JP2004043749A - Hydrogel, gelatinous material and method for controlling diffusion of substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hydrogel capable of suitably controlling the retention and/or diffusion of a valuable substance, a gelatinous material and a method for controlling the diffusion of a substance. <P>SOLUTION: The hydrogel at least contains a hydrogel-forming polymer giving an aqueous solution exhibiting a thermoreversible sol-gel transition to take a sol state at a low temperature and a gel state at a high temperature. The hydrogel satisfies the formulas (D<SB>PR</SB>/D<SB>MB</SB>)≥2 and (D<SB>PR</SB>/D<SB>MG</SB>)≥1.2 wherein D<SB>PR</SB>, D<SB>MB</SB>and D<SB>MG</SB>are diffusion coefficients of phenol red, methyl blue and myoglobin, respectively. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a hydrogel that can be suitably used for holding and / or diffusing (or releasing) various useful substances, a gel body using the hydrogel, and holding and / or holding a useful substance using the hydrogel. The present invention relates to a control method capable of suitably controlling diffusion.
[0002]
By using the hydrogel of the present invention, retention / diffusion of various useful substances (for example, physiologically active substances such as pharmaceuticals) can be suitably controlled so as to conform to various conditions.
[0003]
[Prior art]
Hydrogels containing at least a hydrogel-forming polymer (eg, collagen, agar, alginic acid, hyaluronic acid, etc.) and an aqueous dispersion medium can be used in various industries (eg, chemical, pharmaceutical, food, agricultural, etc.). In addition, it has been widely used for personal and home use. Since hydrogels containing an aqueous dispersion medium are so-called “environmentally friendly materials”, their use is expected to be further expanded in the future.
[0004]
By effectively utilizing the function of holding / diffusion of such a hydrogel, there is a possibility that the holding and / or (or release) of a substance disposed in the hydrogel can be suitably controlled, and thus, it has been conventionally possible. Hydrogels have been used for retention and / or diffusion of various materials. For example, the function of hydrogel retention / diffusion is greatly expected to be applied to a drug delivery system (DDS) that delivers a desired bioactive substance with a desired release profile to a desired location.
[0005]
However, in a conventional hydrogel comprising a hydrogel-forming polymer (collagen, agar, alginic acid, hyaluronic acid, etc.) and an aqueous dispersion medium, the substance to be disposed in the hydrogel It was extremely difficult to control the diffusivity of the material. This is because there are a number of factors that must affect the diffusivity of such substances, and the relationships between these factors are extremely complex, so there is no unified index, and therefore, the result of many trials and errors in the past. Empirically (in a so-called "fumbling" search) only control the retention / diffusion of a substance in a particular hydrogel.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a hydrogel, a gel body, and a method for controlling the diffusion of a substance which have solved the above-mentioned disadvantages of the conventional hydrogel.
[0007]
It is another object of the present invention to provide a hydrogel, a gel body, and a method for controlling the diffusion of a substance, which can appropriately control retention and / or diffusion of a useful substance.
[0008]
[Means for Solving the Problems]
As a result of intensive studies, the present inventor has found that in a hydrogel containing at least a specific hydrogel-forming polymer, the ratio of the diffusion coefficient between the specific substances is different from the function of retaining and / or diffusing various useful substances. Has been found to be extremely useful as an index that directly reflects
[0009]
As a result of further research based on such findings, the present inventor has found that by controlling the ratio of the diffusion coefficient between the above-mentioned specific substances, the retention and / or diffusion of useful substances can be suitably controlled. Is possible.
[0010]
The hydrogel of the present invention is based on the above findings, and more specifically, a hydrogel-forming polymer exhibiting a thermoreversible sol-gel transition in which an aqueous solution thereof becomes a sol state at a low temperature and a gel state at a high temperature. A hydrogel comprising at least:
The ratio of the diffusion coefficients of phenol red (PR), methyl blue (MB) and myoglobin (MG) is (D_PR/ D_MB) ≧ 2 and (D_PR/ D_MG) ≧ 1.2.
According to the present invention, there is further provided a gel body comprising at least a useful substance and a hydrogel disposed around the useful substance; wherein the hydrogel is in a sol state at an aqueous solution at a low temperature and in a gel state at an elevated temperature. And a diffusion coefficient of phenol red (PR), methyl blue (MB), and myoglobin (MG) in the hydrogel, which contains at least a hydrogel-forming polymer exhibiting a thermoreversible sol-gel transition. The ratio is (D_PR/ D_MB) ≧ 2 and (D_PR/ D_MGA) providing a gel body characterized by satisfying ≧ 1.2.
[0011]
According to the present invention, there is further provided a method for controlling retention / diffusion of a useful substance by using a gel body containing at least a useful substance and a hydrogel disposed around the useful substance; Contains at least a hydrogel-forming polymer exhibiting a thermoreversible sol-gel transition in which the aqueous solution becomes a sol state at a low temperature and a gel state at a high temperature, and phenol red (PR), methyl The ratio of the diffusion coefficients of blue (MB) and myoglobin (MG) is (D_PR/ D_MB) ≧ 2 and (D_PR/ D_MG) ≧ 1.2 is provided.
[0012]
According to the findings of the present inventors, the above-mentioned ratio of the diffusion coefficients (D_PR/ D_MB) And (D_PR/ D_MG) Indicates the molecular weight dependence of the diffusion of useful substances based on the hydrophilic / hydrophobic balance of the hydrogel-forming polymer in the hydrogel, and the “sieving effect” due to the network structure of the hydrogel-forming polymer. It is estimated to reflect
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described more specifically with reference to the drawings as necessary. In the following description, “parts” and “%” representing quantitative ratios are based on mass unless otherwise specified.
[0014]
(Hydrogel)
The hydrogel of the present invention contains at least a hydrogel-forming polymer having a sol-gel transition temperature. The hydrogel exhibits a thermo-reversible sol-gel transition that becomes a sol at lower temperatures and a gel at higher temperatures.
[0015]
(Hydrogel-forming polymer)
The “hydrogel-forming polymer” that constitutes the hydrogel of the present invention has a crosslinking structure or a network structure. Based on this structure, a hydrogel is formed by holding a dispersion liquid such as water therein. A polymer having the property of forming a gel. Further, the term "hydrogel" refers to a gel containing at least a crosslinked or network structure composed of a polymer and water supported or held in the structure (a dispersion liquid).
[0016]
The “dispersed liquid” held in the crosslinked or network structure is not particularly limited as long as it is a liquid containing water as a main component. More specifically, the dispersion liquid may be water itself, and may be either an aqueous solution and / or a hydrated liquid. The water-containing liquid preferably contains water in an amount of 80 parts or more, more preferably 90 parts or more, based on 100 parts of the entire water-containing liquid.
[0017]
As long as the object of the present invention is not contradicted, the dispersion liquid may contain an organic solvent (for example, a hydrophilic solvent such as ethanol compatible with water) at a predetermined content.
[0018]
(Sol-gel transition temperature)
In the present invention, “sol state”, “gel state” and “sol-gel transition temperature” are defined as follows. This definition is described in the literature (Polymer @ Journal.18(5), 411-416 (1986)).
[0019]
More specifically, 1 mL of the sol-like hydrogel is placed in a test tube having an inner diameter of 1 cm, and is kept in a water bath at a predetermined temperature (constant temperature) for 12 hours. Thereafter, when the test tube is turned upside down, if the solution / air interface (meniscus) is deformed by the weight of the solution (including the case where the solution flows out), the hydrogel is “ Sol state ". On the other hand, if the solution / air interface (meniscus) does not deform due to the weight of the solution even when the test tube is turned upside down, the hydrogel is considered to be in a “gel state” at the predetermined temperature. Define.
[0020]
In the above measurement, a sol-state hydrogel (solution) having a concentration of, for example, about 8% by mass is used, and the above-mentioned “predetermined temperature” is gradually increased (for example, in steps of 1 ° C.) to change the “sol state” to the “gel state” Is determined as a “sol-gel transition temperature” (in this case, the “predetermined temperature” is decreased in steps of 1 ° C., for example, and the “gel state” is changed to “sol-gel transition temperature”). The temperature at which the transition to the "sol state" may be obtained).
[0021]
(Sol-gel transition temperature)
In the present invention, the definition and measurement of the "sol state", "gel state" and "sol-gel transition temperature" are described in the literature (H. Yoshioka et al., Journal of Macromolecular Science, A31 (1), 113 (1994)). Based on the definition and method described above, it may be obtained as follows.
[0022]
That is, the dynamic elastic modulus of a sample at an observation frequency of 1 Hz is measured by gradually changing the temperature from a low temperature side to a high temperature side (1 ° C./1 minute), and the storage elastic modulus (G ′, elastic term) of the sample is determined. The temperature at the point where the loss elastic modulus (G ", viscous term) is exceeded is defined as the sol-gel transition temperature. Generally, the state where G"> G 'is a sol, and the state where G "<G' is a gel. In measuring the sol-gel transition temperature, the following measurement conditions can be suitably used.
[0023]
<Measurement conditions for dynamic and loss elastic modulus>
Measuring equipment (trade name): Stress control type rheometer CSL700, manufactured by Carri-Med
Concentration of sample solution (or dispersion) (however, as concentration of “polymer compound having sol-gel transition temperature”): 10 (weight)%
Amount of sample solution: about 0.8g
Measurement cell shape / dimension: Acrylic parallel disk (diameter 4.0 cm), gap 600 μm
Measurement frequency: 1Hz
Applied stress: within the linear region.
[0024]
(Suitable sol-gel transition temperature)
In the present invention, the sol-gel transition temperature can be adjusted in a wide range according to the purpose and application.
[0025]
For example, when the cells of an organism are used for culturing cells in vitro, the sol-gel transition temperature should be higher than 0 ° C. and 45 ° C. or lower from the viewpoint of preventing thermal damage to cells and living tissues. More preferably, the temperature is higher than 0 ° C. and 42 ° C. or less (particularly 4 ° C. or more and 40 ° C. or less).
[0026]
A hydrogel having such a suitable sol-gel transition temperature can be easily selected from the specific compounds described below according to the above-mentioned screening method (sol-gel transition temperature measurement method). For example, in a series of operations for regenerating biological tissues and organs using the hydrogel of the present invention, the above-mentioned sol-gel transition temperature (a ° C.) is used to determine the cell / tissue culture temperature (b ° C.) The temperature is preferably set between the temperature at the time of cooling (c ° C.) for seeding, mixing or collecting the tissue. That is, it is preferable that there is a relation of b> a> c between the above three temperatures a ° C., b ° C., and c ° C. Further, (ba) is preferably 1 to 40 ° C, more preferably 2 to 30 ° C, and (ac) is preferably 1 to 40 ° C, more preferably 2 to 30 ° C.
[0027]
(Gel body)
The gel body of the present invention includes at least a useful substance and the above-mentioned hydrogel disposed around the useful substance.
[0028]
(Useful substances)
The useful substance constituting the present invention together with the above-mentioned hydrogel can be retained in the hydrogel, and its retention and / or diffusion (or release) in the hydrogel is useful in some sense. As long as it is not particularly limited. The term "useful in some sense" means that a useful substance is retained and / or diffused (or released) based on its properties (eg, physical action, chemical action, physiological action, coloring, reactivity, etc.). ) Has some sort of externally recognizable influence on the surroundings.
[0029]
This retention in the hydrogel is temporary, as long as the purpose of the retention in the hydrogel (eg, release of useful substances with a given release profile, drug delivery to a given location, etc.) can be achieved. It may be. Furthermore, the composition, state, properties, and the like of the useful substance are not particularly limited as long as retention in such a hydrogel can be achieved. That is, it may be any of a gas, a liquid, and a solid, and may be a mixture (including a solid solution and a solution) as needed. Further, the gel body of the present invention may contain plural and / or plural kinds of useful substances as needed.
[0030]
In terms of the stability of the gel, the useful substance does not substantially undergo a chemical bonding reaction with the hydrogel-forming polymer, at least within the time period in which the useful substance is retained in the hydrogel. Is preferred.
[0031]
(Specific examples of useful substances)
Hereinafter, specific examples of useful substances that can be used in the present invention will be described.
(1) Useful substances based on physical action
For example, various gases, liquids, and solids can be used.
A more specific example is shown below.
Gas: oxygen, air, nitrogen, hydrogen, argon, helium, carbon dioxide, ethylene oxide, etc.
Liquid: Water-compatible ones such as ethanol, methanol, THF, and DMF, and water-incompatible ones such as chloroform, diethyl ether, and silicone oil
solid:¹⁴Radioisotope-labeled compounds such as C,^FifteenN and other stable isotope-labeled compounds, metals or organic substances with various electrical or magnetic properties
[0032]
(2) Useful substances based on chemical action
For example, various reaction reagents can be mentioned.
A more specific example is shown below.
Oxidizing agents such as hydrogen peroxide, periodic acid, permanganate,
Reducing agents such as sodium borohydride, ascorbic acid, aldehydes, sugars,
Oxidases such as cytochrome C oxidase, ascorbate oxidase, glucose oxidase, and amine oxidase, or various enzymes such as reductase, dehydrogenase, oxygenase, and peroxidase;
[0033]
(3) Physiological effects
Various substances are mentioned as described below.
[0034]
(4) Optical properties
Redox indicator: methylene blue, variamine blue B, diphenylamine, ferroin, etc.
pH indicator: phenolphthalein, methyl orange, methyl green, etc.
Fluorescent indicators: umbelliferone, rhodamine B, fluorescein, calcein, etc.
Colored proteins: hemoglobin, myoglobin, cytochrome, etc.
Dyes: Methyl Blue, Congo Red, Direct Yellow, etc.
Pigments: carbon black, iron oxide, titanium oxide, etc.
[0035]
(5) Cosmetics
For example, various antioxidants, cell activators, anti-inflammatory agents, tyrosinase activity inhibitors, humectants, proteins, carotenoids
And so on.
[0036]
A more specific example is shown below.
Active oxygen scavenger: SOD, mannitol, hydroquinone, bilirubin, cholesterol, tryptophan, histidine, quercetin, quercitrin, gallic acid, gallic acid derivative, plant extract containing flavonoids such as ginkgo extract, Gokahi extract, Jashajitsu extract and Jicoppi extract
[0037]
Antioxidants: vitamins A and their derivatives and their salts, vitamins B and their derivatives and their salts, vitamin C and its derivatives and their salts, vitamin D and their derivatives and their salts, Vitamin E and its derivatives and their salts, glutathione and its derivatives and their salts, BHT and BHA
[0038]
Cell activator: Deoxyribonucleic acid and its salts, adenylic acid derivatives such as adenosine triphosphate and adenosine monophosphate and their salts, ribonucleic acids and their salts, guanine, xanthine and their derivatives, and nucleic acids related to their salts Substances; animal-derived extracts such as serum deproteinized extract, spleen extract, placenta extract, cockscomb extract, royal jelly; microorganisms derived from yeast extract, lactic acid bacteria extract, bifidobacterium extract, reishi extract, etc. Extracts from plants such as carrot extract, assembly extract, rosemary extract, oak extract, garlic extract, hinokitiol, cepharanthin; α- or γ-linoleic acid, eicosapentaenoic acid and the like Derivatives, succinic acid and its derivatives and their salts, estradiol and its derivatives and Α-hydroxy acids such as salts thereof, glycolic acid, lactic acid, malic acid, citric acid, salicylic acid and derivatives thereof, and salts thereof.
[0039]
Anti-inflammatory agents: glycyrrhizic acid, glycyrrhetinic acid, mefenamic acid, phenylbutazone, indomethacin, ibuprofen, ketoprofen, allantoin, guaiazulene and their derivatives and their salts, ε-aminocaproic acid, zinc oxide, diclofenac sodium, aloe extract, Salvia extract, Arnica extract, chamomile extract, Birch extract, Hypericum extract, Eucalyptus extract and Mokumoji extract
[0040]
Tyrosinase activity inhibitor: cysteine and its derivatives and salts thereof, Sengoku mosquito extract, Scutellaria extract, Sampens extract, Soybean extract, Touki extract, Ibukittrano extract, Clara extract, Hawthorn extract, Shirahuri extract, Hop extract, Neubara extract and Yoquinin extract
[0041]
Humectant: mucopolysaccharide and / or protein.
Mucopolysaccharides: hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate and salts thereof
Protein: collagen, elastin, keratin and their derivatives and their salts
[0042]
Carotenoids: Derivatives thereof include esters of astaxanthin, for example, amino acid esters such as glycine and alanine, carboxylic acid esters such as acetate and citrate and salts thereof, and inorganic salts such as phosphate and sulfate. Esters and salts thereof, glycosides such as glucoside, or highly unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, unsaturated fatty acids such as oleic acid and linoleic acid, and saturated fatty acids such as palmitic acid and stearic acid. And similar or different diesters selected from fatty acid esters and the like
[0043]
(6) Biological material
For example, various living organisms (microorganisms, plants, animals including humans) themselves, or cells and / or tissues that are part thereof, may be mentioned. A more specific example will be described later.
[0044]
When a biological material is used as a useful substance in a gel, not only the retention / diffusion of the biological material itself in the hydrogel but also the retention / diffusion of a substance released from the biological material is observed. And / or interaction of a plurality or a plurality of types of biological materials may be observed.
[0045]
(Diffusion of useful substances in gel)
The hydrogel of the present invention can arbitrarily control the diffusion rate of the useful substance in the hydrogel. For example, a hydrophilic substance and a hydrophobic substance can be diffused at different diffusion rates depending on their degree of hydrophilicity / hydrophobicity. The diffusion of the water-soluble hydrophilic substance is controlled by a molecular sieving effect of the three-dimensional network structure of the hydrogel-forming polymer. Therefore, in order to decrease the diffusion rate of the water-soluble hydrophilic substance, the concentration of the hydrogel-forming polymer constituting the hydrogel may be increased. The diffusion of a water-soluble hydrophilic substance also depends on the molecular weight of the substance. If the concentration of the hydrogel-forming polymer constituting the hydrogel is constant, the diffusion rate of the substance having a higher molecular weight becomes lower.
[0046]
The diffusion of the water-soluble hydrophobic substance in the hydrogel of the present invention can be caused not only by the molecular sieve effect due to the three-dimensional network structure of the hydrogel-forming polymer, but also by the distribution of the hydrogel-forming polymer to the hydrophobic portion. Because it is affected and is also controlled by the proportion of hydrophobic moieties in the hydrogel-forming polymer, it is usually slower than the diffusion of the water-soluble hydrophilic substance.
[0047]
The diffusion coefficient of the solute in the hydrogel can be determined by the "early-time" approximation method described in the literature (Eric KL Lee, et al, Journal of Membrane Science, 24, 125-143 (1985)). it can. In this method, a process in which a solute uniformly dispersed in a hydrogel plate having a uniform thickness L (cm) elutes from both surfaces of the hydrogel plate is observed with time. When the amount of eluted solute at time t (sec) is Mt and the amount of eluted after infinite time is M∞, M_t/ M_∞In the range of <0.6, the diffusion coefficient D of the solute in the hydrogel (cm²/ Sec), the following equation (1) holds.
M_t/ M_∞= (Dt / π)^1/2× 4 / L (1)
[0048]
Therefore, the diffusion coefficient D can be calculated from the slope of a straight line plotting the elution rate up to the time t with respect to the square root of the elapsed time t.
[0049]
In view of the balance of retention / diffusion (or release) performance of various substances, the hydrogel of the present invention has a ratio of the diffusion coefficient D of phenol red (PR), methyl blue (MB), and myoglobin (MG). , (D_PR/ D_MB) ≧ 2 and (D_PR/ D_MGIt is preferred that ≧ 1.2. More preferable ranges of these values are as follows.
[0050]
(1) (D_PR/ D_MB): More preferably 10 or more, further 20 or more, especially 50 or more
Preferably 1 × 10⁵Below, more preferably 1 × 10⁴Hereinafter, 1 × 10³Less than
[0051]
(2) (D_PR/ D_MG): More preferably 1.5 or more, further 3 or more, especially 5 or more
Preferably 1 × 10⁴Below, more preferably 1 × 10³Hereinafter, 1 × 10²Less than
[0052]
(In the case of collagen etc.)
As described above, collagen, which is a conventional hydrogel-forming polymer, is a hydrophilic polymer, and the hydrophilic / hydrophobic balance can be arbitrarily controlled as in the hydrogel of the present invention. No, it was difficult to control the diffusion rate of the useful substance in collagen.
[0053]
On the other hand, in the case of polylactic acid or polyglycolic acid or the like, on the contrary, the hydrophobicity is strong, and it is still difficult to control the diffusion rate of the useful substance in these polymers.
[0054]
On the other hand, the hydrogel of the present invention can control the balance of hydrophilicity / hydrophobicity substantially arbitrarily as described above, so that the diffusion rate of the useful substance in the gel of the present invention is also reduced. It can be controlled with a substantial degree of freedom.
[0055]
When the gel of the present invention is used together with a known gel-forming polymer such as collagen (that is, as the gel-forming polymer, the polymer used in the present invention and a known gel-forming polymer such as collagen coexist. In the case, the diffusion rate of the chemical mediator can be controlled with a substantial degree of freedom even in a gel containing a known gel-forming polymer such as collagen.
[0056]
(Followability of hydrogel to movement)
The hydrogel of the present invention shows a solid behavior at higher frequencies, while exhibiting a liquid behavior at lower frequencies, in view of the balance of the following properties of the useful substance to be retained. It is preferable to exhibit dynamic behavior. More specifically, the ability to follow the operation of the hydrogel can be suitably measured by the following method.
[0057]
(Measurement method of follow-up performance)
A hydrogel of the present invention containing a hydrogel-forming polymer (1 mL as a hydrogel) is placed in a sol state (a temperature lower than the sol-gel transition temperature) in a test tube having an inner diameter of 1 cm, and the sol-gel transition temperature of the carrier is obtained. The test tube is held for 12 hours in a water bath at a temperature sufficiently higher than the temperature (for example, about 10 ° C. higher than the sol-gel transition temperature) to gel the hydrogel.
[0058]
Next, the time (T) until the solution / air interface (meniscus) is deformed by the weight of the solution when the test tube is turned upside down is measured. Here, 1 / T (sec^-1For lower frequency operation, the hydrogel behaves as a liquid, with 1 / T (sec^-12.) For higher frequency operation, the hydrogel will behave as a solid. In the case of the hydrogel of the present invention, T is 1 minute to 24 hours, preferably 5 minutes to 10 hours.
[0059]
(Steady flow viscosity)
The gel-like properties of the hydrogel of the present invention can be suitably measured by measuring the steady-state flow viscosity. The steady flow viscosity η (eta) can be measured by, for example, a creep experiment.
In the creep experiment, a constant shear stress is applied to the sample, and the time change of the shear strain is observed. In general, in the creep behavior of a viscoelastic body, the shear rate changes with time at the initial stage, but thereafter, the shear rate becomes constant. The ratio between the shear stress and the shear rate at this time is defined as a steady flow viscosity η. This steady flow viscosity is sometimes referred to as Newtonian viscosity. Here, the steady flow viscosity is determined within a linear region that hardly depends on shear stress.
[0060]
The specific measuring method is as follows: a stress control type viscoelasticity measuring device CSL type rheometer (CSL500, manufactured by Carry Med Co., USA) is used as a measuring device, and an acrylic disk (diameter 4 cm) is used as a measuring device. Observe the creep behavior (delay curve) for a measurement time of at least 5 minutes or more. The sampling time is once every second for the first 100 seconds, and once every 10 seconds thereafter.
In determining the "shear stress" (stress) to be applied, the shear angle is applied for 10 seconds and the shift angle is 2 × 10^-3Set to the lowest value that is detected more than rad. At least 20 or more measured values after 5 minutes are used for analysis. The hydrogel of the present invention has a η of 5 × 10 5 at a temperature about 10 ° C. higher than its sol-gel transition temperature.³~ 5 × 10⁶Pa · sec, more preferably 8 × 10³~ 2 × 10⁶Pa · sec, especially 1 × 10⁴Pa × sec or more, 1 × 10⁶It is preferably Pa · sec or less.
[0061]
The above η is 5 × 10³If it is less than Pa · sec, the fluidity becomes relatively high even in a short observation, and the three-dimensional holding of cells and tissues by the gel tends to be insufficient, and it tends to be difficult to function as a carrier. On the other hand, η is 5 × 10⁶When it exceeds Pa · sec, the gel tends to hardly show fluidity even during long-time observation, and the difficulty of following the movement accompanying the regeneration of the biological tissue increases. Also, η is 5 × 10⁶If it exceeds, the gel is more likely to exhibit brittleness, and after a slight pure elastic deformation, the tendency for brittle fracture, which breaks all at once, increases.
[0062]
(Dynamic elastic modulus)
The gel-like properties of the hydrogel of the present invention can be suitably measured also by the dynamic elastic modulus. The gel has an amplitude γ₀, The distortion γ (t) = γ where the frequency is ω / 2π₀When given cosωt (t is time), the constant stress is given by σ₀, Σ (t) = σ where δ is the phase difference₀It is assumed that cos (ωt + δ) is obtained. | G | = σ₀/ Γ₀Then, the ratio (G ″ / G ′) between the dynamic elastic modulus G ′ (ω) = | G | cos δ and the loss elastic modulus G ″ (ω) = | G | sin δ is an index representing the gel-like property. It becomes.
[0063]
The hydrogel of the present invention behaves as a solid for strains of ω / 2π = 1 Hz (corresponding to fast operation) and ω / 2π = 10^-4It behaves as a solid for Hz distortion (corresponding to slow operation). More specifically, the hydrogel of the present invention preferably exhibits the following properties (for details of such elastic modulus measurement, see, for example, Literature: Ryohei Oda, Modern Industrial Chemistry 19, p. 359). , Asakura Shoten, 1985).
When ω / 2π = 1 Hz (frequency at which the gel behaves as a solid), (G ″ / G ′)_s= (Tan δ)_sIs preferably less than 1. This (G "/ G ')_s= (Tan δ)_sIs more preferably 0.8 or less, particularly preferably 0.5 or less.
[0064]
ω / 2π = 10^-4Hz (frequency at which the gel behaves as a liquid), (G "/ G ')_L= (Tan δ)_LIs preferably 1 or more. This (G "/ G ')_L= (Tan δ)_LIs more preferably 1.5 or more, particularly preferably 2 or more.
[0065]
The above (tan δ)_sAnd (tan δ)_L｛(Tanｔδ)_s/ (Tan δ)_L｝ Is preferably less than 1. This ratio ｛(tan δ)_s/ (Tan δ)_L｝ Is more preferably 0.8 or less, particularly preferably 0.5 or less.
[0066]
<Measurement conditions>
Hydrogel-forming polymer concentration: about 8% by mass
Temperature: temperature about 10 ° C. higher than the sol-gel transition temperature of the carrier
Measuring equipment: stress control type rheometer (model name: CSL # 500, manufactured by Carrie Med, USA)
[0067]
(Control of persistence in living body)
The hydrogel of the present invention can optionally control the persistence in a living body (intraperitoneal, subcutaneous, etc.), if necessary. Although the hydrogel of the present invention is mainly intended for use in vitro, depending on the method of use (for example, when a tissue grown using the hydrogel of the present invention is returned to a living body), the hydrogel may be used. This is because it may be preferable to control the in vivo persistence of the gel.
[0068]
If the sol-gel transition temperature of the hydrogel of the present invention is lowered, the hydrogel will remain in the living body for a long time, and if the sol-gel transition temperature is raised, the disappearance of the hydrogel in the living body will be faster. Also, when the concentration of the hydrogel-forming polymer in the hydrogel is increased, the hydrogel will remain in the living body for a long time, and when the concentration of the hydrogel-forming polymer in the hydrogel is reduced, the The hydrogel disappears faster.
[0069]
In the hydrogel of the present invention, when the sol-gel transition temperature of the hydrogel of the present invention is reduced, the storage elastic modulus (G ') of the hydrogel at a biological temperature (37 [deg.] C.) increases. In addition, when the concentration of the hydrogel-forming polymer in the hydrogel is increased, the storage elastic modulus (G ') of the hydrogel at the biological temperature (37C) increases. That is, G ′ at 37 ° C. may be controlled to control the persistence in the living body.
[0070]
In the measurement of G ', the following measurement conditions can be suitably used.
<Measurement conditions for dynamic and loss elastic modulus>
Measuring equipment (trade name): Stress control type rheometer CSL500, manufactured by Carri-Med
Amount of sample solution: about 0.8 kg
Measurement cell shape / dimension: Acrylic parallel disk (diameter 4.0 cm), gap 600 μm,
Measurement frequency: 1Hz
Applied stress: within the linear region.
[0071]
Since the relationship between the remaining time of the hydrogel of the present invention in vivo and G ′ varies depending on the site in the living body, it cannot be said unconditionally. For example, the remaining time in the abdominal cavity and the G ′ at an observation frequency of 1 Hz are not possible. According to the findings of the present inventors, the relationship is as follows.
That is, a desirable range of G ′ for disappearing within 3 days is 10 to 500 Pa, a desirable range of G ′ for remaining for 3 days or more and within 14 days is 200 to 1500 Pa, and G for remaining for 14 days or more. The desirable range of 'is 400 to 10000 Pa.
[0072]
(Fibroblast proliferation)
When the hydrogel of the present invention is used for culturing cells and / or living tissues, a hydrogel based on a hydrogel-forming polymer is used in a hydrogel based on the viewpoint of suppressing contamination by fibroblasts. Preferably, the blast cells do not substantially proliferate. Fibroblasts usually show remarkable proliferation with dendritic morphological changes characteristic of fibroblasts in monolayer culture on cell culture dishes (plates) or in collagen gels (eg, Junpei Enami: Method using cultured cells, see Meiji Watanabe, edited by Extracellular Matrix, Medical Review, Tokyo, 1996, pp. 108-115). On the other hand, in the hydrogel of the present invention, it is easy to prevent the fibroblasts from substantially exhibiting proliferation while maintaining the spherical shape.
[0073]
(Estimated mechanism of suppression of fibroblast proliferation)
The mechanism by which the proliferation of fibroblasts is suppressed in the hydrogel of the present invention is not necessarily clear, but according to the findings of the present inventors, it is estimated as follows.
[0074]
That is, fibroblasts have the property of proliferating two-dimensionally by monolayer culture, that is, by recognizing and adhering to the surface of the support. The structure of a collagen gel is formed by assembling a large number of collagen molecules (molecular weight 300,000) having a length of 300 nm and a diameter of 1.5 nm, and regularly arranging them into a fiber state (collagen fibril) to form a network structure in water. is there. Since this network structure is larger than the wavelength of visible light (400 nm or more), the collagen gel usually looks cloudy. Although collagen gel is used as a three-dimensional culture carrier, fibroblasts recognize the surface of this thick collagen fiber (collagen fibril) as a support and adhere to it. It is estimated that
[0075]
On the other hand, since the hydrogel of the present invention is a hydrogel in which a hydrogel-forming polymer forms a three-dimensional network structure in a molecular state, the heterogeneity of the structure is smaller than the wavelength of visible light. High transparency. Therefore, in the material of the present invention, fibroblasts do not recognize a clear two-dimensional support surface, and as a result, excessive proliferation of fibroblasts is suppressed in the material of the present invention. It is estimated to be.
[0076]
(Evaluation of fibroblast proliferation)
The proliferation of fibroblasts can be evaluated by the following method (for details of this method, for example, Takeshi Yoshikawa, Ken Tsukikawa, St. Marianna University School of Medicine, Vol. 28, No. 4, 161-170 (2000) )).
[0077]
The hydrogel-forming polymer constituting the hydrogel of the present invention is dissolved by stirring in a culture solution, for example, RPMI-1640 (Life @ Technologies, NY, USA) at a low temperature (for example, 4 ° C.), and the normal human lung fiber is dissolved. Blast cells (Normal Human Lung Fibroblasts, NHLF, manufactured by Takara Shuzo Co., Ltd.) at 6 × 10⁴Disperse to a cell density of cells / ml. 0.2 mL of this NHLF dispersion is added to a 24-well plate (material: plastic, one well is about 15 mm in length, 15 mm in width, and about 20 mm in depth; a commercial product, for example, a product name of Becton-Dickinson Co., Ltd .: Multiwell), gelled at 37 ° C., added 0.4 mL of culture solution and added at 37 ° C., 5% CO 2₂Incubate under atmospheric pressure. The state of proliferation of the fibroblasts is confirmed daily (for example, on days 0, 1, 3, and 7) by observation with a phase contrast microscope.
[0078]
(Fibroblast proliferation rate)
Furthermore, the proliferation rate of fibroblasts during the culture period can be measured by a method utilizing the following enzyme activities.
[0079]
For example, after culturing fibroblasts in the hydrogel of the present invention for a predetermined period using a 24-well plate as described above, the carrier is cooled to a temperature lower than its sol-gel transition temperature (for example, sol-gel). The carrier is dissolved by lowering the temperature to 10 ° C. lower than the transition temperature, and 50 μl of WST-8 reagent (manufactured by Dojindo Co., Ltd.) as a reagent for measuring succinate dehydrogenase activity is added to each well.
[0080]
The 24-well plate is reacted at a temperature lower than the sol-gel transition temperature (for example, a temperature lower than the sol-gel transition temperature by 10 ° C., for example, 10 ° C.) for 10 hours, and then stored at about 4 ° C. for 1 hour and completely stored. To a uniform aqueous solution. The aqueous solution is dispensed into a 96-well plate in an amount of 200 μl, and the absorbance (OD (450)) is measured at 450 nm (reference wavelength 620 nm) using a colorimeter for microplate. It has been confirmed that this OD (450) is proportional to the number of viable cells (for example, literature Furukawa, @T., Et al., "High in in vitro-in vitro" histoculture and MTT end point, Int. J. Cancer 51: 489, 1992). That is, the proliferation rate of fibroblasts is determined by the absorbance OD at the start of culture._f= (OD (450)) and absorbance OD after culture_L= (OD (450)) ratio (OD_L/ OD_f).
[0081]
In the present invention, the growth rate P of fibroblasts after culturing at 37 ° C. for 3 days_F= (OD_L/ OD_f) Is preferably in the range of 70% to 200%. This growth rate (OD_L/ OD_f) Is more preferably in the range of 80% to 150%, especially in the range of 90% to 120%.
[0082]
(Relative proliferation of fibroblasts)
In the present invention, in a gel based on a hydrogel-forming polymer, the growth of fibroblasts is suppressed, while the growth of target cells (other than fibroblasts) is not relatively suppressed. Is preferred. More specifically, the growth rate P of the target cell_TAnd the above-mentioned proliferation rate P of fibroblasts_FAnd the ratio (P_T/ P_F) Is preferably 1.1 or more. Furthermore, this ratio (P_T/ P_F) Is preferably 1.5 or more, particularly 2.0 or more. The target cell growth rate P_TCan be obtained as follows.
[0083]
Fibroblast proliferation rate P_FThe above proliferation rate P was changed except that human colon cancer cells (SW-948, trade name: colon adenocarcinoma cell line, manufactured by Dainippon Pharmaceutical Co., Ltd.) were used instead of normal human lung fibroblasts (NHLF) in the measurement._FSimilarly to the measurement, the growth rate P of the cells other than the fibroblasts_T(These growth rates P_TAnd P_FIs measured under the same conditions).
[0084]
Growth rate P measured as above_TAnd P_FThese ratios (P_T/ P_F).
[0085]
(Hydrogel-forming polymer)
The hydrogel-forming polymer that can be used in the hydrogel of the present invention is not particularly limited, as long as it exhibits a thermoreversible sol-gel transition as described above (ie, has a sol-gel transition temperature). From the viewpoint that it is easy to show a suitable sol-gel change at a physiological temperature (about 0 to 42 ° C.), for example, a plurality of blocks having a cloud point in the hydrogel-forming polymer and a hydrophilic It is preferable to achieve a suitable sol-gel transition temperature by adjusting the cloud point of the block, the composition of both blocks, and the hydrophobicity, hydrophilicity, and / or molecular weight of both blocks, respectively.
[0086]
Specific examples of the polymer whose aqueous solution has a sol-gel transition temperature and exhibits a sol state reversibly at a temperature lower than the transition temperature include, for example, a block copolymer of polypropylene oxide and polyethylene oxide. Polyalkylene oxide block copolymers; etherified celluloses such as methylcellulose and hydroxypropylcellulose; and chitosan derivatives (KR Holme. Et al. Macromolecules, 24, 3828 (1991)).
[0087]
As a polyalkylene oxide block copolymer, Pluronic F-127 (trade name, manufactured by BASF Wyandotte Chemicals Co.) gel in which polyethylene oxide is bonded to both ends of polypropylene oxide has been developed. It is known that this highly concentrated aqueous solution of Pluronic F-127 becomes a hydrogel at about 20 ° C. or higher, and becomes an aqueous solution at a lower temperature. However, in the case of this material, a gel state is obtained only at a high concentration of about 20% by mass or more, and even when the temperature is higher than the gelation temperature at a high concentration of about 20% by mass or more, water is further added. And the gel dissolves. In addition, Pluronic F-127 has a relatively small molecular weight, exhibits a very high osmotic pressure in a high gel state of about 20% by mass or more, and easily penetrates a cell membrane, which may adversely affect cells and tissues. There is.
[0088]
On the other hand, in the case of etherified cellulose represented by methylcellulose, hydroxypropylcellulose and the like, the sol-gel transition temperature is usually high and about 45 ° C. or higher (N. Sarkar, J. Appl. Polym. Science,24, 1073, 1979). On the other hand, since the body temperature of the living body is usually around 37 ° C., the etherified cellulose is in a sol state, and it is practically difficult to use the etherified cellulose as a carrier for tissues and organs.
[0089]
As described above, the problems of the conventional polymer in which the aqueous solution has a sol-gel transition point and reversibly shows a sol state at a temperature lower than the transition temperature are as follows: 1) Higher than the sol-gel transition temperature. Once gelled at the temperature, the gel is dissolved when water is further added. 2) The sol-gel transition temperature is higher than the body temperature of the living body (around 37 ° C.), and the body temperature is in the sol state. ) In order to cause gelation, the polymer concentration of the aqueous solution needs to be extremely high.
[0090]
On the other hand, according to the study of the present inventors, a hydrogel-forming polymer having a sol-gel transition temperature preferably higher than 0 ° C. and 42 ° C. or lower (for example, a plurality of blocks having a cloud point, And a hydrophilic block are combined, and the aqueous solution thereof has a sol-gel transition temperature, and a polymer which reversibly shows a sol state at a temperature lower than the sol-gel transition temperature). It has been found that the above problem is solved when a hydrogel is formed.
[0091]
(Suitable hydrogel-forming polymer)
The hydrogel-forming polymer utilizing a hydrophobic bond that can be suitably used as the hydrogel of the present invention is preferably formed by combining a plurality of blocks having a cloud point with a hydrophilic block. The hydrophilic blocks are preferably present for the hydrogel to become water-soluble at a temperature lower than the sol-gel transition temperature, and the plurality of blocks having a cloud point are those in which the hydrogel has a sol-gel transition temperature. It is preferably present to change to a gel state at higher temperatures. In other words, at temperatures above the cloud point, the blocks dissolve the gel because the block with the cloud point dissolves in water below the cloud point and changes to insoluble in water above the cloud point. It acts as a cross-linking point consisting of hydrophobic bonds to be formed. That is, the cloud point derived from the hydrophobic bond corresponds to the sol-gel transition temperature of the hydrogel.
[0092]
However, the cloud point and the sol-gel transition temperature do not necessarily have to match. This is because the cloud point of the above-mentioned “block having a cloud point” is generally affected by the bond between the block and the hydrophilic block.
[0093]
The hydrogel used in the present invention utilizes the property that not only the hydrophobic bond becomes stronger with an increase in temperature but also that the change is reversible with respect to temperature. From the viewpoint that a plurality of crosslinking points are formed in one molecule and a gel having excellent stability is formed, it is preferable that the hydrogel-forming polymer has a plurality of “blocks having a cloud point”.
[0094]
On the other hand, the hydrophilic block in the hydrogel-forming polymer has a function of changing the hydrogel-forming polymer to water-soluble at a temperature lower than the sol-gel transition temperature, as described above. It has the function of forming a hydrogel while preventing the hydrogel from agglomerating and precipitating due to excessive increase in the hydrophobic binding force at a temperature higher than the transition temperature.
[0095]
(Multiple blocks with cloud points)
The block having a cloud point is preferably a block of a polymer having a negative solubility in water-temperature coefficient, and more specifically, polypropylene oxide, a copolymer of propylene oxide and another alkylene oxide, A polymer selected from the group consisting of poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers of N-substituted acrylamide derivatives and N-substituted methacrylamide derivatives, polyvinyl methyl ether, and partially acetylated polyvinyl alcohol Can be preferably used. The above polymer (block having a cloud point) having a cloud point higher than 4 ° C. and not higher than 40 ° C. is a polymer (a compound in which a plurality of blocks having a cloud point and a hydrophilic block are bonded) used in the present invention. ) Is preferred because the sol-gel transition temperature is set to be higher than 4 ° C. and 40 ° C. or lower.
[0096]
Here, the cloud point is measured, for example, by cooling an aqueous solution of about 1% by mass of the above-mentioned polymer (block having a cloud point) into a transparent uniform solution, and then gradually increasing the temperature (heating rate is about 1%). ° C / min), and the point where the solution becomes cloudy for the first time is set as the cloud point.
[0097]
Specific examples of the poly N-substituted acrylamide derivative and the poly N-substituted methacrylamide derivative that can be used in the present invention are listed below.
Poly-N-acroylpiperidine; poly-Nn-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; Poly-N-acryloylpyrrolidine; poly-N, N-ethylmethylacrylamide; poly-N-cyclopropylmethacrylamide; poly-N-ethylacrylamide.
[0098]
The above polymer may be a homopolymer (homopolymer) or a copolymer of a monomer constituting the polymer and another monomer. As the other monomer constituting such a copolymer, any of a hydrophilic monomer and a hydrophobic monomer can be used. In general, copolymerization with a hydrophilic monomer increases the cloud point of the product, and copolymerization with a hydrophobic monomer lowers the cloud point of the product. Accordingly, a polymer having a desired cloud point (for example, a cloud point higher than 4 ° C. and equal to or lower than 40 ° C.) can be obtained also by selecting these monomers to be copolymerized.
[0099]
(Hydrophilic monomer)
Examples of the hydrophilic monomer include N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, and acrylic acid having an hydroxy group. , Methacrylic acid and salts thereof, vinyl sulfonic acid, styrene sulfonic acid and the like, and N, N-dimethylaminoethyl methacrylate N having a basic group, N-diethylaminoethyl methacrylate, N, N-dimethylaminopropyl acrylamide and Examples thereof include, but are not limited to, salts thereof.
[0100]
(Hydrophobic monomer)
On the other hand, as the hydrophobic monomer, acrylate derivatives such as ethyl acrylate, methyl methacrylate, glycidyl methacrylate and methacrylate derivatives, N-substituted alkyl methacrylamide derivatives such as N-n-butyl methacrylamide, vinyl chloride, acrylonitrile, styrene , Vinyl acetate and the like, but are not limited thereto.
[0101]
(Hydrophilic block)
On the other hand, as the hydrophilic block to be bonded to the block having the above cloud point, specifically, methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide , Poly N-methylacrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and salts thereof; poly N, N-dimethylaminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate , Poly N, N-dimethylaminopropylacrylamide and salts thereof.
[0102]
The method for bonding the block having a cloud point and the hydrophilic block is not particularly limited, and for example, it may be obtained as a block copolymer having these blocks, or a graft copolymer or a dendrimer-type copolymer. preferable.
A bond between a block having a cloud point and a hydrophilic block is obtained by introducing a polymerizable functional group (for example, an acryloyl group) into any one of the above blocks and forming a monomer that gives the other block in this manner. It can be carried out by copolymerizing with a block into which a functional group has been introduced. Further, a combination of a block having a cloud point and the above-described hydrophilic block may be obtained by block copolymerization of a monomer that provides a block having a cloud point and a monomer that provides a hydrophilic block. It is possible. Further, the bonding between the block having the cloud point and the hydrophilic block is performed by introducing a reactive group (for example, a hydroxyl group, an amino group, a carboxyl group, an isocyanate group, etc.) into both of them in advance and bonding them together by a chemical reaction. It can also be done by doing. At this time, usually, a plurality of reactive functional groups are introduced into the hydrophilic block.
[0103]
Regarding the bond between a polypropylene oxide having a cloud point and a hydrophilic block, for example, propylene oxide and a monomer (for example, ethylene oxide) constituting “another hydrophilic block” are repeatedly formed by anionic polymerization or cationic polymerization. By sequentially polymerizing, a block copolymer in which polypropylene oxide and a “hydrophilic block” (for example, polyethylene oxide) are bonded can be obtained. Such a block copolymer can also be obtained by introducing a polymerizable group (for example, an acryloyl group) into the terminal of polypropylene oxide and then copolymerizing a monomer constituting a hydrophilic block. Furthermore, the polymer used in the present invention can also be obtained by introducing a functional group capable of binding with a functional group (for example, a hydroxyl group) at the terminal of polypropylene oxide into the hydrophilic block and reacting the functional groups. . The hydrogel-forming polymer used in the present invention can also be obtained by connecting a material such as Pluronic® F-127 (trade name, manufactured by Asahi Denka Kogyo KK) in which polyethylene glycol is bonded to both ends of polypropylene glycol. Can be obtained.
[0104]
The polymer of the present invention in the embodiment including the block having the cloud point, at a temperature lower than the cloud point, since the `` block having the cloud point '' present in the molecule is water-soluble together with the hydrophilic block, It is completely dissolved in water and shows a sol state. However, when the temperature of the aqueous solution of the polymer is heated to a temperature higher than the above cloud point, the "block having a cloud point" existing in the molecule becomes hydrophobic, and the molecules are associated with each other by hydrophobic interaction. I do.
[0105]
On the other hand, since the hydrophilic block is still water-soluble at this time (when heated to a temperature higher than the cloud point), the polymer of the present invention can be used in water to form the hydrophobic association between the blocks having the cloud point in water. To form a hydrogel having a three-dimensional network structure with the crosslinking points. When the temperature of the hydrogel is cooled again to a temperature lower than the cloud point of the “block having a cloud point” present in the molecule, the block having the cloud point becomes water-soluble and the cross-linking points due to hydrophobic association are released. When the hydrogel structure disappears, the polymer of the present invention becomes a complete aqueous solution again. Thus, the sol-gel transition of the polymer of the present invention in a preferred embodiment is based on a reversible change in hydrophilicity and hydrophobicity at the cloud point of a block having a cloud point present in the molecule. Therefore, it has complete reversibility in response to temperature changes.
[0106]
(Solubility of gel)
As described above, the hydrogel-forming polymer of the present invention containing at least a polymer having a sol-gel transition temperature in an aqueous solution is substantially water-insoluble at a temperature (d ° C) higher than the sol-gel transition temperature. And reversibly shows water solubility at a temperature (e ° C.) lower than the sol-gel transition temperature.
[0107]
The above-mentioned high temperature (d ° C) is preferably a temperature higher than the sol-gel transition temperature by 1 ° C or more, and more preferably a temperature higher by 2 ° C or more (particularly 5 ° C or more). The term “substantially water-insoluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (d ° C.) is 5.0 g or less (further 0.5 g or less, particularly 0.1 g or less). ) Is preferable.
[0108]
On the other hand, the above-mentioned low temperature (e ° C.) is preferably 1 ° C. or more (in absolute value) lower than the sol-gel transition temperature, more preferably 2 ° C. or more (particularly 5 ° C. or more). preferable. The term “water-soluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (e ° C.) is preferably 0.5 g or more (more preferably 1.0 g or more). Further, "reversibly water-soluble" means that the aqueous solution of the hydrogel-forming polymer is once gelled (at a temperature higher than the sol-gel transition temperature) even after it is gelled. At lower temperatures, it indicates the above-mentioned water solubility.
The above polymer has a viscosity of 10 to 3,000 mPa · s (10 to 3,000 centipoise), and further 50 to 1,000 mPa · s (50 to 1,000 centipoise) at 5 ° C. in a 10% aqueous solution. Preferably. Such a viscosity is preferably measured, for example, under the following measurement conditions.
[0109]
Viscometer: stress control type rheometer (model name: CSL 500, manufactured by Carry Med, USA)
Rotor diameter: 60mm
Rotor shape: Parallel plate
Measurement frequency: 1 Hz (Hertz)
[0110]
Even if the aqueous solution of the hydrogel-forming polymer of the present invention is gelled at a temperature higher than the sol-gel transition temperature and then immersed in a large amount of water, the gel does not substantially dissolve. The characteristics of the carrier can be confirmed, for example, as follows.
[0111]
That is, 0.15 g of the hydrogel-forming polymer of the present invention is dissolved in 1.35 g of distilled water at a temperature lower than the sol-gel transition temperature (for example, under ice cooling) to prepare a 10 W% aqueous solution. An aqueous solution was poured into a 35 mm plastic Petri dish, and heated to 37 ° C. to form a gel having a thickness of about 1.5 mm in the Petri dish. G). Next, the whole petri dish containing the gel was allowed to stand in water in 250 ml of water at 37 ° C. for 10 hours, and then the mass (g gram) of the whole petri dish containing the gel was measured to determine the dissolution of the gel from the gel surface. Evaluate the presence or absence. At this time, in the hydrogel-forming polymer of the present invention, the mass reduction rate of the gel, that is, (f−g) / f is preferably 5.0% or less, more preferably 1.0%. Or less (especially 0.1% or less).
[0112]
The aqueous solution of the hydrogel-forming polymer of the present invention is gelled at a temperature higher than the above sol-gel transition temperature, and then immersed in a large amount (about 0.1 to 100 times the volume of gel) of water. However, the gel does not dissolve over a long period of time. Such properties of the polymer used in the present invention can be achieved, for example, by the presence of two or more (plural) blocks having a cloud point in the polymer.
[0113]
On the other hand, when a similar gel was prepared using the above-mentioned Pluronic F-127 in which polyethylene oxide was bonded to both ends of polypropylene oxide, the gel was completely dissolved in water after standing for several hours. The inventors have found that
[0114]
From the viewpoint of suppressing the cytotoxicity at the time of non-gelation to the lowest possible level, the concentration with respect to water, that is, {(polymer) / (polymer + water)} × 100 (%) is not more than 20% (further 15%). %, Particularly preferably 10% or less), is preferably used.
[0115]
(Ingredient having physiological action)
The hydrogel or gel body of the present invention may contain a useful substance as described above. Examples of the case where the useful substance has a physiological effect include, for example, antibiotics, anticancer drugs, ECM such as collagen and gelatin, local chemical mediators described below, hormones such as insulin and cell growth factor, foreign genes and the like, or chemicals thereof. Other cells and tissues that secrete mediators, cell growth factors and the like can be mentioned.
[0116]
The amount of such “other components” is an amount that exhibits a predetermined effect, and is used in a gel based on a hydrogel-forming polymer for a predetermined period of time (for example, a period necessary for culturing cells and tissues). Time) There is no particular limitation as long as the amount can be held. Usually, it is preferably about 2 parts or less, more preferably about 1 part or less, based on the hydrogel-forming polymer (10 parts).
[0117]
(Chemical mediator)
Regeneration of living tissue may require not only cells such as progenitor cells but also various chemical mediators such as cell growth factors that promote their differentiation and proliferation. These chemical mediators are normally secreted from the cells themselves. However, in order to promote regeneration efficiently, the carrier for cell / tissue culture of the present invention may contain these chemical mediators in advance and supply them from outside. It is effective.
[0118]
The chemical mediators include: 1) a local chemical mediator that acts only in the immediate vicinity of a cell; 2) a neurotransmitter secreted from a nerve cell and having a very short effective action distance; 3) Examples include hormones that are secreted from endocrine cells and act on target cells throughout the body through the bloodstream or the like, or cell growth factors that are intercellular messengers.
[0119]
Examples of the local chemical mediator 1) include proteins such as nerve cell growth factor, peptides such as chemotactic factor, amino acid derivatives such as histamine, and fatty acid derivatives such as prostaglandin.
[0120]
Examples of the neurotransmitter 2) include low molecular weight substances such as amino acids such as glycine and low molecular peptides such as noradrenaline, acetylcholine and enkephalin.
[0121]
Examples of the cell growth factor or hormone of 3) include fibroblast growth factor (FGF), epithelial growth factor (EGF), vascular endothelial growth factor, and GF. Cell growth factors such as hepatocyte growth factor (HGF), proteins such as insulin, somatotropin, somatomedin, adrenocorticotropic hormone (ACTH), parathyroid hormone (PTH), thyroid stimulating hormone (TSH), or Glycoproteins, TSH-releasing factors, vasopressin, amino acid derivatives such as somatostatin, cortisol, estradiol, tes Steroids such as Suteron and the like.
[0122]
(In vivo tissues and organs)
In the present invention, “biological tissues / organs” refer to tissues, organs, and organs of animals (particularly humans). The living tissues and organs that can be cultured by the hydrogel of the present invention are not particularly limited, but examples include esophagus, stomach, small intestine, large intestine, pancreas, liver, heart, blood vessels, bone, cartilage, nerve, cornea, dermis, and the like. Can be.
[0123]
(Cells / tissues)
Cells and tissues that can be cultured with the hydrogel of the present invention are not particularly limited.
The hydrogel of the present invention can be used particularly effectively for differentiating cells and tissues. Such differentiating cells include, for example, stem cells (stem @ cells) and progenitor cells. Differentiating cells include any of pluripotent, pluripotent, or totipotent cells.
[0124]
(How to repair and regenerate living tissues and organs)
By using the hydrogel of the present invention, for example, cells and tissues can be repaired and regenerated. The method of repair / regeneration is not particularly limited, but it is preferable to use the sol-gel transition of the hydrogel-forming polymer from the viewpoint of facilitating seeding and recovery of the cells and the like.
[0125]
(Embodiment utilizing sol-gel transition)
In such an embodiment utilizing the sol-gel transition, first, cells (for example, stem cells or progenitor cells) or tissues containing them are seeded and mixed in the hydrogel of the present invention. In order to perform such seeding and mixing, for example, a hydrogel-forming polymer constituting the hydrogel of the present invention is added to a culture solution such as RPMI-1640 (Life @ Technologies, NY, USA) at a low temperature. The cells or tissue may be added and suspended by dissolving the hydrogel of the present invention in an aqueous solution (sol) at a temperature lower than its sol-gel transition temperature by stirring and dissolving at 4 ° C., for example. The culture solution used here is not particularly limited, and a culture solution in which target cells (stem cells, progenitor cells, etc.) are easily proliferated and differentiated may be appropriately selected and used. If necessary, the culture medium may contain the aforementioned chemical mediator that promotes the growth and differentiation of the target stem cells or progenitor cells. Further, ECM such as collagen and gelatin may be added.
[0126]
In order to regenerate a living tissue or organ in the hydrogel of the present invention, for example, the suspension is heated to a temperature higher than the sol-gel transition temperature of the hydrogel of the present invention (usually 37 ° C.) to form a gel. After the transformation, the target cells or tissues containing the cells may be cultured at the temperature (usually 37 ° C.).
[0127]
Further, when the hydrogel of the present invention is gelled, it can be gelled by giving a desired shape using a mold having a desired shape. For example, when using cartilage tissue for reconstruction of the ear or nose, the hydrogel of the present invention is cultured in a shape suitable for the application site of the ear or nose, and the cartilage cells are cultured to regenerate the cartilage tissue. It can be used after being molded into a shape.
[0128]
After the target tissue / organ has been regenerated in the hydrogel of the present invention, to recover them from the hydrogel of the present invention, the hydrogel of the present invention containing the target tissue / organ is subjected to the sol-gel transition temperature. The hydrogel of the present invention may be cooled to a lower temperature (for example, 4 ° C.) to return the hydrogel of the present invention to a sol state, and the target tissue / organ may be separated from the hydrogel of the present invention by a usual separation method such as centrifugation.
[0129]
As described above, while the hydrogel of the present invention suppresses the growth of fibroblasts, it does not substantially inhibit (or promote) the growth or differentiation of target cells (stem cells, progenitor cells, etc.). Therefore, the target tissue / organ can be efficiently regenerated in the hydrogel of the present invention.
[0130]
【Example】
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is limited by the claims, and is not limited by the following Examples.
[0131]
Production Example 1
10 g of a polypropylene oxide-polyethylene oxide copolymer (propylene oxide / ethylene oxide average polymerization degree: about 60/180, manufactured by Asahi Denka Kogyo KK: Pluronic F-127) was dissolved in 30 ml of dry chloroform, and phosphorus pentoxide (1 g) was dissolved. In the coexistence, 0.13 g of hexamethylene diisocyanate was added, and the mixture was reacted for 6 hours under reflux at the boiling point. After distilling off the solvent under reduced pressure, the residue was dissolved in distilled water, and subjected to ultrafiltration using an ultrafiltration membrane (Amicon PM-30) having a molecular weight cut off of 30,000 to obtain a high molecular weight polymer and a low molecular weight polymer. Was fractionated. The obtained aqueous solution was frozen to obtain an F-127 high polymer and an F-127 low polymer.
[0132]
The F-127 high polymer obtained above (the hydrogel-forming polymer of the present invention, TGP-1) was dissolved in distilled water at a concentration of 8% by mass under ice-cooling. When this aqueous solution was gradually heated, the viscosity gradually increased from 21 ° C., and solidified at about 27 ° C. to form a hydrogel. When the hydrogel was cooled, it returned to an aqueous solution at 21 ° C. This change was repeatedly observed reversibly.
[0133]
On the other hand, the F-127 low polymer dissolved in distilled water at a concentration of 8% by mass below the freezing point did not gel at all even when heated to 60 ° C or higher.
[0134]
Production Example 2
160 moles of ethylene oxide were added to 1 mole of trimethylolpropane by cationic polymerization to obtain polyethylene oxide triol having an average molecular weight of about 7,000.
[0135]
After dissolving 100 g of the polyethylene oxide triol obtained above in 1000 ml of distilled water, 12 g of potassium permanganate was gradually added at room temperature, and the reaction was allowed to proceed as it was for about 1 hour. After removing the solid by filtration, the product was extracted with chloroform, and the solvent (chloroform) was distilled off under reduced pressure to obtain 90 g of a polyethylene oxide tricarboxyl compound.
[0136]
10 g of the polyethylene oxide tricarboxyl compound obtained as described above and 10 g of a polypropylene oxide diamino compound (propylene oxide average polymerization degree: about 65, manufactured by Jefferson Chemical Co., USA, trade name: Jeffamine D-4000, cloud point: about 9 ° C.) After dissolving in 1,000 ml of carbon tetrachloride and adding 1.2 g of dicyclohexylcarbodiimide, the mixture was reacted for 6 hours under reflux at the boiling point. After cooling the reaction solution and removing solids by filtration, the solvent (carbon tetrachloride) is distilled off under reduced pressure, and the residue is dried under vacuum to obtain a hydrogel of the present invention in which a plurality of polypropylene oxides and polyethylene oxides are bonded. A forming polymer (TGP-2) was obtained. This was dissolved in distilled water at a concentration of 5% by mass under ice-cooling, and its sol-gel transition temperature was measured.
[0137]
Production Example 3
96 g of N-isopropylacrylamide (manufactured by Eastman Kodak Company), 17 g of N-acryloxysuccinimide (manufactured by Kokusan Chemical Co., Ltd.), and 7 g of n-butyl methacrylate (manufactured by Kanto Chemical Co., Ltd.) were dissolved in 4000 ml of chloroform, and nitrogen was dissolved. After the substitution, 1.5 g of N, N′-azobisisobutyronitrile was added, and the mixture was polymerized at 60 ° C. for 6 hours. After the reaction solution was concentrated, it was reprecipitated (reprecipitated) in diethyl ether. After collecting the solid by filtration, it was dried under vacuum to obtain 78 g of poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate).
[0138]
An excess of isopropylamine is added to the poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) obtained above to obtain poly (N-isopropylacrylamide-co-n-butyl methacrylate). Obtained. The cloud point of the aqueous solution of this poly (N-isopropylacrylamide-co-n-butyl methacrylate) was 19 ° C.
[0139]
10 g of the above-mentioned poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) and 5 g of a terminally aminated polyethylene oxide (molecular weight 6,000, manufactured by Kawaken Fine Chemical Co., Ltd.) were mixed with chloroform. It was dissolved in 1000 ml and reacted at 50 ° C. for 3 hours. After cooling to room temperature, 1 g of isopropylamine was added, and the mixture was allowed to stand for 1 hour. Then, the reaction solution was concentrated, and the residue was precipitated in diethyl ether. After collecting the solid matter by filtration, the solid matter is vacuum-dried, and the hydrogel-forming polymer (TGP-3) of the present invention in which a plurality of poly (N-isopropylacrylamide-co-n-butyl methacrylate) and polyethylene oxide are bonded. ) Got.
[0140]
The TGP-3 thus obtained was dissolved in distilled water at a concentration of 5% by mass under ice-cooling, and the sol-gel transition temperature was measured.
[0141]
Production Example 4
(Sterilization method)
2.0 g of the above-described hydrogel-forming polymer (TGP-3) of the present invention was placed in an EOG (ethylene oxide gas) sterilization bag (manufactured by Hogi Medical Co., Ltd., trade name: hybrid sterilization bag), and an EOG sterilizer ( The bag was filled with EOG using an easy pack (manufactured by Inuchi Seieido) and left at room temperature for 24 hours. After further leaving at 40 ° C. for half a day, the EOG was removed from the bag and aerated. The bags were placed in a vacuum dryer (40 ° C.) and sterilized by leaving for half a day with occasional aeration.
[0142]
It was separately confirmed that the sol-gel transition temperature of the polymer did not change by this sterilization operation.
[0143]
Production Example 5
After dissolving 37 g of N-isopropylacrylamide, 3 g of n-butyl methacrylate, and 28 g of polyethylene oxide monoacrylate (molecular weight: 4,000, manufactured by NOF Corporation: PME-4000) in 340 ml of benzene, 2,2 ′ -0.8 g of azobisisobutyronitrile was added and reacted at 60 ° C for 6 hours. The obtained reaction product was dissolved by adding 600 ml of chloroform, and the solution was dropped into 20 L (liter) of ether to precipitate. The resulting precipitate was collected by filtration, and the precipitate was vacuum-dried at about 40 ° C. for 24 hours, then redissolved in 6 L of distilled water, and a hollow fiber ultrafiltration membrane having a molecular weight cut off of 100,000 (H1P100 manufactured by Amicon) was used. -43) and concentrated to 2 l at 10 ° C.
[0144]
The concentrated liquid was diluted by adding 4 l of distilled water, and the dilution operation was repeated. The above-mentioned dilution and ultrafiltration / concentration operations were further repeated five times to remove those having a molecular weight of 100,000 or less. What was not filtered by the ultrafiltration (the one remaining in the ultrafiltration membrane) was recovered and freeze-dried to obtain 60 g of the hydrogel-forming polymer (TGP-4) of the present invention having a molecular weight of 100,000 or more. Obtained.
[0145]
1 g of the hydrogel-forming polymer (TGP-4) of the present invention obtained above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 25 ° C.
[0146]
Production Example 6
The hydrogel-forming polymer (TGP-3) of the present invention of Production Example 3 was dissolved in distilled water at a concentration of 10% by mass, and 5.8 × 10⁵Pa · sec. In the measurement of the steady flow viscosity η, a stress-type rheometer (CSL500) and an acrylic disk (diameter 4 cm) were used as a measuring device, and a sample thickness of 600 μm was used and 10 N / m²Shear stress was applied and creep was observed for 5 minutes after 5 minutes.
[0147]
On the other hand, agar was dissolved in distilled water at a concentration of 2% by mass at 90 ° C., gelled at 10 ° C. for 1 hour, and η at 37 ° C. was measured. The η was the measurement limit of the instrument (1 × 10⁷Pa · sec).
[0148]
Production Example 7
(Evaluation of proliferation of fibroblasts)
After sterilizing the hydrogel-forming polymer (TGP-3) of the present invention prepared in Production Example 3 by the method of Production Example 4, 20% fetal bovine serum was prepared so that the final concentration of the polymer was about 8%. (FCS; manufactured by Dainippon Pharmaceutical Co., Ltd., trade name: Fetal Calf Serum) and antibiotics (LifeTechnologies, trade name: penicillin; final concentration 10,000 U / ml) in RPMI-1640 (manufactured by LifeTechno) in RPMI Dissolved with stirring at 4 ° C. for 24 hours. This operation was performed aseptically.
[0149]
6 × 10 normal human lung fibroblasts (Normal Human Lung Fibroblasts, NHLF, manufactured by Takara Shuzo Co., Ltd.) were added to the above-mentioned hydrogel (TGP-3 / RPMI) of the present invention.⁴The cells were dispersed to a cell density of cells / ml. The NHLF dispersion was dispensed into each well of a 24-well plate [flat bottom multiwell tissue plate (FALCON, Becton Dickinson & Company)], gelled at 37 ° C, and then cultured at 0 ° C. Add 4 ml to 37 ° C, 5% CO₂Cultured under atmospheric pressure. A total of eight 24-well plates were prepared for microscopic observation and measurement of fibroblast proliferation rate, each for the 0th, 1st, 3rd, and 7th days.
[0150]
Separately, 6 × 10 6 cells were added to the above culture medium without using TGP-3 for comparison.⁴An NHLF dispersion liquid was prepared to be dispersed so as to have a cell density of cells / ml. Eight 24-well plates were prepared in the same manner, and the same culture test was performed.
[0151]
As a result of daily observation (0, 1, 3, 7 days) with a phase contrast microscope (Olympus, trade name: IMT-2, 10 times), in the culture of the comparative example (RPMI), fibroblasts started 1 day later. Dendritic growth characteristic of the cells was observed, and the cells became confluent after 7 days, whereas in the hydrogel (TGP-3 / RPMI) of the present invention, the fibroblasts were single cells until 7 days. No growth was observed with the morphology maintained.
[0152]
After a predetermined number of days of culture, the carrier was dissolved by lowering the 24-well plate to 4 ° C., and the WST-8 reagent (Dohin Chemical Co., Ltd.) as a reagent for measuring succinate dehydrogenase activity was placed in each well. ) 50 μl was added. After the 24-well plate was reacted at 4 ° C. for 10 hours, a completely uniform aqueous solution was obtained.
[0153]
The aqueous solution was dispensed at 200 μl into a 96-well plate, and the absorbance (OD (450)) was measured at 450 nm (reference wavelength 620 nm) using a colorimeter for microplate. The proliferation rate of fibroblasts was determined by the absorbance OD at the start of culture (day 0)._f= (OD (450)) and absorbance OD after culture (after 1, 3, 7 days)_L= (OD (450)) ratio (OD_L/ OD_f).
In the hydrogel of the present invention (TGP-3 / RPMI), the proliferation rate (OD) of the fibroblasts after 1, 3 and 7 days_L/ OD_f) Were 105%, 120%, and 125%, respectively, whereas in Comparative Example (RPMI), the proliferation rate (OD) of the fibroblasts was 1, 3, and 7 days later._L/ OD_f) Were 170%, 370%, and 420%, respectively.
[0154]
Example 1
A beagle dog (9.0 to 11.0 kg body weight) was anesthetized with pentobarbital by intravenous anesthesia, the abdomen was opened, and the pancreas was completely removed. When a guide wire having a diameter of 100 μm was inserted into the pancreatic duct, and the pancreatic tissue was excised and removed along the guide wire, only the pancreatic duct could be collected. A pancreatic duct having a length of about 6 cm and a diameter of 150 to 200 μm was minced at a width of 0.5 mm.
[0155]
A solution (hydrogel of the present invention) obtained by dissolving the hydrogel-forming polymer of the present invention (TGP-3) of Production Example 3 at a concentration of 10% by mass in a culture solution (RPMI-1640, manufactured by Life @ Technologies) was used. After cooling to 0 ° C, the above-mentioned pancreatic duct thin section was dispersed, and 0.4 ml was dispensed into each well of a 12-well plate [flat \ multiwell \ tissue \ culture \ plate (FALCON, Becton \ Dickinson \ & Company)] and gel at 37 ° C. After that, 0.4 ml of the culture solution was added, and the mixture was added at 37 ° C. and 5% CO 2.₂Cultured under atmospheric pressure. The culture solution RPMI-1640 contains 10% of dog serum (50 to 100 ml of canine blood collected and allowed to stand at room temperature for 30 minutes to collect the supernatant), 10 mM nicotinamide (manufactured by Sigma, trade name: NIACINAMIDE) ), 10 ng / ml keratinocyte growth factor (KGF; manufactured by PEPROTECH FC Ltd., trade name: Recombinant Keratinocyte Growth Factor).
[0156]
Observation with a stereoscopic microscope (manufactured by Olympus, trade name: inverted system microscope; 100-fold) daily revealed that the cells were divided and proliferated from the outside of the pancreatic duct tissue from day 5 of the culture (see FIG. 5). (Fig. 1). The cell clumps increased in size over time, becoming 150 μm on day 14 and 300 μm on day 30 (FIG. 2). The histological image on day 30 showed that insulin-positive cells were present in the cell clumps and that clusters of islets of Langerhans were formed.
[0157]
By cooling the hydrogel of the present invention containing the cell clumps to 4 ° C. and centrifuging, the cell clumps and the hydrogel of the present invention solubilized by the cooling can be easily separated. Was.
[0158]
Example 2
The costal cartilage of a 4-week-old Lewis male rat was aseptically collected, and the attached soft tissue was removed to remove the cartilage and fragmented. Next, enzyme solution ｛first solution: 0.1% EDTA (manufactured by Nacalai Tesque) / PBS (-) (manufactured by Roman Industries), second solution: 0.25% trypsin-EDTA (manufactured by Gibco) / PBS (−) (Manufactured by Roman Industrial Co., Ltd.), third solution: 0.1% collagenase (manufactured by Wako Pure Chemical Industries) / PBS (+) (manufactured by Gibco Co.) At 37 ° C. for 15 minutes, 1 hour and 3 hours to decompose cartilage tissue.
[0159]
The collected cartilage dispersion was put in a medium containing D-MEM (Dulbecco's Modified Eagle Medium: (manufactured by Gibco) + 10% FCS (Fetal Bovine Serum) + penicillin + streptomycin) for about 2 weeks until confluent. 37 ° C, 5% CO₂Cultured under atmospheric pressure. The resulting culture was treated with 0.25% trypsin / PBS (-) for 5 minutes, and centrifuged at 1000 rpm for 10 minutes to collect chondrocytes.
[0160]
A solution (hydrogel of the present invention) obtained by dissolving the hydrogel-forming polymer of the present invention (TGP-3) of Production Example 3 at a concentration of 10% by mass in a culture solution (RPMI-1640, manufactured by Life @ Technologies) was used. C. and the chondrocytes were cooled to 6 × 10⁵Cells / ml, and dispensed in a volume of 0.4 ml into each well of a 12-well plate [flat bottom multiwell tissue plate (FALCON, Becton Dickinson & Company)] and gelled at 37 ° C. After that, 0.4 ml of the culture solution was added and the mixture was added at 37 ° C. and 5% CO 2.₂Cultured under atmospheric pressure. In addition, as in Example 1, the culture solution RPMI-1640 contained 10% rat serum (a sample obtained by leaving rat blood at room temperature for 30 minutes and collecting the supernatant), 10 mM nicotinamide, and 10 ng / ml keratinocyte growth factor. (KGF) was added.
[0161]
Observation with a stereomicroscope daily revealed that the cells were divided and proliferated from day 5 of culture. The cell clumps increased in size over time, becoming 150 μm on day 14 and 300 μm on day 30. A histological image (Azan staining and Aggrecan immunostaining) on day 30 showed a stained image indicating the formation of a cartilage mass in the cell clumps.
[0162]
By cooling the hydrogel of the present invention containing the cell clumps to 4 ° C. and centrifuging, the cell clumps and the hydrogel of the present invention solubilized by the cooling can be easily separated. Was.
[0163]
Production Example 8
71.0 g of N-isopropylacrylamide and 4.4 g of n-butyl methacrylate were dissolved in 1117 g of ethanol. An aqueous solution in which 22.6 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 773 g of water was added thereto, and the mixture was heated to 70 ° C. in a nitrogen stream. While maintaining the temperature at 70 ° C. in a nitrogen stream, 0.8 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 8 mL of a 10% aqueous solution of ammonium persulfate (APS) were added, and the mixture was stirred and reacted for 30 minutes. Further, 0.8 mL of TEMED and 8 mL of 10% APS aqueous solution were added four times at 30-minute intervals to complete the polymerization reaction. The reaction solution was cooled to 10 ° C. or lower, diluted by adding 5 L of 10 ° C. cold distilled water, and concentrated to 2 L at 10 ° C. using an ultrafiltration membrane having a molecular weight cut off of 100,000.
[0164]
The concentrated solution was diluted by adding 4 L of cold distilled water, and the ultrafiltration and concentration operation was performed again. The above dilution and ultrafiltration / concentration operations were further repeated five times to remove those having a molecular weight of 100,000 or less. What was not filtered by the ultrafiltration (the one remaining in the ultrafiltration membrane) was recovered and freeze-dried to obtain 72 g of the hydrogel-forming polymer (TGP-5) of the present invention having a molecular weight of 100,000 or more. Obtained.
[0165]
1 g of the hydrogel-forming polymer (TGP-5) of the present invention obtained above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 20 ° C.
[0166]
Production Example 9
42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. To this was added an aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 65.1 g of water, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining the temperature at 70 ° C. under a nitrogen stream, 0.4 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 4 mL of a 10% aqueous solution of ammonium persulfate (APS) were added, and the mixture was stirred and reacted for 30 minutes. Further, 0.4 mL of TEMED and 4 mL of a 10% APS aqueous solution were added four times at 30-minute intervals to complete the polymerization reaction. The reaction solution was cooled to 5 ° C. or lower, diluted by adding 5 L of 5 ° C. cold distilled water, and concentrated to 2 L at 5 ° C. using an ultrafiltration membrane having a molecular weight cut off of 100,000.
[0167]
The concentrated solution was diluted by adding 4 L of cold distilled water, and the ultrafiltration and concentration operation was performed again. The above dilution and ultrafiltration / concentration operations were further repeated five times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (the one remaining in the ultrafiltration membrane) was collected and freeze-dried to obtain 40 g of the hydrogel-forming polymer (TGP-6) of the present invention having a molecular weight of 100,000 or more. Obtained.
[0168]
1 g of the hydrogel-forming polymer (TGP-6) of the present invention obtained as described above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 7 ° C.
[0169]
Production Example 10
45.5 g of N-isopropylacrylamide and 0.56 g of n-butyl methacrylate were dissolved in 592 g of ethanol. To this was added an aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 65.1 g of water, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining the temperature at 70 ° C. under a nitrogen stream, 0.4 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 4 mL of a 10% aqueous solution of ammonium persulfate (APS) were added, and the mixture was stirred and reacted for 30 minutes. Further, 0.4 mL of TEMED and 4 mL of a 10% APS aqueous solution were added four times at 30-minute intervals to complete the polymerization reaction. The reaction solution was cooled to 10 ° C. or lower, diluted by adding 5 L of 10 ° C. cold distilled water, and concentrated to 2 L at 10 ° C. using an ultrafiltration membrane having a molecular weight cut off of 100,000.
[0170]
The concentrated solution was diluted by adding 4 L of cold distilled water, and the ultrafiltration and concentration operation was performed again. The above dilution and ultrafiltration / concentration operations were further repeated five times to remove those having a molecular weight of 100,000 or less. Those not filtered by the ultrafiltration (residuals in the ultrafiltration membrane) are collected and freeze-dried to obtain 22 g of the hydrogel-forming polymer (TGP-7) of the present invention having a molecular weight of 100,000 or more. Obtained.
1 g of the hydrogel-forming polymer (TGP-7) of the present invention obtained as described above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 37 ° C.
[0171]
Example 3
The hydrogel-forming polymers TGP-5, TGP-6, and TGP-7 obtained in Production Examples 8, 9, and 10 were each dissolved in physiological saline to prepare a 10 wt% solution. When the respective sol-gel transition temperatures were measured, they were 18 ° C (TGP-5), 5 ° C (TGP-6), and 35 ° C (TGP-7). Each physiological saline solution was cooled to the sol-gel transition temperature or lower, and 20 mL / kg was administered intraperitoneally to 10 6-week-old rats (5 males and 5 females) per group.
[0172]
In the administration method, the abdomen of the rat was shaved with an electric clipper, the administration site was disinfected with ethanol for disinfection, and then administered using a syringe and an indwelling needle (22G). All groups showed the same weight gain as the control group in which physiological saline was administered intraperitoneally at 20 mL / kg, and no abnormal findings were observed during the 7-day observation period. Seven days after the administration, the animals were exsanguinated and killed under ether anesthesia, and all the organs and tissues were examined for abnormalities and for the remaining hydrogel (regeneration material of the present invention) in the abdominal cavity. No abnormalities were observed in any of the organs and tissues in any of the groups. For TGP-5 and TGP-6, hydrogel remained in the abdominal cavity. However, for TGP-7 having a high sol-gel transition temperature of 35 ° C., the hydrogel remained in the abdominal cavity 7 days after administration. I did not admit.
[0173]
Example 4
Hydrogel-forming polymers TGP-5 and TGP-6 obtained in Production Examples 8 and 9 were each dissolved in physiological saline to prepare a 10 wt% solution. Each saline solution was cooled below its sol-gel transition temperature and 1 mL was injected subcutaneously into the back of the rat. In the administration method, the back of the rat was shaved with an electric hair clipper, the administration site was disinfected with ethanol for disinfection, and then administered using a syringe and an indwelling needle (22G).
TGP-5 having a sol-gel transition temperature of 18 ° C. disappeared from the rat subcutaneous day on the 23rd day of administration, and no residual was visually observed. On the other hand, TGP-6 having a sol-gel transition temperature of 5 ° C. was necropsied on the 37th day after administration, and its residual was visually observed (FIG. 3). No abnormal findings were found.
[0174]
Example 5
Each of the hydrogel-forming polymers TGP-5 and TGP-6 obtained in Production Examples 8 and 9 was a physiological solution containing human basic fibroblast growth factor (FGF-b, Cosmo Bio Inc., 100-18B). It was dissolved in saline (FGF-b: 50 pg / mL) to prepare a 10 wt% solution. Each saline solution was cooled below its sol-gel transition temperature and 1 mL was injected subcutaneously into the back of the rat. In the administration method, the back of the rat was shaved with an electric hair clipper, the administration site was disinfected with ethanol for disinfection, and then administered using a syringe and an indwelling needle (22G).
TGP-5 having a sol-gel transition temperature of 18 ° C. disappeared from the rat subcutaneous day on the 23rd day of administration, and no residual was visually observed. On the other hand, when TGP-6 having a sol-gel transition temperature of 5 ° C. was necropsied on the 37th day after administration, the remaining hydrogel (the material for regeneration of the present invention) was visually observed (FIG. 5), and the HE-stained tissue image was obtained. (FIG. 6), formation of countless capillary networks was confirmed around the periphery. This is probably because the growth factor (FGF-b) was gradually released from the regenerated material of the present invention, and the regeneration of capillaries was promoted.
[0175]
Example 6
The hydrogel-forming polymer TGP-5 obtained in Production Example 8 was mixed with a phosphate buffer (1 / 15M, pH7) containing 3 mM phenol red (manufactured by Wako Pure Chemical), and methyl blue (manufactured by Wako Pure Chemical). Phosphate buffer (1/15 M, pH 7) containing 6 mM and phosphate buffer (1/15 M, pH 7) containing 0.9% myoglobin (manufactured by Wako Pure Chemical Industries, Ltd.) It melted under cooling. These solutions were filled in a glass tube having a length of 1.3 mm and an inner diameter of 6 mm, and heated to 37 ° C. to be gelled. These disc-shaped hydrogels were put together with a glass tube into a spectroscopic cell containing 3 mL of a phosphate buffer solution (1/15 M, pH 7), and the absorbance was continuously measured while maintaining the temperature at 37 ° C. and stirring. . The measurement wavelength of the absorbance was 558 nm, 571 nm, and 409 nm for phenol red, methyl blue, and myoglobin, respectively. The elution rate of phenol red, methyl blue, and myoglobin into 3 mL of a phosphate buffer (1/15 M, pH 7) over time was determined by absorbance measurement, and the respective diffusion coefficients were determined from the relationship of equation (1). , 1.6 × 10^-6(Cm²/ Sec), 7.5 × 10^-9(Cm²/ Sec), 2.1 × 10^-7(Cm²/ Sec).
[0176]
The ratio of the diffusion coefficient of phenol red as a water-soluble hydrophilic substance and methyl blue as a water-soluble hydrophobic substance in a TGP-5 hydrogel at 37 ° C. (D_PR/ D_MB) Was 213.
The ratio of the diffusion coefficient of phenol red, a water-soluble hydrophilic low-molecular substance, to myoglobin, a water-soluble hydrophilic high-molecular substance, at 37 ° C. in TGP-5 hydrogel (D_PR/ D_MG) Was 7.6.
[0177]
Comparative Example 1
Phosphorus containing 3 mM of low melting point agarose (Sea @ Prep (registered trademark) agarose, manufactured by BMA (Rockland @ USA), melting point: 50 ° C. or less, gel point: 8 ° C. to 17 ° C.) Phosphate buffer containing 1/1 mM buffer (1 / 15M, pH7), 6 mM methyl blue (manufactured by Wako Pure Chemical), phosphoric acid containing 0.9% myoglobin (manufactured by Wako Pure Chemical) Each of the buffer solutions (1 / 15M, pH7) was heated and dissolved at a concentration of 1 wt%. These solutions were filled in a glass tube having a length of 10 mm and an inner diameter of 2 mm, cooled to 2 ° C., and gelled. These columnar hydrogels were put together with a glass tube into a spectroscopic cell containing 3 mL of a phosphate buffer solution (1/15 M, pH 7), and the absorbance was continuously measured while keeping the temperature at 37 ° C. and stirring. . The measurement wavelength of the absorbance was 558 nm, 571 nm, and 409 nm for phenol red, methyl blue, and myoglobin, respectively. The elution rate of phenol red, methyl blue, and myoglobin into 3 mL of a phosphate buffer (1/15 M, pH 7) over time was determined by absorbance measurement, and the respective diffusion coefficients were determined from the relationship of equation (1). , 4.7 × 10^-6(Cm²/ Sec), 7.1 × 10^-6(Cm²/ Sec), 2.6 × 10^-5(Cm²/ Sec).
The ratio of the diffusion coefficient of phenol red (water-soluble hydrophilic substance) to methyl blue (water-soluble hydrophobic substance) at 37 ° C. in a low melting point agarose hydrogel (D_PR/ D_MB) Was 0.7.
[0178]
The ratio of the diffusion coefficient of phenol red, a water-soluble hydrophilic low-molecular substance, to myoglobin, a water-soluble hydrophilic high-molecular substance, at 37 ° C. in a low melting point agarose hydrogel (D_PR/ D_MG) Was 0.18.
[0179]
Example 7
The hydrogel-forming polymer TGP-5 obtained in Production Example 8 was dissolved in physiological saline, and the polymer concentration was 10% by mass (wt%), 8% by mass (wt%), and 6% by mass (wt%). Was prepared. The respective sol-gel transition temperatures were measured and found to be 18 ° C, 20 ° C, and 22 ° C, respectively. Each physiological saline solution was cooled below its sol-gel transition temperature and administered intraperitoneally to 10 6-week-old rats (5 males and 5 females) in groups of 1 mL / kg. In the administration method, the abdomen of the rat was dehaired with an electric clipper, the administration site was disinfected with ethanol for disinfection, and then administered using a syringe and an indwelling needle (22G). On the 1st, 3rd, 7th, 14th, and 21st days after the administration, two animals (one male and one female) in each group were exsanguinated and killed under ether anesthesia. The remaining material (the material for reproduction of the present invention) was inspected. The hydrogel having a polymer concentration of 6% by mass (wt%) disappeared from the intraperitoneal cavity on day 3 after administration, and the hydrogel having a polymer concentration of 8% by mass (wt%) disappeared from the intraperitoneal cavity on day 14 after administration. The hydrogel having a polymer concentration of 10% by mass (wt%) disappeared from the intraperitoneal cavity 21 days after administration.
[0180]
【The invention's effect】
As described above, when the hydrogel of the present invention is used, retention / diffusion of various useful substances can be suitably controlled.
[0181]
When such a hydrogel is used, for example, as a carrier for cell / tissue culture, fibroblasts do not substantially proliferate, and therefore the target cells / tissues (eg, organs intended for repair / regeneration etc.) And cells of the tissue) can be selectively proliferated, and the regeneration of the target organ or tissue can be effectively achieved.
[0182]
The hydrogel of the present invention gels at a predetermined temperature (for example, human or animal body temperature) to form a three-dimensional network structure, and can retain cell growth factors and the like for a long period of time. Can be promoted.
[0183]
Since the hydrogel of the present invention is composed of a hydrogel-forming polymer that gels at body temperature in a sol state at a low temperature, the hydrogel of the present invention contains stem cells, progenitor cells, or the like in a low-temperature sol state. Tissues and the like can be seeded and mixed, and the carrier is turned into a gel state by gelling at a predetermined temperature (for example, the body temperature of a human or animal) as it is to adhere stem cells and progenitor cells that regenerate tissues and organs. It can function as an artificial carrier for differentiation and morphogenesis.
[0184]
Furthermore, since the hydrogel of the present invention returns to the sol state by cooling to a temperature lower than the sol-gel transition temperature, it can be recovered without substantially damaging tissues and organs regenerated in the gel.
[Brief description of the drawings]
FIG. 1 is a photomicrograph showing clumps in which cells were divided and proliferated from the outside of a pancreatic duct tissue in Example 1 (day 5, magnification × 100).
FIG. 2 is a micrograph showing the cell mass that has increased in size over time in Example 1 (30 days, magnification × 100).
FIG. 3 is a photograph obtained in Example 4 showing the residual TGP-6 with a sol-gel transition temperature of 5 ° C. administered to the back of a rat.
FIG. 4 is a HE (hematoxylin-eosin) stained tissue image (magnification: 100 ×) obtained in Example 4 showing around TGP-6 having a sol-gel transition temperature of 5 ° C. administered to the back of a rat. is there.
FIG. 5 is a photograph obtained in Example 5 showing the remaining TGP-6 having a sol-gel transition temperature of 5 ° C. administered to the back of a rat.
FIG. 6 is an HE-stained tissue image (magnification: 100 ×) obtained in Example 5, showing around TGP-6 at a sol-gel transition temperature of 5 ° C. administered to the back of a rat.

Claims (10)

A hydrogel containing at least a hydrogel-forming polymer exhibiting a thermoreversible sol-gel transition in which the aqueous solution becomes a sol state at a low temperature and a gel state at a high temperature;
The ratio of the diffusion coefficient D of phenol red (PR), methyl blue (MB), and myoglobin (MG) satisfies (D _PR / D _MB ) ≧ 2 and (D _PR / D _MG ) ≧ 1.2. Characterized hydrogel. The hydrogel according to claim 1, wherein the hydrogel-forming polymer is a polymer in which a plurality of blocks having a cloud point and a hydrophilic block are bonded. The hydrogel according to claim 1, wherein the sol-gel transition temperature is higher than 0 ° C. and 42 ° C. or less. The hydrogel according to claim 1, further comprising a chemical mediator. The hydrogel according to any one of claims 1 to 4, wherein the aqueous solution of the hydrogel-forming polymer exhibits substantially water insolubility in a high temperature gel state. The hydrogel according to claim 1, further comprising water. A gel body comprising at least a useful substance and a hydrogel disposed around the useful substance;
The hydrogel contains at least a hydrogel-forming polymer exhibiting a thermoreversible sol-gel transition in which the aqueous solution becomes a sol state at a low temperature and a gel state at a high temperature, and
The ratio of the diffusion coefficients of phenol red (PR), methyl blue (MB), and myoglobin (MG) in the hydrogel is (D _PR / D _MB ) ≧ 2 and (D _PR / D _MG ) ≧ 1.2. A gel body characterized by filling. The gel body according to claim 7, wherein the useful substance is at least one substance selected from the group consisting of a physiologically active substance, a coloring substance, cosmetics, and a biological material. A method for controlling retention / diffusion of a useful substance by using a gel body containing at least a useful substance and a hydrogel disposed around the useful substance,
The hydrogel contains at least a hydrogel-forming polymer exhibiting a thermoreversible sol-gel transition in which the aqueous solution becomes a sol state at a low temperature and a gel state at a high temperature, and
The ratio of the diffusion coefficients of phenol red (PR), methyl blue (MB), and myoglobin (MG) in the hydrogel is (D _PR / D _MB ) ≧ 2 and (D _PR / D _MG ) ≧ 1.2. A control method characterized by satisfying. The control method according to claim 9, wherein the useful substance is recovered by setting the temperature of the hydrogel to a sol state by setting the temperature lower than the sol-gel transition temperature.

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