Method for producing hydrogel having porous structure

Abstract

To provide a novel simpler producing method capable of producing a hydrogel material having a porous structure of μm scale that can permeate substances such as cells and so on.SOLUTION: This is a method for producing a biocompatible hydrogel with a μm scale porous structure by using a freezing and thawing process as a trigger for the porous structuring of initially formed non-porous structured hydrogels, while keeping the total concentration of raw material polymers in the solution within a specified range.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] In recent years, hydrogels having a polymer network structure have been expected to be used not only for medical purposes such as artificial tissues, scaffolding materials for regenerative medicine, sealants, adhesion prevention materials, drug delivery substrates, and contact lenses, but also for a variety of other applications such as sensors and surface coating materials, due to their excellent water retention and biocompatibility properties (e.g., Non-Patent Document 1). In particular, the production of gels having a three-dimensionally connected micrometer-scale porous structure is promising for the development of materials for tissue engineering applications.

[0003] However, in the past, in order to obtain a micrometer-scale porous structure, it was necessary to use a top-down approach, such as microfabricating a prefabricated hydrogel or polymer structure 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 hydrophilic polymer raw material such as biocompatible polyethylene glycol (PEG), a simple preparation technique using a two-liquid mixture is used, but this method can only prepare hydrogels with small nanometer-scale porous structures, making it difficult for cells or substances with micrometer-scale sizes to permeate through them. [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 novel manufacturing method that enables the production, by a simpler technique, of a hydrogel material having a μm-scale porous structure that is permeable to substances such as cells. [Means for solving the problem]

[0006] In response to this problem, the present inventors have found that a biocompatible hydrogel having a micrometer-scale porous structure can be produced by improving a conventional hydrogel production method, and employing a freeze-thaw process as a trigger for forming a porous structure on an initial hydrogel having a non-porous structure once produced while keeping the total concentration of raw material polymers in a solution within a predetermined range, and thus completing the present invention. The production method of the present invention can produce a hydrogel having a micrometer-scale porous structure by a simple method without requiring special equipment or the like. Furthermore, the inventors have also found that the obtained hydrogel not only has a micrometer-scale sponge-like three-dimensional network structure (porous structure), but also has characteristics in terms of physical properties such as excellent substance permeability, low 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 the steps of: a) preparing a first solution by dissolving a compound A in a first solvent; b) preparing a second solution by dissolving compound B in a second solvent; c) a step of mixing the first solution with the second solution to obtain an initial hydrogel having a non-porous structure, wherein 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 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 second solution; d) freezing the initial hydrogel to obtain a frozen hydrogel; and e) treating the frozen hydrogel at a temperature equal to or higher than the higher of the melting points of the first and second solvents to obtain a hydrogel having a porous structure; Including, In both the initial hydrogel and the hydrogel having a porous structure, the compound A and the compound B are crosslinked to each other to form a gel, The first and second solvents are both aqueous solvents; The compound A and the compound B are di-, tri-, tetra- or octa-branched polyethylene glycols (PEGs) having a total of one or more nucleophilic or electrophilic functional groups at the side chains or ends. This provides:

[0008] In addition, in a preferred embodiment, the present invention provides <2> The compound A and / or the compound B is 4×10 4 The above having a weight average molecular weight of <1> 2. The method of claim 1 , <3> The freezing treatment is carried out at a temperature of -10°C or lower for 1 hour or more. <1> 2. The method of claim 1 , <4> When 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, X>Z>Y The above equation satisfies the relation <1> 2. The method of claim 1 , <5> C total The equilibrium swelling degree of the hydrogel having the porous structure prepared so that the swelling ratio is 20 g / L is Q low Let, 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 above equation satisfies the relation <1> 2. The method of claim 1 , <6> The elongational breaking stress of the initial hydrogel (σ max ) to S initialand the breaking stress during elongation of the hydrogel having the porous structure is S, S>S initial The above equation satisfies the relation <1> 2. The method of claim 1 , <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 above equation satisfies the relation <1> 2. The method of claim 1 , <8> The Young's modulus of the initial hydrogel (E max ) to U initial and the Young's modulus of the hydrogel having the porous structure is U, U>U initial The above equation satisfies the relation <1> 2. The method of claim 1 , <9> The fracture energy of the hydrogel having a porous structure is higher than the fracture energy of the initial hydrogel. <1> 2. The method of claim 1 , <10> The compound A is a di-, tri-, tetra-, or octa-branched PEG having a total of two or more nucleophilic functional groups at a side chain or terminal; The 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. <1> 2. The method of claim 1 , <11> 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. <1> The manufacturing method according to This provides: Effect of the Invention

[0009] According to the production method of the present invention, a biocompatible hydrogel having a micrometer-scale porous structure can be obtained simply and efficiently by using a freeze-thaw process as a trigger for forming a porous structure while keeping the total concentration of the raw material 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 does not require the removal of organic solvents or porogens, unlike conventional gelation methods.

[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), and therefore can be a suitable material for cell infiltration and adhesion. In addition, the manufacturing method of the present invention can also impart bioabsorbability to the gel, in which case the gel will not remain in the body after tissue regeneration or repair, and this characteristic can increase the sense of security for both doctors and patients. It is expected that such characteristics will be evaluated as extremely valuable advantages in the field of regenerative medicine. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing the scheme of the production method of the present invention and the changes in the internal structure of the hydrogel in each step. [Diagram 2] FIG. 2 shows confocal laser microscope images of hydrogels prepared at each total polymer concentration. [Diagram 3] Figure 3 shows confocal laser microscope images of hydrogels prepared with different freezing times. [Figure 4] Figure 4 shows a 3D stack of confocal laser microscope images of hydrogels prepared at each total polymer concentration. [Diagram 5] FIG. 5 shows photographs of hydrogels prepared at each total polymer concentration after immersion in India ink. [Figure 6] Figure 6 shows a confocal laser microscope image of a porous hydrogel immersed in a solution containing 10 μm particles. [Figure 7] FIG. 7 is a graph showing the equilibrium swelling of hydrogels prepared at different total polymer concentrations. [Figure 8] Figure 8 shows confocal laser microscope images of the porous hydrogel before and after swelling. [Figure 9] FIG. 9 is a graph showing the breaking stress, breaking elongation, and fracture energy of hydrogels produced at different total polymer concentrations. [Figure 10] FIG. 10 is a graph showing the evaluation results of the breaking stress and stress relaxation property of the hydrogels produced at each total polymer concentration. [Figure 11] 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 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.

[0013] (1) Manufacturing method of 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 a compound A in a first solvent; b) preparing a second solution by dissolving compound B in a second solvent; c) mixing the first solution with the second solution to obtain an initial hydrogel with 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 of the melting points of the first and second solvents to obtain a hydrogel having a porous structure.

[0014] Here, in step c), the total polymer concentration (Ctotal ) 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:

[0015] The hydrogel obtained by the manufacturing method of the present invention has a sponge-like three-dimensional network structure (porous structure) formed by phase separation of the polymers that are the constituents during the manufacturing process (hereinafter, such a structure may be referred to as a "sponge-like porous structure"). The mesh 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, polymer units are not observed in the voids that form the porous structure. That is, the hydrogel as a whole does not have two or more regions in which the density of the polymer is significantly different. In addition, since there are no polymer units in the voids, when the hydrogel is compressed by an external force, it can expel water like a so-called normal sponge. In addition, in the manufacturing process of the present invention, some or all of the polymer units used are not solvent-philic. As a result, a phase-separated structure is obtained that has a non-solvophilic polymer as a constituent.

[0016] 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 the loss modulus G" satisfy the relationship G'≧G". In particular, when the solvent contained in a 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 in each step will be described below.

[0018] 1-1. Compounds A and B (gel components) 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) having multiple branches, such as 2-branched, 3-branched, 4-branched, or 8-branched, and 4-branched polyethylene glycols are particularly preferred. Such gels consisting of a 4-branched polyethylene glycol backbone are generally known as Tetra-PEG gels, and a meshwork network is constructed by an AB-type cross-end coupling reaction between two types of 4-branched polymers, each of which has an electrophilic functional group such as an active ester structure and a nucleophilic functional group such as an amino group at its end (Matsunaga et al., Macromolecules, Vol. 42, No. 4, pp. 1344-1351, 2009). In addition, Tetra-PEG gels can be easily prepared in situ by simple two-liquid mixing of each polymer solution. Using Tetra-PEG technology, it is also possible to control the gelation time by adjusting the pH and ionic strength during hydrogel preparation. The obtained hydrogel is mainly composed of PEG, and therefore has excellent biocompatibility. Compounds A and B can be selected from this branched polyethylene glycol group depending on the final use, shape, etc. of the hydrogel.

[0020] In the manufacturing method of the present invention, compounds A and B crosslink with each other to form a three-dimensional network structure and constitute a hydrogel. Therefore, compounds A and B have a total of two or more nucleophilic functional groups or electrophilic functional groups for linking with each other by crosslinking at the side chain or end of the polyethylene glycol (PEG) skeleton. In this case, the compound A can be a first polymer unit having a total of two or more nucleophilic functional groups at the side chain or end, and the compound B can be a second polymer unit having a total of two 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. These functional groups are preferably present at the end. In addition, the composition may have a content of the first polymer unit (compound A) greater than the content of the second polymer unit (compound B), or the composition may have a content of the second polymer unit greater than the content of 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 use known nucleophilic functional groups as appropriate. 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. By using the same functional groups, 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 may be maleimidyl groups or active ester groups. Examples of the active ester group include N-hydroxy-succinimidyl (NHS) groups and sulfosuccinimidyl groups. Those skilled in the art may use other known electrophilic functional 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. The same functional groups result in uniform reactivity with the nucleophilic functional groups that form crosslinks, 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 or equal to 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] A non-limiting example of a preferred PEG having a nucleophilic functional group at its terminal is a compound represented by the following formula (I) having four branches of a polyethylene glycol backbone and a thiol group at its terminal. [ka]

[0026] n 11 ~n 14may 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. Therefore, in order to obtain a hydrogel 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 from 28 to 227, preferably from 56 to 227, and more preferably from 56 to 114.

[0027] 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 hydrogel having a uniform three-dimensional structure. 11 ~R 14 represents a C1-C7 alkylene group, a C2-C7 alkenylene group, -NH-R 15 -, -CO-R 15 -, -R 16 -OR 17 -, -R 16 -NH-R 17 -, -R 16 -CO2-R 17 -, -R 16 -CO2-NH-R 17 -, -R 16 -CO-R 17 -, R 16 -NH-CO-R 17 -or-R 16 -CO-NH-R 17 - where R 15 R represents a C1-C7 alkylene group. 16 R represents a C1-C3 alkylene group. 17 represents a C1-C5 alkylene group.

[0028] Here, the term "C1-C7 alkylene group" refers to an alkylene group having 1 to 7 carbon atoms which may have branches, and refers to a linear C1-C7 alkylene group or a C2-C7 alkylene group having one or more branches (having 2 to 7 carbon atoms including the branches). Examples of the C1-C7 alkylene group include a methylene group, an ethylene group, a propylene group, and a butylene group. Examples of C1-C7 alkylene groups include -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-, -(CH2)2-C(C2H5)2-, and -(CH2)3C(CH3)2CH2-.

[0029] The term "C2-C7 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 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, for example, compounds represented by the following formula (II) having four branches of a polyethylene glycol backbone and a maleimidyl group at their termini. [ka]

[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, 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 24If 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 from 11 to 569, preferably from 28 to 227, and more preferably from 56 to 114.

[0032] 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, and each represents a C1-C7 alkylene group, a C2-C7 alkenylene group, -NH-R 25 -, -CO-R 25 -, -R 26 -OR 27 -, -R 26 -NH-R 27 -, -R 26 -CO2-R 27 -, -R 26 -CO2-NH-R 27 -, -R 26 -CO-R 27 -, -R 26 -NH-CO-R 27 - or -R 26 -CO-NH-R 27 - where R 25 R represents a C1-C7 alkylene group. 26 R represents a C1-C3 alkylene group. 27 represents a C1-C5 alkylene group.

[0033] 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.).

[0034] 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.

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

[0036] Steps a) and b) are steps of preparing a first and a second solution 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 small amount of alcohol such as ethanol, a polar solvent such as DMSO, or other organic solvents may 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 concentration specified in step c) is satisfied, the compounds A and B may be the same or different, but it is preferable that they are the same.

[0039] 1-3. Process c) Step c) is a step of mixing the first solution with the second solution to prepare a third solution, which crosslinks 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 ) at a ratio such that the concentration is less than the higher of the two concentrations.

[0041] In this specification, the term "viscosity increase characteristic concentration" refers to a 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. Here, ηsp can be determined by a method commonly used by those skilled in the art. The obtained ηsp is plotted on the Y axis and c on the X axis, and linear fitting is performed for the region showing linearity on the low concentration side. When each specific viscosity at an arbitrary concentration (c_arb) obtained from the fitted straight line is ηsp_fit, ηsp at c_arb is ηsp > 1.2 × ηsp_fit The minimum c that satisfies the relationship is defined 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 step c) is in a gelled 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 as disclosed in International Publication WO2007 / 083522. The temperature of the two liquids when mixed is not particularly limited, and may be a temperature at which the precursor units are dissolved and each liquid has fluidity. For example, the temperature of the solutions when mixed may 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 the two liquids are more easily mixed.

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

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

[0047] In the 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, for example, cooling can be performed for a period of 1 hour or more, preferably 2 hours or more, and more preferably 3 hours or more. Typically, the freezing treatment in step d) is performed at a temperature of -20°C or lower for 24 hours or more.

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

[0049] The manufacturing method of the present invention is characterized by the fact that it has been found that a porous PEG gel having pores on the order of μm can be manufactured by simply carrying out a simple process of freezing an initial hydrogel with a non-porous structure in step d) and then melting it in step e). As shown in FIG. 1, when an initial hydrogel is frozen below freezing point, the solvent component (water) present in the gel is formed as ice crystals (frozen hydrogel in the figure). The formation of these ice crystals increases the volume, and as a result, the surrounding PEG network, which is the framework that constitutes the gel, is concentrated. Next, when the gel is melted at a temperature higher than freezing point, spaces (pores) on the order of μm are formed inside the gel, and a hydrogel having a porous structure is obtained.

[0050] Here, "treating at a temperature equal to or higher than the melting point of the solvent" in step e) means performing any treatment that causes the frozen hydrogel to leave the frozen state and reach 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 with 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) Characteristics of porous hydrogel As described above, the hydrogel having a porous structure obtained by the production method of the present invention (porous hydrogel) has a three-dimensional network structure (porous structure) on the μm scale, and is a material suitable for cell infiltration and adhesion. Furthermore, compared with a non-porous hydrogel (initial hydrogel) formed from the same polymer unit, it has characteristics in terms of physical properties such as excellent substance permeability, low swelling, and improved strength.

[0052] The porous hydrogel obtained by the production method of the present invention has a lower transmittance than that of the initial non-porous hydrogel. This is because the polymer components of the porous hydrogel behave as if the polymer is in a phase-separated state in the solvent, and the porous hydrogel is not completely transparent but is opaque. In terms of such transmittance, the porous hydrogel has completely different characteristics from ordinary non-porous hydrogels, which are almost transparent.

[0053] More specifically, when the transmittance of the initial hydrogel obtained in step c) is X, the transmittance of the frozen hydrogel obtained in step d) is Y, and the transmittance of the porous hydrogel obtained in step e) is Z, the porous hydrogel obtained in step e) has the following transmittance: X>Z>Y The following relation is satisfied.

[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 total The equilibrium swelling degree of the hydrogel with a porous structure prepared so that the swelling ratio is 20 g / L is Q low and an arbitrary total polymer concentration (C total ) is the equilibrium swelling degree of the porous hydrogel prepared in step 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 and the breaking stress of the porous hydrogel during 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 in that the initial hydrogel elongation at break (λ max ) to T initial and 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 in that the initial Young's modulus (E max ) to U initial and the Young's modulus of the porous hydrogel is U, 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 degree, elongation stress at break, elongation at break, Young's modulus, and fracture energy 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 the application. Any method known in the art can be used for such processing. For example, in the case of a thin film, a thin film can be obtained by applying the gelling liquid in a fluid state before it is completely solidified onto a flat substrate such as glass. EXAMPLES

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

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

[0063] Tetra-PEG-SH and Tetra-PEG-MA were dissolved in 50 mM citrate-phosphate buffer (CPB) solutions at pH 5, which were prepared separately as the first and second solutions. Solutions with a variety of 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 solutions obtained were mixed in a separate container, degassed and stirred using a planetary centrifugal mixer, and then quickly transferred to a Falcon tube, capped to prevent drying, and left at room temperature for 24 hours.

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

[0066] 2. Freeze-thaw treatment The initial hydrogel obtained above was frozen at -20°C for 0.5 to 24 hours to obtain a frozen hydrogel. Unless otherwise specified, the freezing time was 24 hours in all experiments. The frozen product was 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 Confocal microscope images are shown in Figure 2 for the cases where the total polymer concentration (C total It was found that the lower the molecular weight of the raw 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 a freezing time of 0 to 3 hours, and the results are shown in Figure 3. As a result, it was found that when the freezing time exceeded 0.5 hours, a porous structure on the order of μm was obtained, and this tendency became more pronounced as the freezing time was increased.

[0069] In addition, the internal structure of the gel was analyzed three-dimensionally using images taken with a confocal laser microscope, and it was confirmed that the porous structure was three-dimensionally connected (Figure 4).

[0070] 4. Material permeability of hydrogels The obtained porous hydrogel was subjected to an immersion test in India ink to evaluate its substance 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, it was found that the hydrogel with larger pore size was colored black, and the components of the India ink had permeated into the gel.

[0071] Similarly, an immersion test was performed using particles (Fluoresbrite® YG Carboxylate Microspheres) with a size of 10 μm, similar to that of living cells, suspended in D-PBS(-) at a concentration of 0.05% by weight. Observation with a confocal laser microscope revealed that the particles were distributed within the gel and had excellent permeability (Figure 6).

[0072] 5. Hydrogel Swelling Assessment 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 We defined and plotted (Figure 7). Here, "d 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 Fig. 7) was less swollen than the non-porous initial hydrogel ("AP Gel" in Fig. 7). Specifically, C total The equilibrium swelling degree of the hydrogel with a porous structure prepared so that the PEG concentration is 20 g / L (in the figure) is Q low Let, and any C total The equilibrium swelling degree of the porous hydrogel prepared in step Q is Q<1.2*Q low It was found that the following relation was satisfied.

[0074] Similarly, when the internal structure of the porous hydrogel was examined before and after swelling using a confocal laser microscope, 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 measuring the 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" is the initial non-porous hydrogel, and "FT Gel" is the porous hydrogel after freeze-thaw treatment (same as in 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 shows remarkable stress relaxation property.

[0078] 7. Application to cell culture The obtained porous hydrogel was used for cell culture. First, the following two types of PEG aqueous solutions were prepared to prepare gels for cell culture. The solvent was CPB 50 mM (pH 5) unless otherwise noted. Type 1) GRGDSPC peptide (GenScript) was mixed with the 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 at 1 volume % and incubated at 25 °C for 5 minutes.

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

Claims

1. A method for producing a hydrogel having a porous structure, comprising the steps of: 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) a step of 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 mixing said first solution and said second solution in a ratio such that the concentration of said first solution is equal to or less than the higher of the two concentrations of said first solution and said second solution; d) freezing the initial hydrogel to obtain a frozen hydrogel; and e) treating the frozen hydrogel at a temperature equal to or higher than the higher of the melting points of the first and second solvents to obtain a hydrogel having a porous structure; Including, In both the initial hydrogel and the hydrogel having a porous structure, the compound A and the compound B are crosslinked to each other to form a gel, The first and second solvents are both aqueous solvents; The compounds A and B are di-, tri-, tetra- or octa-branched polyethylene glycols (PEGs) having a total of one or more nucleophilic or electrophilic functional groups at their side chains or ends. Manufacturing method.

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 1 hour or more.

4. When 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, X>Z>Y The method according to claim 1 , which satisfies the following relationship:

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

11. 2. 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.

Images