Gel materials for regenerative medicine

Abstract

[Problem] The problem addressed is to provide novel uses to which a high-molecular-weight gel material with a porous structure on the nanometer scale can advantageously be applied. [Solution] A gel material for regenerative medicine includes a high-molecular-weight gel formed by mutually cross-linking hydrophilic polymer units, and is characterized in that: the high-molecular-weight gel contains water as a solvent, and comprises a three-dimensional mesh structure having two regions, namely, a first region in which the polymer units are densely present, and a second region in which the polymer units are sparsely present; and the mesh size formed by the first region is in the range 1–500 μm.

Description

[Technical field]

[0001] The present invention relates to a gel material for regenerative medicine comprising a polymer gel having a porous structure composed of two regions, a concentrated phase and a dilute phase, of a hydrophilic polymer component, and a kit for producing said gel material. [Background technology]

[0002] In recent years, polymer gels having a network structure have been expected to be used for a variety of purposes, such as sensors and surface coatings, as well as for medical purposes, such as artificial tissues, scaffolding materials for regeneration, sealing, adhesion prevention, drug delivery, and contact lenses, due to their excellent water retention and biocompatibility properties (e.g., Non-Patent Document 1). In particular, for these applications, the development of polymer materials having a micrometer-scale porous structure is desired.

[0003] However, in the past, in order to obtain a micrometer-scale porous structure, it was necessary to use a top-down method, such as microfabrication of pre-prepared gel or polymer structures by lithography, or to prepare a polymer material using a polymer raw material that is insoluble in a solvent. On the other hand, when using a solvent-philic polymer raw material, the material itself either dissolves in the solvent or only a gel with a small nanometer-scale porous structure could be prepared. Furthermore, there have been no reports of the application of such a porous gel material to the regeneration or repair of biological tissue. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sakai et al., Macromolecules, 41, 5379-5384, 2008 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide a gel material composed of a solvent-philic polymer having a μm-scale porous structure, and further to provide a new application to which such a gel material can be suitably applied. [Means for solving the problem]

[0006] As a result of intensive research aimed at solving the above problems, the present inventors have found that a polymer gel obtained by crosslinking a hydrophilic polymer raw material under specific conditions has a unique structure not seen in conventional polymer gels, in which the polymer gel behaves like phase separation in a poor-solvent polymer and forms a sponge-like three-dimensional network structure (micrometer-scale porous structure) consisting of dense regions where the polymer component is densely present and dilute regions where the polymer component is sparsely present, and further that in the regeneration and repair of biological tissues such as bones, the regeneration and repair of biological tissues can be promoted by applying a gel material containing such a polymer gel to the affected area. Based on these findings, the present invention has been completed.

[0007] That is, in one aspect, the present invention provides <1> A gel material for regenerative medicine comprising a polymer gel formed by crosslinking hydrophilic polymer units to each other, the polymer gel containing water as a solvent and having a three-dimensional network structure having two regions, a first region in which the polymer units are densely present and a second region in which the polymer units are sparsely present, the size of the network formed by the first region being 1 to 500 μm. This provides:

[0008] In addition, as a preferred embodiment of the gel material for regenerative medicine of the present invention, <2> The polymer unit has a transmittance lower than the transmittance of the polymer unit before gelation. <1> 2. A gel material for regenerative medicine according to claim 1 ; <3> The above has an osmotic pressure in the range of 1 / 5 to 1 / 2 of the osmotic pressure of the polymer unit before gelation. <1> or <2> 2. A gel material for regenerative medicine according to claim 1 ; <4> Osmotic pressure (Πos ) and elastic pressure (Π el ) is Π el >Π os The above has the relationship <1> ~ <3> 1. A gel material for regenerative medicine according to any one of the preceding claims. <5> The polymer concentration in the first region is 10 to 99% by weight, and the polymer concentration in the second region is 0 to 1% by weight. <1> ~ <4> 1. A gel material for regenerative medicine according to any one of the preceding claims. <6> The polymer content is 5% by weight or less. <1> ~ <5> 1. A gel material for regenerative medicine according to any one of the preceding claims. <7> The polymer unit is a polymer having a polyethylene glycol skeleton or a polyvinyl skeleton. <1> ~ <6> 1. A gel material for regenerative medicine according to any one of the preceding claims. <8> The polymer unit comprises a first polymer unit having one or more nucleophilic functional groups at a side chain or at a terminal, and a second polymer unit having one or more electrophilic functional groups at a side chain or at a terminal. <1> ~ <7> 1. A gel material for regenerative medicine according to any one of the preceding claims. <9> The nucleophilic functional group is selected from the group consisting of a thiol group and an amino group, and the electrophilic functional group is selected from the group consisting of a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group, an imidazoyl group, an acryloyl group, a nitrophenyl group, and a -CO 2 PhNO 2 The above, selected from the group consisting of <8> 2. A gel material for regenerative medicine according to claim 1 ; <10> The above-mentioned method for use in regenerating tissue <1> ~ <9> 1. A gel material for regenerative medicine according to any one of the preceding claims. <11> The tissue is bone, cartilage, skin, or nerve. <10> A gel material for regenerative medicine according to the present invention; and <12> the above <1> ~ <11> A method of treatment using the gel material for regenerative medicine according to any one of the above items. This provides:

[0009] In another aspect, the present invention also relates to a kit for producing the above-mentioned gel material for regenerative medicine, <13>A kit for producing a gel material for regenerative medicine, characterized in that two types of solutions of the following (A) and (B) are stored without being mixed with each other: (A) an aqueous solution containing a hydrophilic first raw material polymer having a concentration less than the overlap concentration and not less than the critical gelation concentration; (B) an aqueous solution containing a hydrophilic second raw material polymer having a concentration less than the overlap concentration and not less than the critical gelation concentration; the solution of (A) and / or (B) further contains a non-reactive polymer having no functional group capable of undergoing a crosslinking reaction with the raw material polymer in the molecule, and the polymer gel obtained by mixing the solutions of (A) and (B) is a polymer gel formed by crosslinking the first raw material polymer and the second raw material polymer with each other, and the polymer gel has a structure composed of two regions: a first region where polymer units derived from the raw material polymer are densely present, and a second region where polymer units derived from the raw material polymer are sparsely present; and <14>A kit for producing a gel material for regenerative medicine, characterized in that two types of solutions of the following (A') and (B') are stored without being mixed with each other: (A') an aqueous solution containing a first gel precursor, wherein the first gel precursor is obtained by crosslinking a hydrophilic raw material polymer under conditions of a concentration less than the overlap concentration and less than the critical gelation concentration, and has a relationship of G'<G" in the storage elastic modulus G' and the loss elastic modulus G"; (B') an aqueous solution containing a second gel precursor, wherein the second gel precursor is obtained by crosslinking a hydrophilic raw material polymer under conditions of a concentration less than the overlap concentration and less than the critical gelation concentration, and has a relationship of G'<G" in the storage elastic modulus G' and the loss elastic modulus G"; the solution of (A') and / or (B') further contains a crosslinking agent, and the polymer gel obtained by mixing the solutions of (A') and (B') is a polymer gel formed by crosslinking the first raw material polymer and the second raw material polymer with each other, and the polymer gel has a structure composed of two regions: a first region where polymer units derived from the raw material polymer are densely present, and a second region where polymer units derived from the raw material polymer are sparsely present; the kit is provided.

[0010] As a preferred embodiment of the kit of the present invention, <15> The raw polymer is a polymer having a polyethylene glycol skeleton or a polyvinyl skeleton. <13> or <14> 2. A kit according to claim 1 ; <16> The raw polymer comprises a first polymer having one or more nucleophilic functional groups at a side chain or at a terminal, and a second polymer having one or more electrophilic functional groups at a side chain or at a terminal. <13> ~ <15> 1. The kit according to any one of the preceding claims. <17> The nucleophilic functional group is selected from the group consisting of a thiol group and an amino group, and the electrophilic functional group is selected from the group consisting of a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group, an imidazoyl group, an acryloyl group, a nitrophenyl group, and a -CO 2 PhNO 2 The above, selected from the group consisting of <16> 2. A kit according to claim 1 ; <18> The non-reactive polymer is polyethylene glycol or cellulose having no crosslinking reactive group. <13> 2. A kit according to claim 1 ; <19> The first gel precursor and the second gel precursor each comprise a first polymer having one or more nucleophilic functional groups at a side chain or at an end, and a second polymer having one or more electrophilic functional groups at a side chain or at an end, the first gel precursor has a higher content of the first polymer than the content of the second polymer, and the second gel precursor has a higher content of the second polymer than the content of the first polymer. <14> 2. A kit according to claim 1 ; <20> The gel precursor has a diameter in the range of 10 to 1000 nm. <14> 2. A kit according to claim 1 ; <21> the above <13> ~ <20> A method for producing a gel material for regenerative medicine in a living body by using the kit according to any one of the above items 1 to 5; and <22> In the treatment of tissue regeneration, <13> ~ <20> Use of the kit according to any one of claims 1 to 5 This provides: Effect of the Invention

[0011] The gel material for regenerative medicine of the present invention can promote the regeneration and repair of biological tissues such as bones by applying the gel material to the affected area. Specifically, as shown in the examples, filling a bone defect with the gel material for regenerative medicine of the present invention can improve bone union and new bone growth in the bone defect.

[0012] Furthermore, by using the kit of the present invention, it is possible to form a gel material for regenerative medicine in situ within a living body, and therefore the kit can be used as a gel material that can be injected into closed or semi-closed cavities within a living body. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a graph showing the time scale of the gelation reaction in the polymer gel of the present invention prepared in Example 2. [Diagram 2] FIG. 2 is a graph showing the change in transmittance during the gelation process of the present invention. [Diagram 3] FIG. 3 is a graph showing the change in transmittance of a comparative gel that does not go through a gel precursor. [Figure 4] FIG. 4 is a graph showing the change over time in the swelling degree of the polymer gel of the present invention. [Diagram 5] FIG. 5 is a graph showing the change in osmotic pressure (Πos) of the polymer gel of the present invention. [Figure 6] FIG. 6 shows fluorescence microscope images of the polymer gel of the present invention (right) and the comparative gel (left). [Figure 7] Figure 7A shows micro-CT images of rat tibias without (a-c) and with (d-f) polymer gel 2, 3, and 4 weeks after bone defect creation. Figure 7B shows HE-stained images of rat tibias without (g and h) and with (i and j) polymer gel 4 weeks after bone defect creation (black bar: 5 mm; i and j are enlarged views of the dashed squares in images g and h, respectively). [Figure 8]FIG. 8 shows micro-CT images of a sham (NTC; negative control), a comparative gel-filled sample ("Tetra-PEG gel"), and a polymer gel-filled sample of the present invention ("Tetra-PEG sponge"), taken 2, 4, and 8 weeks after the creation of a bone defect. [Figure 9] FIG. 9 shows images of the polymer gel of the present invention ("Oligo-TetraPEG gel") and the comparative gel (Tetra-PEG gel) when subcutaneously implanted into a rat. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented without departing from the spirit of the present invention.

[0015] 1. Gel material for regenerative medicine according to the present invention The gel material for regenerative medicine of the present invention is characterized by containing a polymer gel having a sponge-like porous structure on the μm scale.

[0016] In this specification, "regenerative medicine" broadly means a treatment to regenerate or repair biological tissues that are missing, damaged, or functionally impaired due to factors such as birth or disease, injury, accident, aging, etc., and to restore their functions. Therefore, it is not limited to treatment involving transplantation of somatic stem cells or pluripotent stem cells such as iPS cells collected from oneself or another person, or tissues obtained from such cells ("regenerative medicine in the narrow sense").

[0017] Furthermore, in this specification, the "tissue" that is the target of regenerative medicine is not particularly limited as long as it is a biological tissue that requires regeneration or repair, and examples thereof include bone, cartilage, skin, nerves, and organs.

[0018] The polymer gel, which is the main component of the gel material for regenerative medicine, and the method for producing the same will be described in detail below.

[0019] (1) Polymer gel The polymer gel contained in the gel material for regenerative medicine of the present invention is formed by crosslinking hydrophilic polymer units with each other to form a gel, and contains water as a solvent. It is a so-called hydrogel, i) a three-dimensional network structure having two regions, a first region in which the polymer units are densely present and a second region in which the polymer units are sparsely present, ii) The mesh size formed by the first region is characterized by having a size of 1 to 500 μm.

[0020] That is, although the polymer gel of the present invention is formed from solvent-philic polymer units, it behaves as if a poor solvent polymer is in a phase-separated state in the solvent, and has a structure in which two regions with different polymer concentrations are formed, namely, a dense phase (first region) in which the polymer components are densely present in the gel, and a dilute phase (second region) in which the polymer components are sparsely present. The polymer gel forms a sponge-like three-dimensional network structure / porous structure by this phase separation (hereinafter, such a structure may be called a "sponge-like porous structure"), and is also characterized by its network size being on the order of μm, which is much larger than the nm order obtained in conventional gels. Here, the first region is called a "concentrated phase" in the relative sense that the concentration (density) of the polymer units present in that region is higher than the density in the second region. Preferably, the first region has a concentration (density) of about 100 times that of the second region.

[0021] In this specification, the term "gel" generally refers to a dispersion system of a polymer that has high viscosity and has lost fluidity, and in which the storage modulus G' and loss modulus G" satisfy the relationship G'≧G".

[0022] As described above, the polymer gel of the present invention is characterized by having a micrometer-order porous structure. Specifically, the mesh size formed by the first region can be 1 to 500 μm, and is preferably 10 to 100 μm. The mesh size means the length of the long side of the mesh unit (i.e., hole) whose periphery is formed by the first region which is a dense phase. Alternatively, when the mesh unit is substantially circular, it can be the length of its diameter. Inside such a mesh unit, the second region which is a dilute phase and / or the solvent are present.

[0023] Typically, the first region, which is a thick phase, has a polymer concentration of 10 to 99% by weight based on the entire gel including the solvent, and the second region, which is a dilute phase, has a polymer concentration of 0 to 1% by weight. Preferably, the first region has a polymer concentration of 40 to 80% by weight, and the second region has a polymer concentration of 0.01 to 0.1% by weight.

[0024] The polymer content in the entire polymer gel in the present invention is 5% by weight or less, preferably 4% by weight or less, and more preferably 1.5 to 3.0% by weight.

[0025] As described above, water is used as the solvent contained in the polymer gel of the present invention. In this case, the polymer gel containing water becomes a hydrogel. However, in some cases, the solvent may further contain alcohols such as ethanol, or organic solvents such as DMSO.

[0026] The polymer units constituting the polymer gel used in the gel material for regenerative medicine of the present invention and the characteristic physical properties exhibited by the polymer gel will be described below.

[0027] 1-a. Polymer unit The polymer unit used to form the polymer gel in the present invention is a solvent-philic polymer, i.e., a polymer that is soluble in the solvent contained in the gel. That is, in a hydrogel in which the gel contains water as a solvent, the polymer unit is a hydrophilic polymer. As long as the polymer unit can form a gel by a gelling reaction (crosslinking reaction, etc.) in a solution, any polymer unit known in the art can be used depending on the application, shape, etc. of the final gel. More specifically, a polymer unit that can form a network structure, particularly a three-dimensional network structure, in the final gel by crosslinking the polymer units with each other is preferred.

[0028] The hydrophilic polymer used as the polymer unit may preferably be a polymer having a polyethylene glycol skeleton or a polyvinyl skeleton. Representative examples of the polymer having a polyethylene glycol skeleton include polymer species having a plurality of polyethylene glycol skeleton branches, and in particular, polymer species having four polyethylene glycol skeleton branches are preferred. Such a gel consisting of a four-branched polyethylene glycol skeleton is generally known as a Tetra-PEG gel, and a meshwork network is constructed by an AB-type cross-end coupling reaction between two types of four-branched polymers each having an electrophilic functional group such as an active ester structure and a nucleophilic functional group such as an amino group at the end (Matsunaga et al., Macromolecules, Vol. 42, No. 4, pp. 1344-1351, 2009). In addition, the Tetra-PEG gel can be easily prepared in situ by simple two-liquid mixing of each polymer solution, and it is also possible to control the gelation time by adjusting the pH and ionic strength during gel preparation. In addition, since this gel is mainly composed of PEG, it is also excellent in biocompatibility.

[0029] Polymers other than those having a polyethylene glycol skeleton may also be used as long as they can be crosslinked to gel. For example, polymers having a polyvinyl skeleton such as methyl methacrylate may also be used.

[0030] Although not necessarily limited thereto, in order to form a sponge-like porous structure in the final gel, the polymer unit is preferably a means of reacting and crosslinking two kinds of polymer species, a first polymer unit having one or more nucleophilic functional groups at the side chain or end, and a second polymer unit having one or more electrophilic functional groups at the side chain or end. Here, the total number of nucleophilic functional groups and electrophilic functional groups is preferably 5 or more. It is more preferable that these functional groups are present at the end. In addition, the composition may have a content of the first polymer unit greater than that of the second polymer unit, or the composition may have a content of the second polymer unit greater than that of the first polymer unit. As described later, in a preferred embodiment, two or more kinds of gel precursors having different compositions are formed, and then the gel precursors are further crosslinked to obtain a polymer gel.

[0031] Nucleophilic functional groups present in the polymer unit include thiol groups (-SH), amino groups, etc., and those skilled in the art can use known nucleophilic functional groups as appropriate. Preferably, the nucleophilic functional group is a -SH group. The nucleophilic functional groups may be the same or different, but are preferably the same. By using the same functional groups, the reactivity with the electrophilic functional groups that form crosslinks becomes uniform, making it easier to obtain a gel with a uniform three-dimensional structure.

[0032] The electrophilic functional group present in the polymer unit may be an active ester group. Examples of such an active ester group include a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group, an imidazolyl group, an acryloyl group, a nitrophenyl group, a -CO 2 PhNO 2(Ph represents an o-, m-, or p-phenylene group), and those skilled in the art can use other known active ester groups as appropriate. Preferably, the electrophilic functional group is a maleimidyl group. The electrophilic functional groups may be the same or different, but are preferably the same. When the functional groups are the same, the reactivity with the nucleophilic functional groups that form crosslinks becomes uniform, making it easier to obtain a gel with a uniform three-dimensional structure.

[0033] A non-limiting specific example of a preferred polymer unit having a nucleophilic functional group at its terminal is a compound represented by the following formula (I) having four branches of a polyethylene glycol skeleton and a thiol group at its terminal. [ka]

[0034] n 11 ~n 14 may be the same or different. 11 ~n 14 The closer the values ​​of n are, the more uniform the three-dimensional structure can be and the higher the strength will be. Therefore, in order to obtain a gel with high strength, it is preferable that they are the same. 11 ~n 14 If the value of is too high, the gel strength will be weak and 11 ~n 14 If the value of n is too low, the gel is difficult to form due to the steric hindrance of the compound. 11 ~n 14 is an integer value of 25 to 250, preferably 35 to 180, more preferably 50 to 115, and particularly preferably 50 to 60. The molecular weight is 5×10 3 ~5×10 4 Da is 7.5 x 10 3 ~3×10 4 Da is preferred, 1×10 4 ~2×10 4 Da is more preferred.

[0035] In the above formula (I), R 11~R 14 R is a linker moiety that connects the functional group to the core moiety. 11 ~R 14 may be the same or different, but are preferably the same in order to produce a high-strength gel having a uniform three-dimensional structure. 11 ~R 14 is C 1 -C 7 Alkylene group, C 2 -C 7 Alkenylene group, -NH-R 15 -, -CO-R 15 -, -R 16 -OR 17 -, -R 16 -NH-R 17 -, -R 16 -CO 2 -R 17 -, -R 16 -CO 2 -NH-R 17 -, -R 16 -CO-R 17 -, R 16 -NH-CO-R 17 -or-R 16 -CO-NH-R 17 - where R 15 is C 1 -C 7 R represents an alkylene group. 16 is C 1 -C 3 R represents an alkylene group. 17 is C 1 -C 5 Represents an alkylene group.

[0036] Here, "C 1 -C 7 The term "alkylene group" refers to an alkylene group having 1 to 7 carbon atoms, which may be branched, and is a straight-chain C 1 -C 7 Alkylene group or C having one or more branches 2 -C 7 It means an alkylene group (having 2 to 7 carbon atoms, including branches). 1 -C 7Examples of the alkylene group include a methylene group, an ethylene group, a propylene group, and a butylene group. 1 -C 7 An example of an alkylene group is -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -CH(CH 3 )-, -(CH 2 ) 3 -, -(CH(CH 3 )) 2 -, -(CH 2 ) 2 -CH(CH 3 )-, -(CH 2 ) 3 -CH(CH 3 )-, -(CH 2 ) 2 -CH(C 2 H 5 )-, -(CH 2 ) 6 -, -(CH 2 ) 2 -C(C 2 H 5 ) 2 - and -(CH 2 ) 3 C(CH 3 ) 2 CH 2 - etc.

[0037] "C 2 -C 7 The term "alkenylene group" refers to a linear or branched alkenylene group having 2 to 7 carbon atoms and having one or more double bonds in the chain, and examples thereof include a divalent group having a double bond formed by removing 2 to 5 hydrogen atoms from adjacent carbon atoms of the alkylene group.

[0038] On the other hand, non-limiting specific examples of preferred polymer units having an electrophilic functional group at their terminals include, for example, compounds represented by the following formula (II) having four branches of a polyethylene glycol skeleton and a maleimidyl group at their terminals. [ka]

[0039] In the above formula (II), n 21 ~n 24 may be the same or different. 21 ~n 24 The closer the values ​​of n are, the more uniform the gel can be in a three-dimensional structure and the higher the strength, which is preferable, and it is more preferable that they are the same. 21 ~n 24 If the value of is too high, the gel strength will be weak and 21 ~n 24 If the value of n is too low, the gel is difficult to form due to the steric hindrance of the compound. 21 ~n 24 is an integer value of 5 to 300, preferably 20 to 250, more preferably 30 to 180, further preferably 45 to 115, and further preferably 45 to 55. The molecular weight of the second four-branching compound of the present invention is 5×10 3 ~5×10 4 Da is raised to 7.5×10 3 ~3×10 4 Da is preferred, 1×10 4 ~2×10 4 Da is more preferred.

[0040] In the above formula (II), R 21 ~R 24 R is a linker moiety that connects the functional group to the core moiety. 21 ~R 24 may be the same or different, but are preferably the same in order to produce a high-strength gel having a uniform three-dimensional structure. 21 ~R 24 are the same or different, C 1 -C 7 Alkylene group, C 2 -C 7 Alkenylene group, -NH-R 25 -, -CO-R 25 -, -R 26 -OR 27 -, -R 26 -NH-R 27 -, -R26 -CO 2 -R 27 -, -R 26 -CO 2 -NH-R 27 -, -R 26 -CO-R 27 -, -R 26 -NH-CO-R 27 - or -R 26 -CO-NH-R 27 - where R 25 is C 1 -C 7 R represents an alkylene group. 26 is C 1 -C 3 R represents an alkylene group. 27 is C 1 -C 5 Represents an alkylene group.

[0041] In this specification, the alkylene group and the alkenylene group may have one or more optional substituents. Examples of the substituents include, but are not limited to, an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, an acyl group, or an aryl group. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl portion of other substituents containing an alkyl portion (e.g., an alkyloxy group, an aralkyl group, etc.).

[0042] In addition, in the present specification, when a functional group is defined as "optionally having a substituent", the type of the substituent, the substitution position, and the number of the substituent are not particularly limited, and when it has two or more substituents, they may be the same or different. Examples of the substituent include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent.

[0043] 1-b. Properties of polymer gels As described above, the polymer gel used in the regenerative medicine gel material of the present invention has a sponge-like porous structure on the order of μm formed by a dense phase (first region) and a dilute phase (second region) within the gel, and this structure gives it characteristic properties in terms of various physical properties.

[0044] The polymer gel of the present invention has a transmittance lower than that of the polymer unit before gelation. This is because the polymer gel has two regions with different polymer concentrations, a dense phase (first region) and a dilute phase (second region), and behaves as if a poor solvent polymer is in a phase-separated state in the solvent, so that it is not completely transparent but exhibits a cloudy appearance. Preferably, the polymer gel has a transmittance in the range of 90 to 96%. In terms of the transmittance, the polymer gel exhibits characteristics that are completely different from those of ordinary polymer gels, which are almost transparent.

[0045] The polymer gel of the present invention has an osmotic pressure in the range of 1 / 5 to 1 / 2 of the osmotic pressure of the polymer unit before gelation, and also has a lower osmotic pressure than a single-phase polymer gel formed from the same polymer unit.

[0046] In addition, the polymer gel of the present invention has an osmotic pressure (Π os ) and elastic pressure (Π el ) is Π el >Π os This Π el It is better to os The larger the relationship, the more likely the gel is to shrink. el <Π os This contrasts with the tendency for the material to swell, which has a relationship of

[0047] Although not necessarily bound by theory, the polymer gel used in the gel material for regenerative medicine of the present invention has a two-phase separation structure consisting of a dense phase and a dilute phase. os) is lower than that of a normal single-phase polymer gel, while it is understood that the gel tends to shrink due to an increase in elastic pressure. In these respects, it can be said that the polymer gel of the present invention has characteristic properties that are significantly different from conventional polymer gels.

[0048] Furthermore, since the polymer densities of the polymer units in the dense phase and the dilute phase are different as described above, the polymer gel of the present invention can have different water contents in these two regions. Specifically, the polymer gel of the present invention has a water content in the first region (concentrated phase) in the range of 10 to 99%, and a water content in the second region (dilute phase) in the range of 99 to 100%.

[0049] The polymer gel of the present invention can be processed into various shapes, such as a thin film, depending on the application. Any method known in the art can be used for such processing. For example, in the case of a thin film, the gel can be obtained by applying the gel, in a fluid state before it is completely solidified, onto a flat substrate such as glass.

[0050] (2) Method for producing polymer gel Next, a method for producing a polymer gel (gelling step) used in the gel material for regenerative medicine of the present invention will be described. The polymer gel of the present invention can be produced, for example, by the steps shown in the first and second embodiments below, and in either embodiment, it can be produced by crosslinking a solvent-philic raw polymer under conditions below the overlap concentration. By carrying out the gelling step under such conditions, a polymer gel having a micrometer-order pore structure, which was previously difficult to produce, can be produced from a solvent-philic polymer.

[0051] 2-a. First embodiment of the method for producing polymer gel In a first aspect, the method for producing a polymer gel according to the present invention is characterized in that it comprises the following steps: a) crosslinking a solvent-philic starting polymer under conditions below the overlap concentration and below the critical gelation concentration to form a gel precursor; b) A step of crosslinking the gel precursors with each other using a crosslinking agent to obtain the final product, a polymer gel.

[0052] In step a), the raw polymers (polymer units) that will ultimately constitute the polymer gel are reacted in a state immediately before gelation to form gel precursors (polymer clusters) that have a structure that has not yet reached gel formation, i.e., in a sol state. In step b), an appropriate crosslinking agent is then added as desired to further react these gel precursors with each other, and crosslink them three-dimensionally to obtain the final product, the polymer gel. Here, the gel precursor is not necessarily limited to a single type with the same composition, as described later, but multiple gel precursors with different compositions can also be used. In this way, the manufacturing method of the first embodiment is based on the concept of using the gel precursor as an intermediate of the final gel, so to speak.

[0053] In step a), the initial concentration of the raw material polymer is set to be less than the overlap concentration and less than the critical gelation concentration, and by using such an initial concentration of the raw material polymer, a sol state not reaching gelation, preferably a gel precursor having a structure immediately before gelation, can be formed.

[0054] In step a), the initial concentration of the raw polymer is the overlap concentration C * Less than 1 / 3C * Here, the "overlap concentration" (also called "overlap concentration") is the concentration at which polymers in a solvent begin to spatially contact each other, and is generally less than the overlap concentration C * is expressed by the following formula:

number

[0055] Overlap density C * For the calculation method, see, for example, Polymer Physics (by M. Rubinstein and R. Colby). Specifically, for example, it can be calculated by measuring the viscosity of a dilute solution and using the Flory-Fox equation.

[0056] In addition, in step a), the initial concentration of the raw polymer is set to be less than the critical gelation concentration. Here, the "critical gelation concentration" means the minimum concentration of the raw polymer required to achieve gelation in a system in which a three-dimensional gel is constructed by crosslinking the raw polymer, and is also called the minimum gelation concentration. In the present invention, the term critical gelation concentration includes, for example, in a system in which two or more raw polymers are used, not only the case in which the total concentration of the raw polymers does not reach the concentration that leads to gelation, but also the case in which only one raw polymer has a low concentration, i.e., the ratio of the raw polymers is unequal, so that gelation does not occur.

[0057] Generally, the critical gelation concentration (minimum gelation concentration) depends on the type of raw polymer used, but such concentration is known in the art or can be easily determined experimentally by a person skilled in the art. Typically, it is 0.5 to 5% by weight, and the lower limit is about 1 / 5 of the overlap concentration.

[0058] As a method for adjusting the initial concentration of the raw material polymer to a condition below the critical gelation concentration, for example, when two types of polymer units having nucleophilic functional groups or electrophilic functional groups are used as described above, a condition in which they are contained in equivalent amounts but at a low concentration that is not sufficient to cause gelation as a whole can be used, or a condition in which the concentration of one type of polymer unit is low, i.e., non-equivalent, so that gelation does not occur can be used.

[0059] Step a) can typically be carried out by mixing or stimulating a solution containing two types of raw material polymers. It can also be carried out by radical polymerization of monomers using a radical initiator. The concentration, addition rate, mixing rate, and mixing ratio of each solution are not particularly limited and can be appropriately adjusted by those skilled in the art. Also, it will be apparent that even when using three or more types of raw material polymers, similarly, solutions containing the corresponding raw material polymers can be prepared and appropriately mixed. As the solvent for the solution containing the raw material polymer, water, alcohols such as ethanol, DMSO, etc. can be used. When the solution is an aqueous solution, an appropriate pH buffer such as a phosphate buffer can be used.

[0060] As a means of mixing, for example, it can be carried out using a two - liquid mixing syringe as disclosed in International Publication WO2007 / 083522. The temperature of the two liquids during mixing is not particularly limited and may be a temperature at which the precursor units are each dissolved and each liquid has fluidity. For example, the temperature of the solution when mixing can be in the range of 1°C to 100°C. The temperatures of the two liquids may be different, but it is preferable that the temperatures are the same because it is easier to mix the two liquids.

[0061] The gel precursor obtained in step a) has a structure in which the precursor units are bonded or cross - linked to each other, but is formed under conditions where gelation has not yet occurred. Therefore, the gel precursor has a relationship of G’ < G” in terms of the storage modulus G’ and the loss modulus G”. Generally, in a polymer before gelation, the value of the loss modulus G” is larger than the storage modulus G’, and then, with gelation, the magnitude of these physical property values is reversed and G’ becomes larger. And the point where G’ = G” is the so - called gelation point. Therefore, the fact that the gel precursor cluster has G’ < G” means that it is in a sol state and has not yet reached the gelation state. Preferably, at a frequency of 1 Hz, G’ < G” < 100G’.

[0062] Preferably, G" of the gel precursor is in the range of 0.005 to 5 Pa at a frequency of 1 Hz, more preferably in the range of 0.01 to 1 Pa, and even more preferably in the range of 0.01 to 0.5 Pa. These elastic moduli can be calculated by a known method such as dynamic viscoelasticity measurement using a known measuring instrument such as a rheometer.

[0063] The gel precursor in the present invention preferably has a diameter of 10 to 1000 nm, more preferably 50 to 200 nm, and preferably has a highest proportion of gel precursors having a diameter of about 100 nm in the distribution.

[0064] The raw polymer used to form the gel precursor cluster is hydrophilic, i.e., soluble in water, which is the solvent contained in the gel. The detailed description of these is the same as that of the polymer unit in the polymer gel of the present invention described above.

[0065] Next, in step b), the gel precursors obtained in step a) are further reacted with each other and cross-linked three-dimensionally to obtain a polymer gel as the final product. As described above, the gel precursors are formed to be in a state before the gelation point, so that the substituents used for cross-linking in each gel precursor remain unreacted. Therefore, the substituents in the gel precursors can be reacted with the remaining substituents of other gel precursors and further cross-linked to form the final gel.

[0066] The polymer gel finally obtained in step b) is characterized by having a structure composed of two regions, a first region in which the polymer units derived from the raw polymer are densely present, and a second region in which the polymer units derived from the raw polymer are sparsely present. As a result, a polymer gel having a micrometer-order porous structure can be obtained from a solvent-philic raw polymer, as already described in the description of the polymer gel according to the present invention.

[0067] Preferably, in the step b), a crosslinking agent for crosslinking the gel precursors with each other can be added or stimulated. Such a crosslinking agent can be one having the same substituent as the crosslinking group in the raw polymer, or the raw polymer itself can be used as a crosslinking agent and added additionally. For example, in the step a), when a gel precursor is obtained by reacting two kinds of raw polymers having a nucleophilic functional group or an electrophilic functional group in unequal amounts, the gel precursors can be further crosslinked by adding a crosslinking agent having the functional group with the lower concentration. Such a crosslinking agent can be bis(sulfosuccinimidyl) glutarate (BS 2 Examples of the stimuli that can be used include thiol groups such as thiolamine (G), DL-dithiothreitol (DTT), and synthetic peptides with thiol groups at the termini. For example, UV light can be irradiated onto a functional group that undergoes photodimerization (such as a maleimide group) to stimulate crosslinking.

[0068] In a preferred embodiment, step b) can also be carried out in the presence of a non-reactive polymer that does not have a functional group capable of crosslinking with the gel precursor in the molecule. Such a non-reactive polymer is suitable for obtaining a three-dimensional structure such as a two-phase separation of a thick phase and a thin phase. An example of such a non-reactive polymer is a polymer that has the same basic skeleton as the raw polymer but does not have a crosslinking reactive group in the side chain or at the end, such as polyethylene glycol that does not have a crosslinking reactive group. Alternatively, materials such as cellulose and modified cellulose can be used.

[0069] Other reaction solution conditions in step b) are the same as those in step a). Preferably, in step b), the final gel can be obtained within a reaction time of 2 hours or less, and more preferably within a reaction time of 1 hour or less. In general, when preparing a gel containing a low concentration of polymer, a long reaction time is required (for example, about 8 hours when the polymer content is 1% by weight or less, although this depends on the system), whereas the above-mentioned production method allows the gel to be prepared in a much shorter time.

[0070] 2-b. Second embodiment of the method for producing polymer gel In a second aspect, the method for producing a polymer gel according to the present invention is characterized in that it comprises the following steps: c) obtaining the polymer gel by crosslinking solvent-philic raw polymers having a concentration below the overlap concentration and above the critical gelation concentration in the presence of a predetermined concentration of a non-reactive polymer.

[0071] That is, in the production method of the second embodiment, by adding a non-reactive polymer of a predetermined concentration to the solution, the polymer gel of the present invention can be obtained in one step without first going through a gel precursor from the raw polymer as in the first embodiment. Even in this case, by using the raw polymer with a concentration less than the overlap concentration, it is possible to obtain a polymer gel having two regions of a dense phase and a dilute phase and a micrometer-order porous structure.

[0072] Here, the non-reactive polymer is a polymer that does not have a functional group capable of crosslinking with the raw polymer in the molecule, and can be a polymer that has the same basic skeleton as the raw polymer but does not have a crosslinking reactive group in the side chain or at the end, for example, polyethylene glycol that does not have a crosslinking reactive group. Alternatively, materials such as cellulose and modified cellulose can be used. The concentration of the non-reactive polymer is preferably in the range of 1 / 10 to 10 of the raw polymer concentration, more preferably 1 / 5 to 2 / 10. The range is.

[0073] The second embodiment is the same as the first embodiment in that the initial concentration of the raw polymer is less than the overlap concentration, whereas the second embodiment is different from the first embodiment in that the initial concentration of the raw polymer is equal to or greater than the critical gelation concentration, and gelation is carried out in one step.

[0074] Other than that, the reaction solution conditions, mixing method, etc. are the same as those in the first embodiment.

[0075] 2. Kit of the Present Invention In another aspect, the present invention relates to a kit for producing a gel material for regenerative medicine, the kit being based on the above-mentioned method for producing a polymer gel.

[0076] Specifically, in accordance with the above-mentioned aspects of the method for producing a polymer gel, kits of the first and second aspects shown below can be mentioned (although the order is reversed, preferred aspects of these kits correspond to the second and first aspects of the above-mentioned production method, respectively). However, it should be understood that polymer gels used as gel materials for regenerative medicine can be produced without using these kits.

[0077] In a first aspect, the kit of the present invention is characterized in that it contains the following two types of solutions (A) and (B) without being mixed with each other: (A) an aqueous solution containing a hydrophilic first raw polymer having a concentration less than the overlap concentration and greater than or equal to the critical gelation concentration; (B) An aqueous solution containing a hydrophilic second raw material polymer having a concentration less than the overlap concentration and greater than or equal to the critical gelation concentration.

[0078] In addition, in the first aspect, the kit of the present invention further comprises, in the solution (A) and / or (B), a non-reactive polymer that does not have a functional group in its molecule capable of undergoing a crosslinking reaction with the raw material polymer.

[0079] The polymer gel obtained using the kit of the present invention has the following structural characteristics as described above: That is, the polymer gel obtained by mixing the above solutions (A) and (B) is a polymer gel in which the first raw polymer and the second raw polymer are crosslinked with each other, and the polymer gel has a structure composed of two regions, a first region in which polymer units derived from the raw polymer are densely present, and a second region in which polymer units derived from the raw polymer are sparsely present.

[0080] In addition, in a second aspect of the kit of the present invention (corresponding to the first aspect of the production method), the kit is characterized in that the following two types of solutions (A') and (B') are contained without being mixed with each other: (A’) An aqueous solution containing a first gel precursor, wherein the first gel precursor is obtained by crosslinking a hydrophilic raw material polymer under conditions of a concentration lower than the overlap concentration and a concentration lower than the critical gelation concentration, and has a relationship of G’ < G” in the storage elastic modulus G’ and the loss elastic modulus G”; this solution; (B’) An aqueous solution containing a second gel precursor, wherein the second gel precursor is obtained by crosslinking a hydrophilic raw material polymer under conditions of a concentration lower than the overlap concentration and a concentration lower than the critical gelation concentration, and has a relationship of G’ < G” in the storage elastic modulus G’ and the loss elastic modulus G”; this solution.

[0081] In addition, in such a second aspect, the kit of the present invention further includes a non-reactive polymer having no functional group capable of undergoing a crosslinking reaction with the raw material polymer in the solution of (A’) and / or (B’) above in its molecule.

[0082] Also, similar to the above first correspondence, the polymer gel obtained using the kit of the present invention has the following structural characteristics. That is, the polymer gel obtained by mixing the solutions of (A’) and (B’) above is a polymer gel in which the above first raw material polymer and the above second raw material polymer are crosslinked with each other, and the polymer gel has a structure composed of two regions: a first region in which polymer units derived from the raw material polymer are densely present, and a second region in which polymer units derived from the raw material polymer are sparsely present.

[0083] A preferred embodiment of the raw material polymer used in the kit of the present invention is the same as the above polymer unit. For example, preferably, the raw material polymer in the kit of the present invention is a polymer having a polyethylene glycol skeleton or a polyvinyl skeleton. Similarly, in the kit of the present invention, preferably, the raw material polymer is composed of a first polymer having one or more nucleophilic functional groups in its side chain or at its terminal, and a second polymer having one or more electrophilic functional groups in its side chain or at its terminal. Here, the types of the nucleophilic functional group and the electrophilic functional group are also as described above.

[0084] In the second aspect of the kit of the present invention, preferably, the first gel precursor and the second gel precursor each comprise a first polymer having one or more nucleophilic functional groups at a side chain or at an end and a second polymer having one or more electrophilic functional groups at a side chain or at an end, the first gel precursor has a higher content of the first polymer than the content of the second polymer, and the second gel precursor has a higher content of the second polymer than the content of the first polymer. The gel precursor preferably has a diameter in the range of 10 to 1000 nm, more preferably 50 to 200 nm.

[0085] Furthermore, the non-reactive polymer contained in the kit of the present invention can be, for example, polyethylene glycol or cellulose that does not have a cross-linking reactive group.

[0086] The solutions (A), (B), (A') and (B') in the kit of the present invention may contain, as other components, an appropriate pH buffer solution such as a phosphate buffer.

[0087] 3. Treatment Method of the Present Invention As described above, the gel material for regenerative medicine of the present invention can be used in regenerative medicine applications, and is preferably suitable for tissue regeneration. Therefore, the present invention also relates to a treatment method (or tissue regeneration method) using the gel material for regenerative medicine.

[0088] The "tissue" that is the target of the treatment method (or tissue regeneration method) of the present invention is not particularly limited as long as it is a biological tissue that requires regeneration or repair, and examples thereof include bone, cartilage, skin, nerves, organs, etc. Therefore, the treatment method of the present invention is preferably intended for regeneration of bone, cartilage, skin, or nerves, and more preferably for regeneration of bone.

[0089] In the treatment method (or tissue regeneration method) of the present invention, specifically, it is preferable to cover the affected area with the gel material for regenerative medicine by administering, injecting or applying (hereinafter referred to as "administration, etc.") the gel material for regenerative medicine to the affected area requiring regeneration or repair. Alternatively, the gel material for regenerative medicine can be used to fill the space in the affected area (the area to be regenerated or repaired). For example, in the treatment of damage or rupture of bones or nerves, it is expected that the gel material for regenerative medicine will be administered, etc. to the affected area after a procedure such as reconnection of the ruptured or damaged part has been performed. In some cases, such administration, etc. may also create an environment in which the gel material for regenerative medicine is present in the surrounding area of ​​the affected area.

[0090] In the treatment method (or tissue regeneration method) of the present invention, when administering the gel material for regenerative medicine, the gel material for regenerative medicine can be prepared in situ (within a living body) at the affected area or its vicinity where it is needed. In this case, it is preferable to mix two types of polymer solutions (for example, solutions of (A) and (B), or solutions of (A') and (B')) at the affected area or its vicinity using the kit of the present invention based on the above-mentioned method for producing a polymer gel.

[0091] Therefore, the present invention provides a method for producing a gel material for regenerative medicine in vivo using the above kit. In another aspect, the present invention also provides use of the above kit in tissue regeneration treatment.

[0092] When preparing the gel material for regenerative medicine in situ (inside the living body) at the affected area or its vicinity, the gelation time is preferably within 1 hour, more preferably within 10 minutes, and even more preferably within 1 minute. As described above, the gelation time can be adjusted by appropriately setting the polymer concentration, pH, and ionic strength of the polymer solution included in the kit. EXAMPLES

[0093] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. 1 H NMR spectrum was analyzed using a JNM-ECS400 (400 MHz) manufactured by JEOL. Deuterated chloroform was used as the solvent, and tetramethylsilane was used as the internal standard. Molecular weight was determined using a linear positive ion mode of a Bruker Daltonics mass spectrometer Ultraflex III. In the following examples, the unit of g / L for polymer concentration is used, and 1 g / L corresponds to about 0.1% by weight. EXAMPLES

[0094] Synthesis of gel precursor [SHPEG+MAPEG] The gel precursor, which is the precursor in the gelation reaction, was synthesized as follows using SHPEG (tetrathiol-polyethylene glycol) having -SH groups at the ends and MAPEG (tetramaleimidyl-polyethylene glycol) having maleimidyl groups at the ends. These raw polymers SHPEG and MAPEG were both commercially available from NOF Corporation (both molecular weights were 10,000).

[0095] SHPEG and MAPEG were dissolved in 50 mM citrate-phosphate buffer solutions (the ratio of substances was SHPEG / MAPEG = 1 / 1, the total polymer concentration was 20 g / L). The two solutions obtained were mixed in a separate container and degassed and stirred using a centrifugal mixer. The mixture was then quickly transferred to a Falcon tube, capped to prevent drying, and left at room temperature for 12 hours. At this time, multiple samples were prepared containing two types of gel precursors in which either one was contained in excess, so that the molar ratios of SHPEG:MAPEG were 0.78:0.22 and 0.22:0.78.

[0096] The time changes of the storage modulus G’ and the loss modulus G” in the reaction process were observed using a rheometer (25 °C, 1 Hz). At the end point of the reaction, it was confirmed that the obtained gel precursor had a relationship of G’ < G” and was a sol-state polymer cluster that had not yet reached gel formation.

Example

[0097] Synthesis of polymer gel Using the gel precursor synthesized in Example 1, a polymer gel (Oligo-TetraPEG gel) was synthesized as follows.

[0098] The gel precursor solution A with r = 0.78 and the gel precursor solution B with r = 0.22 obtained in Example 1 were each diluted with water to a concentration of 10 g / L (or 20 g / L). The amount of unreacted SH groups in the solution was calculated, and a cross-linking agent (Bis-(sulfosuccinimidyl) glutarate (BS 2 G)) was added, and defoaming and stirring were performed using a rotary-revolution mixer. Then, the mixture was quickly transferred to a Falcon tube, capped to prevent drying, and left standing at room temperature for 5 hours. Observation with a rheometer (25 °C, 1 Hz) was carried out, and at the end point of the reaction, it was confirmed that the gel precursor had a relationship of G’ > G” and that a polymer gel was formed by cross-linking of the gel precursor (Figure 1).

Example

[0099] Transmittance of polymer gel The change in transmittance in the gelation process of Example 2 is shown in Figure 2. The transmittance was calculated from the time change in absorbance at 25 °C and a wavelength of 400 nm after injecting the two liquids of the gel precursor into the cell after mixing. In the figure, the changes in G’ and G” are also shown, and the point where G’ = G” is the gelation point.

[0100] Also, as a comparative example, Figure 3 shows the change in transmittance and the changes in G’ and G” for a single-phase polymer gel obtained by mixing two liquids of a 60 g / L SHPEG solution and a MAPEG solution without passing through the gel precursor.

[0101] 2 and 3, it was found that in a normal gel (FIG. 3) that does not go through a gel precursor, there was almost no change in transmittance before and after gelation (T = about 98%), whereas in the polymer gel of Example 2 (FIG. 2), the transmittance T decreased significantly from about 99% to about 94% upon gelation, and the gel became cloudy. This is because in the polymer gel of the present invention, two regions were generated upon gelation: a dense phase where polymer units are densely present, and a dilute phase where polymer units are sparsely present, resulting in phase separation. EXAMPLES

[0102] Polymer gel swelling test A swelling test was carried out on three types of polymer gels, namely, the polymer gels (a, b) of the present invention and the gel (c) of the comparative example. Gels a and b were synthesized according to Example 2. (a) Oligo-Tetra-PEG gel (10 g / L): Two types of gel precursors were prepared according to Example 2: 20 g / L, r = 0.78; 20 g / L, r = 0.22. Each gel precursor solution was diluted to 10 g / L, and the two liquids were mixed to form a gel. (b) Oligo-Tetra-PEG gel (20g / L): Two types of gel precursors were prepared using the same procedure as in Example 2: 20 g / L, r = 0.78 and 20 g / L, r = 0.22. The gel precursor solutions were mixed together and gelled. (c) Tetra-PEG gel (comparative example): Two liquids, 60 g / L SHPEG solution and MAPEG solution, were mixed and gelled.

[0103] A cylindrical sample (height 7 mm, diameter 15 mm) was prepared, and after the gelation reaction had fully completed (1 day), it was immersed in pure water and the change in swelling degree over time was measured at 25°C. The results are shown in Figure 4.

[0104] In the case of normal gel c, which did not go through a gel precursor, the gel was observed to swell over time, whereas the polymer gel of the present invention showed the characteristic property of shrinking with a long relaxation period. EXAMPLES

[0105] Osmotic pressure test of polymer gel The osmotic pressure (Π os ) and elastic pressure (Π el As comparative examples, the raw polymer before gelation and a normal gel not going through a gel precursor were also measured in the same manner. Polymer gel of the present invention: According to Example 2, gels were prepared under a number of conditions, including gel precursor solution concentrations of 10, 12.5, 15, 17.5, and 20 g / L. Raw polymer: MAPEG solutions of 10, 20, 30, 40, 50, and 60 g / L were used. Comparative Example Gel: The gels used were prepared by mixing two liquids, SHPEG and MAPEG solutions at 10, 20, 30, 40, 50, and 60 g / L, respectively.

[0106] A sample was prepared in a dialysis membrane and dialyzed against a PVP solution (29k, 5-120 g / L) to determine the concentration of PVP at which the gel did not swell from its initial state. os -Π el ) and the osmotic pressure of PVP (Π PVP ) is balanced. el = G') was measured using a rheometer, and the following equation was used to calculate Π os asked for.

number

[0107] The osmotic pressure (Π os ) is plotted in FIG. 5. As a result, the polymer gel of the present invention has a high overlap concentration (C *) below, it was found to have a lower osmotic pressure than both the raw polymer and the comparative gel at the same concentration. EXAMPLES

[0108] Fluorescence microscope image of polymer gel The structures of the gel of the present invention (a) and the comparative gel (b) were observed using a two-photon laser microscope (Zeiss). (a) Oligo-Tetra-PEG gel (10 g / L): Two types of gel precursors were prepared according to Example 2: 20 g / L, r = 0.78; 20 g / L, r = 0.22. Each gel precursor solution was diluted to 10 g / L, and the two liquids were mixed to form a gel. (b) Tetra-PEG gel (comparative example): Two liquids, 60 g / L SHPEG solution and MAPEG solution, were mixed and gelled.

[0109] After the above gel samples (a) and (b) were prepared, they were left to stand in water for 7 days before use. Primary staining: The specimen was immersed in an anti-PEG solution (0.04 g / L), left to stand for 1 hour, and then washed for 15 minutes three times. Secondary staining: The plate was immersed in antibody-AlexaFloar488 (0.04 g / L), left to stand for 1 hour, and then washed for 15 minutes three times.

[0110] The obtained fluorescence microscope image is shown in Figure 6. It was found that the comparative Tetra-PEG gel (left) had a mesh structure on the order of nm, whereas the polymer gel of the present invention (right) had a mesh structure (porous structure) on the order of μm, which is much larger than the structure predicted from the molecular structure. The outer periphery of the μm-order mesh structure is formed by a dense phase in which the polymer is densely present, and the inside is a dilute phase or solvent. EXAMPLES

[0111] Cell adhesion of polymer gels We also evaluated the adhesiveness of cells to the polymer gel of the present invention prepared in Example 2. As a result, we confirmed that the polymer gel of the present invention can efficiently adhere ATDC5 (somatic pluripotent cells) compared to a normal Tetra-PEG gel (comparative example) that does not go through a gel precursor. EXAMPLES

[0112] Application to bone regeneration (1) Verification in a rat tibia bone defect model The effect of the polymer gel having a sponge-like porous structure of the present invention on bone regeneration was examined using a rat tibia bone defect model. After creating a 9 mm bone defect in the tibia of an adult rat, the bone defect site was filled with a polymer gel (Oligo-TetraPEG gel) by the method of Example 2. Specifically, polymer solutions containing SHPEG and MAPEG were injected and mixed into the bone defect site, and gelation was performed in situ.

[0113] The tissues around the defect were then sutured, and the bone regeneration effect at the bone defect was observed over time (Figure 7). As a result of micro-CT analysis, after 4 weeks of bone defect, bone regeneration was incomplete in the non-polymer gel-filled group (control), and no bone union was observed at the bone defect end (Figure 7A, a-c). On the other hand, in the polymer gel-filled group, bone union was observed at the bone defect end 2 weeks after the bone defect was created, and after 4 weeks, most of the bone defect site was replaced by newly formed bone (Figure 7A, d-f). As a result of histological analysis of the bone defect site 4 weeks after the bone defect was created, infiltration of fibrous cells was observed at the bone defect site in the control group, the missing bone end did not heal, and bone regeneration was insufficient (Figure 7B, g and h). On the other hand, in the polymer gel-filled group, the defect site was almost completely replaced by newly formed bone, and the bone end was healed (Figure 7B, i and j).

[0114] (2) Verification in a mouse cranial bone defect model Next, after creating a bone defect with a diameter of 4 mm in the skull of an adult mouse, the effect of the polymer gel (Oligo-TetraPEG gel) was examined in the same manner as in (1) above. In this analysis, one day after creating a bone defect and suturing the tissue surrounding the defect, the polymer gel was filled into the defect site by mixing two polymer solutions containing SHPEG and MAPEG, respectively. As a comparison subject, in addition to the sham group (negative control; NTC), a comparative gel ("c) Tetra-PEG gel" in Example 4) was used. As a result, as shown in FIG. 8, the micro-CT analysis showed that almost no bone regeneration was observed in the sham group and the comparative gel group 12 weeks after the bone defect, whereas a remarkable bone regeneration effect was observed in the Oligo-TetraPEG gel group of the present invention (the "Tetra-PEG sponge" group in FIG. 8). EXAMPLES

[0115] Subcutaneous application A subcutaneous implantation test was carried out on rats for two types of gel, the gel of the present invention (a) and the comparative gel (b). (a) Oligo-TetraPEG gel: Two types of gel precursors were prepared according to Example 2: 20 g / L, r = 0.78; 20 g / L, r = 0.22. Each gel precursor solution was diluted to 10 g / L, and the two liquids were mixed to form a gel. (b) TetraPEG gel (comparative example): Two liquids, 20g / L SHPEG solution and MAPEG solution, were mixed and gelled.

[0116] The back of the rat was shaved under anesthesia, and two incisions were made on the left and right to create subcutaneous pockets. The right side was filled with the gel of the present invention (a), and the left side was filled with the comparative gel (b). Specifically, polymer solutions containing SHPEG and MAPEG were injected and mixed at the relevant sites, and gelation was allowed to occur in situ.

[0117] The wound skin was then sutured. Two weeks after the treatment, the rats were sacrificed, and the implants were extracted and evaluated macroscopically (Figure 9). It was found that soft tissue was formed within the gel of the present invention (a). On the other hand, no new tissue formation was observed in the comparative gel (b).

[0118] The above results demonstrate that the regeneration and repair of biological tissues can be promoted by applying a gel material for regenerative medicine containing the polymer gel having a sponge-like porous structure of the present invention to an affected area.

Claims

1. A gel material for regenerative medicine comprising a polymer gel in which hydrophilic polymer units are crosslinked with each other, The polymer gel is Contains water as a solvent, a three-dimensional network structure having two regions, a first region in which the polymer units are densely present and a second region in which the polymer units are sparsely present; The gel material for regenerative medicine, wherein the mesh size formed by the first region has a size of 1 to 500 μm.

2. The gel material for regenerative medicine according to claim 1 , which has a permeability lower than the permeability of the polymer unit before gelation.

3. 3. The gel material for regenerative medicine according to claim 1, having an osmotic pressure in the range of 1 / 5 to 1 / 2 of the osmotic pressure of the polymer unit before gelation.

4. Osmotic pressure (Π ｏｓ ) and elastic pressure (Π ｅｌ ) is Π ｅｌ >Π ｏｓ The gel material for regenerative medicine according to any one of claims 1 to 3, having the following relationship.

5. The gel material for regenerative medicine according to any one of claims 1 to 4, wherein the polymer concentration in the first region is 10 to 99% by weight and the polymer concentration in the second region is 0 to 1% by weight.

6. The gel material for regenerative medicine according to any one of claims 1 to 5, having a polymer content of 5% by weight or less.

7. The gel material for regenerative medicine according to any one of claims 1 to 6, wherein the polymer unit is a polymer having a polyethylene glycol backbone or a polyvinyl backbone.

8. The polymer unit comprises a first polymer unit having one or more nucleophilic functional groups at a side chain or terminal, and a second polymer unit having one or more electrophilic functional groups at a side chain or terminal. The gel material for regenerative medicine according to any one of claims 1 to 7.

9. The nucleophilic functional group is selected from the group consisting of a thiol group and an amino group, and the electrophilic functional group is selected from the group consisting of a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group, an imidazoyl group, an acryloyl group, a nitrophenyl group, and a -CO ２ PhNO ２ The gel material for regenerative medicine according to claim 8, selected from the group consisting of:

10. The gel material for regenerative medicine according to any one of claims 1 to 9, which is used for tissue regeneration.

11. The gel material for regenerative medicine according to claim 10 , wherein the tissue is bone, cartilage, skin, or nerve.

12. A kit for producing a gel material for regenerative medicine, comprising the following two solutions (A) and (B) stored without being mixed with each other: (A) an aqueous solution containing a hydrophilic first raw material polymer having a concentration less than the overlap concentration and equal to or greater than the critical gelation concentration; (B) an aqueous solution containing a hydrophilic second raw material polymer having a concentration less than the overlap concentration and equal to or greater than the critical gelation concentration; The solution of (A) and / or (B) further contains a non-reactive polymer that does not have a functional group capable of undergoing a crosslinking reaction with the raw material polymer in the molecule, The polymer gel obtained by mixing the solutions (A) and (B) is a polymer gel in which the first raw polymer and the second raw polymer are crosslinked with each other, the polymer gel has a structure composed of two regions, namely, a first region in which the polymer units derived from the raw material polymer are densely present, and a second region in which the polymer units derived from the raw material polymer are sparsely present. The kit.

13. The kit according to claim 12 , wherein the starting polymer is a polymer having a polyethylene glycol backbone or a polyvinyl backbone.

14. The kit according to claim 12 or 13, wherein the raw polymer comprises a first polymer having one or more nucleophilic functional groups at a side chain or end, and a second polymer having one or more electrophilic functional groups at a side chain or end.

15. The nucleophilic functional group is selected from the group consisting of a thiol group and an amino group, and the electrophilic functional group is selected from the group consisting of a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group, an imidazoyl group, an acryloyl group, a nitrophenyl group, and a -CO ２ PhNO ２ 15. The kit of claim 14, selected from the group consisting of:

16. The kit of claim 12, wherein the non-reactive polymer is polyethylene glycol or cellulose that does not have a cross-linking reactive group.

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