JP4187917B2 - Method for producing glycosaminoglycan-collagen complex for tissue regeneration matrix - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention, tissue regeneration matrix for glycosaminoglycans of cartilage, etc. (GAG) - a method of manufacturing a collagen composite.
[0002]
[Prior art]
Although cartilage is an extremely difficult organ to regenerate, the development of cartilage regeneration material is eagerly desired as a countermeasure for joint diseases such as osteoarthritis due to aging and sports disorders. The main component of the cartilage tissue is proteoglycan rich in type II collagen, hyaluronic acid, and chondroitin sulfate chains. To date, hydroxyapatite-type II collagen, hyaluronic acid, and chondroitin sulfate self-assembled bodies have been shown to promote cartilage regeneration to some extent in vivo.
[0003]
In addition, hyaluronic acid has been crosslinked with a crosslinking agent (diamine, diepoxy, epichlorohydrin), crosslinked with a condensing agent (water-soluble carbodiimide), and a hydrophobic group has been introduced into the carboxyl group of hyaluronic acid. Things have been reported.
[0004]
In addition, preparation of glycosaminoglycan (GAG) -collagen complex can be achieved by immobilizing GAG on a collagen matrix obtained by freeze-drying a collagen solution (JSPieper et al., “Biomaterials”, 2000, 21: 581-593) The technique of chemically cross-linking the GAG-collagen complex has been taken (JP-A-6-505642, JP-A-7-196704).
[0005]
[Problems to be solved by the invention]
Regarding the complex of glycosaminoglycan and collagen, a method of immobilizing glycosaminoglycan on a collagen sponge using a condensing agent and a method of crosslinking a polyion complex of glycosaminoglycan and collagen using a condensing agent However, when these methods are used, there is a problem that molding is difficult.
[0006]
The present invention can be used as various tissue regeneration matrices, such as cartilage, liver, blood vessels, nerves, etc., in a system that can be molded in a mold, and exhibits very similar physical properties to each tissue and excellent biological function. The purpose is to develop tissue regeneration materials.
[0007]
[Means for Solving the Problems]
Hyaluronic acid is one of the polysaccharides called glycosaminoglycans, and type II collagen is one of the polycations. It is known that when a hyaluronic acid aqueous solution and a type II collagen hydrochloric acid solution are mixed, a polyion complex (PIC) is formed. This is because the molecular chain of hyaluronic acid is negatively charged (polyanion) due to dissociation of the carboxyl group, while type II collagen is positively charged (polycation) as a whole polymer chain (isoelectric point). 8 or more).
[0008]
Water-soluble carbodiimide (WSC: 1-ethyl-3- (3- (dimethylaminopropyl) carbodiimide), which is a condensing agent, stably activates a carboxyl group under the conditions of pH 4 to 8, and the active intermediate is It is known to react with amino and hydroxyl groups to form amides and esters.
[0009]
As a result of studying preparation of a glycosaminoglycan-polycation complex (hybrid) gel, the present inventor formed a polyion complex between glycosaminoglycan and polycation collagen using a specific salt concentration. Can be obtained by a condensation reaction using WSC by cross-linking with a condensing agent while suppressing the formation of a glycosaminoglycan- collagen complex gel, and various tissues such as cartilage, liver, blood vessels, nerves, etc. It was found that an excellent recycled material can be obtained.
[0010]
The present invention, glycosaminoglycan and tissue regeneration matrix for glycosaminoglycans collagen crosslinked by a condensation reaction - that provides collagen composite.
[0011]
That is , the present invention relates to a method for producing a glycosaminoglycan-collagen complex in which glycosaminoglycan and collagen are crosslinked by a condensation reaction.
By suppressing the formation of a polyion complex between glycosaminoglycan and collagen by the presence of a salt that dissolves in water in a mixed aqueous solution of glycosaminoglycan and collagen, and by a condensation reaction using water-soluble carbodiimide as a condensing agent. A method for producing a glycosaminoglycan- collagen complex for tissue regeneration matrix, which is crosslinked.
[0012]
Further, the present invention, the glycosaminoglycan, which comprises using a combination of water-soluble carbodiimide and 2-hydroxy succinimide as condensing agent - a method for producing a collagen composite.
[0013]
The present invention also provides the glycosaminoglycan described above, wherein the glycosaminoglycan is hyaluronic acid, the collagen is type II collagen, the salt is NaCl, and the salt concentration is 0.4 ± 0.05 M. This is a method for producing a saminoglycan- collagen complex.
[0014]
The present invention also provides the glycosaminoglycan described above, wherein the sol-form aqueous solution before the condensation reaction is poured into a mold, and then the condensation reaction is performed to form the shape of an ear, nose, or cartilage defect. -A method for producing a collagen complex.
[0015]
The present invention also glycosaminoglycan formed by crosslinking - to a matrix of hydrogels collagen composite was washed with water to remove ions of the salt in the matrix, water-soluble carbodiimide, and the by-products A method for producing the glycosaminoglycan- collagen complex described above.
[0016]
The glycosaminoglycan, hyaluronic acid, chondroitin sulfate, heparin, keratan sulfate, Ru can be used both to polysaccharides such as heparan sulfate. The collagen has 10 several types, types have a particularly limited. Using hyaluronic acid (abbreviation HyalA) as glycosaminoglycan representative of the case of using type II collagen as collagen, the present invention will be described in detail below.
[0017]
In the method of the present invention, the conditions for cross-linking are as follows: (1) cross-linking at a salt concentration that does not form PIC, that is, salt concentration that does not form PIC by electrostatic interaction; Crosslinking is performed at pH 4 or higher, which is highly stable.
[0018]
In water containing no salt such as NaCl, hyaluronic acid is negatively charged as a molecule by a carboxyl group (polyanion), while collagen is positively charged as a whole molecule. When a salt-free hyaluronic acid aqueous solution and a salt-free collagen aqueous solution are mixed, precipitation occurs as a polyion complex due to the negative charge of hyaluronic acid and the positive charge of collagen.
[0019]
When an appropriate amount of salt is present when mixing an aqueous solution of hyaluronic acid and collagen, a polyion complex is not formed because ions generated by dissolution of the salt cancel the charge of hyaluronic acid and collagen. The salt used for this principle can be any salt that dissolves in water, for example, NaCl, CaCl ₂ , Na ₂ SO _2, etc. However, the optimum salt concentration may vary slightly depending on the type of salt. The crosslinking with water-soluble carbodiimide is performed at a salt concentration that does not form this polyion complex.
[0020]
Immediately after crosslinking, salt is still present in the gel in the form of salt ions. To remove this salt, wash with a large excess of water. Unless the ions contained in the gel are removed, it is not possible to form a crosslink by a polyion complex. That is, a bond by a covalent bond + electrostatic interaction is not formed.
[0021]
The advantages of crosslinking with a condensing agent are (1) that the condensing agent can be removed by washing after the cross-linking, so that the toxicity of the cross-linking agent does not have to be a problem, and (2) the biodegradable hydrogel has (3) The degree of crosslinking can be controlled (pore size control) by controlling the concentration of the condensing agent, and (4) TGF-, which is a protein that has a function of accelerating tissue repair in a hydrogel. Cytokines such as β (Transforming Growth Factor beta), fibroblast growth factor FGF (accelerating fibroblast growth), vascular endothelial growth factor VEGF (Vascular Endothelial Growth Factor) accelerating proliferation of vascular endothelial cells It is also possible to encapsulate and release it slowly.
[0022]
The hyaluronic acid-type II collagen complex of the present invention has a water content of 90 to 99% by weight, a hyaluronic acid (polyanion) / collagen (polycation) ratio (weight) of 50/50 to 5/95, and a porosity of 90-99% by volume. The crosslink density corresponds to the water content.
[0023]
FIG. 1 is a schematic view of a composite matrix immediately after forming a glycosaminoglycan- collagen ( polycation ) gel by the method of the present invention. Immediately after the formation of the composite matrix, salt (NaCl in the case of FIG. 1) is contained in the composite matrix. Therefore, since the Na ions and Cl ions are present in the matrix, the matrix is crosslinked only by covalent bonds. . Next, the composite matrix is placed in a large excess of water to remove Na ions and Cl ions inside the matrix.
[0024]
FIG. 2 is a schematic diagram showing the structure (covalent bond + electrostatic interaction) when a gel of hyaluronic acid and type II collagen swells in water. This schematic diagram shows the structure after washing the matrix with a large excess of water to remove by-products such as Na ions, Cl ions, water soluble carbodiimide and urea in the matrix. In this case, it is considered that a bond is formed in addition to the covalent bond (amide and ester bond) formed by the water-soluble carbodiimide. This bond is due to the electrostatic interaction between COO- and NH3 ⁺ . This means that since Na ions and Cl ions are removed from the inside of the matrix, the unreacted carboxyl group and amino group form a complex and become an apparent crosslinking point.
[0025]
FIG. 3 is a graph showing an evaluation of PIC formation at various pH of a hyaluronic acid solution by examining the transmittance (%) of light at 500 nm when the mixing ratio of hyaluronic acid and type II collagen is 1: 1. . This figure is an examination of the pH at which a polyion complex is not formed when hyaluronic acid and a collagen aqueous solution (each having a concentration of 1.25%) are mixed at a weight ratio of 1: 1. The horizontal axis represents the pH of the hyaluronic acid aqueous solution, and the vertical axis represents the light transmittance of 500 nm (using a spectrophotometer [UV / Vis spectrometer]).
[0026]
When the transmittance is 0%, it means that no light is transmitted, which indicates that a polyion complex is formed. It shows that a polyion complex is not formed as it approaches 100%. When the pH of the aqueous hyaluronic acid solution is between 6 and 1.5, the transmittance is almost 0%, so that a polyion complex is formed. It can be seen that when PH becomes 1, the transmittance increases to about 70%, so that the polyion complex is not formed.
[0027]
FIG. 4 is also a graph in which PIC formation at various salt concentrations (in the case of FIG. 1, NaCl) was evaluated by examining the transmittance (%) of light at 500 nm. This figure is an examination of the salt concentration at which polyion complexes are not formed when hyaluronic acid and collagen aqueous solution (each having a concentration of 1.25%) are mixed at a weight ratio of 1: 1. . The horizontal axis represents the NaCl concentration contained in the mixed solution of hyaluronic acid and collagen, and the vertical axis represents the light transmittance of 500 nm.
[0028]
When the transmittance is 0%, it means that no light is transmitted, which indicates that a polyion complex is formed. It shows that a polyion complex is not formed so that it approaches 100%. When the NaCl concentration is close to 0.4M, the transmittance sharply increases, and the transmittance shows the maximum value at 0.4M. Therefore, when expressed in terms of 0.4M and its vicinity, numerical values, about 0.4 ± 0.05M. It can be seen that no polyion complex is formed.
[0029]
FIG. 3 and FIG. 4 reveal that the light transmittance (%) is maximized at pH 1 or 0.4 M NaCl concentration, and the formation of PIC is greatly suppressed. The reason for this is that at low pH, the dissociation of the carboxyl group of hyaluronic acid and type II collagen is suppressed (pKa or less), so that a complex cannot be formed with the protonated amino group of type II collagen. Conceivable.
[0030]
On the other hand, the reason why the transmittance has the maximum value when the NaCl concentration is 0.4 M can be considered as follows. That is, it is considered that the addition of salt works to suppress the formation of PIC up to a salt concentration of 0.4M, and collagen precipitates due to the salting out effect when the salt concentration exceeds 0.4M.
[0031]
From the above results, it has been clarified that the optimum conditions under which hyaluronic acid and type II collagen do not form PIC are at pH 1 or a salt concentration of 0.4M. The pH at which the WSC of the condensing agent stably activates the carboxyl group is 4 to 8, and is extremely unstable under pH 1 conditions. Therefore, various concentrations of WSC (0 to 1000 mM) were added under the condition of a salt concentration of 0.4 M for crosslinking.
[0032]
FIG. 5 shows that the obtained gel was washed with a large excess of water to remove the condensing agent, Na ions, and Cl ions from the gel matrix, and the degree of swelling of the lyophilized matrix [Swelling Ratio; It is a graph which shows the relationship between the value of how many times its own weight absorbs water = (weight of wet gel−weight of dry gel) / weight of dry gel] and WSC concentration (mM).
[0033]
This figure shows how the degree of swelling of the complex obtained when the concentration of water-soluble carbodiimide (WSC) is changed when the mixing ratio of hyaluronic acid and collagen is 1: 1. It is a thing. What can be said from this figure is that the content of water contained in the complex can be controlled by controlling the concentration of the condensing agent. A reduction in the degree of swelling means that the content of water contained in the composite is reduced, so that the composite becomes hard.
[0034]
FIG. 6 is a graph showing the relationship between matrix conversion and WSC concentration (mM) when the mixing ratio of hyaluronic acid and type II collagen is changed. This figure shows the results of examining the influence of the concentration of water-soluble carbodiimide (WSC) on the conversion rate. The complex of interest is one having a hyaluronic acid to collagen ratio of 1: 1 to 1: 8. As the concentration of the water-soluble carbodiimide is increased, the concentration reaches approximately 100% at approximately 20 to 30 mM, indicating that a hyaluronic acid-type II collagen complex almost as prepared is obtained.
[0035]
Conversely, it indicates that a complex cannot be obtained quantitatively unless 20 to 30 mM or more of water-soluble carbodiimide is added. When the WSC concentration is 100 mM or more, the conversion rate is further increased. If the molecular weight of hyaluronic acid or the concentrations of hyaluronic acid and collagen are increased, crosslinking is possible even when the water-soluble carbodiimide concentration is lower than 20 mM.
[0036]
FIG. 7 is also a graph showing the relationship between the degree of swelling of the matrix and the WSC concentration (mM). This figure shows that even when the ratio of hyaluronic acid and collagen is changed from 1: 1 to 1: 8, the degree of swelling can be controlled by controlling the concentration of water-soluble carbodiimide.
[0037]
DETAILED DESCRIPTION OF THE INVENTION
In the method of the present invention, when a glycosaminoglycan and collagen ( polycation ) are crosslinked using a water-soluble carbodiimide, crosslinking is performed under conditions that do not form a polyion complex. There are two. One is a pH at which the carboxyl group of glycosaminoglycan (for example, hyaluronic acid) is not dissociated, and the other is an optimum salt concentration at which a polyion complex is not formed. The optimum conditions for not forming a polyion complex are as follows. In the case of a combination of hyaluronic acid and type II collagen, the pH is 1 and the NaCl concentration is 0.4 ± 0.05 M (pH is not adjusted). It is about 5). This value varies somewhat with other combinations of glycosaminoglycan and collagen (for example, chondroitin sulfate and collagen), and the optimum value is determined by each combination.
[0038]
However, when crosslinking is performed using a water-soluble carbodiimide, it is necessary to consider the stability of the water-soluble carbodiimide. Since the pH at which the water-soluble carbodiimide reacts stably is 4 to 8, when a salt is added, crosslinking is possible because the water-soluble carbodiimide is within the range of pH 4 to 8 where the water-soluble carbodiimide is stably present.
[0039]
The water-soluble carbodiimide used is a reagent that stably activates a carboxyl group and reacts with an amino group or a hydroxyl group when the pH is in the range of 4-8. That is, the condition that the water-soluble carbodiimide is stably present and does not form a polyion complex is when the NaCl concentration is 0.4 ± 0.05 M (the pH at this time is approximately 5). The concentration of the water-soluble carbodiimide can be cross-linked even if the molecular weight of glycosaminoglycan or the concentration of glycosaminoglycan and collagen is increased.
[0040]
A combination of 2-hydroxysuccinimide and water-soluble carbodiimide may be used. 2-hydroxysuccinimide is used for increasing the reaction efficiency of the water-soluble carbodiimide, and the concentration of the water-soluble carbodiimide is preferably 1 to 0.1 with respect to 1.
[0041]
The mixing ratio of glycosaminoglycan and collagen can be arbitrarily changed, and changing the mixing ratio changes the physical properties and biological functions. The molecular weight of glycosaminoglycan such as hyaluronic acid can be applied to any molecular weight from tens of thousands to millions in this system.
[0042]
By controlling the concentration of the condensing agent, the content of water contained in the complex can be controlled. When the content of water contained in the composite decreases, that is, the degree of swelling decreases, the composite becomes hard. Whether it is hard or not depends on what type of tissue is made using the composite. For example, when it is desired to rapidly decompose the complex in vivo, it is necessary to lower the concentration of water-soluble carbodiimide and increase the degree of swelling.When a hard material is required, the concentration of water-soluble carbodiimide is reduced. It is necessary to raise. However, since water-soluble carbodiimide is not good for living organisms, it is more preferable to have a low concentration and a high cell growth ability.
[0043]
Immediately after crosslinking, salt ions are present in the form of salt ions (in the case of NaCl, Na ions and Cl ions), so the gel is washed with a large excess of water to remove the salt ions. . As a cleaning method, for example, a gel produced in deionized water having a gel volume of 100 times or more is immersed. By washing, in addition to ions contained in the gel, unreacted water-soluble carbodiimide and by-products such as urea are removed. By removing ions contained in the gel, a bond by a covalent bond + electrostatic interaction is formed.
[0044]
In using the complex of the present invention, a sol-like aqueous solution before the condensation reaction is poured into an appropriate template, and the condensation reaction is performed, so that various shapes such as ears, nose and cartilage defect sites can be obtained. It can be easily formed. As for the method of use, sponge-like or gel-like ones are used. When cartilage is produced in the matrix, chondrocytes are contained. When blood vessels are produced, vascular endothelial cells are introduced and cultured. In addition to being used as a matrix itself, the matrix can also be used as a scaffold for promoting cell proliferation and differentiation. In addition, for use in experiments on cells, etc., after washing in water, it is placed in a phosphate buffer having a salt concentration almost equal to the osmotic pressure in the living body, and the water in the complex is converted into a phosphate buffer. Used after replacement.
[0045]
【Example】
Examples 1-6
Hyaluronic acid (molecular weight = 640,000, manufactured by Seikagaku Corporation) 1.25 w / v% aqueous solution and type II collagen (manufactured by Nitta Gelatin) 1.25 w / v% 0.01 N HCl in a mixed solution of 0.4 M NaCl was added, and a water-soluble carbodiimide aqueous solution as a condensing agent was added dropwise in a state where the pH was about 5. The mixture was sufficiently stirred and degassed, and then allowed to stand at 30 ° C. for 2 hours. Thereafter, the obtained gel was washed with a large excess of water for 2 days to remove the condensing agent, by-products, Na ions, and Cl ions from the matrix of the gel. This was lyophilized for 1 day.
[0046]
The ratio of hyaluronic acid to collagen in each of Examples 1 to 6 and the amount (mM) of added water-soluble carbodiimide were as follows. Example 1 = 1: 8-100, Example 2 = 1: 1-100, Example 3 = 1: 8-20, Example 4 = 1: 4-20, Example 5 = 1: 2-20, Example 6 = 1: 1-20. For example, in Example 1 of 1: 8-100, the ratio of hyaluronic acid to collagen is 1: 8, and the concentration of water-soluble carbodiimide is 100 mM. As controls, TCPS (Tissue Culture PolyStylene; commonly used petri dishes for cell culture) and collagen (cross-linked with 20 mM water-soluble carbodiimide) were used.
[0047]
FIG. 8 shows the results of identification of ester formation by FT-IR for Example 6, the ratio of hyaluronic acid to collagen and the amount of added water-soluble carbodiimide of 1: 1-200 mM, hyaluronic acid and collagen, respectively. Indicates. About Example 6, it can confirm that the covalent bond has formed by chemical reaction.
[0048]
FIG. 9 shows the results obtained by culturing the hyaluronic acid and type II collagen matrix of each example for 7 days at 37 ° C. and counting the number of cells. The hyaluronic acid to collagen ratio of each example— (WSC) Concentration) and the relative growth rate are shown as bar graphs.
[0049]
For the preparation of the composite of each example, a disk-shaped composite having a thickness of 1 mm and a diameter of 15 mm was used. On top of that, 1 × 10 ⁵ chondrocytes taken out from the calf's joint and proliferated were seeded. As the composition of the culture solution at this time, a DMEM medium containing 10% bovine serum (FBS) was used. After seeding the cells on the complex, the culture solution was changed after 4 days, and then the number of cells was counted using Cell Counting Kit (manufactured by Dojin Chemical Co., Ltd.) 3 days later (Total culture days 7 days). did.
[0050]
The data shows the relative number of cells, where the number of cells on TCPS is 1. From this result, the first thing that can be said is that the complex according to the present invention significantly promotes the proliferation of chondrocytes at any ratio (4 to 7 times). Although the number of cells does not increase so much that it is only collagen, it can be seen that it has an excellent cell growth ability by complexing with hyaluronic acid.
[0051]
In addition, the difference in the ratio between hyaluronic acid and collagen is not recognized in this case. The difference in the concentration of water-soluble carbodiimide is not a significant difference. There is a difference in cell morphology. On TCPS and collagen, chondrocytes are in a fibrous form, and on the composite of the example, the chondrocyte has a round shape that is unique to cells. It can be said that the nature is high.
[0052]
The conditions that are most excellent in physical properties and biological functions are those in Example 3. In FIG. 7, when the ratio of hyaluronic acid to collagen is 1: 4 or 1: 8 and the water-soluble carbodiimide is 20 mM, the degree of swelling is minimal. This indicates that the composite contains less water and is harder than the others. That is, it is easy to handle. In addition, in FIG. 9, when Example 3 and Example 4 are compared, it can be said that Example 3 is more biologically active, although there is not much difference in the number of cells. Although Example 6 has the largest number of cells, the strength is weak and it cannot be said to be optimal.
[0053]
【The invention's effect】
The glycosaminoglycan and collagen composite gel of the present invention, such as hyaluronic acid-type II collagen composite gel, can easily form complex shapes such as ears, nose, cartilage defects, etc., and toxicity of the crosslinking agent Therefore, it is expected to be degraded by enzymes in the living body (hyaluronidase, collagenase), and is extremely useful as a tissue regeneration matrix.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing the structure (covalent bond) during gel formation of hyaluronic acid and type II collagen.
FIG. 2 is a schematic diagram showing the structure (covalent bond + electrostatic interaction) of hyaluronic acid and type II collagen gel when swollen in water.
FIG. 3 is a graph in which the formation of PIC at various pH values of a hyaluronic acid solution was evaluated by examining the transmittance (%) of light at 500 nm.
FIG. 4 is a graph in which the formation of PIC at various salt (NaCl) concentrations of a hyaluronic acid solution was evaluated by examining the transmittance (%) of light at 500 nm.
FIG. 5 is a graph showing the relationship between the degree of swelling of hyaluronic acid and type II collagen matrix and the WSC concentration (mM).
FIG. 6 is a graph showing the relationship between the conversion rate of hyaluronic acid and type II collagen matrix and WSC concentration (mM) when the mixing ratio of hyaluronic acid and type II collagen is changed.
FIG. 7 is a graph showing the relationship between the degree of swelling of hyaluronic acid and type II collagen matrix and the WSC concentration (mM) when the mixing ratio of hyaluronic acid and type II collagen is changed.
FIG. 8 is a graph showing the identification of ester formation by FT-IR for the hyaluronic acid and type II collagen matrix of the present invention.
FIG. 9 is a graph showing hyaluronic acid-collagen ratio (WSC concentration) and relative growth rate obtained by culturing chondrocytes using hyaluronic acid and type II collagen matrix obtained in each example.

Claims (5)

In a method for producing a glycosaminoglycan-collagen complex in which glycosaminoglycan and collagen are crosslinked by a condensation reaction,
By suppressing the formation of a polyion complex between glycosaminoglycan and collagen by the presence of a salt that dissolves in water in a mixed aqueous solution of glycosaminoglycan and collagen, and by a condensation reaction using water-soluble carbodiimide as a condensing agent. A method for producing a glycosaminoglycan- collagen complex for tissue regeneration matrix, which comprises crosslinking. The method for producing a glycosaminoglycan- collagen complex according to claim 1, wherein water-soluble carbodiimide and 2-hydroxysuccinimide are used in combination as a condensing agent. Glycosaminoglycan is hyaluronic acid, collagen is type II collagen, a salt is NaCl, glycolide according to claim 1 or 2, wherein the salt concentration is characterized 0.4 ± 0.05 M der Rukoto A method for producing a saminoglycan- collagen complex. The shape of an ear, nose, or cartilage defect is formed by pouring a sol-like aqueous solution before the condensation reaction into a mold and then performing the condensation reaction. Of producing a glycosaminoglycan-collagen complex. Glycosaminoglycan formed by crosslinking - claims the luma trix such a hydrogel collagen composite was washed with water, and removing ions of the salt in the matrix, water-soluble carbodiimide, and the by-products Item 5. The method for producing a glycosaminoglycan- collagen complex according to any one of Items 1 to 4.

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