Production method for hydrogel having porous structure

Abstract

The present invention addresses the problem of providing a new production method with which it is possible to prepare, with a simpler approach, a hydrogel material having a µm-scale porous structure through which substances such as cells can pass.　It has been found that a biocompatible hydrogel having a µm-scale porous structure can be produced by adopting a freezing-thawing process as a trigger for forming the porous structure, with respect to an initial hydrogel having a non-porous structure generated temporarily, while setting, within a prescribed range, the total concentration of a raw material polymer in a solution.

Description

Method for producing hydrogel with porous structure

[0001] The present invention relates to a method for producing a biocompatible hydrogel having a three-dimensionally interconnected porous structure.

[0002] In recent years, hydrogels, which have a polymer network structure, have been expected to be used for a variety of purposes, including medical applications such as artificial tissues, scaffolds for regenerative medicine, sealants, adhesion inhibitors, drug delivery substrates, and contact lenses, as well as sensors and surface coating materials, due to their excellent water retention capacity and biocompatibility (see, for example, Non-Patent Document 1). In particular, the production of gels with a three-dimensionally interconnected micrometer-scale porous structure holds promise for the development of materials for tissue engineering applications.

[0003] However, conventional approaches to obtain micrometer-scale porous structures have required either top-down techniques, such as microfabrication of prefabricated hydrogels or polymer structures using lithography, or the preparation of polymeric materials using solvent-insoluble polymeric raw materials. On the other hand, when using hydrophilic polymeric raw materials such as biocompatible polyethylene glycol (PEG), a simple two-liquid mixing technique has been used. However, this method can only produce hydrogels with nanometer-scale porous structures, making it difficult for cells or substances with micrometer-scale dimensions to penetrate.

[0004] Sakai et al., Macromolecules, 41, 5379-5384, 2008

[0005] Therefore, an object of the present invention is to provide a novel manufacturing method that enables the production of a hydrogel material having a micrometer-scale porous structure that is permeable to substances such as cells, using a simpler method.

[0006] In response to these problems, the inventors have improved conventional hydrogel preparation methods, finding that a biocompatible hydrogel having a micrometer-scale porous structure can be produced by adjusting the total concentration of raw polymers in the solution to a predetermined range and using a freeze-thaw process to trigger the formation of a porous structure in an initial non-porous hydrogel. The present invention was thus completed. The production method of the present invention allows for the production of a hydrogel having a micrometer-scale porous structure using a simple method without requiring specialized equipment. Furthermore, the inventors have also found that the resulting hydrogel not only has a micrometer-scale sponge-like three-dimensional network structure (porous structure), but also has other physical properties, such as superior substance permeability, reduced swelling, and improved strength, compared to non-porous hydrogels formed from the same polymer units.

[0007] That is, in a representative embodiment, the present invention provides: <1> a method for producing a hydrogel having a porous structure, comprising: a) a step of preparing a first solution by dissolving a compound A in a first solvent; b) a step of preparing a second solution by dissolving a compound B in a second solvent; and c) a step of mixing the first solution with the second solution to obtain an initial hydrogel having a non-porous structure, wherein a total polymer concentration (C total ) is the viscosity increase characteristic concentration (C Ａ ) or the compound B's inherent viscosity increase characteristic concentration (C Ｂd) a step of freezing the initial hydrogel to obtain a frozen hydrogel; and e) a step of treating the frozen hydrogel at a temperature equal to or higher than the melting point of the first or second solvent, whichever is higher, to obtain a hydrogel having a porous structure, wherein the initial hydrogel and the hydrogel having a porous structure both form gels by crosslinking the compound A and the compound B with each other, and the first and second solvents are both aqueous solvents; and the compounds A and B are bi-, tri-, tetra- or octa-branched polyethylene glycol (PEG) having a total of one or more nucleophilic functional groups or electrophilic functional groups at side chains or terminals.

[0008] In a preferred embodiment, the present invention provides: <2> the compound A and / or the compound B is 4×10 ４ <3> The manufacturing method according to the above <1>, wherein the freezing treatment is carried out at a temperature of -10°C or lower for 1 hour or more; <4> The manufacturing method according to the above <1>, wherein the relational expression X>Z>Y is satisfied, where X is the transmittance of the initial hydrogel, Y is the transmittance of the frozen hydrogel, and Z is the transmittance of the hydrogel having a porous structure; <5> C total The equilibrium swelling degree of the hydrogel having the porous structure prepared so that the amount of the hydrogel is 20 g / L is defined as Q ｌｏｗ and any C total When the equilibrium swelling degree of the hydrogel having a porous structure prepared in the above step is Q, Q<1.2*Q ｌｏｗ <6> the manufacturing method according to the above <1>, wherein the initial hydrogel has a breaking stress at elongation (σ max ) to S initial and the breaking stress at elongation of the hydrogel having a porous structure is S, S>S initial <7> The manufacturing method according to the above <1>, wherein the elongation at break (λ max ) to Tinitial and the elongation at break of the hydrogel having a porous structure is T, T > T initial <8> the manufacturing method according to the above <1>, wherein the Young's modulus (E max ) to U initial and the Young's modulus of the hydrogel having the porous structure is U, U>U initial <9> The manufacturing method according to the above <1>, wherein the fracture energy of the hydrogel having a porous structure is higher than the fracture energy of the initial hydrogel; <10> The manufacturing method according to the above <1>, wherein the compound A is a bi-, tri-, tetra-, or octa-branched PEG having a total of two or more nucleophilic functional groups at side chains or terminals; and the compound B is a bi-, tri-, tetra-, or octa-branched PEG having a total of two or more electrophilic functional groups at side chains or terminals; <11> The manufacturing method according to the above <1>, wherein 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, and a sulfosuccinimidyl group.

[0009] According to the production method of the present invention, a biocompatible hydrogel with a micrometer-scale porous structure can be easily and efficiently obtained by using a freeze-thaw process as a trigger for forming a porous structure while maintaining the total concentration of the raw polymers within a predetermined range. In particular, the production method of the present invention is advantageous in that it does not require the use of special equipment, and unlike conventional gelation methods, it does not require the removal of organic solvents or porogens.

[0010] Furthermore, the hydrogel obtained by the manufacturing method of the present invention forms a sponge-like, micrometer-scale three-dimensional network structure (porous structure), making it a suitable material for cell infiltration and adhesion. Additionally, the manufacturing method of the present invention can also impart bioabsorbability to the gel, which means that the gel will not remain in the body after tissue regeneration or repair, providing increased peace of mind for both physicians and patients. These properties are expected to be highly valued as advantages in the field of regenerative medicine.

[0011] Figure 1 is a schematic diagram showing the scheme of the manufacturing method of the present invention and the changes in the internal structure of the hydrogel at each step. Figure 2 is a confocal laser microscope image of hydrogels prepared at each total polymer concentration. Figure 3 is a confocal laser microscope image of hydrogels prepared at different freezing times. Figure 4 is a 3D stack image of confocal laser microscope images of hydrogels prepared at each total polymer concentration. Figure 5 is a photograph of hydrogels prepared at each total polymer concentration after immersion in India ink. Figure 6 is a confocal laser microscope image of a porous hydrogel immersed in a solution containing 10 μm particles. Figure 7 is a graph showing the equilibrium swelling degree of hydrogels prepared at each total polymer concentration. Figure 8 is a confocal laser microscope image of a porous hydrogel before and after swelling. Figure 9 is a graph showing the breaking stress, breaking elongation, and fracture energy of hydrogels prepared at each total polymer concentration. Figure 10 is a graph showing the results of evaluating the rupture stress and stress relaxation of hydrogels prepared at various total polymer concentrations. Figure 11 shows confocal laser microscope images of various porous hydrogels seeded with cells. 20 μm and 100 μm indicate the distance (depth) from the surface of the porous hydrogel where the cells were seeded.

[0012] The following describes embodiments of the present invention. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be implemented with appropriate modifications within the scope that does not depart from the spirit of the present invention.

[0013] (1) Method for producing porous hydrogel The production method of the present invention is a method for producing a hydrogel having a porous structure, and includes the following steps a) to e): a) preparing a first solution by dissolving compound A in a first solvent; b) preparing a second solution by dissolving compound B in a second solvent; c) mixing the first solution and the second solution to obtain an initial hydrogel having a non-porous structure; d) freezing the initial hydrogel to obtain a frozen hydrogel; e) treating the frozen hydrogel at a temperature equal to or higher than the higher melting point of the first and second solvents to obtain a hydrogel having a porous structure.

[0014] Here, in step c), the total polymer concentration (C total ) is the viscosity increase characteristic concentration (C Ａ ) or the compound B's inherent viscosity increase characteristic concentration (C Ｂ The method includes mixing the first solution and the second solution in a ratio such that the concentration of the first solution is equal to or less than the higher of the two concentrations of the first solution and the second solution.

[0015] The hydrogel obtained by the production method of the present invention has a sponge-like three-dimensional network structure (porous structure) formed by phase separation of the constituent polymers during the production process (hereinafter, such a structure may be referred to as a "sponge-like porous structure"). The network size is characterized by being on the order of μm, typically 10 to 400 μm, which is much larger than the nm-order obtained in conventional hydrogels. In this hydrogel, no polymer units are observed in the voids forming the porous structure. That is, the hydrogel as a whole does not have two or more regions with significantly different polymer densities. Furthermore, because there are no polymer units in the voids, when the hydrogel is compressed by external force, it can expel water like a so-called ordinary sponge. Furthermore, in the production process of the present invention, some or all of the polymer units used are not solvent-philic. As a result, a phase-separated structure composed of solvent-insensitive polymers is obtained.

[0016] In this specification, the term "gel" generally refers to a dispersion system of polymers that has high viscosity and has lost fluidity, and in which the storage modulus G' and the loss modulus G" satisfy the relationship G'≧G". In particular, when the solvent contained in the gel is an aqueous solvent, the gel is called a "hydrogel".

[0017] The compounds A and B constituting the hydrogel of the present invention and the conditions for the various steps will be explained below.

[0018] 1-1. Compounds A and B (Gel Constituents) Compounds A and B, which are components for forming the hydrogel of the present invention, are hydrophilic polymer species that can form a network structure, particularly a three-dimensional network structure, in the final hydrogel by crosslinking the polymer species with each other.

[0019] More specifically, compounds A and B are polyethylene glycols (PEGs) with multiple branches, such as di-, tri-, tetra-, or octa-branched, with tetra-branched polyethylene glycols being particularly preferred. Gels composed of such tetra-branched polyethylene glycol backbones are generally known as Tetra-PEG gels, in which a mesh-like network is constructed by an AB cross-end coupling reaction between two types of tetra-branched polymers, each of which has an electrophilic functional group, such as an activated ester structure, and a nucleophilic functional group, such as an amino group, at its terminal (Matsunaga et al., Macromolecules, Vol. 42, No. 4, pp. 1344-1351, 2009). Furthermore, Tetra-PEG gels can be easily prepared in situ by simply mixing two polymer solutions. Using Tetra-PEG technology, it is also possible to control the gelation time by adjusting the pH and ionic strength during hydrogel preparation. Because the resulting hydrogels are primarily composed of PEG, they also have excellent biocompatibility. Compounds A and B can be selected from the branched polyethylene glycol group depending on the intended use, shape, etc. of the final hydrogel.

[0020] In the production method of the present invention, compounds A and B crosslink with each other to form a three-dimensional network structure, thereby constituting a hydrogel. Therefore, compounds A and B have a total of two or more nucleophilic or electrophilic functional groups at the side chains or ends of the polyethylene glycol (PEG) backbone to link them together by crosslinking. In this case, compound A can be a first polymer unit having a total of two or more nucleophilic functional groups at the side chains or ends, and compound B can be a second polymer unit having a total of two or more electrophilic functional groups at the side chains or ends. The total number of nucleophilic and electrophilic functional groups is preferably five or more. These functional groups are preferably present at the ends. Furthermore, the composition may have a higher content of the first polymer unit (compound A) than the content of the second polymer unit (compound B), or the composition may have a higher content of the second polymer unit than the first polymer unit.

[0021] Nucleophilic functional groups present in Compounds A and B include thiol groups (-SH) (also called sulfhydryl groups) and amino groups, and those skilled in the art can appropriately use known nucleophilic functional groups. Preferably, the nucleophilic functional groups are thiol groups or amino groups. The nucleophilic functional groups may be the same or different, but are preferably the same. When the functional groups are the same, the reactivity with the electrophilic functional groups that form crosslinks becomes uniform, making it easier to obtain a hydrogel with a uniform three-dimensional structure.

[0022] The electrophilic functional groups present in Compounds A and B can be maleimidyl groups or active ester groups. Examples of active ester groups include an N-hydroxy-succinimidyl (NHS) group and a sulfosuccinimidyl group. Those skilled in the art can appropriately use other known electrophilic functional groups. 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 hydrogel with a uniform three-dimensional structure.

[0023] In addition to the above-mentioned combinations of nucleophilic functional groups and electrophilic functional groups, covalent bond-forming reactions known to those skilled in the art, such as cycloaddition reactions between azides and alkynes and other reactions known as click chemistry, can also be used as appropriate.

[0024] The molecular weight of PEG used as compounds A and B is typically 4×10 ４ The weight average molecular weight of the copolymer may be less than 5×10. 3 ~4×10 ４ , more preferably 1×10 ４ ~4×10 ４ , and more preferably 1×10 ４ ~2×10 ４ In a preferred embodiment, compound A and / or compound B may be 4×10 ４ It has the following weight average molecular weight:

[0025] Non-limiting examples of preferred PEG having a nucleophilic functional group at its terminal include a compound represented by the following formula (I) having four branches of a polyethylene glycol backbone and a thiol group at its terminal.

[0026] 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, resulting in higher strength. Therefore, in order to obtain a high-strength hydrogel, it is preferable that n be the same. １１ ~n １４ If the value of is too high, the strength of the gel 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 28 to 227, preferably 56 to 227, and more preferably 56 to 114.

[0027] In the above formula (I), R １１ ~R １４ is a linker moiety connecting the functional group and the core moiety.１１ ~R １４ may be the same or different, but are preferably the same in order to produce a high-strength hydrogel 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 -O-R 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 represents an alkylene group. １６ is C １ -C ３ represents an alkylene group. １７ is C １ -C ５ represents an alkylene group.

[0028] Here, "C １ -C ７ The term "alkylene group" means 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 ７ Examples of the alkylene group include a methylene group, an ethylene group, a propylene group, and a butylene group. １ -C ７Examples of the alkylene group are -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)-, -(CH2)3-, -(CH(CH3))2-, -(CH2)2-CH(CH3)-, -(CH2)3-CH(CH3)-, -(CH2)2-CH(C2H5)-, -(CH2)6-, -(CH ２ )2-C(C2H5)2-, and -(CH2)3C(CH3)2CH2-.

[0029] "C ２ -C ７ The term "alkenylene group" refers to a branched or straight-chain alkenylene group having 2 to 7 carbon atoms and having one or more double bonds in the chain, and examples thereof include divalent groups having double bonds formed by removing 2 to 5 hydrogen atoms from adjacent carbon atoms of the alkylene group.

[0030] On the other hand, non-limiting specific examples of preferred PEGs having an electrophilic functional group at their termini include compounds represented by the following formula (II), which have four branches of a polyethylene glycol backbone and a maleimidyl group at their termini:

[0031] 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 three-dimensional structure of the hydrogel will be, and the higher the strength will be, so it is preferable, and it is more preferable that they are the same. ２１ ~n ２４ If the value of is too high, the strength of the gel 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 11 to 569, preferably 28 to 227, and more preferably 56 to 114.

[0032] In the above formula (II), R ２１ ~R ２４ is a linker moiety connecting the functional group and the core moiety. ２１ ~R ２４may 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, and C １ -C ７ Alkylene group, C ２ -C ７ Alkenylene group, —NH—R ２５ -, -CO-R ２５ -, -R ２６ -O-R ２７ -, -R ２６ -NH-R ２７ -, -R ２６ -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 represents an alkylene group. ２６ is C １ -C ３ represents an alkylene group. ２７ is C １ -C ５ represents an alkylene group.

[0033] In this specification, alkylene groups and alkenylene groups may have one or more optional substituents. Examples of the substituents include, but are not limited to, alkoxy groups, halogen atoms (which may be fluorine, chlorine, bromine, or iodine atoms), amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and aryl groups. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moieties of other substituents containing an alkyl moiety (e.g., alkyloxy groups, aralkyl groups, etc.).

[0034] Furthermore, in this 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 two or more substituents are present, 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.

[0035] 1-2. Steps a) and b)

[0036] Steps a) and b) are steps of preparing first and second solutions containing compounds A and B, respectively. The solvent in the first and second solutions is typically water, but in some cases, a mixed solvent containing a trace amount of alcohol such as ethanol, a polar solvent such as DMSO, or other organic solvent can also be used. The pH of the first and second solutions is in the range of 1.0 to 10.6, more preferably 2.2 to 8.0.

[0037] The pH of the first and second solutions can be adjusted using a pH buffer known in the art. For example, the pH can be adjusted to the above range by using a citrate-phosphate buffer (CPB) and changing the mixing ratio of citric acid and disodium hydrogen phosphate. In this specification, CPB and McIlvaine buffer are the same.

[0038] The concentrations of compounds A and B in the first and second solutions are adjusted so that the mixed solution (third solution) obtained in step c) described below has a predetermined concentration. As long as the concentrations specified in step c) are satisfied, the concentrations may be the same or different, but it is preferable that the concentrations are the same.

[0039] 1-3. Step c) Step c) is a step of preparing a third solution by mixing the first solution and the second solution, thereby crosslinking compounds A and B to obtain an initial hydrogel with a non-porous structure.

[0040] Here, the mixing of the first solution and the second solution results in a total polymer concentration (C total ) is the characteristic viscosity increase concentration of compound A (C Ａ ) or the characteristic viscosity increase concentration of compound B (C Ｂ ) is used at a ratio that is equal to or less than the higher of the two concentrations.

[0041] As used herein, the "viscosity increase characteristic concentration" refers to the concentration specific to each compound, determined by the following procedure. First, compound solutions of different concentrations (c) are prepared in 50 mM CPB (pH 5) as the solvent. Next, the specific viscosity (ηsp) of each compound solution is determined. ηsp can be determined by methods commonly known to those skilled in the art. The obtained ηsp is plotted on the Y-axis and c on the X-axis, and a linear fit is performed for the region showing linearity on the low concentration side. When the specific viscosity at an arbitrary concentration (c_arb) obtained from the fitted line is defined as ηsp_fit, the minimum c at which ηsp at c_arb satisfies the relationship ηsp > 1.2 × ηsp_fit is determined as the viscosity increase characteristic concentration.

[0042] In an exemplary embodiment, the total polymer concentration in the third solution (C total ) can be 90 g / L or less, preferably 40 g / L or less, more preferably 20 g / L or less, and even more preferably 10 g / L.

[0043] The initial hydrogel formed in this step c) is in a gel state but does not have a micrometer-order porous structure.

[0044] The first solution and the second solution can be mixed using, for example, a two-liquid mixing syringe such as that disclosed in International Publication WO 2007 / 083522. The temperature of the two liquids during mixing is not particularly limited, as long as the precursor units are dissolved and each liquid has fluidity. For example, the temperature of the solutions during 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 they are the same temperature, as this facilitates mixing of the two liquids.

[0045] 1-4. Step d) Step d) is a step of freezing the initial hydrogel with a non-porous structure obtained in step c) to obtain a frozen hydrogel.

[0046] The freezing treatment in step d) can be carried out using a method known in the art, for example, using a cooling device capable of controlling the temperature below freezing point or by immersion in liquid nitrogen.

[0047] In this freezing treatment, the initial hydrogel is cooled to a temperature of preferably -10°C or lower, more preferably -20°C or lower. The cooling time is not particularly limited as long as it is a period during which the initial hydrogel is sufficiently frozen, and can be set appropriately depending on the volume and size of the initial hydrogel, but cooling can be carried out for a period of, for example, 1 hour or more, preferably 2 hours or more, and more preferably 3 hours or more. Typically, the freezing treatment in step d) is carried out at a temperature of -20°C or lower for 24 hours or more.

[0048] 1-5. Step e) Step e) is a step of treating the frozen hydrogel obtained in step d) at a temperature equal to or higher than the melting point of the first and second solvents, whichever is higher, to obtain a hydrogel having a porous structure.

[0049] The manufacturing method of the present invention is characterized by the discovery that a porous PEG gel with micrometer-order pores can be produced by simply subjecting a non-porous initial hydrogel to the freezing treatment in step d) and the subsequent thawing treatment in step e). Without being bound by theory, as shown in Figure 1, when the initial hydrogel is frozen below freezing, the solvent component (water) present in the gel forms ice crystals (represented as "frozen hydrogel" in the figure). The formation of these ice crystals increases the volume, resulting in the concentration of the surrounding PEG network, which is the framework that constitutes the gel. Next, when the gel is melted at a temperature higher than freezing, micrometer-order spaces (pores) are formed within the gel, resulting in a hydrogel with a porous structure.

[0050] Here, "treatment at a temperature equal to or higher than the melting point of the solvent" in step e) means any treatment that removes the frozen hydrogel from its frozen state and brings it into a molten state in which the solvent component in the gel can flow. Therefore, such treatment includes placing the frozen hydrogel itself in an environment equal to or higher than the melting point, or heating it in any device. It also includes immersing the frozen hydrogel in a solvent equal to or higher than the melting point (typically, water or a mixed solvent used as the solvent for the first and second solutions).

[0051] (1) Properties of Porous Hydrogels As described above, the porous hydrogels obtained by the production method of the present invention (porous hydrogels) have a three-dimensional network structure (porous structure) on the μm scale, making them suitable for cell infiltration and adhesion. Furthermore, compared with non-porous hydrogels (initial hydrogels) formed from the same polymer units, they also have other characteristics in terms of physical properties, such as superior substance permeability, reduced swelling, and improved strength.

[0052] The porous hydrogel obtained by the production method of the present invention has a lower transmittance than the initial non-porous hydrogel. This is because the polymer components constituting the porous hydrogel behave as if the polymer in the solvent were in a phase-separated state, resulting in a cloudy appearance rather than complete transparency. In terms of transmittance, the porous hydrogel has completely different properties from ordinary non-porous hydrogels, which are nearly transparent.

[0053] More specifically, the porous hydrogel obtained by the production method of the present invention satisfies the relational expression X>Z>Y, where X is the transmittance of the initial hydrogel obtained in step c), Y is the transmittance of the frozen hydrogel obtained in step d), and Z is the transmittance of the porous hydrogel obtained in step e).

[0054] The porous hydrogel obtained by the production method of the present invention is also characterized in terms of equilibrium swelling degree (Q). Specifically, the porous hydrogel of the present invention is also characterized in terms of equilibrium swelling degree. Specifically, the total polymer concentration (C totalThe equilibrium swelling degree of the hydrogel having a porous structure prepared so that the pore size is 20 g / L is Q ｌｏｗ and an arbitrary total polymer concentration (C total When the equilibrium swelling degree of the porous hydrogel prepared in step (2) is Q, Q<1.2*Q ｌｏｗ The following relation is satisfied.

[0055] In addition, the porous hydrogel obtained by the production method of the present invention has a breaking stress at elongation (σ max Specifically, the porous hydrogel of the present invention is characterized in that the initial hydrogel has a breaking stress at elongation (σ max ) to S initial When the breaking stress of the porous hydrogel at elongation is S, S>S initial The following relation is satisfied.

[0056] In addition, the porous hydrogel obtained by the production method of the present invention has a breaking elongation (λ max Specifically, the porous hydrogel of the present invention is characterized by the elongation at break (λ max ) to T initial When the elongation at break of the porous hydrogel is T, T>T initial The following relation is satisfied.

[0057] In addition, the porous hydrogel obtained by the production method of the present invention has a Young's modulus (E max Specifically, the porous hydrogel of the present invention is characterized by a Young's modulus (E max ) to U initial and the Young's modulus of the porous hydrogel is U, then U>U initial The following relation is satisfied.

[0058] Furthermore, the fracture energy of the porous hydrogel obtained by the production method of the present invention is higher than the fracture energy of the initial hydrogel.

[0059] It will be understood by those skilled in the art that the transmittance, equilibrium swelling, stress at break at elongation, elongation at break, Young's modulus, and energy to break shown above can be measured using techniques commonly used in the art.

[0060] The porous hydrogel obtained by the production method of the present invention can be processed into various shapes, such as a thin film, depending on its intended use. Any method known in the art can be used for such processing. For example, a thin film can be obtained by applying the gelling liquid, while it is still in a fluid state before it completely solidifies, onto a flat substrate such as glass.

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, the polymer concentration is expressed in units of g / L, with 1 g / L corresponding to 0.1% by weight.

[0062] 1. Preparation of non-porous hydrogel (initial hydrogel) using four-branched polyethylene glycol (PEG) The raw polymers used were Tetra-PEG-SH (tetrasulfhydryl-polyethylene glycol) with -SH groups at the terminals and Tetra-PEG-MA (maleimidyl-polyethylene glycol) with maleimidyl groups at the terminals. Both were commercially available from NOF Corporation. Two types of polymers were used: one with a weight-average molecular weight (Mw) of 10,000 and the other with a weight-average molecular weight (Mw) of 20,000.

[0063] Tetra-PEG-SH and Tetra-PEG-MA were dissolved in 50 mM citrate-phosphate buffer (CPB) at pH 5 to prepare separate solutions, designated as the first and second solutions. Solutions with different PEG concentrations were prepared so that the overall polymer concentration after mixing would be in the range of 10 to 80 g / L.

[0064] The two resulting solutions were mixed in separate containers, degassed, and stirred using a planetary centrifugal mixer. The mixture was then quickly transferred to a Falcon tube, capped to prevent drying, and left to stand at room temperature for 24 hours.

[0065] The time course of the storage modulus G' and loss modulus G" of the mixed solution was observed using a rheometer (25°C, 1 Hz), and it was confirmed that an initial gelled hydrogel had been formed.

[0066] 2. Freezing and thawing: The initial hydrogels obtained above were frozen at -20°C for 0.5 to 24 hours to obtain frozen hydrogels. Unless otherwise specified, the freezing time was 24 hours in all experiments. The frozen hydrogels were then left to stand at 25°C for 24 hours and thawed.

[0067] 3. Evaluation of the internal structure of the hydrogel The internal structure of the obtained porous hydrogel was evaluated using a confocal laser microscope. total ) of 10, 20, 40, and 80 g / L, and for the weight-average molecular weight of the raw material PEG used for each of these, 10,000 (10k) and 20,000 (20k) are shown in Figure 2. total It was found that the lower the molecular weight of the raw material PEG, the larger the pore size of the porous structure. total It was found that a porous structure can be easily obtained even in

[0068] Similarly, the internal structure of the hydrogel was observed using a confocal laser microscope after freezing for 0 to 3 hours, and the results are shown in Figure 3. As a result, it was found that a micrometer-order porous structure was obtained when the freezing time exceeded 0.5 hours, and this tendency became more pronounced as the freezing time increased.

[0069] Furthermore, three-dimensional analysis of the internal structure of the gel using images from a confocal laser microscope confirmed that the porous structure was three-dimensionally interconnected (Figure 4).

[0070] 4. Material permeability of hydrogels The obtained porous hydrogels were subjected to an immersion test in India ink to evaluate their material permeability. The colloid size in the India ink ranged from 0.1 to 10 μm. Figure 5 shows an image of the gel after immersion. As a result, the hydrogels with larger pore sizes were colored blacker, indicating that the components of the India ink had permeated into the gel.

[0071] Similarly, an immersion test was conducted using particles (Fluoresbrite® YG Carboxylate Microspheres) with a size of 10 μm, the same size as living cells, suspended in D-PBS(-) at a concentration of 0.05 wt %. Observation with a confocal laser microscope revealed that the particles were distributed throughout the gel, demonstrating excellent permeability (Figure 6).

[0072] 5. Evaluation of Hydrogel Swelling The obtained porous hydrogel was immersed in Dulbecco's phosphate buffered saline (D-PBS(-)) to evaluate the swelling behavior of the gel (FIGS. 7 and 8).

[0073] In Figure 7, the equilibrium swelling degree Q of the hydrogel is expressed as (d eq / d ini ) 3 and plotted it (Fig. 7). eq ” is the diameter of the hydrogel at equilibrium, and “d ini " is the diameter of the hydrogel immediately after preparation. As a result, it was found that the porous hydrogel after the freeze-thaw treatment ("FT Gel" in Figure 7) was less swollen than the non-porous initial hydrogel ("AP Gel" in Figure 7). Specifically, C total The equilibrium swelling degree of the hydrogel with a porous structure prepared so that the PEG concentration (in the figure) was 20 g / L was defined as Q ｌｏｗ and any C total When the equilibrium swelling degree of the hydrogel having a porous structure prepared in the above step is Q, Q<1.2*Q ｌｏｗ It was found that the following relation is satisfied.

[0074] Similarly, when the internal structure of the porous hydrogel was examined using a confocal laser microscope before and after swelling, it was confirmed that the porous structure was maintained even after swelling (Figure 8).

[0075] 6. Evaluation of Mechanical Strength of Hydrogel Next, the mechanical strength of the obtained porous hydrogel was evaluated by a uniaxial tensile test and a uniaxial compression test.

[0076] Uniaxial tensile tests were performed using an Autograph AG-X plus (Shimadzu Corporation), and the results of measurements of breaking stress, breaking elongation, and fracture energy are shown in Figure 9. The same test was performed five times for each hydrogel, and the average values ​​and their standard deviations were plotted. In the figure, "AP Gel" represents the non-porous initial hydrogel, and "FT Gel" represents the porous hydrogel after freeze-thaw treatment (similar to Figure 10). As a result, it was confirmed that the porous hydrogel had improved both in strength and toughness.

[0077] Similarly, the measurement results of the breaking stress and stress relaxation property obtained by the uniaxial compression test are shown in Figure 10. As a result, it was confirmed that the porous hydrogel can withstand higher compression and exhibits remarkable stress relaxation property.

[0078] 7. Application to cell culture Cell culture was performed using the obtained porous hydrogel. First, to prepare gels for cell culture, the following two types of PEG aqueous solutions were prepared. Unless otherwise specified, the solvent was CPB 50 mM (pH 5). Type 1) GRGDSPC peptide (GenScript) was mixed with Tetra-PEG-MA solution to a concentration of 0.8 mM and incubated at 25°C for 5 minutes. Type 2) Alexa Fluor TM 594 C5 Maleimide (1 g / L DMSO solution) was mixed with the Tetra-PEG-SH solution to a concentration of 1% by volume and incubated at 25°C for 5 minutes.

[0079] These solutions were sterilized using a 0.22 μm filter. Equal volumes of filtered PEG solution were mixed and incubated in a rectangular silicone mold (15 mm × 35 mm, 3 mm high) at 25°C for 24 hours. The formed gel was subjected to a freeze-thaw treatment. The frozen-thawed hydrogel was removed from the mold, immersed in D-PBS(-), and incubated at 25°C for 24 hours. The gel was then cut into disks (10 mm diameter, 3 mm thick), placed in a 24-well plate, and immersed in DMEM. Human dermal fibroblasts (passage 7) were cultured in a 10-cm dish at 37°C under 5% CO2. After cell detachment, the cells were seeded on the prepared gel disks and cultured at 37°C under 5% CO2 for 7 days. Following the LIVE / DEAD™ Viability / Cytotoxicity Kit protocol, cell culture gels were immersed in DMEM containing 1 μM calcein AM and incubated at 37°C under 5% CO2 for 10 minutes. After washing with D-PBS(-), the hydrogels were observed under a confocal laser microscope. Excellent cell spreading was confirmed in the porous gels (Figure 11).

Claims

1. A method for producing a hydrogel having a porous structure, comprising: a) preparing a first solution by dissolving a compound A in a first solvent; b) preparing a second solution by dissolving a compound B in a second solvent; and c) mixing the first solution with the second solution to obtain an initial hydrogel with a non-porous structure, wherein the total polymer concentration (C total ) is the viscosity increase characteristic concentration (C Ａ ) or the compound B specific viscosity increase characteristic concentration (C Ｂ d) a step of freezing the initial hydrogel to obtain a frozen hydrogel; and e) a step of treating the frozen hydrogel at a temperature equal to or higher than the melting point of the first and second solvents to obtain a hydrogel having a porous structure, wherein the initial hydrogel and the hydrogel having a porous structure are both formed by crosslinking the compound A and the compound B with each other, and both the first and second solvents are aqueous solvents; and the compounds A and B are bi-, tri-, tetra- or octa-branched polyethylene glycols (PEGs) having a total of one or more nucleophilic functional groups or electrophilic functional groups at their side chains or ends.

2. The compound A and / or the compound B is 4×10 ４ 2. The process of claim 1 having a weight average molecular weight of:

3. The method according to claim 1, wherein the freezing treatment is carried out at a temperature of -10°C or lower for one hour or more.

4. The method according to claim 1, wherein the transmittance of the initial hydrogel is X, the transmittance of the frozen hydrogel is Y, and the transmittance of the hydrogel having a porous structure is Z, satisfying the following relationship: X>Z>Y.

5. C total The equilibrium swelling degree of the hydrogel having the porous structure prepared so that the swelling rate is 20 g / L is Q ｌｏｗ And, any C total When the equilibrium swelling degree of the hydrogel having a porous structure prepared in the above step is Q, Q<1.2*Q ｌｏｗ The method according to claim 1 , which satisfies the following relationship:

6. The elongation breaking stress (σ max ) to S initial and the breaking stress at elongation of the hydrogel having a porous structure is S, S>S initial The method according to claim 1 , which satisfies the following relationship:

7. The elongation at break of the initial hydrogel (λ max ) to T initial and the elongation at break of the hydrogel having a porous structure is T, T>T initial The method according to claim 1 , which satisfies the following relationship:

8. Young's modulus of the initial hydrogel (E max ) to U initial and the Young's modulus of the hydrogel having a porous structure is U, U>U initial The method according to claim 1 , which satisfies the following relationship:

9. The method according to claim 1, wherein the fracture energy of the hydrogel having the porous structure is higher than the fracture energy of the initial hydrogel.

10. The method according to claim 1, wherein compound A is a di-, tri-, tetra- or octa-branched PEG having a total of two or more nucleophilic functional groups at the side chain or terminal; and compound B is a di-, tri-, tetra- or octa-branched PEG having a total of two or more electrophilic functional groups at the side chain or terminal.

11. The method of claim 1, wherein 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, and a sulfosuccinimidyl group.

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