness. Furthermore, while porous gels produced using solid porogens generally tend to have reduced mechanical strength, the gel composition 100 of the present embodiment is made porous by utilizing liquid-liquid phase separation, and therefore the porogen is a liquid (sol) or an uncrosslinked gel. Therefore, the resulting porous gel can maintain high mechanical strength.

[0059] The fine phase-separated 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 cylindrical (columnar) structure, and a lamellar (plate-like) structure. Here, the term "fiber structure" refers to a structure in which either one or both of the first and second phases extend in a fibrous form with a diameter of about several μm (less than 10 μm) to form a network. The term "gyroid structure" refers to a structure in which either one or both of the first and second phases extend in a branch-like (bone-like) form thicker than a fiber (diameter 10 μm or more) to form a network. In the fiber structure and the gyroid structure, both the first and second phases are 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 (islands, discontinuous phase) is dispersed in the other phase (sea, continuous phase). Note that FIG. 1 shows a fiber structure in which a fiber phase (first phase 21) extends fibrously in a matrix phase (second phase 22) to form a network, but this structure is merely an example, and the phase separation structure of the gel composition 100 is not limited to this.

[0060] When gel composition 100 is used as a cell scaffold material or a medical material, the phase-separated structure preferably includes a fibrous (fibrous) structure or a gyroid structure, and more preferably includes a fibrous (fibrous) structure. The fibrous (fibrous) structure and the gyroid structure can form interconnected pores when the gel composition is made porous, and these interconnected pores can promote the diffusion of nutrients and oxygen and cell migration. In particular, the fibrous structure can form many interconnected pores of an appropriate size (narrowness) (diameter: about several micrometers, about 1 μm to 9 μm), which can prevent excessive outflow of the culture medium compared to a gyroid structure (diameter of interconnected pores: about several tens of micrometers, about 10 μm to 99 μm), thereby further promoting cell culture.

[0061] The type of phase-separated structure formed in a gel composition is determined by the types of the first polymer and 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, for example, create a phase diagram and, based on it, select a gelling agent that forms a desired phase-separated structure.

[0062] 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.

[0063] The amount of photopolymerization agent added to the gelling agent and the resulting gel composition of this embodiment is not particularly limited and may be adjusted as appropriate. For example, the ratio (PI / (PM1+PM2)) of the mass of the photopolymerization initiator (PI) to the total mass (PM1+PM2) of the first polymer and the second polymer can be 0.1% to 10% by mass, or 1% to 3% by mass.

[0064] The gelling agent of this embodiment may be composed only of the first polymer, the second polymer, and the photopolymerization initiator. Furthermore, the gelling agent of this embodiment and the resulting gel composition may contain other components as needed. For example, the other component may contain a water-containing solvent (hereinafter, occasionally referred to as an "aqueous solvent"). By dissolving the first polymer and the second polymer in an aqueous solvent, the gelling agent of this embodiment can be used as an injectable gel, a photocurable composition for 3D printers, an adhesive for biological tissue, and the like, as described below.

[0065] Depending on the intended use, 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 vaccine carrier, the gelling agent may contain a drug or protein. Examples of drugs and proteins 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.

[0066] Furthermore, the gelling agent of this embodiment and the resulting gel composition may contain a decellularized matrix, which is the matrix structure of biological tissue, as another component (additive). By including extracellular matrix components that remain 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. Examples of decellularized matrices that can be used include decellularized matrices prepared from organs such as the bladder, heart, liver, pancreas, and small intestine. Gel compositions containing additives such as decellularized matrices can be used as cell scaffolding materials, medical materials, and the like.

[0067] 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, as long as the effects of the present invention are achieved.

[0068] The gel composition (typically a hydrogel) obtained in this embodiment contains a gelling agent, an aqueous solvent, and, if necessary, other additives. 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 from the viewpoint of increasing the strength of the gel composition, it may be, for example, 1% by mass to 20% by mass, and preferably 5% by mass to 10% by mass.

[0069] The method of using the gelling agent of this embodiment (i.e., the method of producing a gel composition) is not particularly limited. For example, the method of producing a gel composition includes the steps of mixing a gelling agent with an aqueous solvent to prepare a mixture (gelling agent solution) and irradiating the mixture with ultraviolet light. In the mixture, a first phase 21 and a second phase 22 are formed by liquid-liquid phase separation, and by irradiating this with ultraviolet light, the ethylenically unsaturated groups of the second polymer are polymerized, crosslinking the second phase 22 and producing a gel composition (typically a hydrogel). The wavelength, intensity, irradiation time, etc. of the ultraviolet light to be irradiated are not particularly limited and can be selected appropriately, but may be within the following ranges, for example: ultraviolet light wavelength: 350 nm to 450 nm, intensity: 50 mJ / cm 2 ~5000mJ / cm 2 , Irradiation time: 10 seconds to 100 seconds.

[0070] <Injectable gel> A gelling agent solution containing the gelling agent of this embodiment and an aqueous solvent can be used as an injectable gel that can be injected into a desired location (for example, an affected area) using a syringe or the like.

[0071] As mentioned above, the click reaction between thiol groups (SH groups) and vinyl sulfone groups (VS groups) is highly reactive. For this reason, in the injectable gel disclosed in Non-Patent Document 3, three types of drugs - a UPy drug in which UPy-modified gelatin is dissolved in an aqueous solvent, an SH drug in which an SH-modified polymer is dissolved in an aqueous solvent, and a VS drug in which a VS-modified polymer is dissolved in an aqueous solvent - were prepared and stored in separate containers, and when used, the three drugs had to be injected from separate syringes and mixed at the injection site (three-component injectable gel).

[0072] In contrast, the reactivity of the ethylenically unsaturated group in the second polymer of this embodiment is not as high as that of the click reaction, and the polymerization reaction does not occur until irradiation with ultraviolet light. Therefore, the injectable gel of this embodiment can be a one-component injectable gel in which all components of the gelling agent are mixed with an aqueous solvent. Compared to three-component injectable gels, one-component injectable gels are easier to handle and less expensive in all aspects of production, management, and use.

[0073] The aqueous solvent used in the injectable gel is not particularly limited as long as it contains water, and examples thereof include ultrapure water, physiological saline, buffer solutions of various inorganic salts such as boric acid, phosphoric acid, and carbonate, and mixtures thereof. Furthermore, the aqueous solvent may be a water-soluble solvent with low toxicity to living organisms (e.g., ethanol), or a mixture of such a solvent with water. When it is envisioned that the injectable gel will be injected into a living organism, for example, as a biological tissue adhesive, or that the produced gel composition will be used for cell culture, etc., the aqueous solvent is preferably a buffer solution. The aqueous solvent may be composed of only one type of solvent, or a mixture of two or more types of solvents.

[0074] The injectable gel may be composed of only a gelling agent and an aqueous solvent, or may contain other additives that may be contained in the gel composition described above, as needed.

[0075] The concentration of the first polymer (UPy polymer) in the injectable gel may be, for example, 0.5% to 15% by mass, or 2.5% to 7.5% by mass. Furthermore, when the concentration of the UPy polymer is 2.5% by mass or more, or 5% by mass or more, a gel phase-separated structure with a fibrous structure is easily obtained. Furthermore, the concentration of the second polymer in the injectable gel may be, for example, 0.5% to 15% by mass, or 2.5% to 7.5% by mass.

[0076] In order to easily form a uniform gel, it is preferable that the concentration of the first polymer (C1) in the injectable gel is close to the concentration of the second polymer (C2), for example, the ratio (C1) / (C2) may be 0.8 to 1.2.

[0077] The injectable gel may be prepared and used, for example, as follows. First, a gelling agent is dissolved or dispersed in an aqueous solvent to prepare an injectable gel. The method of dissolving or dispersing is not particularly limited, and any known method can be used. Next, the prepared injectable gel (solution) is applied (injected) into a target area (affected area) in a living body using a syringe or the like, and the applied injectable gel is then irradiated with ultraviolet light. The irradiation with ultraviolet light causes a crosslinking reaction (polymerization of ethylenically unsaturated groups) to produce a gel composition (see Figures 1 and 5).

[0078] <Photocurable composition for 3D printers> As described above, the gelling agent solution (one-component solution) containing the gelling agent of this embodiment and an aqueous solvent does not undergo a polymerization reaction until irradiated with ultraviolet light. Furthermore, the gelling agent solution of this embodiment can be ejected from a syringe or the like (good injectability). Therefore, the gelling agent solution of this embodiment can be used as a photocurable composition for 3D printers.

[0079] For example, a 3D printer 50 shown in FIG. 12 can be used as the 3D printer. The 3D printer 50 includes a storage unit (chamber) 52 capable of storing a photocurable composition 51, a discharge unit (print head, nozzle) 53 capable of discharging the stored photocurable composition 51, and a UV light irradiation unit (e.g., an LED module) 55 that irradiates the photocurable composition 51 discharged from the discharge unit 53 with ultraviolet light (UV light) 54. The photocurable composition 51 is discharged from the discharge unit 53 and crosslinked by irradiating it with UV light 54 from the UV light irradiation unit 55. By repeating this process, a desired structure (a three-dimensional gel composition) 56 can be produced from the photocurable composition 51.

[0080] The composition and manufacturing method (preparation method) of the photocurable composition for 3D printers (gelling agent solution) 51 are the same as those of the injectable gel described above, and the preferred embodiments are also the same.

[0081] The 3D printer is not limited to the above configuration. For example, a stereolithography 3D printer using a known modeling method, such as a DLP type, LCD type, laser type (SLA method), or inkjet type, can be used.

[0082] 5. Uses of gelling agents and gel compositions The gel composition obtained using the gelling agent of this embodiment is derived from a biopolymer and therefore has high biocompatibility, biodegradability, and cell adhesiveness. Furthermore, because the gel composition has a phase-separated structure, it has high mechanical strength and adhesiveness, and furthermore, it becomes porous when placed under moist conditions (e.g., in vivo). Therefore, the gel composition of this embodiment is excellent, for example, as a cell scaffold material.

[0083] The gelling agent of this embodiment can be used as a biological tissue adhesive to be applied to a diseased site or a tissue defect site. For example, as shown in FIG. 5, a one-component injectable gel containing the gelling agent of this embodiment can be applied to a diseased site or a tissue defect site using a syringe or the like, and then irradiated with UV light to form a gel composition. The gel composition becomes porous when placed in a moist environment (in vivo), which allows cells to easily infiltrate and promotes tissue regeneration. Cells, etc. can also be encapsulated in the gelling agent.

[0084] As such, the gelling agent and gel composition of the present embodiment can be used as medical materials for wound covering, muscle tissue regeneration, bone regeneration, nerve regeneration, ischemia treatment, diabetes treatment, heart disease treatment, etc. Furthermore, by culturing cancer cells using the gel composition of the present embodiment, it becomes possible to construct a cancer tissue model, which can be expected to be applied to drug discovery screening.

[0085] [Second embodiment] In the first embodiment, the first polymer has a UPy group and the second polymer has an ethylenically unsaturated group, but the present invention is not limited to this form. In this embodiment, the first polymer has both a UPy group and an ethylenically unsaturated group, and the second polymer has neither a UPy group nor a crosslinkable group (e.g., an ethylenically unsaturated 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.

[0086] 1. First polymer The first polymer of this embodiment is a polymer in which a UPy group and an ethylenically unsaturated group have been introduced into a first biopolymer. The first biopolymer, the Upy group, and the ethylenically unsaturated group may be the same as those in the first embodiment, and preferred embodiments are also the same.

[0087] In the first polymer (e.g., gelatin), the molar ratio of the ethylenically unsaturated group (X), the ureidopyrimidinone group (UPy), and the primary amino group (A) may be, for example, (X) / (UPy) / (A)=(40-60) / (35-55) / (0-20) in order to increase the strength of the gel composition.

[0088] The concentration (introduction rate of functional groups) of each functional group (UPy group, X group) in the first polymer can be determined by the method described in the first embodiment, and the molar ratio of the functional groups described above can be calculated therefrom.

[0089] The method for producing (synthesizing) the first polymer into which the UPy group and the X group have been introduced is not particularly limited, and the first polymer can be synthesized by a known method. For example, first, a UPy-modified polymer is synthesized by introducing a ureidopyrimidinone group into the amino group of a first biopolymer (e.g., gelatin) by the method described in the first embodiment (see FIG. 3). The X group is introduced into the UPy-modified polymer by the method described in the first embodiment, thereby obtaining a first polymer into which the UPy group and the X group have been introduced.

[0090] The first polymer may consist of only one type of polymer, or may include two or more types of polymers.

[0091] 2. Second polymer The second polymer of this embodiment can be the second biopolymer described in the first embodiment. The second polymer may also 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.

[0092] 3. Photopolymerization initiator As the photopolymerization initiator of this embodiment, the same one as in the first embodiment can be used, and the preferred embodiments are also the same.

[0093] 4. Gelling agent and gel composition The gelling agent of this embodiment contains a first polymer having both a UPy group and an ethylenically unsaturated group, a second polymer having neither a UPy group nor a crosslinkable group (e.g., an ethylenically unsaturated group), a photopolymerization initiator, and, if necessary, other additives. Examples of the other additives include those similar to those in the first embodiment, and preferred embodiments are also similar. Like the first embodiment, the gelling agent of this embodiment can be used in injectable gels, photocurable compositions for 3D printers, adhesives for biological tissues, and the like.

[0094] When the gelling agent of this embodiment is mixed with an aqueous solvent, it does not form a homogeneous phase, but rather undergoes fine liquid-liquid phase separation between a phase containing the first polymer and a phase containing the second polymer. By irradiating this liquid-liquid phase-separated sol solution with ultraviolet light to cause gelation, a gel composition having a fine phase-separated structure with a first phase containing the first polymer and a second phase containing the second polymer is obtained. In this embodiment, the first phase containing UPy groups has a crosslinked structure. When placed under a humid condition (e.g., in vivo), the second phase without a crosslinked structure is dissolved and removed, forming voids. In this way, using the gelling agent of this embodiment, a porous gel composed of a first phase (first gel containing UPy groups) with a crosslinked structure can be produced.

[0095] As with the first embodiment, the gel composition of this embodiment can take various forms, such as a fiber (fibrous) structure or a gyroid structure. For example, a phase diagram represented by the ratio of the first polymer to the second polymer can be created, and based on this, a person skilled in the art can select a gelling agent that can form a desired phase-separated structure. 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. [Example]

[0096] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0097] 1. Gelling agent Four types of gelators 1 to 4 were prepared. Each of gelators 1 to 4 was composed of two polymers selected from polymers (0) to (III) as shown in Table 1, and a photopolymerization initiator (LAP, Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) was added to each of them.

[0098] The polymers (0) to (III) shown in Table 1 are as follows. Polymer (0): Raw gelatin (unmodified gelatin) Polymer (I): Methacryloylated gelatin Polymer (II): UPylated gelatin Polymer (III): UPy-methacryloylated gelatin Polymers (I), (II), and (III) were synthesized by introducing functional groups into polymer (0) as described below.

[0099] (1) Synthesis of polymer (I) (methacryloylated gelatin) Raw material gelatin (polymer (0)) (pig skin gelatin, weight-average molecular weight 100,000, Sigma-Aldrich Japan LLC) was dissolved in bicarbonate buffer at a concentration of 100 mg / mL and heated to 50°C. The solution was then adjusted to pH 9 with 5 M sodium hydroxide or 6 M hydrochloric acid. A predetermined amount of methacrylic anhydride (Sigma-Aldrich Japan LLC) was added and mixed, and the mixture was stirred at 50°C for 1 hour. The pH was then readjusted to 7.4 to terminate the reaction. The mixture was then dialyzed in water for 3 days, and the product was recovered by lyophilization. The amount of methacrylic anhydride added was 1 molar equivalent per molar equivalent of the amino group content of the raw material gelatin (311 μmol / g, measured by the TNBS method).

[0100] <Measurement of methacryloyl group introduction rate> The methacryloyl group introduction rate was calculated from the following formula using the primary amino group concentration (mol / g) of raw gelatin and the primary amino group (residual amino group) concentration (mol / g) of methacryloylated gelatin quantified by the TNBS method. The methacryloyl group introduction rate was 71 mol%.

[0101] Methacryloyl group introduction rate (mol%) = [(primary amino group concentration of raw gelatin) - (primary amino group concentration of methacryloylated gelatin)] / (primary amino group concentration of raw gelatin) × 100

[0102] (2) Synthesis of polymer (II) (UPylated 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, manufactured by Sigma-Aldrich Japan KK) was dispersed in 1,6-diisocyanatohexane (78.3 mmol, 13.163 g, manufactured by Tokyo Chemical Industry Co., Ltd.), heated to 100 °C for reaction, and continuously stirred for 16 hours. After cooling to 25 °C, 10 times the amount of n-hexane was added to precipitate the product. The obtained precipitate was filtered and recovered, 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.

[0103] Next, the raw material gelatin (polymer (0)) 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 was added to the gelatin solution, and UPy-modified gelatin was synthesized by stirring at 20 - 30 °C for 16 - 24 hours (see Fig. 3). After the reaction was completed, reprecipitation treatment was performed with an ethanol / ethyl acetate mixed solvent, washed with cold chloroform and cold ethanol, and then dried under reduced pressure to obtain the target powder. The amount of the ureidopyrimidinone derivative solution added was such that the UPy group was 0.5 molar equivalent relative to 1 molar equivalent of the amino group amount (311 μmol / g, measured by the TNBS method) of the raw material gelatin.

[0104] <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-modified gelatin by the TNBS method. The UPy group introduction rate was 48 mol%.

[0105] UPy group introduction rate (mol%) =[(Primary amino group concentration of raw material gelatin) - (Primary amino group concentration of UPy-modified gelatin)] / (Primary amino group concentration of raw material gelatin) × 100

[0106] (3) Synthesis of polymer (III) (UPy-methacryloylated gelatin) UPy-methacryloylated gelatin was synthesized by introducing methacryloyl groups into the primary amino groups of the previously synthesized UPy-gelatin in the same manner as for the methacryloylated gelatin described above. Note that methacrylic anhydride was added in an amount equivalent to 1 molar equivalent of the amino groups of the UPy-gelatin.

[0107] The primary amino group concentrations (mol / g) of raw gelatin, UPy-modified gelatin, and UPy-methacryloylated gelatin were quantified by the TNBS method, and the molar ratio (X) / (UPy) / (A) of methacryloyl groups (X), ureidopyrimidinone groups (UPy), and primary amino groups (A) in UPy-methacryloylated gelatin was calculated from the obtained values. The molar ratio (X) / (UPy) / (A) was 52 / 48 / 0 (zero).

[0108] [Table 1]

[0109] 2. Evaluation (1) Microscopic observation of gel composition Gel compositions were prepared using gelators 1 to 4 shown in Table 1 and observed by confocal laser scanning microscopy (CLSM). Furthermore, the prepared gel compositions were immersed in phosphate buffer solution (PBS), and the gel compositions after immersion were observed. For CLSM observation, one polymer of each gelator was fluorescently labeled with Cy5.5, and the other polymer was fluorescently labeled with fluorescein.

[0110] <Preparation of Gel Composition 1> Polymers (0) and (1) of gelator 1 shown in Table 1 were each dissolved in phosphate buffered saline (PBS, pH 7.4) at 150 mg / mL (15 wt%). After heating at 50°C, the pH was adjusted to 7.0-8.0 to obtain two types of gelatin solutions. After heating the respective gelatin solutions at 37°C for 30 minutes, equal amounts were mixed, and a photopolymerization initiator (LAP) was added to prepare a pre-gel solution (final concentration of LAP was 1 mg / mL). This pre-gel solution was dropped onto a substrate using a pipette and irradiated with UV light (wavelength: 365 nm, output: 100 mJ / cm). 2 , irradiation time: 30 seconds) to prepare gel composition 1.

[0111] <Preparation of Gel Compositions 2 to 4> Gel compositions 2 to 4 were prepared in the same manner as gel composition 1 above, except that polymers 2 to 4 shown in Table 1 were used instead of polymers (0) and (I) of gelling agent 1.

[0112] <Microscopic observation> 5 mL of PBS was added to the prepared Gel Compositions 1 to 4, and the mixture was allowed to stand for 24 hours. Confocal laser scanning microscope (CLSM) photographs of Gel Compositions 1 to 4 before and after immersion in PBS are shown in FIG.

[0113] As can be seen from the photograph "before immersion in PBS" in Figure 6, phase separation occurred in gel compositions 2 and 4, and they had a fibrous structure in which the fiber phase extended fibrously within the matrix phase to form a network. On the other hand, no phase separation occurred in gel compositions 1 and 3 (a homogeneous phase was formed). In Gel Composition 2, the polymer (II) having UPy groups formed a fiber phase. The matrix phase was a phase of the polymer (I) having methacryloyl groups, which was crosslinked by UV irradiation. The polymer (III) having UPy groups also formed a fiber phase in gel composition 4. Since the polymer (III) has methacryloyl groups, the fiber phase in gel composition 4 was crosslinked by UV irradiation.

[0114] As can be seen from the photograph "after immersion in PBS" in Figure 6, the non-crosslinked phase of gel compositions 2 and 4 dissolved in PBS and became porous upon immersion in PBS. In the photographs "after immersion in PBS" of gel compositions 2 and 4, the black areas (dark colored areas) are voids where no gel composition is present (pores formed by immersion in PBS). On the other hand, gel compositions 1 and 3 did not become porous. In Gel Composition 2, the non-crosslinked fiber phase dissolved in PBS, leaving the matrix phase. As a result, a gel composition in which fibrous pores (capillaries) were formed was obtained. In Gel Composition 4, the non-crosslinked matrix phase dissolved in PBS, leaving behind the fiber phase, resulting in a gel composition that stretched like fibers to form a network.

[0115] (2) Evaluation of cell invasiveness Pregel solutions were prepared using gelling agents 1 to 4 using the same method as described above in "(1) Microscopic observation of gel compositions." 100 μL of each pregel solution was added to a cell culture insert (pore diameter: 8 μm, 24 wells) and gelled by UV irradiation. 1 × 10 normal human skin-derived fibroblasts were added to the obtained gel compositions. 4 Cells were seeded at a density of 100 cells / insert and cultured for 72 hours in an incubator at 37°C with 5% CO2. After culture, each sample was fixed with formalin, stained for actin and nuclei, and observed under a fluorescent microscope to determine the cell invasion distance into the gel composition. The invasion distance was quantified using ImageJ (image processing software provided by the National Institutes of Health (NIH)) based on the microscopic images, measuring the distance from the interface between the gel and culture medium to the invaded cells. The results are shown in Figure 7.

[0116] As shown in Figure 7, cells showed the highest infiltration rate in gel composition 2. This is presumably due to the porosity of gel composition 2, which has a phase-separated structure, caused by the culture medium during cell culture. From these results, it is presumed that gel composition 2 also exhibits good cell infiltration in vivo (under wet conditions), and that, for example, when used as a biological tissue adhesive, it will promote tissue regeneration. Furthermore, gel composition 4, which has a phase-separated structure, also exhibited a higher average infiltration distance and better cell infiltration than gel compositions 1 and 3, which have a homogeneous phase. This is also presumably due to the porosity of gel composition 4.

[0117] The reason for the difference in cell infiltration (infiltration distance) between gel compositions 2 and 4, both of which have a porous structure, is presumed to be as follows: In gel composition 4, which is composed of a fibrous phase, the voids formed between the fibers were easily collapsed, resulting in increased density (reduced porosity), making it difficult for cells to infiltrate (the infiltration distance was short). On the other hand, the fibrous pores (capillaries) formed in gel composition 2 were difficult to collapse and maintained their porosity, making it easy for cells to infiltrate (the infiltration distance was long).

[0118] (3) Injectability evaluation Pregel solutions were prepared using gelators 2 and 4 using a method similar to that described in "(1) Microscopic Observation of Gel Compositions" above. Each pregel solution was filled into a 1 mL syringe whose weight had been measured in advance. A 27 G needle was attached to the syringe, and an injection test of the pregel solution was performed using a compression tester (Shimadzu Corporation). In the injection test, the maximum pressure was set to 50 N, and the solution was injected at a rate of 100 mm / min. The weight of the syringe after injection was measured, and the ratio (%) of the weight of the pregel solution injected from the syringe to the weight of the pregel solution filled in the syringe was calculated and used as the injectability. The results are shown in Figure 8. As shown in Figure 8, the pregel solutions prepared from gelators 2 and 4 exhibited high injectability of over 90%.

[0119] (4) Degradability test Pregel solutions were prepared using gelators 2 and 4 using a method similar to that described in "(1) Microscopic Observation of Gel Compositions" above. Each pregel solution was irradiated with UV light to gel it, producing 1 mm-thick hydrogel sheets. The sheets were then cut into disks using an 8 mm diameter punch. The disk-shaped samples were immersed in a solution containing 1 mg / mL collagenase (120 U / mL, manufactured by Nacalai Tesque) and allowed to stand at 37°C for 1, 4, and 8 hours. After removal, the samples were lyophilized and weighed. The ratio (%) of the weight of the sample after immersion in the collagenase solution (weight after lyophilization) to the weight of the sample before immersion in the collagenase solution (weight after lyophilization) was calculated and shown in Figure 9. As shown in Figure 9, the gel compositions prepared using gelators 2 and 4 were degraded by collagenase, demonstrating biodegradability.

[0120] (5) Adhesion test In accordance with ASTM-F2392-04R, adhesion evaluation was performed using collagen casing as a model tissue for evaluating tissue adhesiveness. First, a 3 mm diameter pinhole was created in a 35 mm diameter collagen casing. Pregel solutions of gelling agents 2 and 4 were prepared using the same method as described above in "(1) Microscopic observation of gel compositions." 300 μL of each pregel solution was dropped onto the collagen casing, gelled by UV irradiation, and then the pressure resistance was measured. The higher the pressure resistance, the higher the adhesiveness of the gel composition. The results are shown in Figure 10.

[0121] Gel compositions 2 and 4 had pressure resistance strength sufficient for practical use as tissue adhesives. Gel composition 2 in particular exhibited high adhesiveness. The reason for the higher adhesiveness of gel composition 2 compared to gel composition 4 is presumed to be because the methacryloyl group introduction rate (mol%) of polymer (I) in gel composition 2 was higher than the methacryloyl group introduction rate (mol%) of polymer (III) in gel composition 4. Compared to gel composition 4, gel composition 2 had a higher crosslink density, which is presumed to have increased gel composition strength and improved adhesive strength.

[0122] (6) Measurement of cell viability First, disk-shaped samples (hydrogel sheets) were prepared using gelators 2 and 4 in the same manner as in the above-mentioned "(4) Degradability test." Each disk-shaped sample was immersed in 1 mL of DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin) and left to stand at 37°C for 24 hours, and the supernatant was collected. Next, mouse fibroblasts (L929 cells) were added to 1 × 10 4 The cells were seeded at a concentration of 1000 cells / well (96-well plate) and cultured for 24 hours in an incubator at 37°C and 5% CO2. 100 μL of the collected supernatant was added to each well and cultured for an additional 24 hours. A control well to which no supernatant was added was also cultured in the same manner. After culture, the number of viable cells was counted using WST-8 reagent, and the cell viability of gel compositions 2 and 4 was calculated, assuming the number of cells in the control as 100%. The results are shown in Figure 11.

[0123] As shown in FIG. 11, both the samples of Gel Composition 2 and Gel Composition 4 exhibited high cell viability, confirming that they had high cytocompatibility.

[0124] (7) Fabrication of structures (gel compositions) using a 3D printer, cell culture tests, and microscopic observations First, a pre-gel solution was prepared using gelator 2 in the same manner as in "(1) Microscopic observation of gel composition" above. For CLSM observation, polymers (I) and (II) were fluorescently labeled in the same manner as in "(1) Microscopic observation of gel composition."

[0125] The prepared pre-gel solution was used as a photocurable composition 51 for a 3D printer in a 3D printer (BioX, Cellink) (see FIG. 12). The photocurable composition (pre-gel solution) 51 could be ejected without any problems from the 3D printer 50, and a grid-shaped structure (gel composition) 56 could be produced (nozzle: 27G, print speed: 20 mm / s, pressure: 80 kPa, wavelength: 405 nm, UV irradiation time: 15 seconds).

[0126] Next, the prepared structure (gel composition) 56 was immersed in PBS for 24 hours, and the immersed structure 56 was observed using a confocal laser scanning microscope (CLSM). As shown in FIG. 12, a porous structure with fibrous pores (capillaries) was formed in the structure 56 after immersion in PBS, similar to the gel composition prepared by dropping from a pipette in "(1) Microscopic observation of gel composition." In the CLSM image of FIG. 12, a mottled pattern of light and shade can be seen in the lattice-like gel composition. The dark areas of the mottled pattern represent voids (pores, capillaries) where no gel composition is present.

[0127] <Cell culture test> Using the same method as described above in "(1) Microscopic Observation of Gel Composition," a pre-gel solution was prepared using gelling agent 2, and mouse myoblasts (C2C12) were dispersed therein to prepare a photocurable composition for 3D printers. This cell-containing photocurable composition was ejected from a 3D printer and irradiated with UV light using the same method as described above to produce a lattice-shaped structure (gel composition). The structure (gel composition) was immersed in culture medium and cultured for 48 hours. After culture, the cells were stained for actin, and the structure was observed using a confocal laser scanning microscope (CLSM).

[0128] In the CLSM image of the lattice-like gel composition in Figure 13, the light-colored (white) areas of the shading pattern represent stained cells. As can be seen from Figure 13, cells adhered and spread within the gel composition, demonstrating that this material functions as a cell scaffold. [Industrial Applicability]

[0129] 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 medical applications, such as medical materials and cell scaffold materials. [Explanation of symbols]

[0130] 100 Gel composition 21. A first phase comprising a first polymer 22 Second phase containing second polymer 50 3D printers 51 Photocurable composition for 3D printers (gelling agent solution) 52 Storage 53 Discharge part 54 Ultraviolet light (UV light) 55 UV light irradiation section 56 Structures (three-dimensional gel compositions)

Claims

1. A gelling agent, 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; a photopolymerization initiator; 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 an ethylenically unsaturated group.

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

3. The gelling agent according to claim 1 or 2, wherein the second biopolymer is gelatin.

4. The gelling agent according to any one of claims 1 to 3, wherein a second polymer has the ethylenically unsaturated group.

5. The gelling agent according to any one of claims 1 to 3, wherein a first polymer has the ureidopyrimidinone group and the ethylenically unsaturated group.

6. The gelling agent according to any one of claims 1 to 5, wherein the ethylenically unsaturated group is at least one selected from the group consisting of an acryloyl group and a methacryloyl group.

7. The gelling agent according to any one of claims 1 to 6, which is a photocurable gelling agent.

8. The gelling agent according to any one of claims 1 to 7, which is capable of forming a gel composition having a phase-separated structure.

9. The gelling agent according to claim 8 , wherein the gel composition becomes porous in an aqueous solvent.

10. An injectable gel comprising the gelling agent according to any one of claims 1 to 9.

11. The injectable gel of claim 10, which is a one-component type.

12. An adhesive for biological tissue, comprising the gelling agent according to any one of claims 1 to 9.

13. A photocurable composition for 3D printers, comprising the gelling agent according to any one of claims 1 to 9.

14. A gel composition comprising the gelling agent according to any one of claims 1 to 9.

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

16. In an aqueous solvent, The gel composition according to claim 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 constituted by said one phase is formed.

17. A cell scaffold material comprising the gel composition according to any one of claims 14 to 16.

18. A medical material comprising the gel composition according to any one of claims 14 to 16.

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