JP2009279337A - Hard tissue regeneration device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new hard tissue regeneration device using osteoblast. <P>SOLUTION: The hard tissue regeneration device is a complex of an osteoblast and a honeycomb porous body having through-holes with 1 μm-14 μm pore diameter comprising calcium phosphate produced by the osteoblast and a non-water-soluble polymer. The device is used for a joint treatment and a hard tissue regeneration treatment with respect to the disease of a bone and cartilage, and urges the excellent growth of the osteoblast and bone regeneration by allowing the honeycomb porous body with excellent biocompatibiliy to be a basic skeleton. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a composite of osteoblasts, calcium phosphate produced by osteoblasts and a honeycomb-shaped porous body having specific micropores, and a method for producing the same, useful as a device for regenerating hard tissue. More specifically, the present invention relates to a hard tissue that is a composite of osteoblasts, calcium phosphate gel produced by osteoblasts, and a honeycomb-like porous body having through holes with a pore diameter of 1 μm to 14 μm made of a water-insoluble polymer. The present invention relates to a playback device and a manufacturing method thereof.

  With the progress of an aging society, hard tissue diseases such as bone, cartilage, and teeth represented by osteoporosis and joint diseases, especially bone tissue diseases are increasing. These diseases have become serious social problems because they significantly damage human QOL in addition to the increase in geriatric medical expenses.

  As one of the treatment methods for hard tissue diseases, particularly bone tissue diseases, a tissue engineering method for repairing a bone defect has attracted attention. In this method, a hard tissue, typically bone, formed using osteoblasts is transplanted into a diseased site, and the hard tissue itself is regenerated in vivo.

  In order to grow osteoblasts and regenerate hard tissue, a culture substrate on which osteoblasts can attach and grow on them, a so-called scaffold, is required. A typical osteoblast proliferation scaffold is a carrier for cell culture made of hydroxyapatite (calcium phosphate gel), but in order to promote efficient regeneration of hard tissue by osteoblasts, various improvements to the carrier, Ingenuity has been made.

  As such an example, an osteoblast culture support made of hydroxyapatite adsorbed with a polysaccharide (Patent Document 1) and a diameter of 100 to 500 μm formed by engagement of a fibrous calcium phosphate compound A porous calcium phosphate compound sheet (Patent Document 2) containing a pore continuum composed of pores and having an overall porosity in the range of 70 to 99%, a molding composed of calcium phosphate having a particle size of 300 to 1000 μm Examples thereof include a cell culture support (Patent Document 3) characterized in that pores having a diameter of 100 to 300 μm are formed with a porosity of 50 to 80%.

  It is said that a scaffold using such hydroxyapatite is generally a culture substrate (matrix) in which pores having a diameter of 100 μm or more are formed (see, for example, Patent Document 2). However, in order to form pores having a desired size, it is necessary to control the crystal growth of hydroxyapatite. However, it is generally not easy to control the crystal growth of an inorganic substance.

  On the other hand, several scaffolds using chemical substances other than hydroxyapatite have been proposed. For example, a porous carrier formed in a three-dimensional shape using a protein such as collagen or a saccharide such as hyaluronic acid, and the porous carrier is formed so as to surround the porous carrier and communicates with the outside. And a tissue regeneration base material (Patent Document 4) provided with a support formed of a polymer such as polylactic acid that is supported in the above state. And having a substantially continuous polymer phase having a highly interconnected bimodal distribution consisting of circular open pores of about 50 to about 500 μm and circular open pores of less than 20 μm, wherein the pores are There has also been proposed a biocompatible porous scaffold (Patent Document 5) that is regularly and linearly arranged in the walls of large pores.

  Such a base material or scaffold has a complicated structure such as a porous body and a support, or pores having different diameters regularly arranged, and its production requires a complicated operation.

The inventors of the present invention have referred to a thin film (hereinafter referred to as a honeycomb-shaped porous body) called a honeycomb structure, a honeycomb-patterned film, or a honeycomb-shaped porous body, in which a large number of fine pores made of a water-insoluble polymer are regularly arranged. Have been reported to be a good scaffold in in vitro culture of hepatocytes, cardiomyocytes, chondrocytes, and epithelial cells (eg, Patent Document 6, Patent Document 7, Patent Document 8, etc.) . On the other hand, with respect to tumor cells, it has been confirmed that the honeycomb-like porous body suppresses the proliferation thereof (for example, Patent Document 9).
JP-A-8-149976 JP 2003-93052 A JP-A-6-343456 International Patent Application Publication WO2003 / 011353 Pamphlet JP-T-2002-541925 JP 2002-334959 A JP 2002-152005 A JP 2007-6987 A JP 2005-52526 A

  It is an object of the present invention to provide a new hard tissue regeneration device using osteoblasts.

  In the present invention, instead of the culture substrate having pores of several tens to several hundreds of μm that have been reported so far, a honeycomb-like porous body having a minute pore size of about 1 μm to several tens of μm is used as osteoblasts. Can be used as a scaffold for, and by growing osteoblasts on the honeycomb porous body and generating calcium phosphate crystals, the honeycomb porous body and the osteoblast and calcium phosphate can be used as a skeleton. The inventors found that a composite with a honeycomb porous body was formed, and completed the following inventions.

(1) A device for regenerating a hard tissue, which is a composite of osteoblasts, calcium phosphate produced by osteoblasts, and a honeycomb-like porous body having a through-hole having a pore diameter of less than 15 μm made of a water-insoluble polymer.

(2) The hard tissue regeneration device according to (1), wherein the honeycomb porous body has a pore size of 1 μm to 14 μm.

(3) The hard tissue regeneration device according to (1) or (2), wherein the through-holes of the honeycomb-like porous body communicate with surrounding through-holes existing in the planar direction.

(4) The hard tissue regeneration device according to any one of (1) to (3), wherein calcium phosphate produced by osteoblasts enters at least part of the through-holes of the honeycomb-shaped porous body.

(5) The hard tissue regeneration device according to any one of (1) to (4), wherein the osteoblast is an osteoblast derived from a mammal that requires regeneration of hard tissue.

(6) an osteoblast comprising a step of inoculating osteoblasts into a honeycomb porous body having a through-hole having a pore diameter of less than 15 μm, and a step b) of culturing the osteoblasts to produce calcium phosphate; A method for producing a composite of calcium phosphate produced by osteoblasts and a honeycomb-shaped porous body having a through-hole having a pore diameter of less than 15 μm and comprising a water-insoluble polymer.

(7) The manufacturing method according to (6), wherein the honeycomb porous body has a pore size of 1 μm to 14 μm.

  The device for regenerating a hard tissue according to the present invention can be used for a hard tissue regeneration treatment for joint treatment and bone / cartilage disease, and a bone-like porous body having excellent biocompatibility as a basic skeleton, Good cell proliferation and bone regeneration can be promoted.

  The present invention relates to a device for regenerating hard tissue, which is a composite of osteoblasts, calcium phosphate produced by osteoblasts, and a honeycomb-like porous body having a through-hole having a pore diameter of less than 15 μm made of a water-insoluble polymer. A device for regenerating hard tissue is used for regenerative treatment of hard tissue diseases such as bone, cartilage, and teeth typified by osteoporosis and joint diseases. It can be understood as a graft that can promote tissue regeneration safely and without side effects.

  As the osteoblast, either an osteoblast directly collected from bone tissue in a living body or an osteoblast derived by differentiation from a bone marrow stem cell collected from bone marrow under appropriate conditions may be used. For example, Bellows et al. (Calcifi Tissue Int., 1986, 38, 143-154), Harris et al. (Prostate, 1994, Vol. 24, 204-211, for example) P.) Or Bokhari et al. (Biomaterials, 2005, Vol. 26, No. 25, p. 5198-5208) and can be collected and used by methods known to those skilled in the art. As osteoblasts, for example, osteoblasts induced to differentiate from bone marrow stem cells based on the method of Bokhara et al. (Biomaterials, 2005, Vol. 26, No. 25, pages 5198-5208) may be used. . Since the complex according to the present invention can be used as a device for regenerating hard tissue, osteoblasts are derived from osteoblasts derived from patients undergoing regenerative treatment of hard tissues (from bone marrow stem cells collected from the bone marrow of patients under appropriate conditions). (Including osteoblasts induced to differentiate below).

  The calcium phosphate constituting the present invention is produced by the osteoblast itself as it grows. Therefore, the present invention is different from the technique of forming the scaffold itself using calcium phosphate or hydroxyapatite as in the conventional techniques described in Patent Documents 1 to 3. The composite according to the present invention is preferably a composite in which at least a part of the polymer surface of the honeycomb-like porous body constituting the composite is covered with calcium phosphate, and at least one of the through holes of the honeycomb-like porous body. A complex in which calcium phosphate is contained in the part is preferable (see FIG. 4).

  The honeycomb-like porous body made of a water-insoluble polymer constituting the composite of the present invention is a porous thin film made of a polymer (polymer), and minute pores oriented in the vertical direction of the film are films. Is provided in a honeycomb shape (honeycomb shape). A porous thin film in which pores are provided in such a regular arrangement such as a honeycomb shape is completely different from a normal porous body having irregular pores in which the pore diameter, shape, or depth of the pores vary. Understood as a different structure.

  The pores of the honeycomb-like porous body constituting the composite of the present invention are desirably holes (through holes) penetrating the membrane in the vertical direction. This through hole is preferably in communication with a surrounding through hole existing in the plane direction of the thin film and inside the film. Although it is inferred, the through-holes in the honeycomb-shaped porous body and the communication structure between the through-holes exert an interaction between osteoblasts growing on the honeycomb-shaped porous body, or supply of culture medium and dissolved oxygen It is considered that the waste products are discharged smoothly.

  As the honeycomb-like porous body constituting the composite of the present invention, a honeycomb-like porous body having a through-hole having a pore diameter of less than 15 μm, preferably a through-hole having a pore diameter of 1 μm to 14 μm is used. The reason is unknown, but the proliferation of osteoblasts and the expression of bone regeneration markers when the honeycomb porous body having a through-hole having a pore diameter of 15 μm or more is used as a scaffold is a flat membrane having no through-hole. Almost no difference is observed from the above (hereinafter referred to as flat film). Therefore, when using the honeycomb-like porous body as a scaffold for osteoblast culture, it is considered that the pore diameter needs to be at least less than 15 μm, preferably in the range of 1 to 14 μm. In addition, although it is reasoning, the above-mentioned specific pore size is a pore size of several tens of μm to several hundreds of μm that has been required in the conventional scaffold for the culture proliferation of osteoblasts described in Patent Documents 1 to 5. Due to the large difference, the structural feature of the microporous pores with uniform and high regularity of the honeycomb-shaped porous body is a suitable stimulus for the proliferation of osteoblasts that cannot be provided by the conventionally used scaffolds. It is presumed that

  The honeycomb-like porous body constituting the composite of the present invention is described in JP-A-8-311231, JP-A-2001-157475, JP-A-2002-347107, or JP-A-2002-335949. It can be produced by a method based on a production principle in which water droplets are condensed on the surface of a polymer water-insoluble organic solvent solution and the water droplet is used as a mold. In particular, a water-insoluble organic solvent, for example, a water-insoluble polymer in which the water-insoluble polymer is dissolved in a water-insoluble organic solvent having a surface tension γL of 50 dyn / cm or less, and the concentration of the water-insoluble polymer is 0.1 g / L to 20 g / L. When the surface tension of the organic solvent solution is γS, and the surface tension γL of the water-insoluble organic solvent to be applied and the surface tension γLS between the substrate and the solvent, the relationship of γS−γLS> γL is satisfied. A water-insoluble organic solvent solution of a water-insoluble polymer applied on the substrate is placed in an airflow that is applied to the surface of the substrate and further controlled in a relative humidity range of 30 to 99% and a wind speed of 0.01 to 20 m / sec. Those produced by evaporating the solvent are preferred.

  The water-insoluble organic solvent has a surface tension of 50 dyn / cm or less, and is water-insoluble to such an extent that it can retain water droplets condensed on the surface of the solution. An organic solvent having a boiling point of 90 ° C. Examples of such water-insoluble organic solvents include halogenated hydrocarbons such as carbon tetrachloride, dichloromethane and chloroform, aromatic hydrocarbons such as benzene, toluene and xylene, esters such as ethyl acetate and butyl acetate, and methyl isobutyl ketone. And water-insoluble ketones, carbon disulfide, and the like.

  The water-insoluble polymer is not particularly limited as long as it is a polymer that is insoluble in water and soluble in the above water-insoluble organic solvent, or can be dissolved in a water-insoluble organic solvent in the presence of a suitable surfactant. Can be appropriately selected and used. Examples thereof include biodegradable polymers such as polylactic acid and polyhydroxybutyric acid, aliphatic polycarbonates, amphiphilic polymers, photofunctional polymers, and electronic functional polymers.

  Specific examples include conjugated diene polymers such as polybutadiene, polyisoprene, styrene-butadiene copolymer, acrylonitrile-butadiene-styrene copolymer; polyε-caprolactone; polyurethane; cellulose acetate, celluloid, cellulose nitrate, Cellulosic polymers such as acetyl cellulose and cellophane; polyamide polymers such as polyamide 6, polyamide 66, polyamide 610, polyamide 612, polyamide 12, polyamide 46; polytetrafluoroethylene, polytrifluoroethylene, perfluoroethylene-propylene Fluoropolymer such as copolymer; polystyrene, styrene-ethylene-propylene copolymer, styrene-ethylene-butylene copolymer, styrene-isoprene copolymer, chlorinated polymer Styrene polymers such as len-acrylonitrile-styrene copolymer, methacrylic acid ester-styrene copolymer, styrene-acrylonitrile copolymer, styrene-maleic anhydride copolymer, acrylate-acrylonitrile-styrene copolymer Polyethylene, chlorinated polyethylene, ethylene-α-olefin copolymer, ethylene-vinyl acetate copolymer, ethylene-vinyl chloride copolymer, ethylene-vinyl acetate copolymer, polypropylene, olefin-vinyl alcohol copolymer, Olefin polymers such as polymethylpentene; formaldehyde polymers such as phenol resin, amino resin, urea resin, melamine resin, and benzoguanamine resin; polybutylene terephthalate, polyethylene terephthalate, polyethylene naphthalate Polyester polymers such as polyester; epoxy resins; (meth) acrylic polymers such as poly (meth) acrylic acid esters, poly-2-hydroxyethyl acrylate, methacrylic acid ester-vinyl acetate copolymers; norbornene resins; Silicon resin; polymers of hydroxycarboxylic acids such as polylactic acid, polyhydroxybutyric acid, and polyglycolic acid. These may be used alone or in combination.

  The polymer constituting the honeycomb porous body constituting the composite of the present invention does not necessarily need to be biodegradable, but as a water-insoluble polymer for producing a hard tissue regeneration device, polystyrene, Examples thereof include polylactic acid and poly (ε-caprolactone).

  The honeycomb porous body constituting the composite of the present invention is preferably prepared including an amphiphilic polymer. Preferred amphiphilic polymers include polyethylene glycol / polypropylene glycol block copolymers, amphiphilic resins having an acrylamide polymer as a main chain skeleton and a dodecyl group as a hydrophobic side chain and a lactose group or a carboxyl group as a hydrophilic side chain, Amphiphilic resins with hydrophilic groups such as ion complexes of anionic polymers such as heparin, dextran sulfate, and nucleic acids (DNA and RNA) and long-chain alkylammonium salts, water-soluble proteins such as gelatin, collagen, and albumin Amphiphilic resins such as lactic acid-polyethylene glycol block copolymer, poly ε-caprolactone-polyethylene glycol block copolymer, polymalic acid-polymalic acid alkyl ester block copolymer, and the like are included. Not.

  Examples of specific combinations of the water-insoluble organic solvent and the water-insoluble polymer include, for example, polyalkyl methacrylate or polyalkyl acrylate such as polystyrene, polycarbonate, polysulfone, polyethersulfone, polyalkylsiloxane, and polymethyl methacrylate. For polymers selected from the group consisting of polybutadiene, polyisoprene, poly-N-vinylcarbazole, polylactic acid, poly-ε-caprolactone, polyalkylacrylamide, and copolymers thereof, carbon tetrachloride, dichloromethane, A combination of organic solvents such as chloroform, benzene, toluene, xylene and carbon disulfide can be used. For polymers selected from the group consisting of acrylates, methacrylates and copolymers thereof having a fluorinated alkyl side chain, fluorocarbon solvents such as AK-225 (Asahi Glass Co., Ltd.), trifluorobenzene, etc. The use of fluoroethers also gives good results. Among these, it can be appropriately selected and used in consideration of solubility in the water-insoluble polymer specifically used.

  Further, when manufacturing a honeycomb-shaped porous body using a fluorine-based polymer in which hydrogen in a side chain of polyacrylate or methacrylate having a fluorinated alkyl side chain is substituted with fluorine, a fluorine-based organic solvent (AK- 225) also gives good results.

  In preparing the honeycomb-shaped porous body constituting the composite of the present invention, the concentration of the water-insoluble polymer in the water-insoluble organic solvent solution is 0.1 g / L to 20 g / L, particularly 0.5 g / L to 10 g. / L is preferable.

  The substrate on which the water-insoluble organic solvent solution of the water-insoluble polymer is applied is, for example, paper, glass plate, paper laminated with plastic (eg, polyethylene, polypropylene, polystyrene, etc.), metal plate (eg, aluminum, zinc, copper) Etc.), plastic film (eg, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose nitrate, polyethylene terephthalate, polyethylene, polystyrene, polypropylene, polycarbonate, polyvinyl acetal, etc.), silicon plate, etc. Is mentioned.

  Further, the substrate to which the water-insoluble organic solvent solution of the water-insoluble polymer is applied includes the surface tension γS of the substrate surface, the surface tension γL of the water-insoluble organic solvent to be applied, and the surface tension γLS between the substrate and the solvent. It is desirable to select and use a substrate satisfying the relationship of γS−γSL> γL. This is because the wettability of the water-insoluble organic solvent solution of the water-insoluble organic solvent solution to the water-insoluble organic solvent itself can affect the thickness of the liquid film formed on the substrate. The substrate preferably has a high affinity with the water-insoluble organic solvent solution of the water-insoluble polymer to be applied. Specifically, a substrate having a surface exhibiting a surface tension that can be expressed by the above formula using the surface tension γL of the water-insoluble organic solvent as an index may be used. Preferable examples of such a substrate include a glass plate, a silicon plate, or a metal plate.

  It is also possible to use a substrate whose surface has been processed to increase the affinity with a water-insoluble organic solvent solution. Such improvement of the wettability of the substrate surface can be achieved by a method known per se in accordance with the substrate and the water-insoluble organic solvent to be used, such as silane coupling treatment or thiol compound for glass or metal substrates, respectively. A monomolecular film forming method or the like can be used. For example, as a substrate when a hydrophobic organic solvent such as chloroform is used as the water-insoluble organic solvent, it is preferable to use a sufficiently cleaned Si substrate, a glass substrate whose surface is modified with an alkylsilane coupling agent, or the like. Further, when a fluorine-based solvent is used, it is preferable to use a Teflon (registered trademark) substrate or a glass substrate modified with a fluorinated alkylsilane coupling agent or the like.

  The liquid film thickness when a water-insoluble organic solvent solution of a water-insoluble polymer is applied to the substrate to form a liquid film of the solution is 50 μm to 5 mm, preferably 2 mm or less. As a method of applying a water-insoluble organic solvent solution of a water-insoluble polymer to a substrate, in addition to a method of dropping the solution on the substrate, a bar coat, a dip coat, a spin coat method, and the like can be given. Any of continuous types can be used.

  The water-insoluble organic solvent solution of the water-insoluble polymer applied to the substrate surface is preferably evaporated by contacting with air having a relative humidity of 30% or more. By setting the evaporation rate of the solvent to a rate at which the liquid film thickness at the time of application of the water-insoluble organic solvent solution of the water-insoluble polymer to the substrate surface decreases to 50 μm within 20 minutes, A honeycomb-like porous body can be prepared.

  The evaporation rate is such that an air layer flow of 0.1 L (liter) / min or more is substantially parallel to or upward from the surface of the liquid film of the water-insoluble organic solvent solution of the water-insoluble polymer applied on the substrate. Forming and evaporating the water-insoluble organic solvent, heating a substrate coated with a water-insoluble organic solvent solution of a water-insoluble polymer below the boiling point of the water-insoluble organic solvent and below the dew point of the air in contact with the liquid film (for example, Berche The water-insoluble organic solvent of the water-insoluble polymer is evaporated under a reduced pressure that does not exceed the boiling point of the water-insoluble organic solvent and the dew point of the air in contact with the liquid film. It can be achieved by a method of applying a solvent solution to a substrate and evaporating a water-insoluble organic solvent. Here, the dew point refers to a temperature at which water vapor contained in air at a certain temperature reaches saturation and condenses, and is a value determined with respect to relative humidity and absolute temperature.

  As a suitable example, the relative humidity is 30 to 99% and the wind speed is 0.01 to 20 m, substantially parallel to or upward from the liquid film of the water-insoluble organic solvent solution of the water-insoluble polymer applied to the substrate. It is preferable to place a liquid film of a water-insoluble organic solvent solution of a water-insoluble polymer in an air stream adjusted within a range of / second.

  By the above method, the honeycomb-like porous body used in the present invention can be produced. At this time, the pore diameter can be further finely adjusted by adjusting the amount of the water-insoluble organic solvent solution of the water-insoluble polymer applied on the substrate. For example, when using a solution in which poly (ε-caprolactone) is dissolved in chloroform so as to be 5 mg / mL, by changing the amount to 1.5 mL, 2.5 mL, and 5.0 mL of the same solution, A honeycomb-like porous body having through holes of 3 to 4, 5 to 6, and 10 to 11 μm can be produced.

  Further, this honeycomb-like porous body can be stretched by, for example, pinching both ends with a micromanipulator or tweezers or by hand and pulling in the extension direction. Such a stretched honeycomb-like porous body Can be used in the present invention as long as the specific pore diameter described above is maintained. Stretching may be uniaxial stretching, biaxial stretching, or triaxial stretching. The elongation ratio in the stretching direction is not particularly limited, but is preferably in the range of 1.1 to 10 times.

  The amphiphilic substance contained in the honeycomb-shaped porous body is preferably removed from the honeycomb-shaped porous body prior to coating the honeycomb-shaped porous body with the extracellular matrix. The removal of the amphiphile can be performed by immersing the honeycomb-shaped porous body in 1-propanol for several minutes to 10 minutes. The honeycomb porous body used in the present invention is preferably sterilized by UV irradiation, heat treatment, ethanol treatment or the like.

  Osteoblast culture in the honeycomb porous body produced by the above method is performed by inoculating the honeycomb porous body with osteoblasts isolated from a living body or induced to differentiate from bone marrow stem cells under appropriate conditions. What is necessary is just to culture. By this culture, the inoculated osteoblasts proliferate while producing calcium phosphate on the honeycomb-shaped porous body, and a honeycomb having a through-hole having a pore diameter of less than 15 μm made of calcium phosphate and water-insoluble polymer produced by osteoblasts. To form a complex with the porous material.

  That is, the present invention includes a step a) of seeding osteoblasts on a honeycomb porous body having through-holes having a pore diameter of less than 15 μm, and culturing osteoblasts on the honeycomb porous body to form the honeycomb porous body. Production of a composite of osteoblasts, calcium phosphate produced by osteoblasts, and a honeycomb porous body having a through-hole having a pore diameter of less than 15 μm made of a water-insoluble polymer, including the step b) of generating calcium phosphate on the body surface A method is provided.

  Isolation of osteoblasts from a living body can be performed according to the above-mentioned known methods. As osteoblasts, for example, osteoblasts induced to differentiate from bone marrow stem cells based on the method of Bokhara et al. (Biomaterials, 2005, Vol. 26, No. 25, pages 5198-5208) may be used. . In the present invention, any of osteoblasts derived from various animals represented by mammals such as rats, mice, rabbits, and pigs can be used, but the use of human osteoblasts is particularly preferred. In particular, since the complex according to the present invention can be used as a device for regenerating hard tissue, osteoblasts are suitable for osteoblasts derived from patients undergoing hard tissue regenerative treatment (from bone marrow stem cells collected from the bone marrow of patients). Including osteoblasts induced to differentiate under conditions).

For inoculation of osteoblasts, an appropriate buffer or medium, preferably Williams'E medium, F-10 medium, RPMI 1640 medium, Eagle's MEM medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums About 1.0 × 10 ³ to 1.0 × per unit area (cm ² ) of the honeycomb-shaped porous body prepared in a well plate or a suitable container. The suspension can be added to the honeycomb porous body so as to be 10 ⁶ . After the osteoblasts are added to the honeycomb-shaped porous body, the osteoblasts proliferate by incubation for 21 to 42 days at pH 6.0 to 8.0, 30 to 40 ° C., and 5% CO ₂ atmosphere. A complex according to the present invention is formed. During culture, it is preferable to perform medium exchange at an appropriate interval.

  A typical structure of the composite produced by the present invention is that osteoblasts cover a honeycomb-like porous body in which calcium phosphate crystals are deposited or intruded on the polymer surface or pores. This composite is not in a state where the osteoblasts and the honeycomb porous body are simply in contact with each other, and the osteoblasts can be easily washed even if the composite is washed with water or a buffer solution. It can be used as a device for transplanting to a hard tissue disease site as it is to regenerate a hard tissue defect. In addition, the complex of the present invention can also be used as a tool for simply searching for a substance that promotes hard tissue regeneration for osteoblasts and for searching and evaluating other therapeutic agents for bone diseases.

  Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

1) Preparation of honeycomb-like porous body and flat membrane Poly (ε-caprolactone) (PCL, manufactured by Wako Pure Chemical Industries, Ltd., molecular weight 70,000 to 100,000), which is a biodegradable polymer, and amphiphilic poly Acrylamide polymer (a copolymer consisting of Cap, dodecylacrylamide, 6-acrylamide-n-caproic acid and 2,2′-azobis (isobutyronitrile)) dissolved in chloroform at a weight ratio of 10: 1 to 5 mg / mL did. Cover a glass petri dish (inner diameter: 90 mm) with a cover glass having a diameter of 14 mm, cast a 1.5 mL, 2.5, 5.0, and 7.5 mL chloroform solution containing the PCL on each glass, and a relative humidity of 80%. Chloroform was evaporated under an atmosphere with a flow rate of 2.0 L / min. Through the above operation, honeycomb-like porous bodies having through holes with pore diameters of 3 to 4, 5 to 6, 10 to 11, and 15 to 16 μm were prepared. FIG. 1 shows an enlarged photograph of a honeycomb-shaped porous body having through-holes having a pore diameter of 10 to 11 μm, which will be described later, using a scanning electron microscope.

After the chloroform has completely evaporated, the cover glass is removed from the glass petri dish, immersed in 1-propanol for 10 minutes to remove the amphiphilic polymer, further washed with ethanol, and vacuumed to remove the residual solvent. For 3 hours. Thereafter, UV irradiation (about 40 mW / cm ² ) was performed for 3 hours to produce a sterilized honeycomb porous body.

1 mL of a chloroform solution having a PCL and Cap (weight ratio of 10: 1) concentration of 10 mg / mL was cast on a cover glass placed on the stage of a spin coater (MIKASA 1H-D7), and the condition was 1,000 rpm for 15 seconds. Then, the stage was rotated to produce a flat film having a uniform film thickness. The flat membrane was immersed in 1-propanol for 10 minutes to remove the amphiphilic polymer, further washed with ethanol, and placed under vacuum for 3 hours to remove residual solvent. Then, in order to sterilize, UV irradiation (about 40 mW / cm < ² >) was performed for 3 hours, and the sterilized PCL flat film was produced.

2) Preparation of osteoblasts Skulls were excised and recovered from male Wistar rats (Charles River Laboratories) weighing 200-250 g (6-8 weeks old), and Bokhari et al. (Biomaterials, 2005, Vol. 26, No. 26). 25, 5198-5208), osteoblasts were isolated and suspended in DMEM medium.

3) Cultivation of osteoblasts The four types of honeycomb-like porous bodies and flat membranes prepared in 1) were placed in a 24-well plate to which 1.0 mL of DMEM medium was added, and the osteoblasts prepared in 2) from above. 2 × 10 ⁵ cells / DMEM medium was added. The cells were cultured for 28 days under conditions of 37 ° C. and 5% CO ₂ . The medium was replaced with fresh medium after 48 hours.

  After completion of 4 weeks of culture, osteoblasts on the honeycomb-shaped porous body were subjected to an index of bone formation by the method of Pearse et al. (Pearse AG E, “Histochemistry-Theoretical and Applied, 1968, published by Churchill, London) et al. Were stained with alkaline phosphatase (blue) and calcium (red), and observed with an optical microscope (Fig. 2), and 9 different points per honeycomb porous body and flat membrane with a 10x objective lens Observation and image analysis were performed using Image J (NIH) to quantify the amount of bone formation (FIG. 3).

  As a result, it was confirmed that osteoblasts covered the honeycomb-like porous body in the honeycomb-like porous body having a through-hole having a pore diameter of 3 to 4 μm, a through-hole having a pore diameter of 5 to 6 μm, and a through-hole of 10 to 11 μm. It was done. Moreover, the highest bone forming ability was recognized in the honeycomb-like porous body having through holes having a pore diameter of 3 to 4 μm.

4) Observation with a scanning electron microscope A 2.5% glutaraldehyde solution was prepared by removing trypsin treatment on the honeycomb-shaped porous body having through-holes having a pore diameter of 3 to 4 μm after culturing to remove surface osteoblasts. And then dehydrated stepwise using 20% to 100% ethanol. Further, after replacing the organic solvent with tert-butyl alcohol, the honeycomb-shaped porous body was frozen and dried using a freeze vacuum drying apparatus (Hitachi ES-2030), and then an ion sputtering apparatus (Hitachi E-1030) was used. It was covered with platinum palladium and observed using a scanning electron microscope (Hitachi S-3500N).

  As a result, precipitation of calcium phosphate crystals was observed on the polymer surface and through-hole portions of the honeycomb-like porous body (FIG. 4).

2 is a scanning electron micrograph of a honeycomb-like porous body (pore diameter 10 to 11 μm) produced in Example 1. FIG. It is an optical microscope photograph when the alkaline phosphatase (blue) and calcium (red) which are the indexes of bone formation are dye | stained with respect to the composite_body | complex produced in Example 1. FIG. Panel a has a flat membrane, panel b has a pore diameter of 3 to 4 μm, panel c has a pore diameter of 5 to 6 μm, panel d has a pore diameter of 10 to 11 μm, and panel e includes a honeycomb porous body having through holes having a pore diameter of 15 to 16 μm. It is a complex. 2 is a graph showing the osteogenic ability of osteoblasts in the composite prepared in Example 1. FIG. It is a scanning electron micrograph of what removed the surface osteoblast from the composite_body | complex containing the honeycomb porous body which has a through-hole of 3-4 micrometers of hole diameters.

Claims (7)

  A device for regenerating hard tissue, which is a composite of osteoblasts, calcium phosphate produced by osteoblasts, and a honeycomb-shaped porous body having a through-hole having a pore diameter of less than 15 μm made of a water-insoluble polymer.   The device for hard tissue regeneration according to claim 1, wherein the pore size of the honeycomb-shaped porous body is 1 µm to 14 µm.   The device for hard tissue regeneration according to claim 1 or 2, wherein the through-holes of the honeycomb-shaped porous body communicate with surrounding through-holes existing in the planar direction.   The hard tissue regeneration device according to any one of claims 1 to 3, wherein calcium phosphate produced by osteoblasts enters at least part of the through-holes of the honeycomb-shaped porous body.   The hard tissue regeneration device according to any one of claims 1 to 4, wherein the osteoblast is an osteoblast derived from a mammal that requires regeneration of a hard tissue.   Osteoblasts and osteoblasts comprising a step of inoculating osteoblasts into a honeycomb porous body having a through-hole having a pore diameter of less than 15 μm, and a step b) of culturing the osteoblasts to produce calcium phosphate A method for producing a composite of calcium phosphate produced by a honeycomb-shaped porous body having a through-hole having a pore diameter of less than 15 μm and comprising a water-insoluble polymer.   The manufacturing method according to claim 6, wherein the honeycomb porous body has a pore diameter of 1 μm to 14 μm.