JP7582610B2 - Method for producing biomaterials, method for treating scaffolds, treatment liquid and aqueous solution - Google Patents

Description

The present invention relates to a method for producing a biomaterial, a method for treating a scaffold, a treatment liquid, and an aqueous solution.

In recent years, extracellular matrices derived from decellularized animal tissues, i.e., decellularized extracellular matrices, have been attracting attention as biomaterials. Because decellularized extracellular matrices are derived from living organisms, they are less likely to cause rejection in the body, and research and commercialization of such matrices as adhesion inhibitors and transplant materials for various organs or blood vessels is underway (Patent Documents 1 to 3).

Special Publication No. 2013-536738 JP 2016-144470 A International Publication No. 2018/186502

However, after extensive research, the inventors found that there is still room for improvement in the biocompatibility of biomaterials using decellularized extracellular matrix. For example, when a material obtained by culturing endothelial cells and fibroblasts using the decellularized extracellular matrix as a scaffold is transplanted into a living body, blood perfusion can cause blood clots and block blood vessels.

The present invention has been made in consideration of the above circumstances, and aims to provide a method for producing a biomaterial with improved biocompatibility, or a method for treating a scaffold to improve biocompatibility. The present invention also aims to provide a treatment liquid or aqueous solution that can improve the biocompatibility of a biomaterial.

The method for producing a biomaterial of the present invention includes a step of treating a scaffold containing a decellularized extracellular matrix with a hydroxyl-containing polymer having a group represented by formula (I) below.
*-O-Z...(I)
(In formula (I), Z is a monovalent group in which two or more hydrogen atoms of a monovalent hydrocarbon group having 3 to 8 carbon atoms have been substituted with hydroxyl groups, and * represents the bonding position of the monovalent group.)

It is preferable that the scaffold comprises cells engrafted on the decellularized extracellular matrix.

The method for producing a biomaterial of the present invention preferably further comprises a step of recellularizing the treated scaffold.

The decellularized extracellular matrix is preferably derived from an organ or blood vessel.

The decellularized extracellular matrix is preferably derived from the liver or a part of the liver.

It is preferable that the hydroxyl group-containing polymer contains a structural unit derived from a monomer having a group represented by formula (I) and an ethylenically unsaturated group.

In the method for producing a biomaterial of the present invention, it is preferable to carry out the treatment with a solution containing the above-mentioned hydroxyl group-containing polymer.

In the method for producing a biomaterial of the present invention, it is preferable that the decellularized extracellular matrix has a portion derived from a blood vessel, and the treatment is performed by injecting the solution into the lumen of the portion derived from the blood vessel, or by circulating the solution within the lumen of the portion derived from the blood vessel.

The method for treating a scaffold of the present invention comprises the step of treating a scaffold comprising a decellularized extracellular matrix with a hydroxyl-containing polymer having a group represented by formula (I) below.
*-O-Z...(I)
(In formula (I), Z is a monovalent group in which two or more hydrogen atoms of a monovalent hydrocarbon group having 3 to 8 carbon atoms have been substituted with hydroxyl groups, and * represents the bonding position of the monovalent group.)

The treatment solution of the present invention is a treatment solution for treating a scaffold containing a decellularized extracellular matrix, and contains a hydroxyl group-containing polymer having a group represented by the following formula (I):
*-O-Z...(I)
(In formula (I), Z is a monovalent group in which two or more hydrogen atoms of a monovalent hydrocarbon group having 3 to 8 carbon atoms have been substituted with hydroxyl groups, and * represents the bonding position of the monovalent group.)

The aqueous solution of the present invention contains a hydroxyl-containing polymer having a group represented by the following formula (I) and a buffer.
*-O-Z...(I)
(In formula (I), Z is a monovalent group in which two or more hydrogen atoms of a monovalent hydrocarbon group having 3 to 8 carbon atoms have been substituted with hydroxyl groups, and * represents the bonding position of the monovalent group.)

According to the present invention, it is possible to provide a method for producing a biomaterial with improved biocompatibility, or a method for treating a scaffold to improve biocompatibility. In addition, according to the present invention, it is possible to provide a treatment liquid or aqueous solution that can improve the biocompatibility of a biomaterial.

The method for producing a biomaterial of this embodiment includes a step of treating a scaffold containing a decellularized extracellular matrix with a hydroxyl-containing polymer having a group represented by the following formula (I): The method for producing a biomaterial of this embodiment is carried out outside the body (i.e., ex vivo or in vitro).
*-O-Z...(I)
(In formula (I), Z is a monovalent group in which two or more hydrogen atoms of a monovalent hydrocarbon group having 3 to 8 carbon atoms have been substituted with hydroxyl groups, and * represents the bonding position of the monovalent group.)
According to such a production method, it is possible to provide a biomaterial having superior biocompatibility, and in particular, it is likely to be possible to provide a biomaterial having superior antithrombotic properties.

In the following, the decellularized extracellular matrix may be referred to as decellularized ECM, and the hydroxyl-containing polymer having a group represented by the above formula (I) may be referred to as hydroxyl-containing polymer (I).

<Decellularized ECM>
The scaffold of this embodiment is a material for engrafting and culturing cells, and includes a decellularized extracellular matrix. The decellularized ECM is an extracellular matrix remaining after removing living cells from a tissue or organ derived from a living body (i.e., decellularization). The animal is preferably a mammal, such as a human, a rat, a mouse, a guinea pig, a marmoset, a rabbit, a dog, a cat, a sheep, a pig, a horse, a cow, a goat, a monkey, a chimpanzee, etc., and these may be immunodeficient animals.

The decellularized ECM may be derived from an animal organ or tissue, specifically from an animal's internal organs, blood vessels, or skin (or dermis). Organs include the heart, intestines, trachea, lungs, liver, kidneys, stomach, pancreas, esophagus, etc. The decellularized ECM may be derived from a part of these organs, or from the entirety of them.

Blood vessels include veins, arteries, portal veins, capillaries, etc.

The method for producing decellularized ECM is not particularly limited, and can be appropriately selected depending on the organ or tissue, animal species, etc. used. Specific examples include cell membrane destruction by mechanical stimuli such as high pressure, freezing, electroporation, and osmotic pressure changes, washing and cell destruction with a decellularization solution, etc. These methods may be used alone or in combination. When used in combination, for example, a method in which an animal's organ or tissue is frozen and then thawed to destroy the cell membrane of the cells contained in the organ or tissue, and the organ or tissue is perfused with a decellularization solution to further destroy and wash away the cells, can be mentioned.

The temperature at which the organ or tissue is frozen may be, for example, -10°C or lower, -20°C or lower, -30°C or lower, -40°C or lower, -50°C or lower, -60°C or lower, -70°C or lower, -80°C or lower, -90°C or lower, or -100°C or lower. The temperature at which the organ or tissue is frozen may be, for example, -150°C to -10°C, -130°C to -15°C, or -100°C to -20°C. The temperature at which the frozen organ or tissue is thawed may be, for example, 4°C to 50°C, 4°C to 45°C, or 4°C to 40°C.

Examples of the decellularization solution include an aqueous solution in which a surfactant, an acidic compound, an alkaline compound, an enzyme, a chelating agent, or an alcohol is dissolved in water, physiological saline, or the like. As the surfactant, either an ionic surfactant or a nonionic surfactant may be used, or these may be used in combination. Examples of the ionic surfactant include anionic surfactants such as sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl, and Triton (registered trademark) X-200, and zwitterionic surfactants such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Examples of the nonionic surfactant include Triton (registered trademark) X-100, n-dodecyl-β-D-maltoside (DDM), digitonin, Tween (registered trademark) 20, and Tween (registered trademark) 80. After perfusion, the obtained decellularized ECM may be washed with water or physiological saline to wash away the decellularization solution. Examples of enzymes include nucleic acid degrading enzymes such as DNase and RNase, proteolytic enzymes such as trypsin and collagenase, and α-galactosidase.

The decellularized ECM may be one that has been subjected to a protein cross-linking treatment. If the decellularized ECM has been subjected to a protein cross-linking treatment, the mechanical strength of the decellularized ECM is improved, which is preferable. The protein cross-linking treatment may be a treatment using an external cross-linking agent that bonds with two or more protein molecules to cross-link the protein molecules together. The protein cross-linking treatment may also be a treatment in which protein molecules are reacted with each other to form bonds such as disulfide bonds between amino acid residues contained in the protein molecules to directly cross-link them.

The scaffold may contain cells engrafted on the decellularized ECM. That is, the decellularized ECM contained in the scaffold may be recellularized in advance before being treated with the hydroxyl group-containing polymer (I). Examples of cells engrafted on the decellularized ECM include endothelial cells, epithelial cells, fibroblasts, parenchymal cells, and the like. In particular, when the decellularized ECM is derived from the liver or a part of the liver, examples of cells that form liver tissue include vascular endothelial cells, bile duct epithelial cells, hepatic parenchymal cells, hepatic progenitor cells, Kupffer cells, similar endothelial cells, and stellate cells.
The method of recellularization is not particularly limited, and examples thereof include the following methods. First, the decellularized ECM sterilized by gamma radiation is enclosed in a closed chamber, and the circulatory circuit is connected to the vein and portal vein. While circulating the cell culture medium, various cells (liver parenchymal cells, vascular endothelial cells, etc.) are loaded from the blood vessels into the decellularized ECM according to a specific order, injection speed, and injection pressure. After several days of circulatory culture, the various cells take an ordered three-dimensional structure and are attached to the decellularized ECM, resulting in recellularization.

The scaffold of this embodiment may contain components other than decellularized ECM. Examples of such components include hydrogel. When the scaffold contains hydrogel, the mechanical stability of the scaffold is increased, and the shape of the scaffold is easily maintained even after it is implanted in a living body.

A hydrogel is a gel containing water and a polymer. The polymer is not limited as long as it is a biocompatible polymer, and may be crosslinked or uncrosslinked. Specifically, examples of the polymer include hydrophilic polymers such as polyacrylamide, polyacrylic acid or its salts, polyhydroxyethyl methacrylate, polyvinyl alcohol, polylactic acid, and polyglycolic acid, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, polysaccharides such as starch, glycogen, agarose, pectin, and cellulose, proteins such as collagen, gelatin, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, and Matrigel (registered trademark), and glycoproteins such as proteoglycans. The hydrogel can be placed at the desired position of the decellularized ECM using a syringe or the like.

The decellularized ECM of this embodiment may be sterilized. Sterilization methods may be known methods, such as sterilization with antibiotics (penicillin, ampicillin, tetracycline, streptomycin, gentamicin, ampholycin B, etc.), autoclave sterilization, UV irradiation sterilization, gamma ray irradiation sterilization, ozone sterilization, ethylene oxide gas (EOG) sterilization, or combinations thereof.

<Hydroxyl Group-Containing Polymer (I)>
The hydroxyl-containing polymer (I) of this embodiment has a group represented by the above formula (I). In formula (I), the carbon number of Z is preferably 3 to 6, and more preferably 3 to 5. The number of hydroxyl groups that Z has is preferably 2 or 3, and more preferably 2.

The hydroxyl-containing polymer (I) is not particularly limited as long as it is a polymer having a group represented by the above formula (I). The hydroxyl-containing polymer (I) may be a polyester compound, a polyether compound, a polyamide compound, a polyimide compound, a polyamideimide compound, a polyamic acid compound, a polyvinyl compound, or a combination thereof. The hydroxyl-containing polymer (I) may be a homopolymer or a copolymer. The group represented by formula (I) may be bonded to the end of the hydroxyl-containing polymer (I) or to a portion other than the end. When the hydroxyl-containing polymer (I) has a main chain and a graft chain, the group represented by formula (I) may be bonded to the main chain or to the graft chain. In addition, a polymer obtained by introducing a group represented by formula (I) into a natural polymer such as gelatin, hyaluronic acid, or alginic acid can also be used as the hydroxyl-containing polymer (I).

The solubility of the hydroxyl-containing polymer (I) in 100 ml of water at 25°C is preferably 5 g or more, and more preferably 10 g or more. There is no particular upper limit to the solubility of the hydroxyl-containing polymer (I) in water at 25°C, and the polymer may be one that can be mixed with water in any ratio.

The hydroxyl-containing polymer (I) preferably contains 0.0001 mol/g or more of the group represented by formula (I), more preferably 0.001 mol/g or more, even more preferably 0.0025 mol/g or more, and even more preferably 0.005 mol/g or more. The unit mol/g is the number of moles of the group represented by formula (I) per gram of the hydroxyl-containing polymer (I).

It is preferable that the hydroxyl-containing polymer (I) contains a structural unit derived from a monomer having a group represented by formula (I) and an ethylenically unsaturated group (hereinafter also referred to as monomer (I)). In addition, the "structural unit derived from monomer (I)" refers not only to a structural unit obtained by polymerizing monomer (I), but also to a structural unit obtained by a method other than polymerizing monomer (I), as long as the structural unit has the same structure as the structural unit obtained by polymerizing monomer (I). For example, a structural unit obtained by deprotecting the hydroxyl group in the Z group of monomer (I) after polymerization of monomer (I) is the same structural unit as that obtained by polymerizing monomer (I), and is therefore included in the "structural unit derived from monomer (I)". In addition, when the Z group of monomer (I) has multiple hydroxyl groups, a monomer in which two of the hydroxyl groups form a cyclic ether structure through intramolecular dehydration is polymerized, and then the structural unit obtained by hydrolyzing the cyclic ether structure with an acid or the like to return it to a hydroxyl group is included in the "structural unit derived from monomer (I)" because it has the same structure as when monomer (I) is polymerized.

The monomer (I) is preferably a compound represented by the following formula (IA):

（式（ＩＡ）において、Ｚは、式（Ｉ）のＺと同じ意味であり、Ｒは、水素原子又はメチル基を表す。）

(In formula (IA), Z has the same meaning as Z in formula (I), and R represents a hydrogen atom or a methyl group.)

（式（ＩＩ）において、Ｒ^１は－ＣＨ（ＯＨ）ＣＨ_２ＯＨ又は－ＣＨ_２ＯＨであり、Ｒ^２は、水素原子又は－ＣＨ_２ＯＨであり、Ｒ^３は、水素原子又は－ＣＨ_２ＯＨであり、＊は式（ＩＩ）で表される基の結合位置を表す。） Z is preferably a group represented by the following formula (II).

(In formula (II), R ¹ is -CH(OH)CH ₂ OH or -CH ₂ OH, R ² is a hydrogen atom or -CH ₂ OH, R ³ is a hydrogen atom or -CH ₂ OH, and * indicates the bonding position of the group represented by formula (II).)

As the monomer (I), glycerol mono(meth)acrylate is preferred.

The structural unit derived from monomer (I) also includes a structural unit derived from a monomer that can be (co)polymerized and then treated with an acid to generate a hydroxyl group, such as monomer (IB) below.

（式（ＩＢ）中、Ｒ^１１は、水素原子又はメチル基を表し、Ｒ^１３及びＲ^１４は、それぞれ独立に水素原子又は炭素数１～５のアルキル基を表す）

(In formula (IB), R ¹¹ represents a hydrogen atom or a methyl group, and R ¹³ and R ¹⁴ each independently represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.)

When the monomer (IB) is polymerized and then treated with acid, the dioxolane ring is hydrolyzed, generating two hydroxyl groups in the structural unit.

The hydroxyl group-containing polymer (I) may be a copolymer of the monomer (I) and another monomer, such as a compound having an ethylenically unsaturated group and an alkylene glycol group or a polyalkylene glycol group.
Further, examples of other monomers include neutral hydrophilic monomers such as (meth)acrylamide, N-alkyl(meth)acrylamides (for example, N,N-dimethyl(meth)acrylamide), N-vinylpyrrolidone, N-vinylpyrrolidinone, N-vinylacetamide, N-vinylformamide, hydroxyethyl(meth)acrylate, and hydroxypropyl(meth)acrylate; anionic hydrophilic monomers such as (meth)acrylic acid or a salt thereof, maleic acid or a salt thereof, fumaric acid or a salt thereof, itaconic acid or a salt thereof, (meth)acrylamidomethylpropanesulfonic acid or a salt thereof, vinylphosphonic acid or a salt thereof, and vinylbenzoic acid or a salt thereof; and cationic hydrophilic monomers such as 3-aminopropyl(meth)acrylamide (APMA), (meth)acrylamidopropyltrimethylammonium chloride (MAPTAC), and N,N-dimethylaminoethyl(meth)acrylate.
Examples of other monomers include hydrophobic monomers such as methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, methoxyethyl (meth)acrylate, and styrene, and reactive group-containing monomers such as glycidyl (meth)acrylate, isopropenyl oxazoline, vinyl oxazoline, and maleic anhydride.
The other monomers may be used alone or in combination of two or more.
The content of the structural unit derived from a monomer having a carboxy group and an ethylenically unsaturated group in the hydroxyl group-containing polymer (I) may be less than 1% by mass, 0.8% by mass or less, or 0.5% by mass or less, based on the total amount of the hydroxyl group-containing polymer (I). The structural unit derived from a monomer having a carboxy group and an ethylenically unsaturated group may be a structural unit obtained by polymerizing the monomer, but may also be a structural unit obtained by a method other than polymerizing the monomer, as long as it has the same chemical structure (for example, a structural unit obtained by polymerizing a corresponding ester compound, hydrolyzing the ester group to give a carboxy group, etc.).

An example of a compound having an ethylenically unsaturated group and an alkylene glycol group or a polyalkylene glycol group is a compound represented by the following formula (III).

In the above formula (III), A represents a hydrogen atom or _an organic group having 1 to 20 carbon atoms, X represents -C(=O)-, _-CH2- , _-CH2CH2- , _-CH2CH2CH2- or a single bond _, Q represents an alkylene group _having 2 to 20 carbon atoms, n represents a repeating unit of an alkylene oxide group and is a number from 1 to 200, and _Ak represents a hydrogen atom or an organic group having 1 to 20 carbon atoms which may have a substituent.

Each of the A and A _k is preferably a hydrogen atom or an organic group having 1 to 12 carbon atoms, more preferably a hydrogen atom or an organic group having 1 to 8 carbon atoms, and more preferably a hydrogen atom or an organic group having 1 to 4 carbon atoms. When the organic group has a substituent, it is more preferable that the number of carbon atoms including the substituent is within the above range. Each of the A and A _k is even more preferably a hydrogen atom or a methyl group.

The above Q is preferably an alkylene group having 2 to 5 carbon atoms, and more preferably an ethylene group, an n-propylene group, or an isopropylene group. Q may contain only one type of alkylene group, or may contain two or more types of alkylene groups. When Q contains an ethylene group, it is preferable that 50 to 100 mol % of all alkylene glycol units in the polyalkylene glycol group are ethylene groups, more preferably 80 to 100 mol % are ethylene groups, and more preferably 90 to 100 mol % are ethylene groups.

m may be the average number of moles added for the entire compound represented by formula (III), preferably 1 to 100, more preferably 5 to 80, and even more preferably 10 to 60.
When X is represented by -C(=O)-, examples of the compound include adducts of alkylene oxides having 1 to 100 carbon atoms to (meth)acrylic acid: esters of (meth)acrylic acid and alkoxypolyalkylene glycols obtained by adding alkylene oxides having 1 to 100 carbon atoms to alkyl alcohols having 1 to 30 carbon atoms, such as methanol, ethanol, 2-propanol, and 1-butanol.
When X is represented by -CH ₂ -, -CH ₂ CH ₂ -, or -CH ₂ CH ₂ CH ₂ -, examples of the compound include compounds having a structure in which 1 to 100 moles of alkylene oxide are added to unsaturated alcohols such as allyl alcohol, methallyl alcohol, 2-buten-1-ol, 2-methyl-2-buten-1-ol, 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, 2-methyl-3-buten-2-ol, and vinyl alcohol, and one or more of these may be used.
Further, examples of the compound represented by formula (III) include polyalkylene oxide chain-containing (meth)acrylic acid esters such as 2-ethylhexyl carbitol (meth)acrylate, and alkoxyalkyl (meth)acrylic acid esters such as methoxybutyl (meth)acrylate, methoxyethyl (meth)acrylate, and ethoxybutyl (meth)acrylate.

The content of the structural unit derived from the monomer (I) in the hydroxyl-containing polymer (I) may be 1 to 100% by mass, or may be 5 to 100% by mass. When the hydroxyl-containing polymer (I) contains a structural unit derived from a compound having an ethylenically unsaturated group and an alkylene glycol group or a polyalkylene glycol group, the content of the structural unit may be 30 to 95% by mass based on the total amount of the hydroxyl-containing polymer (I).

For example, when the hydroxyl-containing polymer (I) has structural units derived from the monomer (I), a method for producing the hydroxyl-containing polymer (I) includes polymerizing a monomer composition containing the monomer (I) and, as an optional component, another monomer for copolymerization with the monomer (I).

A polymerization initiator can be used when polymerizing the monomer composition.

As the polymerization initiator, a radical polymerization initiator (particularly a thermal polymerization initiator) is preferable, and examples of the peroxide-based initiator include benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, diisopropyl peroxydicarbonate, cyclohexanone peroxide, t-hexylperoxy-2-ethylhexanoate (trade name: Perhexyl O (registered trademark)), 1,1-di(t-hexylperoxy)cyclohexane (trade name: Perhexa HC (registered trademark)), cumene hydroperoxide, t-butyl hydroperoxide, methyl ethyl ketone peroxide, and diisopropylbenzene hydroperoxide. Polymerization initiators: azo compound-based polymerization initiators such as 2,2'-azobisisobutyronitrile, 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2,3-dimethylbutyronitrile), 2,2'-azobis-(2-methylbutyronitrile), 2,2'-azobis(2,3,3-trimethylbutyronitrile), 2,2'-azobis(2-isopropylbutyronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 2,2'-4-methoxy-2,4-dimethylvaleronitrile), 2-(carbamoylazo)isobutyronitrile, 4,4'-azobis(4-cyanovaleric acid), and dimethyl-2,2'-azobisisobutyrate.
These may be used alone or in combination of two or more kinds.

The amount of the polymerization initiator used is not particularly limited, but is usually preferably 0.001 to 20 parts by mass, more preferably 0.01 to 10 parts by mass, per 100 parts by mass of the raw material monomers of polymer (A).

The polymerization initiator may be added to the monomer composition all at once before the reaction is carried out, or may be added in two or more portions during the reaction (divided addition). In the case of divided addition, a portion of the polymerization initiator may be added to the monomer composition to start the reaction, and then the polymerization initiator may be intermittently added to the remaining monomer composition during the reaction.

The monomer composition may contain a solvent. That is, the monomer composition may be polymerized in the presence of a solvent. The solvent is not particularly limited, and may include water and organic solvents, with water being preferred. When the monomer contains a solvent, the content of the monomer in the monomer composition (the content of monomer (I), or, when other monomers are used, the total amount of monomer (I) and other monomers) may be 0.1 to 50% by mass, 0.5 to 30% by mass, or 1 to 10% by mass. In this case, the hydroxyl group-containing polymer (I) is obtained as a solution of the hydroxyl group-containing polymer (I).

The weight average molecular weight of the hydroxyl group-containing polymer (I) is not particularly limited, but may be 5,000 to 2,000,000 in order to further increase water solubility. The weight average molecular weight can be measured by gel permeation chromatography (GPC).

<Methods for producing biomaterials and treating scaffolds>
In the method for producing a biomaterial of this embodiment, a scaffold containing decellularized ECM is treated with the above-mentioned hydroxyl-containing polymer (I). For example, a method for treating the scaffold may be to contact the surface of the scaffold with the hydroxyl-containing polymer (I).

The hydroxyl-containing polymer (I) may be brought into contact with the scaffold in the form of a solution (treatment liquid) in which the hydroxyl-containing polymer (I) is dissolved in a solvent. The solvent in the solution is preferably water or a mixed solvent of water and a hydrophilic solvent. In other words, the solution may be an aqueous solution. The proportion of water in the solvent in the aqueous solution is preferably 80% by mass or more, more preferably 90% by mass or more, and even more preferably 95% by mass or more.

The pH of the aqueous solution of this embodiment can be appropriately selected depending on the type of organ or tissue from which the decellularized ECM is derived, but is preferably 6.5 to 8.0, and more preferably 7.0 to 7.5. The aqueous solution of this embodiment may be one in which the hydroxyl-containing polymer (I) is dissolved in physiological saline.

The content of the hydroxyl group-containing polymer (I) in the aqueous solution may be 0.01 to 50% by mass, 0.05 to 30% by mass, or 0.05 to 10% by mass. The content of the solvent in the aqueous solution may be 50% by mass or more, 70% by mass or more, or 90% by mass or more.

The aqueous solution contains a buffer. The buffer is not particularly limited, but examples thereof include borate buffer, phosphate buffer, carbonate buffer, citrate buffer, acetate buffer, Tris buffer, etc. These buffers may be used alone or in any combination of two or more. Examples of borate buffer include boric acid, or borates such as alkali metal borate and alkaline earth metal borate. Examples of phosphate buffer include phosphoric acid, or phosphates such as alkali metal phosphate and alkaline earth metal phosphate. Examples of carbonate buffer include carbonic acid, or carbonates such as alkali metal carbonate and alkaline earth metal carbonate. Examples of citrate buffer include citric acid, or alkali metal citrate and alkaline earth metal citrate. In addition, hydrates of borates or phosphates may be used as borate buffers or phosphate buffers. More specific examples of the borate buffer include boric acid or a salt thereof (sodium borate, potassium tetraborate, potassium metaborate, ammonium borate, borax, etc.); the phosphate buffer includes phosphoric acid or a salt thereof (disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, trisodium phosphate, dipotassium phosphate, calcium monohydrogen phosphate, calcium dihydrogen phosphate, etc.); the carbonate buffer includes carbonic acid or a salt thereof (sodium hydrogen carbonate, sodium carbonate, ammonium carbonate, potassium carbonate, calcium carbonate, potassium hydrogen carbonate, magnesium carbonate, etc.); the citrate buffer includes citric acid or a salt thereof (sodium citrate, potassium citrate, calcium citrate, sodium dihydrogen citrate, disodium citrate, etc.); the acetate buffer includes acetic acid or a salt thereof (ammonium acetate, potassium acetate, calcium acetate, sodium acetate, etc.). Among these buffers, the phosphate buffer (particularly a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate) is preferred.

The content of the buffering agent in the aqueous solution may be appropriately selected depending on the type of organ or organs that are the raw material for the decellularized ECM, the animal species, etc., and is not particularly limited. Based on the total volume of the aqueous solution, the total content of the buffering agent is 0.01 to 15 w/v%, preferably 0.05 to 10 w/v%, more preferably 0.1 to 7.5 w/v%, and particularly preferably 0.5 to 5 w/v%.

When the scaffold is treated using the treatment solution, for example, the scaffold may be immersed in the treatment solution or the treatment solution may be brought into contact with a desired position of the scaffold. The desired position includes a portion that comes into contact with blood during blood perfusion at the time of transplantation or after transplantation, for example, a portion of the decellularized ECM derived from a blood vessel (artery, vein, portal vein, capillary blood vessel, etc.). When the decellularized ECM has a portion derived from a blood vessel, a specific method of contacting the treatment solution includes injecting the treatment solution into the lumen of the portion derived from a blood vessel with a syringe or the like, or circulating the solution within the lumen of the portion derived from a blood vessel. Circulation may be performed by transporting the treatment solution with a pump or the like. The portion derived from a blood vessel of the decellularized ECM may have endothelial cells or the like engrafted thereon before treatment with the hydroxyl group-containing polymer (I).
The surface of the scaffold may be modified by the hydroxyl group-containing polymer (I) by the above treatment. In addition, the above treatment may not only modify the surface, but also modify the interior of the scaffold by penetrating the hydroxyl group-containing polymer (I) into the interior.

The biomaterials produced by the manufacturing method of this embodiment can be used as transplant materials, artificial organs, and bioreactors, without any particular limitations.

<Synthesis Example 1>
A 200 mL glass separable flask equipped with a thermometer, a gas inlet tube, a reflux condenser, and a stirrer was charged with 10 g of glycerol monoacrylate (hereinafter referred to as GLMA) and 86 g of pure water, and the temperature was raised to 97° C. while stirring under a nitrogen stream. Next, 1 g of a 0.5% by mass aqueous solution of azo-based initiator VA086 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to start polymerization. The start of polymerization was confirmed by a reflux state caused by polymerization heat, and while heating to maintain the reflux state, 1 g of a 0.5% by mass aqueous solution of VA086 was added 2 hours, 4 hours, and 6 hours after the start of polymerization. The reaction was completed 8 hours after the start of polymerization, and an aqueous solution of polyglycerol acrylate (hereinafter referred to as PGLMA) (solid content 10% by mass) was obtained.

<Synthesis Example 2>
In a 200 mL glass separable flask equipped with a thermometer, a gas inlet tube, a reflux condenser, and a stirrer, 60 g of pure water was charged and heated to 80° C. under stirring. Next, 3 g of glycerol monomethacrylate (hereinafter referred to as GLMMA), 36 g of Shin-Nakamura Chemical's methoxypolyethylene glycol #400 acrylate (hereinafter referred to as AM-90G), 20 g of a 2 mass% aqueous sodium persulfate solution (hereinafter referred to as 2% NaPS), 0.76 g of a 35 mass% aqueous sodium hydrogensulfite solution (hereinafter referred to as 35% SBS), and 20 g of pure water were dropped from separate dropping nozzles into the pure water under stirring. The dropping times of each solution were 180 minutes for GLMMA and 180 minutes for AM-90G, 210 minutes for 2% NaPS, 180 minutes for 35% SBS, and 180 minutes for pure water. The above-mentioned droppings were all started at the same time, and the dropping speed was constant and continuously dropped. After the droppings were all completed, the reaction solution was kept at 80°C for another 30 minutes to mature and complete the polymerization. The obtained reaction solution was diluted with pure water to prepare an aqueous solution of a copolymer (a copolymer of GLMMA and AM-90G) with a solid content concentration of 10% by mass.

Example 1
The liver removed from the rat was perfused with phosphate buffered saline to drain the remaining blood in the blood vessels. The rat liver was then frozen and stored at -80°C, and thawed when used. The decellularized ECM was prepared from the thawed rat liver by the perfusion method. The decellularized ECM was then sterilized by gamma irradiation. 20 ml of a solution obtained by diluting 10% by mass of PGLMA aqueous solution 100 times with phosphate buffered saline (PBS) was injected into the prepared rat decellularized ECM, and incubated at 37°C for 1 hour to prepare a sample. 10 ml of blood was collected from a pig, and immediately injected into the part derived from the blood vessel in the obtained sample, and allowed to stand for 10 minutes. After standing, 30 ml of PBS was perfused to wash out the blood, and the remaining blood in the decellularized ECM and the formation of a blood clot were compared. The observation results are summarized in Table 1.

Example 2
The same operation as in Example 1 was carried out, except that "20 ml of a solution obtained by diluting a 10% by mass aqueous solution of PGLMA 100 times with PBS" in Example 1 was changed to "20 ml of a solution obtained by diluting a 10% by mass aqueous solution of the copolymer of Synthesis Example 2 10 times with PBS". The observation results are summarized in Table 1.

<Comparative Example 1>
The same operations as in Example 1 were carried out, except that "20 ml of a solution obtained by diluting a 10% by weight aqueous solution of PGLMA 100 times with PBS" in Example 1 was changed to "20 ml of PBS." The observation results are summarized in Table 1.

<Comparative Example 2>
The same operations as in Example 1 were carried out except that "20 ml of a solution obtained by diluting a 10% by mass aqueous solution of PGLMA 100 times with PBS" was used instead of "20 ml of a solution obtained by diluting a 10% by mass aqueous solution of polyvinylpyrrolidone (PVP) 10 times with PBS". The observation results are summarized in Table 1. Polyvinylpyrrolidone is a polymer that is frequently used as a biocompatible material.

Example 3
A decellularized ECM of a porcine liver was prepared by carrying out the same method as in Example 1 on a liver extracted from a pig. The prepared decellularized ECM was subjected to gamma ray sterilization, and recellularization was carried out to prepare a recellularized sample. An artificial blood vessel was connected to each of the portal vein and vein-derived portions of the prepared sample, and a treatment liquid was transferred through the artificial blood vessel with a pump to circulate within the sample. The treatment liquid was a 100-fold dilution of an aqueous solution of PGLMA (solid content 10% by mass) of Synthesis Example 1 with physiological saline. The portal vein and vein of the treated sample were anastomosed side-to-side to the portal vein and vein of a donor pig, and blood was reperfused into the sample. Angiography was performed 2 hours after the start of reperfusion, confirming blood flow within the sample, and no thrombus was observed.

Claims (10)

A method for producing a biomaterial, comprising a step of treating a scaffold containing a decellularized extracellular matrix with a hydroxyl-containing polymer having a structural unit derived from glycerol mono(meth)acrylate . The method for producing a biomaterial according to claim 1, wherein the scaffold comprises cells engrafted on the decellularized extracellular matrix. The method for producing a biomaterial according to claim 1 or 2, further comprising a step of recellularizing the treated scaffold. The method for producing a biomaterial according to any one of claims 1 to 3, wherein the decellularized extracellular matrix is derived from an organ or blood vessel. The method for producing a biomaterial according to any one of claims 1 to 4, wherein the decellularized extracellular matrix is derived from the liver or a part of the liver. The method for producing a biomaterial according to any one of claims 1 to 5 , wherein the treatment is performed with a solution containing the hydroxyl group-containing polymer. The method for producing a biomaterial according to claim 6, wherein the decellularized extracellular matrix has a portion derived from a blood vessel, and the treatment is carried out by injecting the solution into the lumen of the portion derived from a blood vessel or circulating the solution within the lumen of the portion derived from a blood vessel. 1. A method for treating scaffolding, comprising the steps of:
A method comprising the step of treating a scaffold comprising a decellularized extracellular matrix with a hydroxyl-containing polymer having structural units derived from glycerol mono(meth)acrylate . A treatment solution for treating a scaffold comprising a decellularized extracellular matrix, comprising:
A treatment liquid comprising a hydroxyl group-containing polymer having a structural unit derived from glycerol mono(meth)acrylate . The processing solution according to claim 9 , further comprising a buffer .