WO2020261653A1

WO2020261653A1 - Porous body, method for producing porous body, method for transplanting porous body, and cell support

Info

Publication number: WO2020261653A1
Application number: PCT/JP2020/008957
Authority: WO
Inventors: 優史丸山; 広貴佐久間; 正樹松森
Original assignee: 株式会社日立製作所
Priority date: 2019-06-25
Filing date: 2020-03-03
Publication date: 2020-12-30
Also published as: JP2021003007A

Abstract

The present invention provides: a porous body which has communicating holes having a three-dimensional net-like structure and is useful as a scaffold for cells or a biocompatible material; a method for producing a porous body; a method for transplanting a porous body; and a cell support using the porous body. A porous body (1) has communicating holes (20) formed in a base material, wherein each of the communicating holes (20) is formed from pores each having an aspect ratio of 10 or more or is formed by the connection of the pores, and the base material is formed from a biocompatible material. A method for producing a porous body comprises the steps of: precipitating sacrificial templates (120) each having an aspect ratio of 10 or more in a medium; forming a base material layer (10) that serves as a base material around the sacrificial templates (120); and removing the sacrificial templates (120) from the formed base material layer (10). A method for transplanting a porous support comprises transplanting the porous body (1) into the body of a non-human animal. A cell support comprises the porous body (1) and cells supported on the porous body (1).

Description

Porous body, method for producing a porous body, method for transplanting a porous body, and a cell carrier

The present invention relates to a porous body useful as a cell scaffold or a biocompatible material, a method for producing a porous body, a method for transplanting a porous body, and a cell carrier using the same.

In the field of regenerative medicine, treatments and treatments for transplanting artificial organs and tissues are performed for functional deterioration and loss of organs and tissues. Some artificial organs and artificial tissues utilize the function of transplanted cells in the living body of the transplant destination. In some types of cell transplantation, endocrine cells and cells that exhibit a paracrine effect are transplanted into the patient's body. A cell scaffold material that serves as a scaffold for cell adhesion and proliferation is used as a cell device that supports such transplanted cells.

In general, cell scaffolding materials are required to have low toxicity and performance that does not inhibit cell function. Further, it is necessary to have a structure in which the cells are stably supported and do not fall off, oxygen and nutrients are sufficiently supplied to the supported cells, and waste products from the cells are appropriately excreted. In addition, it is required to have a high porosity so that it can be integrated with a living tissue after transplantation, and to have a property of being easily processed and molded into a desired shape.

Conventionally, hydrogel has been widely studied as a cell scaffolding material. As a means for processing and molding a hydrogel, a gel 3D printer that performs modeling using a gel has also been developed. However, hydrogels are not suitable for densifying cells and have a drawback that the dead volume becomes large with respect to the cells to be supported. In addition, since the substance diffusion characteristics are not always excellent, the supply of oxygen and nutrients and the excretion of waste products are poor, which is disadvantageous for the exertion of cell functions.

As a cell scaffold material, a porous body having a large number of communication holes in a base material such as resin is also being studied. Porous bodies are advantageous for densifying cells and are suitable for reducing the occupied volume of cell devices. In addition, since it has excellent substance diffusion characteristics, it is also advantageous in terms of oxygen / nutrient supply and excretion of waste products. However, since the porous body is not suitable for bottom-up molding such as using a 3D printer, it is not easy to process and mold it into a desired shape.

Conventionally, as a method of forming a porous body, a method of adding a sacrificial template (porogen) for molding pores to a resin raw material, polymerizing the resin, and then removing the sacrificial template to form pores has been known. There is.

For example, Patent Document 1 describes a technique using a phase separating agent that is compatible with an olefin polymer and phase-separates from a cured product of the olefin polymer. Further, Patent Document 2 describes a method for preparing a bioreabsorbable dressing using sieved Porogen particles.

Japanese Unexamined Patent Publication No. 2012-124434 Special Table 2010-508977

A porous body having communication holes communicating with the outside in the base material can be used as a cell scaffold by introducing cells into the communication holes. However, when a porous body is used as a cell scaffold, it is necessary to have the ability to support cells at high density and the ability to maintain the supported cells for a long period of time. The communication pores form discrete network-structured stomata throughout the porous body so that the supported cells are dispersed without being densely packed and sufficient oxygen and nutrients are supplied to the individual cells. It is desirable to do. It is desired that the diameter of the communication hole is such that the cells can reach the deep part and do not easily fall off after the cells adhere to the deep part.

Further, a porous body having communication holes communicating with the outside in the base material can be used as a biocompatible material such as an artificial tissue instead of a cell scaffold. However, when a porous body is used as a biocompatible material, a high degree of substance permeability may be required for coexistence with a living body. When the porous body is used as a component of a cell device, an auxiliary device to the circulatory system, a filter, or the like, a structure having high substance permeability such that the communication holes form a three-dimensional network structure is desired.

However, there is a current situation in which it is not easy to appropriately mold and balance the communication holes having such a shape and structure and the desired outer shape according to the purpose of the porous body. It is difficult to form communication holes with an appropriate shape and structure while molding a porous body into a desired outer shape by conventional modeling technology using general Porogen particles and bottom-up modeling technology using a 3D printer. Is. The more complicated the shape of the porous body and the more the dead volume of the porous body is reduced, the more difficult it becomes to form the porous body, and the more useful cell scaffolds and biocompatible materials cannot be obtained.

Therefore, the present invention has a three-dimensional network structure of communication holes and is useful as a cell scaffold or a biocompatible material, a porous body, a method for producing a porous body, a method for transplanting a porous body, and the like. It is an object of the present invention to provide a cell carrier using.

In order to solve the above problems, the porous body according to the present invention is a porous body having communication holes in the base material, and the communication holes are pores having an aspect ratio of 10 or more, or the fine particles. It is formed by connecting the pores, and the base material is made of a biocompatible material.

Further, the method for producing a porous body according to the present invention is a method for producing a porous body having communication holes in a base material, which comprises a step of precipitating a sacrificial template having an aspect ratio of 10 or more in a medium. A step of forming a base material layer to be the base material around the sacrificial template and a step of removing the sacrificial template from the formed base material layer are included.

Further, in the method for transplanting a porous carrier according to the present invention, the above-mentioned porous body is transplanted into the body of an animal other than humans.

Further, the cell carrier according to the present invention includes the porous body and cells supported on the porous body.

According to the present invention, a porous body having three-dimensional network-structured communication holes and useful as a cell scaffold or a biocompatible material, a method for producing a porous body, a method for transplanting a porous body, and the like. A cell carrier using the above can be provided.

It is a figure which shows typically an example of the manufacturing method (precipitation step) of a porous body. It is a figure which shows typically an example of the manufacturing method (precipitation step) of a porous body. It is a figure which shows typically an example of the manufacturing method (precipitation step) of a porous body. It is a figure which shows typically an example of the manufacturing method (precipitation step) of a porous body. It is a figure which shows typically an example of the manufacturing method (base material layer formation process) of a porous body. It is a figure which shows typically an example of the manufacturing method (base material layer formation process) of a porous body. It is a figure which shows typically an example of the manufacturing method (removal process) of a porous body. It is a figure which shows typically an example of the manufacturing method (removal process) of a porous body. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows another example of the manufacturing method of a porous body schematically. It is a figure which shows typically the specific example of the manufacturing method of a porous body. It is a figure which shows typically the specific example of the manufacturing method of a porous body. It is a figure which shows typically the specific example of the manufacturing method of a porous body. It is a figure explaining the anastomosis of a porous body to a blood vessel. It is a figure explaining the anastomosis of a porous body to a blood vessel.

Hereinafter, the porous body according to the embodiment of the present invention, the method for producing the porous body, the method for transplanting the porous body, and the cell carrier using the porous body will be described. It should be noted that the following description shows specific embodiments of the present invention, and the present invention is not limited to these descriptions, and those skilled in the art are skilled in the art within the scope of the technical idea disclosed in the present specification. Various changes and modifications can be made by. The same reference numerals may be given to common configurations in the following figures, and duplicate description may be omitted.

<Porous medium>
The porous body 1 according to the present embodiment (see FIG. 3B) has a large number of communication holes 20 communicating with the outside of the porous body 1 in the base material constituting the main body of the porous body 1. The communication hole 20 is formed by connecting long pores having a large aspect ratio (ratio of length to diameter) in a vertical cross-sectional view, or long pores having a large aspect ratio.

In the porous body 1, a large number of communication holes 20 form a three-dimensional network structure in the base material. Due to the network structure of a large number of communication holes 20, it is possible to comprehensively introduce cells into the deep part of the porous body 1 and to supply sufficient oxygen and nutrients to the cells supported in the deep part. become. The three-dimensional network structure includes a structure in which the communication holes 20 intersect each other, a structure in which the communication holes 20 are oriented in the same direction or in different directions without intersecting each other, and a structure in which these structures are mixed. Etc. are included.

The porous body 1 according to the present embodiment is manufactured by using a sacrificial template 120 (see FIG. 1D) for molding a communication hole 20 having a desired shape and structure. The sacrificial template 120 is removed after molding the communication holes 20 and does not constitute the finished product of the porous body 1. As the sacrificial template 120, a long precipitate having a large aspect ratio (ratio of length to diameter) in the vertical cross-sectional view is generated in the medium and used for forming the communication hole 20.

1A to 1D, FIGS. 2A to 2B, and FIGS. 3A to 3B are diagrams schematically showing an example of a method for producing a porous body.
As shown in FIGS. 1A to 1D, FIGS. 2A to 2B, and FIGS. 3A to 3B, in the method for producing a porous body according to the present embodiment, a long sacrificial template 120 having a large aspect ratio is deposited in a medium. Precipitation step (see FIGS. 1A to 1D), and a base layer layer forming step (see FIGS. 2A to 2B) for forming a base material layer 10 as a base material for a porous body around the sacrificial template 120, and sacrifice. It includes a removal step (see FIGS. 3A-3B) of removing the sacrificial template 120 from the substrate layer 10 formed around the template.

Conventionally, as a method for producing a porous body using a sacrificial template (porogen), a method using spherical particles having a small aspect ratio is known. Spherical porogen is mixed with a solution in which a resin raw material is dissolved, the resin is polymerized or cured, and then porogen is removed by dissolution, or spherical porogen is mixed in metal powder and the metal powders are heat-treated. There is known a method of removing pologene by burning it while sintering it with.

Since the generally used spherical porogen has good dispersibility, it can be relatively easily mixed with the precursor solution, precursor powder, etc. used for producing a porous body. Further, since the precursor solution, precursor powder, etc. after mixing the pologene show relatively high fluidity, the pologene is not biased when a molding die is used to mold the porous body into a desired outer shape. Can be dispersed.

However, spherical pologene is disadvantageous in that it forms communication holes in the porous body. In order to form communication holes that communicate with the outside of the porous body, it is necessary to bring spherical porogens into contact with each other in the precursor solution, precursor powder, etc., so the precursor solution, precursor powder, etc. may be pressurized. , Need to be compressed. However, when such an operation is applied, the raw material flows and the unsolidified material is deformed, so that it is difficult to form the porous body into a desired outer shape while keeping the pologenes in contact with each other.

In addition, since spherical porogens vary in all directions in the precursor solution, precursor powder, etc., it is difficult to place them in the target position in contact with each other. Unnecessarily dense pologens result in the formation of high-density closed pores when the pologens are removed. As a result, the shape of the communication holes in the vicinity is not maintained, and the mechanical strength of the porous body is locally reduced.

Also, spherical porogens tend to form non-uniform communication holes with non-constant diameters (inner widths). If the communication hole has a thick or thin part, the cells introduced into the communication hole will stay in a specific place, and the oxygen / nutrient supply and waste product release will be different, so that it is porous. It is not appropriate for the quality of the body. Further, since the spherical porogen having a small aspect ratio has a small specific surface area, there is a problem that the specific surface area of the porous body becomes smaller than the porosity.

On the other hand, when a long sacrificial template having a large aspect ratio is used as in the present embodiment, the sacrificial templates can be easily contacted with each other in the precursor solution, precursor powder, etc. used for producing the porous body. Can be made to. Further, by aligning the shapes of a large number of sacrificial templates, it is possible to stably form a large number of communication holes having a high uniformity in diameter (inner width) and a constant shape.

However, long sacrificial templates with a large aspect ratio tend to be entangled with each other when mixed with the precursor solution, precursor powder, etc. used in the production of porous materials. When the sacrificial templates are entangled with each other, the fluidity of the sacrificial template becomes low in the precursor solution, the precursor powder, and the like. If the fluidity of the sacrificial template is low, the sacrificial template will not be properly placed at the desired position in the molding die and will be biased when a molding die is used to form the porous body into a desired outer shape. It becomes difficult to form communication holes with an appropriate distribution in the porous body formed into a desired outer shape.

However, when the manufacturing method of precipitating the sacrificial template in a medium such as a precursor solution as in the present embodiment is used, the contact between the sacrificial templates and the spatial arrangement / orientation of the sacrificial templates can be controlled relatively freely. Can be done. It is also possible to deposit the sacrificial template in a molding die for molding the porous body into a desired outer shape, and it is possible to accurately control the arrangement of the communication holes with respect to the outer shape of the porous body. Therefore, it is possible to stably form communication holes having a three-dimensional network structure that are discretely stretched over the entire porous body.

As shown in FIGS. 1A to 1D, in the precipitation step, the state / property of the precursor solution 100 containing the raw material of the sacrificial template 120 is adjusted in order to precipitate the long sacrificial template 120 having a large aspect ratio in the medium. In addition, a three-dimensional network structure composed of a long sacrificial template 120 having a large aspect ratio is semi-spontaneously formed in the precursor solution 100 as a precipitation medium.

FIG. 1A shows a state in which the precursor solution 100 containing the raw material of the sacrificial template 120 is prepared in the liquid tank. FIG. 1B shows a state in which the nucleus 110 of the sacrificial template 120 is formed in the precursor solution 100. FIG. 1C shows a state in which a long sacrificial template 120 having a large aspect ratio is precipitated in the precursor solution 100. FIG. 1D shows the sacrificial template 120 forming a three-dimensional network structure obtained from the precursor solution 100.

As shown in FIG. 1A, the precursor solution 100 can be prepared in a liquid tank having an appropriate shape. 1A to 1D show a method in which the prepared sacrificial template 120 is once taken out from the liquid tank containing the precursor solution 100 and used for producing the porous body 1. However, the production of the porous body 1 can be performed continuously in the same liquid tank without taking out the prepared sacrificial template 120 from the liquid tank once.

When the prepared sacrificial template 120 is once taken out from the liquid tank and used for manufacturing the porous body, the liquid tank containing the precursor solution 100 is the outer shape of the porous body 1 to be manufactured as long as the three-dimensional structure of the sacrificial template 120 fits. It may have the same shape as the above, that is, a shape that functions as a molding mold for the porous body 1, or may have a shape different from the outer shape of the porous body 1 to be manufactured.

The sacrificial template 120 can be formed of any of low molecular weight compounds, high molecular weight compounds, inorganic compounds, metals and the like. As the precursor solution 100, when the sacrificial template 120 is formed of a low molecular weight compound, when it is formed of a high molecular weight compound such as a low molecular weight compound which is a raw material, or when it is formed of an inorganic compound such as a monoma, a prepolymer, or a high molecular weight compound. When it is formed of a metal such as a metal salt or a metal alkoxide, the metal salt, the metal oxide or the like can be prepared by dissolving and dispersing each in an appropriate solvent.

As shown in FIGS. 1B and 1C, the core 110 of the sacrificial template 120 and the long sacrificial template 120 having a large aspect ratio have an appropriate number density required for forming the communication holes 20 in the precursor solution 100. It can be precipitated as follows. Although the spherical nucleus 110 is shown in FIG. 1B, the form of precipitation is not limited to crystallization from the liquid phase, but may be phase separation of solid components such as spherical, fibrous, and amorphous. Good. As an operation for precipitating the solid component, an appropriate operation can be used depending on the type of the raw material of the sacrificial template 120.

As an operation for precipitating a solid component, when the sacrificial template 120 is formed of a low molecular weight compound, an operation of lowering the solubility of the low molecular weight compound or the like can be used. Solubility can be adjusted by either a method of changing exogenous factors such as temperature, polarity, pH, or salt concentration of the precursor solution 100, or a method of chemically changing intrinsic factors such as coordinating property and amphipathicity of raw materials. You may.

Further, as the operation of precipitating the solid component, when the sacrificial template 120 is formed of a polymer compound, an operation of polymerizing a monoma or a prepolymer, an operation of cross-linking the polymer compound, an operation of phase-separating the polymer compound from the liquid, etc. Can be used. The polymerization or cross-linking may be carried out by any of a method of adding a polymerization agent or a cross-linking agent, a method of thermal polymerization, a method of photopolymerization and the like. Phase separation can be performed by either a method of changing exogenous factors such as temperature, polarity, pH and salt concentration of the precursor solution 100, or a method of chemically changing intrinsic factors such as coordinating property and hostility of raw materials. You may operate it.

Further, as the operation of precipitating the solid component, when the sacrificial template 120 is formed of the inorganic compound, an operation of lowering the solubility of the inorganic compound, an operation of reacting and generating an insoluble inorganic compound, and the like can be used. Solubility can be adjusted by either a method of changing exogenous factors such as temperature, polarity, pH, or salt concentration of the precursor solution 100, or a method of chemically changing intrinsic factors such as coordinating property and amphipathicity of raw materials. You may. The formation of the insoluble inorganic compound may be carried out by any of a precipitation reaction, a redox reaction, a sol-gel reaction and the like.

Further, as the operation of precipitating the solid component, when the sacrificial template 120 is formed of metal, an operation of chemically reducing and precipitating the metal with a reducing agent, an operation of electrochemically reducing and precipitating the metal, and the like can be used. .. The operation of electrochemically reducing and precipitating can be performed, for example, by putting the raw material and the electrolyte of the sacrificial template 120 in a liquid tank and energizing between the electrodes immersed in the liquid tank. By such an operation, a reducing metal such as a dendritic crystal or an acicular crystal can be precipitated on the electrode.

As the low molecular weight compound forming the sacrificial template 120, a compound having a high temperature dependence of solubility and a large solubility difference within the operating temperature range of the precursor solution 100 is preferable. With such a low molecular weight compound, the precipitation of the sacrificial template 120 can be started only by lowering the temperature of the precursor solution 100. Therefore, the number density of the core 110 of the sacrifice template 120 and the shape of the sacrifice template 120 can be easily controlled. Further, since it is not necessary to add an unnecessary component to the precursor solution 100, it is possible to simplify the process of washing the precipitate.

Specific examples of low molecular weight compounds include urea, urea derivatives, thiourea, and thiourea derivatives. Examples of the derivative include phenyl (thio) urea, alkyl-substituted phenyl (thio) urea, alkoxy-substituted phenyl (thio) urea, benzoyl (thio) urea, benzyl (thio) urea, and substituted derivatives thereof. With these compounds, water can be used as the solvent. These compounds are highly temperature dependent for solubility in water and can form a crystalline sacrificial template 120. Therefore, the number density of the core 110 of the sacrifice template 120 and the shape of the sacrifice template 120 can be easily controlled. Further, since water serves as a good solvent, the polarity of the precursor solution 100 can be easily adjusted by adjusting the amount of the poor solvent added.

As the polymer compound forming the sacrificial template 120, a compound that is solidified by the polymerization of a monomer or a prepolymer, or a compound that undergoes phase separation due to a large change in solubility due to an interaction between molecules or the like is preferable. With such a polymer compound, the number density of the nuclei 110 of the sacrificial template 120 and the shape of the sacrificial template 120 can be precisely controlled by changing the type and concentration of the raw material to be added.

Specific examples of the polymer compound include acrylic resins such as polymethylacrylate, polymethylmethacrylate, polyacrylic acid, polymethacrylic acid, polyacrylamide and polydimethylacrylamide, polyolefins such as polyethylene and polypropylene, and polystyrene. .. When a combination of polystyrene and polymethyl methacrylate or the like is used, phase separation occurs due to a decrease in compatibility with the liquid phase, so that the sacrificial template 120 can be formed with a specific morphology.

As the inorganic compound forming the sacrificial template 120, a compound having a high temperature dependence of solubility and a large solubility difference within the operating temperature range of the precursor solution 100 is preferable. With such an inorganic compound, the precipitation of the sacrificial template 120 can be started only by lowering the temperature of the precursor solution 100. Therefore, the number density of the core 110 of the sacrifice template 120 and the shape of the sacrifice template 120 can be easily controlled. Further, since it is not necessary to add an unnecessary component to the precursor solution 100, it is possible to simplify the process of washing the precipitate.

Specific examples of inorganic compounds include alum and the like. Examples of alum include potassium aluminum alum, ammonium aluminum alum, ammonium iron alum, and ammonium chrome alum. With these compounds, water can be used as the solvent. These compounds are highly temperature dependent for solubility in water and can form a crystalline sacrificial template 120. Therefore, the number density of the core 110 of the sacrifice template 120 and the shape of the sacrifice template 120 can be easily controlled.

As the metal forming the sacrificial template 120, a metal forming a metal salt having high solubility and a metal having a large reduction potential are preferable. With such a metal, the number density of the core 110 of the sacrificial template 120 and the shape of the sacrificial template 120 can be precisely controlled by changing the redox potential of the precursor solution 100.

Examples of the solvent of the precursor solution 100 include water, alcohols such as methanol, ethanol and isopropyl alcohol, ethers such as diethyl ether and dibutyl ether, ketones such as acetone, methyl ethyl ketone and cyclohexanone, ethyl acetate and the like. Fatty acid esters, aromatic hydrocarbons such as benzene, toluene, xylene, and dichlorobenzene, aliphatic hydrocarbons such as hexane, cyclohexane, and dodecane, chloroform, dichloroethane, dimethylfuran, dimethylsulfoxide, dimethylacetamide, Various solvents such as dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran and acetonitrile and supercritical fluids can be used.

In the precipitation step, an additive for controlling the morphology of the sacrificial template 120, a seed crystal that promotes precipitation and nucleation of the sacrificial template 120, a nucleating agent, and the like can be added to the precursor solution 100. As the additive, for example, a surfactant, a flocculant, a dispersant, a complexing agent and the like can be used.

Further, in the precipitation step, a base material or the like for controlling the direction of crystal growth of the sacrificial template 120, the frequency of formation of the nuclei 110, the orientation of crystals and molecules, and the like can be put into the liquid tank containing the precursor solution 100. However, it is also possible to provide an inner surface structure or the like for controlling these. As the base material and the inner surface structure, for example, one having a predetermined crystal face exposed, or one having undergone surface treatment such as attachment of a substance such as metal or chemical modification can be used.

When the sacrificial template 120 is deposited in the precursor solution 100, it may form only a long straight portion having a large aspect ratio, or it may form a non-linear portion in addition to a long straight portion having a large aspect ratio. It may be formed. Examples of the non-linear portion include a bifurcated portion branched into two or more from the straight portion, an intersecting portion where the straight portions intersect, a bending portion, a curved portion, and the like.

The cross-sectional shape of the sacrifice template 120 may be circular, elliptical, rectangular, polygonal, amorphous, or the like. Further, the cross-sectional shape of the sacrificial template 120 may be a similar shape for each precipitate or a non-similar shape for each precipitate.

The sacrificial template 120 preferably has an aspect ratio (ratio of length to diameter) of 10 or more in a vertical cross-sectional view. The aspect ratio of the sacrifice template 120 is defined as the ratio of the mean length of the straight line portion to the mean diameter. The aspect ratio is calculated only for the linear portion excluding the non-linear portion when the sacrificial template 120 forms the non-linear portion. The average value of the length and the diameter is calculated from the measurement results of the precipitates having a predetermined number of samples or more and the measurement results of the arbitrary part of the straight part as the measurement point.

As shown in FIG. 1C, the sacrifice template 120 is a structure having a three-dimensional network structure by connecting long straight parts having a large aspect ratio or long straight parts having a large aspect ratio in a vertical cross-sectional view. The precipitate can be grown and formed in the precursor solution 100 so as to be. The prepared sacrificial template 120 can be taken out from the liquid tank containing the precursor solution 100 and subjected to a washing treatment, a drying treatment, or the like, if necessary.

As shown in FIGS. 2A to 2B, in the base material layer forming step, a precursor solution containing a raw material of the base material is formed in order to form the base material layer 10 which is the base material of the porous body around the sacrificial template 120. An operation for adjusting the state / property of 200 is added, and the periphery of the sacrificial template 120 is covered with the base material layer 10. By forming the base material layer 10, the shape of the communication hole 20 formed when the sacrificial template 120 is removed is fixed, and a matrix constituting the main body of the porous body 1 is formed.

FIG. 2A shows a state in which the precursor solution 200 containing the raw material of the base material layer 10 and the sacrificial template 120 are prepared in the liquid tank. FIG. 2B shows a state in which the base material layer 10 is formed around the sacrificial template 120.

As shown in FIG. 2A, the precursor solution 200 and the sacrificial template 120 can be prepared in a liquid tank having an appropriate shape. 2A to 2B show a method of forming a base material layer 10 having a thickness such that the outline of the sacrificial template 120 can be seen around the prepared sacrificial template 120. However, the base material layer 10 can also be formed in a bulk shape that completely fills the periphery of the sacrificial template 120.

The liquid tank containing the precursor solution 200 may have the same shape as the outer shape of the porous body 1 to be manufactured, that is, a shape that becomes a molding mold of the porous body 1 as long as the three-dimensional structure of the sacrificial template 120 fits. However, the shape may be different from the outer shape of the porous body 1 to be manufactured.

Although FIGS. 2A to 2B show an example in which the base material layer 10 is formed in the liquid phase, the base material layer 10 can be used for the type of raw material of the sacrificial template 120 and the type of raw material for the base material. Depending on the situation, it may be formed in the gas phase by using spraying of the precursor solution or the like, or may be formed in the packed phase by using sintering, welding or the like of the precursor powder.

The base material layer 10 can impart biocompatibility to the porous body 1, and is a polymer compound or an inorganic compound as long as the immobilization of the shape of the communication hole 20 by the sacrificial template 120 and the removal of the sacrificial template 120 are not hindered. , Metal, etc. can be used. As the precursor solution 200, when the base material layer 10 is formed of a polymer compound, when it is formed of an inorganic compound such as monoma, prepolyma, or polymer compound, or when it is formed of a metal such as a metal salt or metal alkoxide, it is a metal salt. , Metal oxidants and the like can be prepared by dissolving and dispersing each in an appropriate solvent.

As a method for forming the base material layer 10, when the base material layer 10 is formed of a polymer compound, a reaction of polymerizing a monomer or a prepolymer, a reaction of cross-linking a polymer compound, or the like can be used. The polymerization or cross-linking may be carried out by any of a method of adding a polymerization agent or a cross-linking agent, a method of thermal polymerization, a method of photopolymerization and the like.

Further, as a method for forming the base material layer 10, when the base material layer 10 is formed of an inorganic compound, any of a sol-gel method, a liquid phase method, a solid phase method and the like can be used. When the base material layer 10 is formed of a metal, a method of chemically reducing and precipitating the metal with a reducing agent, a method of electrochemically reducing and precipitating the metal, and the like can be used.

As shown in FIG. 2B, in the base material layer 10, the shape of the communication hole 20 is fixed and the mechanical strength of the porous body 1 is ensured by covering the periphery of the sacrificial template 120 to some extent. As long as it is formed, it may be formed so as to cover the entire sacrifice template 120, or may be formed so as to cover only a part of the sacrifice template 120.

As a method for forming the base material layer 10, an appropriate method using the solidification of the base material raw material, the curing reaction, or the like can be used depending on the type of the base material raw material. As the raw material of the base material, a material that causes precipitation or adsorption on the surface of the sacrificial template 120 may be used.

The base material layer 10 serving as the base material of the porous body 1 can be provided in an appropriate structure depending on the use of the porous body 1. The base material of the porous body 1 may have a non-porous structure having substantially no fine closed pores other than the communication holes 20 forming the three-dimensional network structure, or may have a three-dimensional network structure. It may have a porous structure having fine closed air holes in addition to the communication holes 20 to be formed.

Further, the base material layer 10 serving as the base material of the porous body 1 may be provided in a bulk shape so as to fill the periphery of the sacrificial template 120 or between the adjacent sacrificial templates 120, or as shown in FIG. 2B. A mesh pattern that fills only the periphery of the sacrificial template 120 and does not fill the space between adjacent sacrificial templates 120, that is, the outer wall portion having a thickness such that the base material layer 10 can see the outline of the sacrificial template 120 around the sacrificial template 120. Can also be formed into a cancellous bone shape in which the outer wall portions of adjacent sacrificial templates 120 are bridged by columnar portions.

When the base material layer 10 serving as the base material of the porous body 1 is provided in a bulk shape, the porous body 1 having relatively high strength can be obtained. On the other hand, when the base material layer 10 serving as the base material of the porous body 1 is provided in a mesh shape, both the inner and outer sides of the communication hole 20 become a surface on which cells easily adhere, so that the density is high with respect to the inner and outer sides of the communication hole 20. Cells can be supported. For example, it is possible to support an endothelial cell or the like inside the communication hole 20 and a connective tissue cell or the like on the outside of the communication hole 20 to form an artificial organ / artificial tissue close to a living tissue.

The base material of the porous body 1 may be formed of a material having bioabsorbability, or may be formed of a material having no bioabsorbability. If the material has bioabsorbability, when the porous body 1 is transplanted into a living body, the porous body 1 is decomposed with the passage of time, so that it is not necessary to remove the transplanted porous body 1 after use. Become. If the material has bioabsorbability, the communication hole 20 may disappear in the living body at an early stage, but if the inside of the communication hole 20 is covered with endothelial cells or the like, the shape of the communication hole 20 may be maintained. On the other hand, if the material does not have bioabsorbability, it is necessary to remove the porous body 1 after use, but since the communication holes 20 are difficult to disappear in the living body, the function of the porous body 1 can be maintained for a long period of time. Can be maintained.

Specific examples of bioabsorbable materials include proteins, peptides, polyamino acids, polysaccharides, polyesters, polyamides and the like. As the material having bioabsorbability, derivatives of these, crosslinked products, copolymers containing them, and the like can also be used. Further, as a material having bioabsorbability, an inorganic substance such as metallic magnesium, calcium carbonate or hydroxyapatite or a metal can also be used. These materials may not exhibit bioabsorbability due to cross-linking or coating.

Examples of proteins, peptides and polyamino acids include collagen, gelatin, α-polylysine, ε-polylysine, polyglutamic acid, polyaspartic acid, fibrin, fibroin and the like. Examples of the polysaccharide include chitin and chitosan. Examples of the polyester include polylactic acid, polycaprolactam, polydioxanone, polyglycolic acid, polyhydroxybutyrate and the like.

Specific examples of materials that do not have bioabsorbability include acrylic resin, fluororesin, polysaccharides, polyolefins, silicones, polystyrenes, polyesters, polyurethanes, polycarbonates, and polyimides. As a material having no bioabsorbability, derivatives of these, crosslinked products, copolymers containing these, and the like can also be used. Further, as a material having no bioabsorbability, an inorganic substance such as titanium, silica or zirconia or a metal can be used. As the material having no bioabsorbability, an organic polymer material is preferable from the viewpoint of specific gravity, hardness, moldability, combination with the sacrificial template 120, and the like.

Examples of the acrylic resin include poly (2-hydroxyethyl acrylate), poly (2-hydroxyethyl methacrylate), poly (methoxyethyl acrylate), poly (methoxyethyl methacrylate), polymethyl acrylate, polymethyl methacrylate, and polyacrylic acid. , Polymethacrylic acid, polyacrylamide, polydimethylacrylamide and the like.

Examples of the fluororesin include polytetrafluoroethylene, polyvinylidene fluoride, and a tetrafluoroethylene / hexafluoropropylene copolymer. Examples of the polysaccharide include agarose, cellulose, hydroxyethyl cellulose, hydrokipropyl cellulose and the like. Examples of the polyolefin include polyethylene, polypropylene, polymethylpentene and the like. Examples of the silicone include polydimethylsiloxane and silicone resin.

The base material of the porous body 1 may be blended with a filler for various purposes such as increasing the strength, reducing the weight of the porous body 1, increasing the amount, and preventing blocking. The filler may have a spherical shape, a plate shape, a flake shape, a needle shape, a fibrous shape, an indefinite shape, or the like, a solid shape, or a hollow shape. The filler can be blended, for example, by forming the base material layer 10 in a state of being dispersed in the precursor solution 200.

Specific examples of fillers include silica, alumina, zirconia, talc, clay, mica, graphite, carbon black, carbon fiber, glass, glass fiber, glass balloon, silas balloon, calcium carbonate, magnesium carbonate, aluminum hydroxide, and hydroxide. Examples thereof include magnesium, titanium oxide, iron oxide, calcium oxide, magnesium oxide, cellulose fiber, aramid fiber, and polyamide fiber.

As shown in FIG. 2B, the base material layer 10 may form the base material of the porous body 1 at the stage of being laminated on the surface of the sacrificial template 120 as long as the shape of the communication hole 20 is fixed. After laminating on the surface of the sacrificial template 120, post-treatments such as a drying treatment for removing the solvent of the precursor solution 200, a polymerization treatment and a cross-linking treatment for solidifying the base material layer 10, and a foaming treatment for making the base material layer 10 porous are performed. The base material of the porous body 1 may be formed at this stage. The complex of the formed sacrificial template 120 and the base material layer 10 is taken out from the liquid tank containing the precursor solution 200, if necessary, and chamfered and cut out for the purpose of improving the removability of the sacrificial template 120. It can also be processed.

As shown in FIGS. 3A to 3B, in the removing step, in order to remove the sacrificial template 120 from the base material layer 10 formed around the sacrificial template 120, an operation of adjusting the state / property of the sacrificial template 120 is performed. In addition, a porous body 1 having communication holes 20 whose shape is fixed by the base material layer 10 is obtained.

FIG. 3A shows a state in which the composite of the sacrificial template 120 and the base material layer 10 is prepared in the liquid tank. FIG. 3B shows a state in which the sacrificial template 120 is removed from the composite of the sacrificial template 120 and the base material layer 10.

As shown in FIG. 3A, the removal of the sacrificial template 120 can be performed in the liquid phase. Note that FIGS. 3A to 3B show an example in which the sacrificial template 120 is not once taken out from the liquid tank containing the precursor solution 200 used for forming the base material layer 10, but is continuously treated in the same liquid tank. ing. However, the sacrificial template 120 may be taken out from the liquid tank containing the precursor solution 200 and processed in another liquid tank, depending on the type of raw material of the sacrificial template 120. It can also be processed in the gas phase or the packed phase.

As a method of removing the sacrificial template 120, for example, a method of melting the sacrificial template 120, a method of heat melting, a method of sublimation, a method of chemical reaction decomposition, a combination thereof, etc., depending on the type of the raw material of the sacrificial template 120, etc. Can be used.

The removal rate of the sacrifice template 120 when removing the sacrifice template 120 is not particularly limited. The removal rate of the sacrificial template 120 is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, from the viewpoint of increasing the porosity of the porous body. .. The removal rate of the sacrificial template 120 can be determined as the dry weight of the porous body 1 from which the sacrificial template 120 has been removed with respect to the dry weight of the composite of the sacrificial template 120 and the base material layer 10.

As shown in FIG. 3B, the sacrificial template 120 is removed from the composite of the sacrificial template 120 and the base material layer 10, and post-treatments such as drying treatment, polymerization treatment, cross-linking treatment, and foaming treatment are performed as necessary. , A porous body 1 in which communication holes 20 having a desired shape and structure are formed can be obtained. The communication holes 20 of the porous body 1 have a three-dimensional network structure formed by connecting long pores having a large aspect ratio or long pores having a large aspect ratio in a vertical cross-sectional view. .. The produced porous body 1 can be subjected to cleaning treatment, drying treatment, surface treatment, molding processing and the like, if necessary.

The communication hole 20 of the porous body 1 preferably has an aspect ratio (ratio of length to diameter) of 10 or more in a vertical cross-sectional view. By forming such a communication hole 20, a cell scaffold or a biocompatible material can be obtained in which only small molecules such as oxygen and nutrients can move without passing through large cells. Further, when the cells are supported in the communication holes 20, a three-dimensional network structure in which the cells are less likely to be biased or dropped can be obtained. Therefore, a cell scaffold that improves the engraftment rate of supported cells and the function of cells can be obtained.

The aspect ratio of the communication hole 20 is defined as the ratio of the average length of the straight line portion to the average diameter. When the communication hole 20 forms a non-linear portion, the aspect ratio is calculated only for the straight portion excluding the non-linear portion. The average value of the length and the diameter is calculated from the measurement results of the communication holes 20 and the portions having a predetermined number of samples or more, with an arbitrary portion of the straight portion as a measurement point. The aspect ratio of the communication hole 20 does not have to be the same as the aspect ratio of the sacrificial template 120 used, and the base material may be partially poorly formed, deformed, broken, or swollen.

In the porous body 1, the volume ratio of the communication holes 20 having an aspect ratio of 10 or more is preferably 50% by volume or more, preferably 60 volumes, based on the total volume of all the pores existing in the base material. It is more preferably 70% by volume or more, and further preferably 70% by volume or more. When the volume fraction of the communication holes 20 having a large aspect ratio is high, a large number of communication holes 20 have a high uniformity of diameter (inner width) and a network structure having many straight portions. Therefore, it is possible to prevent the cells introduced into the communication hole 20 from staying at a specific location and causing a difference in the supply of oxygen / nutrients and the release of waste products.

The communication hole 20 is not particularly limited in the average length, the average diameter, the cross-sectional shape, the bending angle / curvature of the non-linear portion, and the like. The average length, average diameter, cross-sectional shape, bending angle / curvature of the non-linear portion of the communication hole 20 can be determined by the application of the porous body 1, the function of the communication hole 20, the type of raw material of the base material, and the porous body 1. It can be provided under appropriate conditions depending on the type of cells to be carried.

When the communication hole 20 is used as a capillary-like structure for the purpose of transporting small molecules, it is preferable to provide the communication hole 20 so that the average diameter is 10 μm or more and 1000 μm or less. With such a diameter, the communication hole 20 can transport only small molecules such as oxygen and nutrients without passing through large cells, and the cells supported in the three-dimensional network structure are biased. It becomes difficult for it to fall off.

The porous body 1 may have closed pores in addition to the communication holes 20 that communicate with the outside of the porous body 1. The closed pores are caused by partial poor formation of the base material, air bubbles inevitably generated / mixed at the stage of forming the base material, elution of the base material when the sacrificial template 120 is removed, and the like. It may be one that is positively formed by a foaming treatment or the like.

The porosity of the porous body 1 is preferably 20% or more, more preferably 30% or more, still more preferably 50% or more. The porosity of the porous body 1 is preferably 99% or less, more preferably 95% or less, still more preferably 90% or less. The higher the porosity of 20% or more, the larger the amount of cells supported on the porous body 1. In addition, the supply of oxygen and nutrients and the release of waste products are improved. On the other hand, the lower the porosity is 99% or less, the easier it is to secure the mechanical strength of the porous body 1.

The surface of the base material and the inner surface of the communication holes 20 of the porous body 1 may be surface-treated. Examples of the surface treatment include chemical modification treatment, plasma treatment, corona discharge treatment, ultraviolet irradiation treatment, ultraviolet irradiation / ozone treatment, and the like. When the chemical modification treatment is performed, hydrophilicity, biocompatibility, etc. are improved, cell adhesion of the base material is improved, and functionality is imparted to the base material itself, depending on the components used for the chemical modification. be able to. Further, according to plasma treatment, corona discharge treatment, ultraviolet irradiation treatment, and ultraviolet irradiation / ozone treatment, hydrophilicity can be improved and functional groups for chemical modification can be introduced.

As the chemical modification treatment, when the base material is a charged material, electrostatic interaction, etc., the base material has a reactive functional group, covalent bond, etc., the base material is a hydrophobic material. In some cases, hydrophobic interactions and the like can be utilized, respectively. Further, when the base material is a material having a molecular recognition site that specifically binds to a specific molecule, a molecule that is specifically recognized can be used. The chemical modification treatment can be performed by various methods such as a coating coating method, a dip coating method, and a spray coating method. The chemical modification treatment may be carried out using one kind of component or a plurality of kinds of components.

Ingredients used in the chemical modification treatment are derived from adhesion factors / extracellular matrix (ECM) such as collagen, fibronectin, bitronectin, laminin, cadoherin, α-polylysine, and ECM such as gelatin and chemically modified gelatin. Ingredients, components that form specific bonds such as biotin, avidin, streptavidin, protein A, protein G, protein L, antibody, epidermal growth factor (EGF), basic fibroblast growth factor (Basic Fibroblast Growth Factor: bFGF), bone morphogenetic protein (BMP), erythropoietin (EPO), insulin, insulin-like growth factor, transferase, interleukin, tumor necrosis factor (TNF), etc. Physiologically active substances such as growth factors, differentiation factors, hormones and cytokines, antibacterial components such as silver nanoparticles, antioxidant components such as glutathione, vitamin E, catalase and peroxidase, active oxygen trapping components, active oxygen decomposing components and , Metal peroxides, sustained-release oxygen components such as urea hydrogen peroxide, anti-inflammatory components, anti-fibrotic components and the like.

Further, as a component used for the chemical modification treatment, a blocking agent for the functional group of the base material can also be used. Specific examples of the blocking agent include ethanolamine for an activated carboxyl group, a glycidyl group and the like. When a blocking agent is used, it is possible to prevent contamination of the porous 1 due to adsorption / bonding of unnecessary components.

4A to 4E are diagrams schematically showing another example of a method for producing a porous body.
As shown in FIGS. 4A to 4E, the porous body 1 has a precipitation step (see FIGS. 4A to 4C), a base material layer forming step (see FIG. 4D), and a removing step (see FIG. 4E). It can also be performed continuously in the same liquid tank. As the precursor solution 300, a solution containing the raw material of the base material in addition to the raw material of the sacrificial template 120 is used.

FIG. 4A shows a state in which a precursor solution 300 containing the raw material of the base material in addition to the raw material of the sacrificial template 120 is prepared in the liquid tank. FIG. 4B shows a state in which the nucleus 110 of the sacrificial template 120 is formed in the precursor solution 300. FIG. 4C shows a state in which a long sacrificial template 120 having a large aspect ratio is precipitated in the precursor solution 300. FIG. 4D shows a state in which the base material layer 10 is formed around the sacrificial template 120. FIG. 4E shows a state in which the sacrificial template 120 is removed from the composite of the sacrificial template 120 and the base material layer 10.

As shown in FIGS. 4A to 4E, the precursor solution 300 can be prepared in a liquid tank having an appropriate shape. 4A to 4E show a method of forming a base material layer 10 having a thickness such that the outline of the sacrificial template 120 can be seen around the prepared sacrificial template 120. However, the base material layer 10 may be formed in a bulk shape that fills the periphery of the sacrificial template 120 or between adjacent sacrificial templates 120. The liquid tank in which the precursor solution 300 is placed may have the same shape as the outer shape of the porous body 1 to be manufactured, that is, a shape that becomes a molding mold of the porous body 1, or the porous body 1 to be manufactured. The shape may be different from the outer shape of.

When each step is continuously performed in the same liquid tank, the raw material of the sacrificial template 120 and the raw material of the base material may be appropriately combined as long as the precipitation conditions are different from each other and the sacrificial template 120 can be removed. Can be used. For example, a water-soluble low-molecular-weight compound such as urea, a urea derivative, thiourea, a thiourea derivative, or myoban is used as a raw material for the sacrificial template 120, and a protein / peptide / polyamino acid, an acrylic resin, or many other as a raw material for the base material. When a water-soluble polymer compound such as a saccharide is used, precipitation or removal of the sacrificial template 120 and formation of the base material layer 10 can be started simply by changing the temperature using water as a solvent. Therefore, it is possible to avoid the trouble of replacement and the risk of damage to the sacrificial template 120.

5A to 5E are diagrams schematically showing another example of a method for producing a porous body.
As shown in FIGS. 5A to 5E, the porous body 1 can also be produced by using a template material 150 having a different shape and forming method from the long sacrificial template 120 having a large aspect ratio.

The template material 150 is used to mold the internal space 50 having a desired shape and structure in the base material of the porous body 1. When a template material 150 different from the sacrificial template 120 is put in the medium and the sacrificial template 120 is deposited, a long communication hole 20 having a large aspect ratio and an internal space communicating with the plurality of communication holes 20 are formed in the base material. A porous body 1 having 50 and 50 is obtained. In addition, in FIGS. 5A to 5E, the internal space 50 is provided in a structure completely covered with the base material of the porous body 1, but the internal space 50 may be provided in a structure communicating with the outside. ..

FIG. 5A shows a state in which the precursor solution 300 containing the raw material of the base material in addition to the raw material of the sacrificial template 120 and the template material 150 are prepared in the liquid tank. FIG. 5B shows a state in which the nucleus 110 of the sacrificial template 120 is formed in the precursor solution 300. FIG. 5C shows a state in which a long sacrificial template 120 having a large aspect ratio is precipitated in the precursor solution 300. FIG. 5D shows a state in which the base material layer 10 is formed around the sacrificial template 120 and the template material 150. FIG. 5E shows a state in which the sacrificial template 120 and the template material 150 are removed from the composite of the sacrificial template 120, the template material 150, and the base material layer 10.

As shown in FIG. 5A, the precursor solution 300 containing the raw material of the base material in addition to the raw material of the sacrificial template 120 and the template material 150 can be prepared in a liquid tank having an appropriate shape. In FIGS. 5A to 5E, a liquid tank having the same shape as the outer shape of the porous body 1 to be manufactured, that is, a shape that becomes a molding mold for the porous body 1, is used. However, a liquid tank having a shape different from the outer shape of the porous body 1 to be manufactured may be used.

The template material 150 can be formed of any of a polymer compound, an inorganic compound, a metal, and the like as long as it can be removed from the precursor solution 300 after the sacrificial template 120 is precipitated. The shape, size, texture, etc. of the template material 150 are not particularly limited. The template material 150 can be prepared as a preformed molded product using the same material as the sacrificial template 120 or a material different from the sacrificial template 120.

As the raw material for forming the template material 150, polystyrene, polysaccharides and the like are preferable. Examples of the polysaccharide include agarose, hydroxyethyl cellulose, hydrokipropyl cellulose and the like. When polystyrene is used, molding is relatively easy, and it can be easily dissolved in a solvent such as toluene or limonene. Further, when a polysaccharide is used, it is often easily dissolved in a solvent such as water or warm water, so that the template material 150 can be easily removed.

As shown in FIGS. 5B and 5C, the core 110 of the sacrificial template 120 and the long sacrificial template 120 having a large aspect ratio are necessary for forming the communication holes 20 in the precursor solution 300 and on the surface of the template material 150. It can be precipitated so as to have an appropriate number density. Although the spherical nucleus 110 is shown in FIG. 5B, the form of precipitation is not limited to crystallization from the liquid phase, but may be phase separation of solid components such as spherical, fibrous, and amorphous. Good. As an operation for precipitating the solid component, an appropriate operation can be used depending on the type of the raw material of the sacrificial template 120.

The template material 150 may be provided with a surface structure for controlling the direction of crystal growth of the sacrificial template 120, the frequency of formation of nuclei 110, the orientation of crystals and molecules, and the like. As the surface structure, for example, a structure in which a predetermined crystal plane is exposed, or a structure in which a substance such as a metal is attached or a surface treatment such as chemical modification is applied can be used. When such a surface structure is provided, the sacrificial template 120 can be deposited on the surface of the template material 150, so that the porous body 1 in which the communication holes 20 are connected to the internal space 50 can be obtained.

As shown in FIG. 5C, the sacrifice template 120 is a structure having a three-dimensional network structure by connecting long straight parts having a large aspect ratio or long straight parts having a large aspect ratio in a vertical cross-sectional view. Therefore, the precipitate can be grown and formed in the precursor solution 100 so that a complex in contact with or entangled with the template material 150 is formed. The prepared sacrificial template 120 and the template material 150 can be taken out from the liquid tank containing the precursor solution 300 and subjected to cleaning treatment, drying treatment and the like, if necessary.

As shown in FIG. 5D, in the base material layer 10, the shapes of the communication holes 20 and the internal space 50 are fixed by covering the periphery of the sacrificial template 120 and the template material 150 to some extent, and the porous body 1 As long as the mechanical strength of the sacrificial template 120 or the template material 150 is ensured, it may be formed so as to cover the entire sacrificial template 120 or the template material 150, or may be formed so as to cover only a part of the sacrificial template 120 or the template material 150. .. In FIGS. 5A to 5E, the bulk-shaped base material layer 10 that completely fills the periphery of the prepared sacrificial template 120 and the template material 150 is formed, but the contours of the sacrificial template 120 and the template material 150 can be seen. A base layer 10 having a thickness may be formed.

As shown in FIG. 5E, the sacrificial template 120 and the template material 150 are removed from the composite of the sacrificial template 120, the template material 150, and the base material layer 10, and if necessary, a drying treatment, a polymerization treatment, a cross-linking treatment, and foaming are performed. When post-treatment such as treatment is performed, a porous body 1 in which the communication holes 20 having the desired shape and structure and the internal space 50 are formed can be obtained. The communication holes 20 of the porous body 1 are formed by connecting long pores having a large aspect ratio or long pores having a large aspect ratio in a vertical cross-sectional view, and are formed with respect to the internal space 50. It becomes a connected three-dimensional network structure. The produced porous body 1 can be subjected to cleaning treatment, drying treatment, surface treatment, molding processing and the like, if necessary.

6A to 6C are diagrams schematically showing specific examples of a method for producing a porous body.
As shown in FIGS. 6A to 6C, the porous body 1 can also form a blood vessel-like structure by the communication holes 20 so as to have high compatibility with the living body to be transplanted. 6A to 6C show an example in which an artificial organ is formed by using a columnar template material 160 having a shape and a different forming method from the long sacrificial template 120 having a large aspect ratio.

The columnar template material 160 is used for molding a columnar space 60 having a diameter larger than that of the communication hole 20 and having a shape and structure substantially crossing the porous body 1 in the base material of the porous body 1. When a columnar template material 160 different from the sacrificial template 120 is placed in the medium to deposit the sacrificial template 120, a long communication hole 20 having a large aspect ratio and a columnar structure communicating with the plurality of communication holes 20 are deposited in the base material. A porous body 1 having a space of 60 is obtained.

FIG. 6A shows a state in which the columnar template material 160 is arranged in the molding die 500 for molding the porous body 1 into a desired outer shape. FIG. 6B shows a state in which the base material layer 10 is formed around the sacrificial template 120 and the columnar template material 160. FIG. 6C shows a state in which the sacrificial template 120 and the columnar template material 160 are removed from the composite of the sacrificial template 120, the columnar template material 160, and the base material layer 10, and the molding die 500 is removed.

As shown in FIG. 6A, as the liquid tank used for precipitating the sacrificial template 120 and forming the base material layer 10, a molding die 500 for molding the porous body 1 into a desired outer shape can be used. In the molding dies 500 shown in FIGS. 6A to 6C, a joining piece 40 that can be fitted with the columnar template material 160 is attached to the side wall thereof so as to project laterally from the inside of the molding dies 500.

The mold 500 can be formed of any of glass, plaster, soft resin, hard resin, ceramic, metal and the like. In FIGS. 6A to 6C, an artificial organ-like molding die is shown as an example of the molding die 500. However, the shape, size, and the like of the molding die 500 are not particularly limited. The molding die 500 may be provided as a closed mold or a split mold as long as the columnar template material 160 can be attached and the sacrificial template 120 and the columnar template material 160 can be removed. The joint piece 40 can be provided in a tubular shape, a split tubular shape, or the like according to the molding die 500.

The mold 500 is a material that can be easily crushed such as plaster, a material that can be easily deformed such as a soft resin, a material that can dissolve the mold 500 itself, and a material that can heat-melt the mold 500 itself. It is preferable to form by the above. Further, the mold release agent may be applied to the inner surface of the molding die 500.

When the molding die 500 is formed of a crushable material, the produced porous body 1 can be easily taken out by crushing the molding die 500. Further, when the molding die 500 is formed of a deformable material, the produced porous body 1 can be easily taken out by deforming the molding die 500. Even when the outer shape of the porous body 1 is complicated, damage at the time of taking out can be avoided.

As the molding die 500, a molding die formed three-dimensionally using a 3D printer is preferably used. When a medium such as a precursor solution is prepared in the molding die 500 three-dimensionally molded using a 3D printer and the base material layer 10 is formed in the shape of the molding die 500, the porous body 1 having a complicated shape is produced with good productivity. Can be manufactured at.

As a three-dimensional modeling method, an appropriate method such as powder sintering modeling, resin melt modeling, stereolithography, and inkjet modeling can be used depending on the type of material of the molding die 500. According to the three-dimensional modeling using a 3D printer, a molding die 500 having a hollow structure, an internal independent structure, and the like, and a molding die 500 suitable for attaching a columnar template material 160 and the like can be accurately and stably manufactured.

As shown in FIG. 6A, the columnar template material 160 is fitted and supported by the joint piece 40, and is arranged so as to substantially traverse the inside of the molding die 500. When the columnar template material 160 is arranged in this way, a columnar space 60 having a shape and structure substantially traversing the porous body 1 is formed, so that an arterial-like / vein-like blood vessel-like structure is formed in the porous body 1. be able to.

The columnar template material 160 has a diameter larger than that of the communication hole 20, and the shape, size, texture, and the like are not particularly limited as long as the columnar template material 160 has a shape and structure that substantially traverses the porous body 1. Like the template material 150, the columnar template material 160 can be prepared as a preformed molded body using the same material as the sacrificial template 120 or a material different from the sacrificial template 120.

As shown in FIG. 6B, the sacrifice template 120 is a structure having a three-dimensional network structure by connecting long straight parts having a large aspect ratio or long straight parts having a large aspect ratio in a vertical cross-sectional view. Therefore, the precipitate can be grown and formed in the precursor solution so that a complex in contact with or entangled with the columnar template material 160 is formed in the molding die 500.

As shown in FIG. 6C, the sacrificial template 120 and the columnar template material 160 are removed from the composite of the sacrificial template 120, the columnar template material 160, and the base material layer 10, and if necessary, a drying treatment, a polymerization treatment, and a cross-linking treatment are performed. When post-treatment such as foaming treatment is performed, a porous body 1 in which the communication holes 20 having the desired shape and structure and the columnar space 60 are formed can be obtained. The communication holes 20 of the porous body 1 are formed by connecting long pores having a large aspect ratio or long pores having a large aspect ratio in a vertical cross-sectional view, and are formed with respect to the columnar space 60. It becomes a connected three-dimensional network structure.

After the porous body 1 is produced, the molding die 500 can be separated / removed from the porous body 1 by leaving the bonded piece 40 in a state of being bonded to the base material. The junction piece 40 can be formed of, for example, a biocompatible material such as polytetrafluoroethylene or polyester. When such a joint piece 40 is provided, the joint piece 40 can function as an artificial blood vessel.

7A-7B are diagrams illustrating anastomosis of a porous body into a blood vessel.
As shown in FIGS. 7A to 7B, the porous body 1 having a blood vessel-like structure formed by the communication holes 20 can be connected to the blood vessel 80 of the transplant destination so that the communication holes 20 form a blood circulation path.

FIG. 7A shows a state in which the porous body 1 is transplanted into a living body in which the blood vessel 80 is present. FIG. 7B shows a state in which the blood vessel 80 at the transplant destination and the junction piece 40 of the porous body 1 are connected.

The porous body 1 can be surgically anastomosed to the blood vessel 80 to be transplanted via the junction piece 40 bonded to the base material. When such a porous body 1 is connected to the blood vessel 80 of the transplant destination, the blood vessel-like structure formed by the communication hole 20 and the columnar space 60 becomes a part of the blood circulation pathway on the living body side, so that the porous body 1 is formed. Blood flow to the supported cells is ensured. Therefore, the engraftment rate of cells supported on the porous body 1 and the function of cells can be improved.

<Device>
The porous body 1 according to the present embodiment can be used as a device by combining it with a base material or other elements other than cells. Applications of the device include a transplant device that functions in vivo, a cell device that supports cells and utilizes the functions of cells, a bioreactor for recovering components secreted by cells, and the like.

The transplanted device can be transplanted into a human body or an animal body other than humans to function as a cell device, a bioreactor, or the like in the living body. When the transplant device is transplanted, if the communication hole 20 is connected to the circulatory system such as the blood vessel of the transplant destination, the three-dimensional network structure of the porous body 1 can be effectively used as a substance transport route. On the other hand, cell devices and bioreactors can also be used in vitro.

Other elements to be composited include a base material for controlling the direction of crystal growth of the sacrificial template 120, the frequency of formation of nuclei 110, the orientation of crystals and molecules, and a reinforcing material for reinforcing the shape of the porous body 1. , Sensors that measure the external and internal environment of the porous body 1, batteries that supply power to electronic components in the device, electrodes used for electrical stimulation of sensor components and living organisms, and porous bodies. 1 Heater that adjusts the external environment and internal environment, chips that function the device, drug sustained-release agents that can administer drugs to living organisms, oxygen sustained-release agents that supply oxygen to cells carried in the device, etc. Can be mentioned.

<Cell carrier>
The porous body 1 according to the present embodiment can be used as a cell carrier by introducing cells into the communication hole 20 and supporting the cells on the porous body 1. The cell carrier can be transplanted into a human body or an animal body other than humans and used as a cell scaffold for utilizing cell functions in the living body. Examples of cell functions include secretion of physiologically active substances such as hormones and cytokines and nutrients, metabolism / decomposition of specific substances, and substitution of normal cell functions.

The cell carrier can be used for in vitro applications in addition to transplantation into a living body. For example, it can be used for maintenance culture of cells, expansion culture for proliferating cells, induction culture for differentiating and inducing cells, and the like.

In the porous body 1 used as a cell carrier, cells may be introduced in vitro before transplantation, or cells may be introduced in vivo after transplantation. If the porous body 1 is provided with a communication hole 20 capable of allowing cells to enter, the recipient cells can migrate and adhere to the inside of the porous body 1 even after transplantation. , Can function as a cell scaffold in vivo.

The type, origin, morphology, etc. of the cells to be supported are not particularly limited. The cells may be any of primary cells collected directly from a tissue or the like, pre-cultured cultured cells, proliferative cell lines, recombinant cells, ES cell-derived cells, iPS cell-derived cells, or the like. .. Further, it may be a single free cell or a cell aggregate (spheroid).

Specific examples of cells include vascular endothelial cells, hepatocytes, bile duct cells, renal cells, mesenchymal stem cells, nerve cells, pancreatic islands, pancreatic α cells, pancreatic β cells, established α cells, established β cells, and the like. Examples include pancreatic cells.

The cells to be supported may be adherent cells or floating cells, but adherent cells are preferable because they are easily retained in the porous body 1.

According to the above-mentioned porous body, the method for producing the porous body, the method for transplanting the porous body, and the cell carrier using the above, the base of the porous body is based on the sacrificial template having a large aspect ratio precipitated in the medium. Since the material is provided with communication holes formed by pores having a large aspect ratio, cells can be supported densely and discretely inside the porous body, and the cells can be maintained healthy for a long period of time. .. In addition, the outer shape of the porous body and the shape and structure of the communication holes can be freely shaped according to the space and application of the transplant destination, and such modeling can be easily performed. In addition, excellent substance permeability can be obtained even when cells are not supported. Therefore, a method for producing a porous body having three-dimensional network-structured communication holes and useful as a cell scaffold or a biocompatible material, a method for transplanting a porous body, and a cell carrier using the same can be obtained. ..

Although the present invention has been described above, the present invention is not limited to the above-described embodiments and modifications, and various modifications can be made without departing from the spirit of the present invention. For example, the present invention is not necessarily limited to those having all the configurations included in the above-described embodiments and modifications. Part of the configuration of a certain embodiment or modification is replaced with another configuration, a part of the configuration of a certain embodiment or modification is added to another configuration, or a configuration of a certain embodiment or modification. You can omit a part of.

Hereinafter, the present invention will be specifically described with reference to examples, but the technical scope of the present invention is not limited thereto.

<Example 1>
In Example 1, a porous body was prepared using thiourea as a raw material for a sacrificial template and a crosslinked product of polyamine and polycarboxylic acid as a base material for the porous body.

Thiourea (0.9 g), ε-polylysine (0.1 g), and polyacrylic acid (0.2 g) were added to 4 mL of water, and this solution was heated to 80 ° C. to obtain a uniform solution. Then, this solution was cooled to precipitate acicular crystals of thiourea, which is a sacrificial template. Subsequently, the solution in which the sacrificial template was precipitated was freeze-dried to remove the solvent, and a substrate layer composed of ε-polylysine and polyacrylic acid was formed around the acicular crystals of thiourea. Then, the complex of the sacrificial template and the base material layer was treated with methanol to elute the sacrificial template thiourea. Then, it was treated with a methanol solution of 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride (DMT-MM), which is a condensing agent, and ε-. Polylysine and polyacrylic acid were crosslinked. The porous substrate obtained in the container was washed with water, and then the excess carboxylic acid was neutralized to obtain a porous body.

The porosity of the porous body according to Example 1 was 60%. The average aspect ratio of the communication holes in the porous body was 20.

The porous body according to Example 1 was surface-modified by the following procedure. A 0.1% by mass α-polylysine solution was added to the prepared porous body, and the mixture was allowed to stand at room temperature for 12 hours to introduce α-polylysine by electrostatic interaction on the surface where excess carboxylic acid was present. .. It was confirmed that α-polylysine did not dissociate from the surface of the porous body even when washed with a phosphate buffer having a pH of 7.4.

<Example 2>
In Example 2, a porous body was prepared using urea as a raw material for the sacrificial template and a crosslinked body of an acrylic polymer as a base material for the porous body.

Urea (3.3 g) was added to 4 mL of water, and this solution was heated to 80 ° C. to obtain a uniform solution. Then, this solution was cooled to precipitate acicular crystals of urea, which is a sacrificial template. Subsequently, the solvent is removed from the solution in which the sacrificial template is precipitated, a mixed solution containing glycidyl methacrylate, ethylene glycol dimethacrylate, and a thermal polymerization initiator is added to the container filled with the sacrificial template, and the mixture is heated to 60 ° C. A substrate layer made of a crosslinked acrylic polymer was formed around the acicular crystals of urea by polymerization. Then, the complex of the sacrificial template and the substrate layer was treated with methanol to elute the sacrificial template urea. The porous base material obtained in the container was washed with water to obtain a porous body.

The porosity of the porous body according to Example 2 was 75%. The average aspect ratio of the communication holes in the porous body was 50.

The porous body according to Example 2 was surface-modified by the following procedure. A 1% by mass gelatin solution was added to the prepared porous body, and the mixture was reacted at 60 ° C. for 12 hours to introduce gelatin on the surface where the glycidyl group was present by reaction with an amine.

<Example 3>
In Example 3, a porous body was prepared using phenylurea as a raw material for the sacrificial template, a crosslinked body of modified collagen as a base material for the porous body, and expanded polystyrene as a template material for molding the internal space. ..

Phenylurea (0.7 g) was added to 4 mL of water, and this solution was heated to 80 ° C. to obtain a uniform solution. Then, this solution was cooled to precipitate acicular crystals of phenylurea, which is a sacrificial template. Subsequently, the solvent is removed from the solution in which the sacrificial template is precipitated, a mixed solution of collagen and sodium chloride at 4 ° C. is added to a container filled with the sacrificial template, and the mixture is heated to 37 ° C. to gel. It was. Then, this gel was freeze-dried to remove the solvent, and a base material layer made of collagen was formed around the acicular crystals of phenylurea. Then, the complex of the sacrificial template and the base material layer was treated with methanol to denature collagen and elute the sacrificial template phenylurea. Then, it was treated with a cross-linking agent to cross-link the denatured collagens. The porous base material obtained in the container was washed with water to obtain a porous body.

The porosity of the porous body according to Example 3 was 60%. The average aspect ratio of the communication holes in the porous body was 30.

<Example 4>
In Example 4, a porous body having a blood vessel-like structure (see FIG. 6C) having a molded outer shape was produced by using a molding die three-dimensionally molded using a 3D printer and a columnar template material.

The columnar template material was formed by cutting expanded polystyrene. As the molding die, an organ-shaped molding die composed of two upper and lower parts was three-dimensionally molded using plaster as a raw material using a 3D printer. As shown in FIG. 6A, a columnar template material was attached to the lower mold, and the upper mold was placed over the columnar template material to form an organ-shaped plaster mold. This gypsum mold is heated to 60 ° C., and an aqueous solution at 80 ° C. containing thiourea, ε-polylysine, monoma acrylate and 4,4'-azobis (4-cyanovaleric acid) (AVCA) is injected to monoma acrylate. Was polymerized. Then, this aqueous solution was slowly cooled to precipitate acicular crystals of thiourea, which is a sacrificial template. Subsequently, the gypsum mold on which the sacrificial template was precipitated was freeze-dried to remove the solvent, and a substrate layer composed of ε-polylysine and polyacrylic acid was formed around the acicular crystals of thiourea. Then, expanded polystyrene, which is a columnar template material, was treated with toluene and eluted. Subsequently, the complex of the sacrificial template and the substrate layer was treated with a methanol solution of DMT-MM as a condensing agent to crosslink ε-polylysine and polyacrylic acid. Then, the complex of the sacrificial template and the base material layer was treated with methanol to elute the sacrificial template thiourea. The porous body obtained in the container is taken out by crushing a plaster mold, the surface of the porous body is coated with a coating solution in which highly viscous silicone macromonoma is dissolved, and the monoma is polymerized and cured to obtain the porous body. It was.

The porous body according to Example 4 has a capillary-like structure by the sacrificial template and an arterial / vein-like structure by the columnar template material, and liquid leakage occurs except for the arterial / vein-like part by the columnar template material. There was no structure.

<Example 5>
In Example 5, a cell carrier was prepared using the porous body according to Example 1 which had undergone surface modification and cells.

The surface-modified porous body according to Example 1, cells, and medium were placed in a deep dish. Then, the dish was swirled and stirred at a rotation speed of 30 rpm for 12 hours to introduce cells into the porous body. After the cells had settled inside the porous body, the swirling stirring was stopped and the culture was started in a static culture. During the static culture, the medium was exchanged every two days, and the number of cells in the collected medium was determined based on the glucose consumption.

As a result, regardless of whether V79 cells or mesenchymal stem cells were supported as cells, cell proliferation proceeded and reached approximately confluence 5 days after the transition to static culture. It was confirmed that the surface-modified porous body according to Example 1 had sufficient biocompatibility.

<Comparative example 1>
In Comparative Example 1, a needle-shaped magnesium salt was used as a raw material for the sacrificial template, and a crosslinked acrylic polymer was used as a base material for the porous body, and as in Example 4, it had a blood vessel-like structure having a molded outer shape. A porous body (see FIG. 6C) was prepared.

The gypsum mold was heated to 60 ° C. in the same manner as in Example 4, and a mixed solution containing acicular magnesium salt, glycidyl methacrylate, ethylene glycol dimethacrylate, and a thermal polymerization initiator was injected. As a result, acicular magnesium salts aggregated around the columnar template material near the site where the mixed solution was injected. When the gypsum mold was heated to polymerize the monoma acrylate, and then the acicular magnesium salt was eluted and then the gypsum mold was crushed, the vicinity of the site where the acicular magnesium salt was agglomerated was of mechanical strength. Damaged due to lack. The bottom side of the plaster mold was filled with needle-shaped magnesium salt, while the other parts were not filled with needle-shaped magnesium salt.

<Comparative example 2>
In Comparative Example 2, urea was used as a raw material for the sacrificial template, and a crosslinked product of polyamine and polycarboxylic acid was used as the base material for the porous body. The base material was cured and then the sacrificial template was precipitated. Was produced.

Urea (3 g), ε-polylysine (0.1 g), polyacrylic acid (0.2 g), DMT-MM (0.01 g) are added to 4 mL of water, and this solution is heated to 80 ° C. to ε. -Polylysine and polyacrylic acid were crosslinked. Then, this solution was slowly cooled to precipitate acicular crystals of urea, which is a sacrificial template. Subsequently, the solution in which the sacrificial template was precipitated was freeze-dried to remove the solvent, and then treated with methanol to elute urea, which is the sacrificial template. When the porous substrate obtained in the container was washed with water, the crosslinked body absorbed water and returned to its original shape, so that the communication holes molded by the sacrificial template were lost.

<Comparative example 3>
In Comparative Example 3, similarly to Example 3, phenylurea was used as a raw material for the sacrificial template, and a crosslinked product of modified collagen was used as a base material for the porous body, and a short sacrificial template having a small aspect ratio was precipitated to be porous. A template was prepared.

Phenylurea (0.7 g) and calcium chloride for growing phenylurea crystals to a low aspect ratio were added to 4 mL of water, and this solution was heated to 80 ° C. to obtain a uniform solution. Then, this solution was cooled to precipitate acicular crystals of phenylurea, which is a sacrificial template. As a result, the distribution of the sacrificial template in solution was biased. Subsequently, the solvent is removed from the solution in which the sacrificial template is precipitated, a mixed solution of collagen and sodium chloride at 4 ° C. is added to a container filled with the sacrificial template, and the mixture is heated to 37 ° C. to gel. It was. Then, this gel was freeze-dried to remove the solvent, and a base material layer made of collagen was formed around the acicular crystals of phenylurea. Then, the complex of the sacrificial template and the base material layer was treated with methanol to denature collagen and elute the sacrificial template phenylurea. Then, it was treated with a cross-linking agent to cross-link the denatured collagens. When the porous base material obtained in the container was washed with water, the porous body collapsed due to lack of mechanical strength.

1 Porous body 10 Base material layer 20 Communication hole 40 Bonding piece 50 Internal space 60 Columnar space 100 Precursor solution 110 Nuclear 120 Sacrificial template 150 Template material 160 Columnar template material 200 Precursor solution 300 Precursor solution 500 Molding mold

Claims

A porous body having communication holes in the base material,
The communication holes are formed by pores having an aspect ratio of 10 or more, or by connecting the pores.
The base material is a porous body made of a biocompatible material.
The porous body according to claim 1.
In the communication holes, the proportion of the pores having an aspect ratio of 10 or more is 50% by volume or more.
The porous body is a porous body in which the porosity of the space occupied by the communication holes is 30% or more.
The porous body according to claim 1.
A porous body having the communication holes and internal spaces communicating with the plurality of communication holes in the base material.
The porous body according to any one of claims 1 to 3.
The base material is a porous body made of a material that does not have bioabsorbability.
The porous body according to claim 4.
The material is a porous body which is acrylic resin, fluororesin, polysaccharide, polyolefin, silicone, polyester, polyurethane, polycarbonate, polyimide, silica, or zirconia.
The porous body according to any one of claims 1 to 3.
The base material is a porous body made of a material having bioabsorbability.
The porous body according to claim 6.
The material is collagen, gelatin, α-polylysine, ε-polylysine, polyglutamic acid, polyaspartic acid, polylactic acid, polycaprolactam, polydioxanone, derivatives thereof, crosslinked or copolymers, or porous metal magnesium. body.
A method for producing a porous body having communication holes in a base material.
A process of precipitating a sacrificial template having an aspect ratio of 10 or more in a medium,
A step of forming a base material layer to be the base material around the sacrifice template, and
A method for producing a porous body, which comprises a step of removing the sacrificial template from the formed base material layer.
The method for producing a porous body according to claim 8.
The sacrificial template is a method for producing a porous body formed of urea, a urea derivative, thiourea, or a thiourea derivative.
The method for producing a porous body according to claim 8.
A method for producing a porous body, in which an additive for controlling the precipitation direction of the sacrificial template is added to the medium.
The method for producing a porous body according to claim 8.
A method for producing a porous body in which a template material different from the sacrificial template is put in the medium to precipitate the sacrificial template.
The method for producing a porous body according to claim 8.
A method for producing a porous body, which comprises a step of surface-modifying the base material from which the sacrificial template has been removed.
The method for producing a porous body according to claim 8.
The medium is prepared in a molding die three-dimensionally modeled using a 3D printer.
A method for producing a porous body in which the base material layer is formed in the shape of the molding mold.
A method for transplanting a porous body having communication holes in a base material.
A method for transplanting a porous body according to any one of claims 1 to 7, wherein the porous body is transplanted into the body of an animal other than humans.
The porous body according to any one of claims 1 to 7.
A cell carrier comprising cells supported on the porous body.