JP4342338B2 - Three-dimensional porous structure and manufacturing method thereof - Google Patents

Description

  The invention of this application relates to a polymer three-dimensional porous structure and a method for producing the same.

  Materials characterized by regularly arranged microstructures are semiconductor low dielectric constant materials, scattering layers for electronic displays, magnetic recording materials, photonic crystals, catalyst carriers, DNA chips, protein chips, micromachines, microchannels, chemicals It is a promising material that is being studied for various uses such as valves and cell culture substrates. In particular, in the promotion of regenerative medicine, which is attracting attention as the next-generation medical technology, it is necessary to provide a three-dimensional scaffold using a fine structure for appropriate cell growth. The balance of cells, growth factors, and scaffolds is important for the proper induction of biological organization, but the extracellular matrix, which is the scaffold, plays a role as a field for cell proliferation and differentiation, and for communication between cells. It is known that It is known that the scaffold base material affects not only the surface chemical properties but also the behavior such as cell adhesion, division, proliferation, migration and differentiation not only by its fine structure. Furthermore, it is considered that a three-dimensional scaffold is necessary to promote three-dimensional biological organization (Non-Patent Document 1-2).

However, up to now, various microstructural processing techniques on the surface of the substrate have been studied and developed, but none of them has realized a high-performance three-dimensional structure. On the other hand, as a method for producing an industrially useful two-dimensional structure, a method for producing a regular mesoscopic pattern structure such as dots, lines, and honeycombs in a polymer has been proposed (Patent Document 1). This method is an inexpensive and simple method using moisture condensation on a cast membrane of a hydrophobic organic solvent solution of polymer, but the evaporation of the organic solvent and moisture condensation in the atmosphere proceed simultaneously in a non-equilibrium manner. The body is limited to a single layer, that is, a two-dimensional pattern structure, and production of a three-dimensional microstructure using a polymer has not been realized so far.
State-of-the-art of regenerative medical engineering, Yoshito Seki, CMC Publishing, 2002 RP Lanza, R. Langer, J. Vacanti, eds, Principle of Tissue Engineering, Academic Press, 2000 JP 2001-157574 A

  An object of the invention of this application is to provide a three-dimensional porous structure exhibiting a uniform structure pattern having various utilities from the above background.

  The inventors of this application eagerly studied to realize a three-dimensional structure on the basis of the two-dimensional microstructure manufacturing technology that has already been proposed, and obtained the following findings. Based on this, the three-dimensional structure of the invention of this application and its manufacturing method have been completed.

  That is, it is considered that the structure already proposed by the inventor causes condensation on the surface of the polymer solution, and the condensed water droplets are closely packed, so that the polymer is deposited in a space other than the water droplets. In addition, it is considered that the condensed water droplets are two-dimensionally arranged on the surface of the polymer solution, that is, become a pore template, and a structure exhibiting a uniform fine pore pattern is produced. Therefore, the inventors of this application think that a three-dimensional microstructure exhibiting a uniform structure pattern can be realized if the condensed water droplets serving as pore templates can be three-dimensionally arranged in an organic solvent. First, a layer of condensed water droplets generated on the surface of the polymer solution was submerged in the polymer solution, and condensed water droplets were generated again on the surface of the polymer solution. By repeating this, it was possible to produce a three-dimensional microstructure.

From the above, according to the invention of the present application, first, an organic solvent solution of a polymer is cast on the surface of the substrate, and the organic solvent is evaporated, and moisture in the atmosphere is condensed at the same time. Provided is a method for producing a three-dimensional porous structure of a polymer, characterized in that the water droplets are evaporated after being arranged in multiple dimensions. And second, polymer for making this structure will provide a production method which comprises one or more biodegradable polymers least.

Third , the three-dimensional arrangement of condensed water droplets is performed by blowing high-humidity air, and fourth , the high-humidity air blowing is intermittently performed. Provide a method.

Still the invention of this application, the fifth, the a porous structure is any polymer 3D having a manufactured honeycomb structured by the manufacturing method, less of a honeycomb having a pore size or thickness Both of them provide a structure characterized by having a range of 0.01-100 μm, and sixth , a structure characterized in that the honeycomb structure has a multilayer structure of 2-20 I will provide a.

And seventh, there is also provided a cell culture scaffold comprising any one of the above structures as at least a part of the structure.

  The invention of this application provides a novel fine porous structure and a method for producing the same. As a result, in addition to cell culture substrates, various biodiagnostic chips such as DNA chips and protein chips, battery membrane materials such as separators and ion exchange membranes, catalyst carriers, optical materials such as displays and optical waveguides, anisotropic materials In addition to conductive solid conductive materials, semiconductor materials, and various recording media, useful carriers such as various catalysts, enzymes, antibodies, and drugs are provided.

  An essential feature of the invention of this application is that water droplets, which serve as a template for pores of the porous structure, are closely packed in a polymer solution in three dimensions. That is, condensation is generated by evaporation of the organic solvent solution of the polymer cast on the substrate surface, and the fine water droplet particles are formed by the interaction due to the difference in polarity between the generated water droplet and the polymer solution, and the sedimentation operation of the water droplet into the solution. This is a technique for close packing in a polymer solution. The three-dimensional porous structure of this application is preferably a structure having a honeycomb structure, which is considered to be due to the hexagonal close-packing of water droplets as a mold. In addition, water droplets are not necessarily high-purity water, and the physical properties of the water droplets are slightly changed, such as absorbing gas molecules after dew condensation, so that the structure to be produced takes the form of closest packing that does not become a honeycomb structure. There may be.

  In the invention of this application, the “substrate” is a portion that is a basis for manufacturing a structure, and is not included in the structure.

  Furthermore, in the invention of this application, moisture essentially implied for the terms “humidity” and “condensation”, and the terms “moisture” and “water droplets” refer to a liquid mainly composed of water. However, it is not necessarily limited to 100% pure water.

  In the invention of this application, “air” may be a relatively inert gas, and is not necessarily limited to a standard composition of air.

  In the embodiment of the invention shown below, water is used as the water droplets to be condensed, and the hydrophobic organic solvent solution of the mixed polymer of the hydrophobic polymer and the amphiphilic polymer is exemplified as the polymer solution. Any combination that can achieve the closest packing of fine water droplets by the interaction between the target water droplet and the polymer solution, that is, the difference in polarity between the water droplet and the polymer solution, is limited to the exemplified combinations. is not.

  Hydrophobic polymers used in the production of the structure of the invention of this application are general-purpose polymers such as polystyrene, polymethylhexadecylsiloxane, and polyvinylcarbazole, engineering plastics such as polysulfone, polymethyl methacrylate, polytetrahydrofurfuryl methacrylate, and polybutyl methacrylate. (Meth) acrylic polymers such as styrene-butadiene elastomers, polyurethane polymers, aliphatic polycarbonates such as polybutylene carbonate and polyethylene carbonate, polylactic acid, lactic acid-glycolic acid copolymers, polyhydroxybutyric acid, polycaprolactone, polyethylene Biodegradable polymers such as enadipate and polybutylene adipate are preferred from the viewpoint of solubility in organic solvents. These may be used alone or in combination.

  The amphiphilic polymer used for producing the structure of the invention of this application has a polyethylene glycol / polypropylene glycol block copolymer or acrylamide polymer as a main chain skeleton, a dodecyl group as a hydrophobic side chain, a lactose group as a hydrophilic side chain, or Amphiphilic polymer having carboxyl group, amphiphilic polymer by ion complex of anionic polymer such as heparin, dextran sulfate, DNA or RNA and long chain alkyl ammonium salt, or water-soluble such as gelatin, collagen, albumin and chitosan An amphiphilic polymer having a protein as a hydrophilic group can be used.

  Since the organic solvent for preparing the polymer solution used for the production of the structure of the invention of this application is required to form fine water droplet particles on the surface of the polymer solution, the organic solvent needs to have hydrophobicity. is there. Examples of the organic solvent include halogen-based organic solvents such as chloroform, dichloromethane and dichloroethane, aromatic hydrocarbons such as benzene, toluene and xylene, esters such as ethyl acetate and butyl acetate, and water-insoluble ketones such as methyl isobutyl ketone. For example, carbon disulfide can be used. These organic solvents may be used individually by 1 type, and the mixed solvent which combined multiple may be used.

  The polymer concentration in preparing the polymer solution used for the production of the structure of the invention of this application can be 0.01-20 w%, preferably 0.1-20 w%, combining the hydrophobic polymer and the amphiphilic polymer. -10 w%. If the polymer concentration is less than 0.01 w%, the mechanical strength of the structure is insufficient, and if it is 20 w% or more, a sufficient three-dimensional structure cannot be obtained. Furthermore, the weight composition ratio of the hydrophobic polymer to the amphiphilic polymer is preferably between 99: 1 and 50:50. When the weight ratio of the amphiphilic polymer to the total polymer is less than 1%, it is difficult to obtain a uniform porous structure, and when it exceeds 50%, the stability of the structure, particularly mechanical stability, is affected.

  The three-dimensional arrangement operation of the condensed water droplets in the production of the structure of the invention of this application is performed by a method in which a solvent having a specific gravity smaller than that of water is selected and the water droplets condensed on the polymer solution surface are submerged in the solution. Can do. That is, this is a method in which the supply of moisture from the atmosphere is temporarily interrupted during the formation of condensation, and the condensation water droplets are submerged into the solution due to the specific gravity difference between the water droplets and the surrounding solution. Or you may use the method of sinking a water droplet by spraying gas on the surface of a dew condensation solution. By spraying, the condensed water droplets sink and the solution surface is exposed, and even if it does not completely sink, new condensation can be generated on the solution surface newly generated in the gap between the condensed water droplets. In the method by gas blowing, it is preferable to use high-humidity air, but any gas having a humidity sufficient to cause condensation on the polymer solution surface may be used. An active gas may be used.

  Substrates used for the production of the structure of the invention of this application include inorganic materials such as glass, metal, ITO substrate and silicon wafer, and high resistance to organic solvents such as polypropylene, polyethylene, polyetherketone and fluororesin. Liquids such as molecules, water, liquid paraffin and liquid polyethers can be used.

  The three-dimensional porous structure provided by the invention of this application may be produced on a substrate and used as it is together with the substrate, or may be peeled off to use only the structure. Since the film-like three-dimensional structure produced on the substrate is structurally stable and self-supporting, it can be easily peeled off from the substrate and used as a self-supporting film. In addition, it can be used after being subjected to various processing according to conventional techniques, and its usage and application are not particularly limited. For example, it is possible to extend the structure and use an extended pore array structure. The stretching method is also not particularly limited, and stretching using tweezers or manual work or stretching using a micromanipulator may be used. Furthermore, it is possible to produce a fine protrusion structure on the surface. The modification method of the fine protrusion structure is not particularly limited, and the fine protrusion structure on the modification surface can be both on the structure surface (non-substrate side) and on the substrate side. As a structure having a fine protrusion structure, a method of producing a structure using a substrate on which a groove serving as a mold of the fine protrusion structure is carved and using it as a film by peeling off the substrate, or a physical peeling operation using an adhesive tape, etc. It is possible to use a structure or the like that has been subjected to surface modification.

  In the invention of this application, the following may be considered as a mechanism for forming a porous structure. That is, the temperature of the surface of the polymer solution is lowered by the latent heat of evaporation of the organic solvent contained in the cast polymer solution, and moisture in the atmosphere aggregates on the surface of the polymer solution, so-called condensation occurs. The surface tension of the condensed water droplets decreases due to the interaction between the condensed water droplets and the hydrophilic group of the amphiphilic polymer contained in the polymer solution. As a result, the condensed water droplets repeatedly associate to form large droplets. Without stabilization, the fine particles are stabilized. As the solvent evaporates, water droplets condensed in hexagonal form are arranged in a close-packed form, and when these water droplets are evaporated, a honeycomb-like three-dimensional porous structure in which polymers are regularly arranged is obtained. The structure of the invention of this application is preferably produced in an atmosphere with a relative humidity of 50-95%. When the relative humidity is 50% or less, sufficient condensation cannot be obtained, and when it is 95% or more, it is difficult to control condensation generation.

  The pore size and layer thickness of the three-dimensional porous structure provided by the invention of this application change the evaporation rate of the solvent, the rate of condensation formation, and the interaction of the condensation water droplets with the amphiphilic polymer contained in the polymer solution. Can be controlled. That is, the pore size and layer thickness can be controlled by various conditions such as the concentration of the polymer solution, the type of solvent, the type and composition ratio of the polymer, and the cast thickness to the substrate. Also, depending on the relative humidity, temperature, spraying method (for example, continuous, intermittent, wind pressure change, etc., change in spraying flow pattern) and atmospheric pressure of the atmosphere when three-dimensionally arranging the atmosphere and condensed water droplets in multiple layers In addition, it is possible to control the growth of condensed water droplets that determine the porous structure, and thus control the pore diameter and layer thickness. In the three-dimensional porous structure provided by the invention of this application, at least one of the pore diameter or the layer thickness of the honeycomb of each layer is preferably 0.01 to 100 μm.

  Hereinafter, although the details of the invention of this application are specifically shown using examples, the invention of this application is not limited to these examples.

  A chloroform solution in which polystyrene (Aldrich) having a weight average molecular weight of 200,000 and dodecylacrylamide-ω-carboxyhexylacrylamide (compound 1: Cap) represented by the following formula, which is an amphiphilic acrylamide, are mixed at a weight ratio of 10: 1 ( A total polymer concentration of 4 mg / l) was prepared. 7 ml (about 10.5 g) of this polymer solution was cast on a glass petri dish having a diameter of 10 cm, high-humidity air having a relative humidity of 50% was blown at a flow rate of 3 l / min, and evaporation of the chloroform solvent was started. After 240 seconds (solution remaining amount: 3.5 g), high-humidity air blowing was stopped, and the glass petri dish was covered to stop the evaporation of chloroform. After maintaining this state for 60 seconds, high-humidity air with a relative humidity of 70% was blown at a flow rate of 3 l / min to evaporate chloroform. Finally, water was sufficiently evaporated to produce a three-dimensional porous structure. The outline of the above method is shown in FIG. The result of electron microscope observation of the surface of the obtained three-dimensional porous structure is shown in FIG.

  A three-dimensional porous structure was produced in the same manner as in Example 1 using polymethyl methacrylate (Aldrich) having a weight average molecular weight of 350,000 instead of the polystyrene in Example 1. FIG. 3 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  A three-dimensional porous structure was produced in the same manner as in Example 1 using polycarbonate having a weight average molecular weight of 29,000 instead of the polystyrene in Example 1. FIG. 4 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  A three-dimensional porous structure was produced in the same manner as in Example 1 using polytetrahydrofurfuryl methacrylate (Scientific Polymer Products) having a weight average molecular weight of 240,000 instead of the polystyrene in Example 1. FIG. 5 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  A three-dimensional porous structure was produced in the same manner as in Example 1, except that poly (ε-caprolactone) (Wako Pure Chemical Industries) having a viscosity average molecular weight of 40,000 was used in place of the polystyrene in Example 1. FIG. 6 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  In place of the polystyrene of Example 1, a poly (glycolic acid-lactic acid) copolymer (composition ratio 50:50) (Aldrich) having a weight average molecular weight of 40,000 to 75,000 was used. A three-dimensional porous structure was produced by the operation. FIG. 7 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  A three-dimensional porous structure was produced in the same manner as in Example 1, except that polysulfone having a weight average molecular weight of 80,000 was used in place of the polystyrene in Example 1. FIG. 8 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  Only the evaporation time after the start of evaporation of the chloroform solvent in Example 4 was changed. Evaporation was stopped when the remaining amount of the solution reached 5.0 g, 4.0 g, 3.5 g, and 3.0 g, respectively, and a three-dimensional honeycomb structure was produced in the same manner as in Example 4. A three-dimensional porous structure was obtained except when the chloroform solution remaining amount was 5.0 g. FIG. 9 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  It is an electron microscopic image of the surface of a structure obtained when the remaining amount of the solution is 5.0 g (a), 4.0 g (b), 3.5 g (c), and 3.0 g (d), respectively.

  The retention time for stopping the evaporation of the chloroform solvent in Example 8 was changed. When the remaining amount of the chloroform solution at the time of stopping the evaporation in Example 8 was 3.0 g, the state where the evaporation of the chloroform was stopped by covering the glass petri dish was held for 0 seconds, 30 seconds, 60 seconds, and 120 seconds, respectively. After that, a three-dimensional porous structure was produced in the same manner as in Example 8. A three-dimensional porous structure was obtained except when the holding time was 0 second. FIG. 10 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  It is the electron microscope image of the structure surface obtained by holding time 0 second (a), 30 second (b), 60 second (c), and 120 second (d), respectively.

  The three-dimensional porous structure obtained when the residual amount of the chloroform solution at the time of stopping evaporation in Example 8 was 3.0 g, and then peeled off after applying the adhesive tape to the structure film surface to produce a fine protrusion structure did. As a result of observation from an oblique direction with an electric microscope, fine protrusion structures were produced on both the peeling tape side and the substrate glass petri dish side. FIG. 11 shows the result of electron microscope observation of the surface of the obtained three-dimensional porous structure.

  Electron microscopic images of the surface of the fine protrusion structure on both the peeling tape side (c) and the substrate glass side (d), and the structure before the peeling treatment (a and b, a is directly above, b is an observation from an angle of 55 degrees Image).

It is the figure which showed the outline about the manufacturing method of the three-dimensional structure of invention of this application. 2 is an electron microscopic image of the surface of a three-dimensional porous structure obtained from a mixed polymer solution of polystyrene and dodecylacrylamide-ω-carboxyhexylacrylamide (Cap) in Example 1. FIG. It is an electron microscope image of the surface of the three-dimensional porous structure obtained by Example 2 (polymethylmethacrylate and Cap). It is an electron microscope image of the surface of the three-dimensional porous structure obtained by Example 3 (polycarbonate and Cap). It is an electron microscopic image of the surface of the three-dimensional porous structure obtained by Example 4 (polytetrahydrofurfuryl methacrylate and Cap). It is an electron microscope image of the surface of the three-dimensional porous structure obtained by Example 5 (poly ((epsilon) -caprolactone) and Cap). It is an electron microscope image of the surface of the three-dimensional porous structure obtained by Example 6 (poly (glycolic acid-lactic acid) copolymer and Cap). It is an electron microscopic image of the surface of the three-dimensional porous structure obtained by Example 7 (polysulfone and Cap). It is an electron microscope image at the time of changing the chloroform solution residual amount shown in Example 8. FIG. It is an electron microscope image at the time of changing the retention time of the chloroform evaporation stop shown in Example 9. FIG. It is an electron microscope image at the time of producing by adhesive tape peeling.

Claims (7)

It is characterized by casting an organic solvent solution of a polymer on the substrate surface, evaporating the organic solvent, condensing moisture in the atmosphere, and further arranging the condensed water droplets in three dimensions and then evaporating the water droplets. A method for producing a three-dimensional porous structure of a polymer. The production method according to claim 1, wherein the polymer contains at least one biodegradable polymer. 3. The manufacturing method according to claim 1, wherein the three-dimensional arrangement of condensed water droplets is performed by blowing high-humidity air. The manufacturing method according to claim 3, wherein the high-humidity air is intermittently sprayed. A porous structure which is a three-dimensional polymer body having a honeycomb structure manufactured by the manufacturing method according to any one of claims 1 to 4, wherein at least one of the pore diameter or the layer thickness of the honeycomb is 0.01 to 100 µm. A three-dimensional porous structure characterized by being in a range. 6. The three-dimensional porous structure according to claim 5 , wherein the honeycomb structure has a multilayer structure of 2-20. A cell culture scaffold comprising the three-dimensional porous structure according to claim 5 or 6 as at least a part of the constitution.